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1. Timm  A, Steinbüchel  A,     ( 1992 )

Cloning and molecular analysis of the poly(3-hydroxyalkanoic acid) gene locus of Pseudomonas aeruginosa PAO1.

European journal of biochemistry 209 (1)
PMID : 1396693  :   DOI  :   10.1111/j.1432-1033.1992.tb17256.x    
Abstract >>
From genomic libraries, the polyhydroxyalkanoate gene locus of Pseudomonas aeruginosa PAO1 was cloned and characterised at the molecular level. Two genes coding for polyhydroxyalkanoate synthases, phaC1Pa and phaC2Pa, a polyhydroxyalkanoate depolymerase gene, phaDPa, and four adjacent open reading frames (ORF1, ORF2, ORF3 and ORF4) were identified from the nucleotide sequence. Two transcriptional start sites, which were preceded by sequences resembling the Escherichia coli consensus sequences for sigma 54 and sigma 70 promoters, were identified experimentally upstream of phaC1Pa, which was shown by Northern blot analysis to constitute an operon together with phaDPa. A third putative promoter resembling the E. coli consensus sequence for sigma 70-dependent promoters was proposed upstream of phaC2Pa, which is in a bicistronic operon with ORF3. Investigations of rpoN-negative mutants of related strains revealed that polyhydroxyalkanoate accumulation from gluconate required an intact rpoN locus in P. aeruginosa. Complementation experiments revealed multiple evidence that either polyhydroxyalkanoate synthase is involved in polyhydroylkanoate accumulation from gluconate as well as from octanoate. The P. aeruginosa PAO1 polyhydroxyalkanoate gene locus was expressed in the polyhydroxyalkanoate-negative mutant Alcaligenes eutrophus PHB-4 and in the poly(3-hydroxybutyrate)-accumulating strain P. oleovorans DSM1045. It conferred on the latter the ability to synthesize and accumulate polyhydroxyalkanoates consisting of medium-chain-length 3-hydroxyalkanoic acids from unrelated substrates in addition to poly(3-hydroxybutyrate). The sequence of the putative translational product of ORF1 was similar to those of the leukotoxin repressor of Pasteurella haemolytica and to the ORF9 product of Azotobacter vinelandii, and that of ORF4 was similar to the algP product of P. aeruginosa and to eukaryotic histone H1 proteins. The proteins of ORF2 and ORF3 appear to be previously unidentified.
KeywordMeSH Terms
Cloning, Molecular
Genes, Bacterial
2. Duong  F, Lazdunski  A, Cami  B, Murgier  M,     ( 1992 )

Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways.

Gene 121 (1)
PMID : 1427098  :   DOI  :   10.1016/0378-1119(92)90160-q    
Abstract >>
A genetic locus implicated in the synthesis and secretion of alkaline protease (APR) in Pseudomonas aeruginosa has been previously described [Guzzo et al., J. Bacteriol. 172 (1990) 942-948]. The nucleotide sequence of the DNA fragment encoding these functions was determined and revealed the existence of five open reading frames: aprA, the structural gene encoding APR; aprI, which encodes a protease inhibitor; and aprD, aprE, aprF whose products are involved in protease secretion. The AprD, AprE and AprF proteins share significant homology with proteins implicated in secretion of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin. These results provide further evidence for the existence of a specialized secretory system widespread among Gram- bacteria.
KeywordMeSH Terms
Multigene Family
3. Ishimoto  KS, Lory  S,     ( 1992 )

Identification of pilR, which encodes a transcriptional activator of the Pseudomonas aeruginosa pilin gene.

Journal of bacteriology 174 (11)
PMID : 1317379  :   DOI  :   10.1128/jb.174.11.3514-3521.1992     PMC  :   PMC206036    
Abstract >>
Two regulatory mutants of Pseudomonas aeruginosa, R1 and RA, that affect transcription of the pilin gene were isolated. This was done by introducing a plasmid carrying a fusion of the pilin gene's promoter with the lacZ gene into a bank of P. aeruginosa DNA mutagenized with the transposon Tn5G. The block in pilin expression in these mutants was shown to be at the level of transcription, since these mutants did not synthesize either pilin mRNA or pilin antigen. A restriction fragment derived from the R1 mutant that contains the entire transposon plus flanking chromosomal DNA was cloned and used as a probe to screen a cosmid library of P. aeruginosa DNA. Cosmids that could complement the pilin expression defect in both R1 and RA were isolated. The gene inactivated in R1 was sequenced. This gene, designated pilR, encodes an approximately 50-kDa polypeptide which exhibits significant similarity to the NtrC family of response regulators of the two-component regulatory system. PilR contains the amino-terminal aspartic acid residues which are conserved among the response regulators, suggesting that pilin gene transcription is regulated via a phosphotransfer mechanism in which PilR is phosphorylated by an as yet unidentified protein kinase.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
4. Mabilat  C, Lourençao-Vital  J, Goussard  S, Courvalin  P,     ( 1992 )

A new example of physical linkage between Tn1 and Tn21: the antibiotic multiple-resistance region of plasmid pCFF04 encoding extended-spectrum beta-lactamase TEM-3.

Molecular & general genetics : MGG 235 (1)
PMID : 1331747  :   DOI  :   10.1007/bf00286188    
Abstract >>
The genetic environment of plasmid-borne blaTEM mutant genes, encoding nine distinct TEM-type extended-spectrum beta-lactamases, was studied in transconjugants from clinical isolates of enterobacteria. Colony hybridization with probes specific for tnpA and tnpR of Tn3, tnpA and tnpI of Tn21, aacA4, and IS15, and restriction endonuclease analysis of plasmid DNA indicated that the structural genes for the enzymes were always associated with intact or deleted variants of the Tn3 family. Four of the nine blaTEM variants, which account for 62% of 222 isolates in a molecular epidemiological study, were associated with replicons indistinguishable from the epidemic Inc7-M plasmid pCFF04 that carries the blaTEM-3 gene. This suggests that mutant genes were selected from the same prototype plasmid carrying penicillinase genes blaTEM-1 or -2. A 6.6 kb DNA fragment of pCFF04 containing blaTEM-3 was characterized by amplification mapping and sequencing. The results obtained indicated that blaTEM-3 was present on a copy of Tn1 interrupted at the start codon of the transposase by a DNA sequence reminiscent of the inverted repeats of class II transposons. This partial Tn1 copy was in turn, inserted into the transposase gene of a Tn21-like transposon containing an integron expressing an aacA4 gene. The presence of an integron can account for the various assortments of aminoglycoside resistance genes found associated with blaTEM-3.
KeywordMeSH Terms
DNA Transposable Elements
Genetic Linkage
R Factors
5. Zumft  WG, Dreusch  A, Löchelt  S, Cuypers  H, Friedrich  B, Schneider  B,     ( 1992 )

Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase.

European journal of biochemistry 208 (1)
PMID : 1324835  :   DOI  :   10.1111/j.1432-1033.1992.tb17156.x    
Abstract >>
The nosZ genes encoding the multicopper enzyme nitrous oxide reductase of Alcaligenes eutrophus H16 and the type strain of Pseudomonas aeruginosa were cloned and sequenced for structural comparison of their gene products with the homologous product of the nosZ gene from Pseudomonas stutzeri [Viebrock, A. & Zumft, W. G. (1988) J. Bacteriol. 170, 4658-4668] and the subunit II of cytochrome-c oxidase (COII). Both types of enzymes possess the CuA binding site. The nosZ genes were identified in cosmid libraries by hybridization with an internal 1.22-kb PstI fragment (NS220) of nosZ from P. stutzeri. The derived amino acid sequences indicate unprocessed gene products of 70084 Da (A. eutrophus) and 70695 Da (P. aeruginosa). The N-terminal sequences of the NosZ proteins have the characteristics of signal peptides for transport. A homologous domain, extending over at least 50 residues, is shared among the three derived NosZ sequences and the CuA binding region of 32 COII sequences. Only three out of nine cysteine residues of the NosZ protein (P. stutzeri) are invariant. Cys618 and Cys622 are assigned to a binuclear center, A, which is thought to represent the CuA site of NosZ and is located close to the C terminus. Two conserved histidines, one methionine, one aspartate, one valine and two aromatic residues are also part of the CuA consensus sequence, which is the domain homologous between the two enzymes. The CuA consensus sequence, however, lacks four strictly conserved residues present in all COII sequences. Cys165 is likely to be a ligand of a second binuclear center, Z, for which we assume mainly histidine coordination. Of 23 histidine residues in NosZ (P. stutzeri), 14 are invariant, 7 of which are in regions with a degree of conservation well above the 50% positional identity between the Alcaligenes and Pseudomonas sequences. Conserved tryptophan residues are located close to several potential copper ligands. Trp615 may contribute to the observed quenching of fluorescence when the CuA site is occupied.
KeywordMeSH Terms
Genes, Bacterial
6. Collis  CM, Hall  RM,     ( 1992 )

Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase.

Journal of bacteriology 174 (5)
PMID : 1311297  :   DOI  :   10.1128/jb.174.5.1574-1585.1992     PMC  :   PMC206553    
Abstract >>
Deletion of individual antibiotic resistance genes found within the variable region of integrons is demonstrated. Evidence for gene duplications and rearrangements resulting from the insertion of gene units at new locations is also presented. Deletion, duplication, and rearrangement occur only in the presence of the integron-encoded DNA integrase. These events are precise and involve loss or gain of one or more complete insert units or gene cassettes. This confirms the recent definition of gene cassettes as consisting of the gene coding sequences, all except the last 7 bases of the 59-base element found at the 3' end of the gene, and the core site located 5' to the gene (Hall et al., Mol. Microbiol. 5:1941-1959, 1991) and demonstrates that individual gene cassettes are functional units which can be independently mobilized. Both deletions and duplications can be generated by integrase-mediated cointegrate formation followed by integrase-mediated resolution involving a different pair of sites. However, deletion occurs 10 times more frequently than duplication, and we propose that the majority of deletion events are likely to involve integrase-dependent excision of the gene unit to generate a circular gene cassette. The implications of these findings in understanding the evolution of integrons and the spread of antibiotic resistance genes in bacterial populations is discussed.
KeywordMeSH Terms
7. Bissonnette  L, Roy  PH,     ( 1992 )

Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria.

Journal of bacteriology 174 (4)
PMID : 1310501  :   DOI  :   10.1128/jb.174.4.1248-1257.1992     PMC  :   PMC206418    
Abstract >>
Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.
KeywordMeSH Terms
8. Gamper  M, Ganter  B, Polito  MR, Haas  D,     ( 1992 )

RNA processing modulates the expression of the arcDABC operon in Pseudomonas aeruginosa.

Journal of molecular biology 226 (4)
PMID : 1325563  :   DOI  :   10.1016/0022-2836(92)91044-p    
Abstract >>
Anaerobic growth of Pseudomonas aeruginosa on arginine depends on the arcDABC operon encoding the enzymes of the arginine deiminase pathway. The co-ordinate, anaerobic induction of these enzymes requires the FNR-like regulatory protein ANR, which activates the arc promoter lying upstream from arcD. By Northern hybridization experiments, three abundant arcA, arcAB and arcABC transcripts and three minor arcDA, arcDAB and arcDABC transcripts could be detected. The 5' ends of the arcA, arcAB and arcABC mRNAs were determined by S1 and primer extension mapping. These 5' ends appear to be generated by endonucleolytic cleavage (processing) in arcD mRNA rather than by a second promoter; this was concluded from the effects of insertion and deletion mutations in arcD. Intergenic inverted repeats between arcA and arcB as well as between arcB and arcC were shown to be involved in the formation of 3' ends of arc transcripts. Deletion of either intergenic region in the P. aeruginosa chromosome led to the loss of the arcA or arcAB transcript, respectively. Dot blot experiments revealed that arc mRNAs extracted from the wild-type strain had similar chemical half-lives in the arcA, arcB and arcC regions, ranging from 16 to 13 minutes. The half-life of arcD mRNA, by contrast, was significantly shorter, suggesting that this mRNA segment may be destabilized by the processing cuts within arcD. Deletion of the putative intergenic stem-loop structures did not result in a dramatic loss of arc mRNA stability. Thus, the intergenic hairpin structures do not contribute importantly to the overall mRNA stability; they might act primarily as partial transcription terminators and locally protect the 3' ends from exonuclease action. The expression levels of the four Arc proteins correlated approximately with the relative abundance of the corresponding mRNA segments. In conclusion, mRNA processing and, presumably, partial termination of transcription contribute to differential gene expression within the arc operon.
KeywordMeSH Terms
Amino Acid Transport Systems
Antiporters
Gene Expression Regulation, Bacterial
Genes, Bacterial
Phosphotransferases (Carboxyl Group Acceptor)
RNA Processing, Post-Transcriptional
9. Toleman  MA, Biedenbach  D, Bennett  D, Jones  RN, Walsh  TR,     ( 2003 )

Genetic characterization of a novel metallo-beta-lactamase gene, blaIMP-13, harboured by a novel Tn5051-type transposon disseminating carbapenemase genes in Europe: report from the SENTRY worldwide antimicrobial surveillance programme.

The Journal of antimicrobial chemotherapy 52 (4)
PMID : 12951335  :   DOI  :   10.1093/jac/dkg410    
Abstract >>
In 2001, as part of the SENTRY worldwide antimicrobial surveillance programme, Pseudomonas aeruginosa 86-14571A was isolated at a hospital in Rome from a cancer patient with a bloodstream infection. The isolate was resistant to all antibiotics except amikacin, and displayed an imipenem MIC of 64 mg/L that decreased to 8 mg/L in the presence of EDTA. The resistance determinant was investigated. The resistant determinant was cloned in Escherichia coli using a shotgun cloning approach. Sequence analysis revealed the presence of a novel IMP-type metallo-beta-lactamase (MBL) gene, blaIMP-13. This encoded a protein displaying most identity to IMP variants: 93% and 92.3% identity, respectively, to IMP-8 and IMP-2 (previously identified in Italy). The protein had 19 amino acid changes from IMP-2 and 17 amino acid changes from IMP-8. The blaIMP-13 gene was found as a gene cassette in the first position of a class 1 integron. A 25 bp inverted repeat sequence IRi was identified 174 bp upstream of the class I integrase, which suggests that the integron is found on a Tn402-like transposon, or defective transposon derivative. This element, in turn, is located in the transposition locus (tnp region) of a Tn21 subfamily transposon that showed most identity to Tn5051, a transposon recently identified from a strain of Pseudomonas putida isolated in New York. Interestingly, the insertion point of the Tn402-like transposon and the sequence of the Tn5051-like genes were identical to those of the genetic element harbouring blaVIM-2 recently identified in Poland. The resistance determinant of P. aeruginosa 86-14571A is a novel IMP-type MBL carried on a composite transposon responsible for wide geographical dissemination of MBL genes in Europe.
KeywordMeSH Terms
10. Bröms  JE, Forslund  AL, Forsberg  A, Francis  MS,     ( 2003 )

Dissection of homologous translocon operons reveals a distinct role for YopD in type III secretion by Yersinia pseudotuberculosis.

Microbiology (Reading, England) 149 (Pt 9)
PMID : 12949185  :   DOI  :   10.1099/mic.0.26322-0    
Abstract >>
The homologous pcrGVHpopBD and lcrGVHyopBD translocase operons of Pseudomonas aeruginosa and pathogenic Yersinia spp., respectively, are responsible for the translocation of anti-host effectors into the cytosol of infected eukaryotic cells. In Yersinia, this operon is also required for yop-regulatory control. To probe for key molecular interactions during the infection process, the functional interchangeability of popB/yopB and popD/yopD was investigated. Secretion of PopB produced in trans in a deltayopB null mutant of Yersinia was only observed when co-produced with its native chaperone PcrH, but this was sufficient to complement the yopB translocation defect. The Yersinia deltayopD null mutant synthesized and secreted PopD even in the absence of native PcrH, yet this did not restore YopD-dependent yop-regulatory control or effector translocation. Thus, this suggests that key residues in YopD, which are not conserved in PopD, are essential for functional Yersinia type III secretion.
KeywordMeSH Terms
11. Sabtcheva  S, Galimand  M, Gerbaud  G, Courvalin  P, Lambert  T,     ( 2003 )

Aminoglycoside resistance gene ant(4')-IIb of Pseudomonas aeruginosa BM4492, a clinical isolate from Bulgaria.

Antimicrobial agents and chemotherapy 47 (5)
PMID : 12709326  :   DOI  :   10.1128/aac.47.5.1584-1588.2003     PMC  :   PMC153341    
Abstract >>
The ant(4')-IIb gene of Pseudomonas aeruginosa BM4492, which encodes an aminoglycoside 4'-O-adenylyltransferase, was identified as a coding sequence of 756 bp corresponding to a protein with a calculated mass of 27,219 Da. Analysis of the deduced sequence indicated that the protein was related to aminoglycoside 4'-O-adenylyltransferases IIa and Ia found in P. aeruginosa and gram-positive bacteria, respectively. The enzyme conferred resistance to amikacin and tobramycin but not to dibekacin, gentamicin, or netilmicin. The ant(4')-IIb gene had a chromosomal location in five of six clinical isolates of P. aeruginosa tested and was plasmid borne in the remaining strain. The ant(4')-IIb gene was detected by PCR in some clinical strains of P. aeruginosa from the same hospital but not in members of other bacterial genera.
KeywordMeSH Terms
12. Corbella  ME, Puyet  A,     ( 2003 )

Real-time reverse transcription-PCR analysis of expression of halobenzoate and salicylate catabolism-associated operons in two strains of Pseudomonas aeruginosa.

Applied and environmental microbiology 69 (4)
PMID : 12676709  :   DOI  :   10.1128/aem.69.4.2269-2275.2003     PMC  :   PMC154809    
Abstract >>
Pseudomonas aeruginosa JB2 can use 2-chlorobenzoate (2-CBa), 3-CBa, 2,3-dichlorobenzoate (2,3-DCBa), and 2,5-DCBa as sole carbon and energy sources, whereas strain 142 can only grow on 2-CBa and 2,4-DCBa. Both strains, however, harbor the same halobenzoate 1,2-dioxygenase (ohbAB) and chlorocatechol (clcABD) degradation genes necessary for the metabolism of ortho-CBas. In addition, the hybABCD operon, encoding a salicylate 5-hydroxylase, is also found in both strains. The expression of ohbAB, hybABCD, and clcABD operons was measured in cultures grown on different CBas as the sole carbon source and also in glucose-grown cells supplemented with CBas as inducers. A method to standardize real-time reverse transcription-PCR experimental data was used that allows the comparison of semiquantitative mRNA accumulation in different strains and culture conditions. In both strains, the ohb and hyb systems were induced in cells grown on 2-CBa or DCBas, whereas clc was induced only by DCBas. Repression by catabolite was observed both on ohb and clc systems in glucose-grown cells. Chlorocatechol 1,2-dioxygenase activity in JB2 was detected even in clc-repressed conditions, confirming the presence of additional isofunctional genes previously detected in P. aeruginosa 142. Although similar levels of induction of ohbAB were observed in strain JB2 grown on either benzoate, monochlorobenzoates, or DCBas, the ohbAB operon of strain 142 was only strongly induced by growth on 2-CBa and, to a lesser extent, on 2,4-DCBa. This observation suggests that regulation of the ohbAB operon may be different in both strains. The concomitant induction of ohb and hyb by CBas may allow the formation of hybrid halobenzoate dioxygenase(s) composed of Ohb/Hyb dioxygenase subunits and Hyb ferredoxin/ferredoxin reductase components.
KeywordMeSH Terms
Operon
Reverse Transcriptase Polymerase Chain Reaction
13. de Chial  M, Ghysels  B, Beatson  SA, Geoffroy  V, Meyer  JM, Pattery  T, Baysse  C, Chablain  P, Parsons  YN, Winstanley  C, Cordwell  SJ, Cornelis  P,     ( 2003 )

Identification of type II and type III pyoverdine receptors from Pseudomonas aeruginosa.

Microbiology (Reading, England) 149 (Pt 4)
PMID : 12686625  :   DOI  :   10.1099/mic.0.26136-0    
Abstract >>
Pseudomonas aeruginosa produces, under conditions of iron limitation, a high-affinity siderophore, pyoverdine (PVD), which is recognized at the level of the outer membrane by a specific TonB-dependent receptor, FpvA. So far, for P. aeruginosa, three different PVDs, differing in their peptide chain, have been described (types I-III), but only the FpvA receptor for type I is known. Two PVD-producing P. aeruginosa strains, one type II and one type III, were mutagenized by a mini-TnphoA3 transposon. In each case, one mutant unable to grow in the presence of the strong iron chelator ethylenediaminedihydroxyphenylacetic acid (EDDHA) and the cognate PVD was selected. The first mutant, which had an insertion in the pvdE gene, upstream of fpvA, was unable to take up type II PVD and showed resistance to pyocin S3, which is known to use type II FpvA as receptor. The second mutant was unable to take up type III PVD and had the transposon insertion in fpvA. Cosmid libraries of the respective type II and type III PVD wild-type strains were constructed and screened for clones restoring the capacity to grow in the presence of PVD. From the respective complementing genomic fragments, type II and type III fpvA sequences were determined. When in trans, type II and type III fpvA restored PVD production, uptake, growth in the presence of EDDHA and, in the case of type II fpvA, pyocin S3 sensitivity. Complementation of fpvA mutants obtained by allelic exchange was achieved by the presence of cognate fpvA in trans. All three receptors posses an N-terminal extension of about 70 amino acids, similar to FecA of Escherichia coli, but only FpvAI has a TAT export sequence at its N-terminal end.
KeywordMeSH Terms
Bacterial Outer Membrane Proteins
Oligopeptides
Receptors, Cell Surface
14. Clarke  L, Moore  JE, Millar  BC, Garske  L, Xu  J, Heuzenroeder  MW, Crowe  M, Elborn  JS,     ( 2003 )

Development of a diagnostic PCR assay that targets a heat-shock protein gene (groES) for detection of Pseudomonas spp. in cystic fibrosis patients.

Journal of medical microbiology 52 (Pt 9)
PMID : 12909651  :   DOI  :   10.1099/jmm.0.05077-0    
Abstract >>
Laboratory detection of Pseudomonas spp., in particular Pseudomonas aeruginosa, remains an important assay in the management of patients with cystic fibrosis (CF). As the groES and groEL genes of P. aeruginosa have now been cloned and their nucleotide sequences determined, the aim of this study was to develop a novel PCR assay for the detection of Pseudomonas spp. from patients with CF by employing conserved primer regions of the groE heat-shock protein domain gene. A PCR assay was designed that targeted a 536 bp region of the groE gene to detect Pseudomonas spp. PCR amplification of genomic DNA from extracted organisms generated an amplicon of the expected size (approx. 536 bp) for all P. aeruginosa (n = 60), Pseudomonas putida, Pseudomonas fluorescens and Pseudomonas stutzeri isolates examined, but did not produce a positive amplicon for several other genera and species that are commonly isolated from the sputum of CF patients. RFLP analysis of the amplicons of all P. aeruginosa isolates demonstrated a single RFLP type that consisted of three bands at approximately 80, 190 and 250 bp; direct sequencing of the amplicons demonstrated the presence of a single sequence type, indicating the highly conserved nature of this region. In addition, the assay successfully produced a positive signal from primary non-selective plates of three known P. aeruginosa culture-positive CF patients, but was unable to generate a signal in a further six CF patients who had no history of infection with P. aeruginosa or other Pseudomonas spp. This assay is recommended to detect the presence of Pseudomonas spp., including P. aeruginosa, from primary culture plates that originate from laboratory analysis of CF patients' sputum, particularly at review, in those patients with no previous history of Pseudomonas infection or those who appear to be transiently colonized by this organism. Employment of such molecular methodologies, in conjunction with routine clinical sputum cultures, may provide improved information on the microbial status of CF patients, which will aid clinicians in both optimum patient management in terms of antibiotic regimes and CF centre infection-control practices.
KeywordMeSH Terms
15. Toleman  MA, Rolston  K, Jones  RN, Walsh  TR,     ( 2003 )

Molecular and biochemical characterization of OXA-45, an extended-spectrum class 2d' beta-lactamase in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 47 (9)
PMID : 12936985  :   DOI  :   10.1128/aac.47.9.2859-2863.2003     PMC  :   PMC182593    
Abstract >>
As part of the CANCER Antimicrobial Surveillance Program in North America, a clinical strain of Pseudomonas aeruginosa, strain 07-406, isolated in Texas was found to be resistant to all antimicrobials except polymyxin B. Genetic analysis of this isolate identified two unique extended-spectrum beta-lactamase genes. One, bla(VIM-7), encoded a metallo-beta-lactamase (unpublished data), and the other, bla(OXA-45), described here, encoded a class D extended-spectrum beta-lactamase. bla(OXA-45) was isolated on a Sau3A1 genomic fragment of 1.8 kb and encodes a protein of 264 amino acids with the highest identities to OXA-18 (65.9%), OXA-9 (42.8%), OXA-22 (40.2%), OXA-12 (38.6%), and OXA-29 (35.2%) but weak identities with other class D beta-lactamases. bla(OXA-45) was found to be harbored on a 24-kb plasmid in a region that displays high identities with a section of the 43-kb genomic island of Salmonella enterica serovar Typhimurium DT104. Biochemically OXA-45 is most similar to OXA-18 in its substrate profile and inhibition by clavulanic acid and is a member of the 2d' class of beta-lactamases.
KeywordMeSH Terms
16. Riccio  ML, Docquier  JD, Dell'Amico  E, Luzzaro  F, Amicosante  G, Rossolini  GM,     ( 2003 )

Novel 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ic, from a Pseudomonas aeruginosa integron.

Antimicrobial agents and chemotherapy 47 (5)
PMID : 12709352  :   DOI  :   10.1128/aac.47.5.1746-1748.2003     PMC  :   PMC153335    
Abstract >>
A novel gene, aac(3)-Ic, encoding an AAC(3)-I aminoglycoside 3-N-acetyltransferase, was identified on a gene cassette inserted into a Pseudomonas aeruginosa integron that also carries a bla(VIM-2) and a cmlA7 gene cassette. The aac(3)-Ic gene product is 59 and 57% identical to AAC(3)-Ia and AAC(3)-Ib, respectively, and confers resistance to gentamicin and sisomicin.
KeywordMeSH Terms
Integrons
17. Tsuge  T, Taguchi  K, Seiichi  T, Doi  Y,     ( 2003 )

Molecular characterization and properties of (R)-specific enoyl-CoA hydratases from Pseudomonas aeruginosa: metabolic tools for synthesis of polyhydroxyalkanoates via fatty acid beta-oxidation.

International journal of biological macromolecules 31 (4��5��)
PMID : 12568928  :  
Abstract >>
The use of (R)-specific enoyl-coenzyme A (CoA) hydratase (PhaJ) provides a powerful tool for polyhydroxyalkanoate (PHA) synthesis from fatty acids or plant oils in recombinant bacteria. PhaJ provides monomer units for PHA synthesis from the fatty acid ss-oxidation cycle. Previously, two phaJ genes (phaJ1(Pa) and phaJ2(Pa)) were identified in Pseudomonas aeruginosa. This report identifies two new phaJ genes (phaJ3(Pa) and phaJ4(Pa)) in P. aeruginosa through a genomic database search. The abilities of the four PhaJ(Pa) proteins and the (R)-3-hydroxyacyl-acyl carrier protein [(R)-3HA-ACP] dehydrases, FabA(Pa) and FabZ(Pa), to supply monomers from enoyl-CoA substrates for PHA synthesis were determined. The presence of either PhaJ1(Pa) or PhaJ4(Pa) in recombinant Escherichia coli led to the high levels of PHA accumulation (as high as 36-41 wt.% in dry cells) consisting of mainly short- (C4-C6) and medium-chain-length (C6-C10) 3HA units, respectively. Furthermore, detailed characterizations of PhaJ1(Pa) and PhaJ4(Pa) were performed using purified samples. Kinetic analysis revealed that only PhaJ4(Pa) exhibits almost constant maximum reaction rates (V(max)) irrespective of the chain length of the substrates. The assay for stereospecific hydration revealed that, unlike PhaJ1(Pa), PhaJ4(Pa) has relatively low (R)-specificity. These hydratases may be very useful as monomer-suppliers for the synthesis of designed PHAs in recombinant bacteria.
KeywordMeSH Terms
18. Walsh  TR, Toleman  MA, Hryniewicz  W, Bennett  PM, Jones  RN,     ( 2003 )

Evolution of an integron carrying blaVIM-2 in Eastern Europe: report from the SENTRY Antimicrobial Surveillance Program.

The Journal of antimicrobial chemotherapy 52 (1)
PMID : 12805257  :   DOI  :   10.1093/jac/dkg299    
Abstract >>
As part of the SENTRY Antimicrobial Surveillance Program, an imipenem-resistant Pseudomonas aeruginosa strain (81-11963A) was isolated from the blood culture of a female neonate institutionalized at the local children's hospital in Warsaw, Poland. Cloning of an imipenem resistance determinant revealed it to be a VIM-2 metallo-beta-lactamase, but sequence analysis of DNA adjacent to blaVIM-2 revealed it to have a unique gene context. Downstream of the blaVIM-2 gene resides an aacA4 gene encoding the AAC(6')-Ib aminoglycoside acetyltransferase. The integron containing blaVIM-2 shows high similarity to that reported from In58 in France but was novel in that it possessed a gene cassette with a 59 truncated base element only 19 base pairs (bp) long, consisting of a conserved core site and an inverse core site separated by only 5 bp. This appears to be the first report of a metallo-beta-lactamase gene arising from a pathogenic strain in Eastern Europe.
KeywordMeSH Terms
19. Dean  CR, Visalli  MA, Projan  SJ, Sum  PE, Bradford  PA,     ( 2003 )

Efflux-mediated resistance to tigecycline (GAR-936) in Pseudomonas aeruginosa PAO1.

Antimicrobial agents and chemotherapy 47 (3)
PMID : 12604529  :   DOI  :   10.1128/aac.47.3.972-978.2003     PMC  :   PMC149306    
Abstract >>
Pseudomonas aeruginosa strains are less susceptible to tigecycline (previously GAR-936; MIC, 8 micro g/ml) than many other bacteria (P. J. Petersen, N. V. Jacobus, W. J. Weiss, P. E. Sum, and R. T. Testa, Antimicrob. Agents Chemother. 43:738-744, 1999). To elucidate the mechanism of resistance to tigecycline, P. aeruginosa PAO1 strains defective in the MexAB-OprM and/or MexXY (OprM) efflux pumps were tested for susceptibility to tigecycline. Increased susceptibility to tigecycline (MIC, 0.5 to 1 micro g/ml) was specifically associated with loss of MexXY. Transcription of mexX and mexY was also responsive to exposure of cells to tigecycline. To test for the emergence of compensatory efflux pumps in the absence of MexXY-OprM, mutants lacking MexXY-OprM were plated on medium containing tigecycline at 4 or 6 micro g/ml. Resistant mutants were readily recovered, and these also had decreased susceptibility to several other antibiotics, suggesting efflux pump recruitment. One representative carbenicillin-resistant strain overexpressed OprM, the outer membrane channel component of the MexAB-OprM efflux pump. The mexAB-oprM repressor gene, mexR, from this strain contained a 15-bp in-frame deletion. Two representative chloramphenicol-resistant strains showed expression of an outer membrane protein slightly larger than OprM. The mexCD-OprJ repressor gene, nfxB, from these mutants contained a 327-bp in-frame deletion and an IS element insertion, respectively. Together, these data indicated drug efflux mediated by MexCD-OprJ. The MICs of the narrower-spectrum semisynthetic tetracyclines doxycycline and minocycline increased more substantially than did those of tigecycline and other glycylcyclines against the MexAB-OprM- and MexCD-OprJ-overexpressing mutant strains. This suggests that glycylcyclines, although they are subject to efflux from P. aeruginosa, are generally inferior substrates for P. aeruginosa efflux pumps than are narrower-spectrum tetracyclines.
KeywordMeSH Terms
Tetracycline Resistance
20. Spencer  DH, Kas  A, Smith  EE, Raymond  CK, Sims  EH, Hastings  M, Burns  JL, Kaul  R, Olson  MV,     ( 2003 )

Whole-genome sequence variation among multiple isolates of Pseudomonas aeruginosa.

Journal of bacteriology 185 (4)
PMID : 12562802  :   DOI  :   10.1128/jb.185.4.1316-1325.2003     PMC  :   PMC142842    
Abstract >>
Whole-genome shotgun sequencing was used to study the sequence variation of three Pseudomonas aeruginosa isolates, two from clonal infections of cystic fibrosis patients and one from an aquatic environment, relative to the genomic sequence of reference strain PAO1. The majority of the PAO1 genome is represented in these strains; however, at least three prominent islands of PAO1-specific sequence are apparent. Conversely, approximately 10% of the sequencing reads derived from each isolate fail to align with the PAO1 backbone. While average sequence variation among all strains is roughly 0.5%, regions of pronounced differences were evident in whole-genome scans of nucleotide diversity. We analyzed two such divergent loci, the pyoverdine and O-antigen biosynthesis regions, by complete resequencing. A thorough analysis of isolates collected over time from one of the cystic fibrosis patients revealed independent mutations resulting in the loss of O-antigen synthesis alternating with a mucoid phenotype. Overall, we conclude that most of the PAO1 genome represents a core P. aeruginosa backbone sequence while the strains addressed in this study possess additional genetic material that accounts for at least 10% of their genomes. Approximately half of these additional sequences are novel.
KeywordMeSH Terms
Genetic Variation
Genome, Bacterial
Oligopeptides
Sequence Analysis, DNA
Water Microbiology
21. Zhang  H, Ishige  K, Kornberg  A,     ( 2002 )

A polyphosphate kinase (PPK2) widely conserved in bacteria.

Proceedings of the National Academy of Sciences of the United States of America 99 (26)
PMID : 12486232  :   DOI  :   10.1073/pnas.262655199     PMC  :   PMC139203    
Abstract >>
Synthesis of inorganic polyphosphate (poly P) from the terminal phosphate of ATP is catalyzed reversibly by poly P kinase (PPK, now designated PPK1) initially isolated from Escherichia coli. PPK1 is highly conserved in many bacteria, including some of the major pathogens such as Pseudomonas aeruginosa. In a null mutant of P. aeruginosa lacking ppk1, we have discovered a previously uncharacterized PPK activity (designated PPK2) distinguished from PPK1 by the following: synthesis of poly P from GTP or ATP, a preference for Mn2+ over Mg2+, and a stimulation by poly P. The reverse reaction, a poly P-driven nucleoside diphosphate kinase synthesis of GTP from GDP, is 75-fold greater than the forward reaction, poly P synthesis from GTP. The gene encoding PPK2 (ppk2) was identified from the amino acid sequence of the protein purified near 1,000-fold, to homogeneity. The 5'-end is 177 bp upstream of the annotated genome sequence of a "conserved hypothetical protein"; ppk2 (1,074 bp) encodes a protein of 357 aa with a molecular mass of 40.8 kDa. Sequences homologous to PPK2 are present in two other proteins in P. aeruginosa, in two Archaea, and in 32 other bacteria (almost all with PPK1 as well); these include rhizobia, cyanobacteria, Streptomyces, and several pathogenic species. Distinctive features of the poly P-driven nucleoside diphosphate kinase activity and structural aspects of PPK2 are among the subjects of an accompanying report.
KeywordMeSH Terms
22. Tungpradabkul  S, Senapin  S, Panyim  S,     ( 1998 )

PCR-based method for isolation of the flagellin genes from Pseudomonas species.

The Journal of general and applied microbiology 44 (3)
PMID : 12501433  :  
Abstract >>
N/A
KeywordMeSH Terms
23. Pournaras  S, Tsakris  A, Maniati  M, Tzouvelekis  LS, Maniatis  AN,     ( 2002 )

Novel variant (bla(VIM-4)) of the metallo-beta-lactamase gene bla(VIM-1) in a clinical strain of Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 46 (12)
PMID : 12435718  :   DOI  :   10.1128/aac.46.12.4026-4028.2002     PMC  :   PMC132756    
Abstract >>
A Pseudomonas aeruginosa isolate highly resistant to carbapenems was collected from a patient with postsurgical cerebrospinal infection in Greece. The isolate carried a class 1 integron that contained as a sole cassette the gene bla(VIM-4), a novel variant of bla(VIM-1), with one nucleotide difference resulting in a Ser-to-Arg change at amino acid position 175 of the VIM-1 enzyme. This is the first detection of a VIM-1 variant after its appearance in Italy.
KeywordMeSH Terms
24. Toleman  MA, Simm  AM, Murphy  TA, Gales  AC, Biedenbach  DJ, Jones  RN, Walsh  TR,     ( 2002 )

Molecular characterization of SPM-1, a novel metallo-beta-lactamase isolated in Latin America: report from the SENTRY antimicrobial surveillance programme.

The Journal of antimicrobial chemotherapy 50 (5)
PMID : 12407123  :   DOI  :   10.1093/jac/dkf210    
Abstract >>
The gene encoding the metallo-beta-lactamase SPM-1 was cloned from a genomic library of Pseudomonas aeruginosa strain 48-1997 A. The insert carrying spm-1 possessed a GC content of 47%, indicating that it is of non-Pseudomonas origin. Upstream of spm-1 there is a small open reading frame (ORF), which is homologous to the LysR family of proteins (69% identity to the LysR protein from Salmonella enterica serovar Typhimurium). Downstream of spm-1 there is the start of an ORF, the product of which shows close homology with the GroEL-type proteins from Xanthomonas campestris. No transmissible element could be identified upstream or downstream of spm-1. The spm-1 gene is carried on a plasmid that can transform both Escherichia coli and P. aeruginosa to ceftazidime resistance. SPM-1 contains the classic metallo-beta-lactamase zinc-binding motif HXHXD and shows the highest identity (35.5%) to IMP-1. SPM-1 is a distinctly different metallo-beta-lactamase from VIM and IMP and, accordingly, represents a new subfamily of mobile metallo-beta-lactamases. The predicted molecular weight of the protein was 27 515 Da, significantly higher than that of IMP (25 041 Da) or VIM (25 322 Da). SPM-1 possesses a unique loop of 23 residues that accounts for the higher molecular mass.
KeywordMeSH Terms
25. Larbig  KD, Christmann  A, Johann  A, Klockgether  J, Hartsch  T, Merkl  R, Wiehlmann  L, Fritz  HJ, Tümmler  B,     ( 2002 )

Gene islands integrated into tRNA(Gly) genes confer genome diversity on a Pseudomonas aeruginosa clone.

Journal of bacteriology 184 (23)
PMID : 12426355  :   DOI  :   10.1128/jb.184.23.6665-6680.2002     PMC  :   PMC135438    
Abstract >>
Intraclonal genome diversity of Pseudomonas aeruginosa was studied in one of the most diverse mosaic regions of the P. aeruginosa chromosome. The ca. 110-kb large hypervariable region located near the lipH gene in two members of the predominant P. aeruginosa clone C, strain C and strain SG17M, was sequenced. In both strains the region consists of an individual strain-specific gene island of 111 (strain C) or 106 (SG17M) open reading frames (ORFs) and of a 7-kb stretch of clone C-specific sequence of 9 ORFs. The gene islands are integrated into conserved tRNA(Gly) genes and have a bipartite structure. The first part adjacent to the tRNA gene consists of strain-specific ORFs encoding metabolic functions and transporters, the majority of which have homologs of known function in other eubacteria, such as hemophores, cytochrome c biosynthesis, or mercury resistance. The second part is made up mostly of ORFs of yet-unknown function. Forty-seven of these ORFs are mutual homologs with a pairwise amino acid sequence identity of 35 to 88% and are arranged in the same order in the two gene islands. We hypothesize that this novel type of gene island derives from mobile elements which, upon integration, endow the recipient with strain-specific metabolic properties, thus possibly conferring on it a selective advantage in its specific habitat.
KeywordMeSH Terms
Genetic Variation
Genome, Bacterial
Proteins
26. DiGiandomenico  A, Matewish  MJ, Bisaillon  A, Stehle  JR, Lam  JS, Castric  P,     ( 2002 )

Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity.

Molecular microbiology 46 (2)
PMID : 12406226  :   DOI  :   10.1046/j.1365-2958.2002.03171.x    
Abstract >>
The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O-antigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.
KeywordMeSH Terms
Bacterial Proteins
27. Ho  SE, Subramaniam  G, Palasubramaniam  S, Navaratnam  P,     ( 2002 )

Carbapenem-resistant Pseudomonas aeruginosa in malaysia producing IMP-7 beta-lactamase.

Antimicrobial agents and chemotherapy 46 (10)
PMID : 12234862  :   DOI  :   10.1128/aac.46.10.3286-3287.2002     PMC  :   PMC128803    
Abstract >>
We have isolated and identified a carbapenem-resistant Pseudomonas aeruginosa strain from Malaysia that produces an IMP-7 metallo-beta-lactamase. This isolate showed high-level resistance to meropenem and imipenem, the MICs of which were 256 and 128 micro g/ml, respectively. Isoelectric focusing analyses revealed pI values of >9.0, 8.2, and 7.8, which indicated the possible presence of IMP and OXA. DNA sequencing confirmed the identity of the IMP-7 determinant.
KeywordMeSH Terms
beta-Lactam Resistance
28. Huston  WM, Jennings  MP, McEwan  AG,     ( 2002 )

The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition.

Molecular microbiology 45 (6)
PMID : 12354238  :   DOI  :   10.1046/j.1365-2958.2002.03132.x    
Abstract >>
Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild-type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild-type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.
KeywordMeSH Terms
29. Dubois  V, Arpin  C, Noury  P, Quentin  C,     ( 2002 )

Clinical strain of Pseudomonas aeruginosa carrying a bla(TEM-21) gene located on a chromosomal interrupted TnA type transposon.

Antimicrobial agents and chemotherapy 46 (11)
PMID : 12384376  :   DOI  :   10.1128/aac.46.11.3624-3626.2002     PMC  :   PMC128703    
Abstract >>
A clinical isolate of Pseudomonas aeruginosa was found to produce a clavulanic acid-inhibited extended-spectrum beta-lactamase with a pI of 6.4. PCR, cloning, and sequencing experiments showed that the corresponding bla(TEM-21) gene was part of a chromosomally located Tn801 transposon disrupted by an IS6100 element and adjacent to an aac(3)-II gene.
KeywordMeSH Terms
30. Partridge  SR, Collis  CM, Hall  RM,     ( 2002 )

Class 1 integron containing a new gene cassette, aadA10, associated with Tn1404 from R151.

Antimicrobial agents and chemotherapy 46 (8)
PMID : 12121911  :   DOI  :   10.1128/aac.46.8.2400-2408.2002     PMC  :   PMC127381    
Abstract >>
The carbenicillin, gentamicin, kanamycin, streptomycin, spectinomycin, sulfonamide, and tobramycin resistance determinants found on Pseudomonas aeruginosa plasmid R151 have previously been shown to translocate to another plasmid, R388, and it was inferred that a transposon, Tn1404, carried the resistance determinants. Sequencing of the cassette array from the plasmid known as R388::Tn1404 revealed two known gene cassettes, oxa10 and aadB, and a previously unidentified cassette determining resistance to streptomycin and spectinomycin, here designated aadA10, in the order oxa10-aadB-aadA10. These cassettes replaced the dfrB2-orfA cassette array of R388, indicating that movement of the resistance determinants from R151 to R388 resulted from recombinational exchange between two class 1 integrons rather than transposition. The AadA10 protein is most closely related to AadA6 (85% identical) and AadA7 (80% identical). The aadA10 cassette found here has only a simple site containing a 7-bp spacer derived from attI1 in place of a 59-base element and is likely to represent a derivative of the complete cassette. IntI1-mediated deletion of the aadA10 cassette was not detected, indicating that this single simple site is either inactive or only weakly active.
KeywordMeSH Terms
31. Choi  JY, Sifri  CD, Goumnerov  BC, Rahme  LG, Ausubel  FM, Calderwood  SB,     ( 2002 )

Identification of virulence genes in a pathogenic strain of Pseudomonas aeruginosa by representational difference analysis.

Journal of bacteriology 184 (4)
PMID : 11807055  :   DOI  :   10.1128/jb.184.4.952-961.2002     PMC  :   PMC134824    
Abstract >>
Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.
KeywordMeSH Terms
Fimbriae Proteins
Genes, Bacterial
32. Girlich  D, Naas  T, Leelaporn  A, Poirel  L, Fennewald  M, Nordmann  P,     ( 2002 )

Nosocomial spread of the integron-located veb-1-like cassette encoding an extended-pectrum beta-lactamase in Pseudomonas aeruginosa in Thailand.

Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 34 (5)
PMID : 11807680  :   DOI  :   10.1086/338786    
Abstract >>
The beta-lactamase gene content and epidemiology of ceftazidime-resistant Pseudomonas aeruginosa isolates (24% of the total number of P. aeruginosa isolates) were investigated at a University Hospital in Thailand during a 4-month period in 1999. Of 33 nonrepetitive clinical isolates, 31 produced a VEB-1-like clavulanic acid-inhibited extended-spectrum beta-lactamase (ESBL). These isolates belonged to different pulsed-field gel electrophoresis types and subtypes. In 1 case, the bla(VEB-1)-like gene was plasmid located. The bla(VEB-1)-like genes were present as a gene cassette on class 1 integrons that varied in size and structure. In most cases, the veb-1 cassette was associated with an arr-2 cassette (rifampin resistance), aminoglycoside resistance gene cassettes, and an oxa-10-like cassette encoding a narrow-spectrum oxacillinase-type beta-lactamase. The present study indicates that ESBLs may be endemic in P. aeruginosa and illustrates that integrons are efficient means for their spread.
KeywordMeSH Terms
33. Raymond  CK, Sims  EH, Kas  A, Spencer  DH, Kutyavin  TV, Ivey  RG, Zhou  Y, Kaul  R, Clendenning  JB, Olson  MV,     ( 2002 )

Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa.

Journal of bacteriology 184 (13)
PMID : 12057956  :   DOI  :   10.1128/jb.184.13.3614-3622.2002     PMC  :   PMC135118    
Abstract >>
The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the P. aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.
KeywordMeSH Terms
Genetic Variation
34. Iyobe  S, Kusadokoro  H, Takahashi  A, Yomoda  S, Okubo  T, Nakamura  A, O'Hara  K,     ( 2002 )

Detection of a variant metallo-beta-lactamase, IMP-10, from two unrelated strains of Pseudomonas aeruginosa and an alcaligenes xylosoxidans strain.

Antimicrobial agents and chemotherapy 46 (6)
PMID : 12019129  :   DOI  :   10.1128/aac.46.6.2014-2016.2002     PMC  :   PMC127220    
Abstract >>
The gene bla(IMP-10) of a variant metallo-beta-lactamase, IMP-10, had a single base replacement of G by T at nucleotide 145, which led to an amino acid alteration of Val49 to Phe compared to the IMP-1 enzyme, indicating that IMP-10 was a point mutation derivative of IMP-1. Highly purified enzymes revealed that IMP-10 was different from IMP-1 in its extremely low hydrolyzing activities for penicillins, such as benzylpenicillin, ampicillin, and piperacillin.
KeywordMeSH Terms
35. Gibb  AP, Tribuddharat  C, Moore  RA, Louie  TJ, Krulicki  W, Livermore  DM, Palepou  MF, Woodford  N,     ( 2002 )

Nosocomial outbreak of carbapenem-resistant Pseudomonas aeruginosa with a new bla(IMP) allele, bla(IMP-7).

Antimicrobial agents and chemotherapy 46 (1)
PMID : 11751148  :   DOI  :   10.1128/aac.46.1.255-258.2002     PMC  :   PMC126979    
Abstract >>
Pseudomonas aeruginosa isolates from an outbreak in Canada were highly resistant to carbapenems and ceftazidime but not piperacillin. They produced a novel integron-associated metallo-beta-lactamase, designated IMP-7, with 91% identity to IMP-1. bla(IMP-7) was not detected with standard bla(IMP)-specific primers, owing to mismatches in the forward primer.
KeywordMeSH Terms
Disease Outbreaks
36. Comer  JE, Marshall  MA, Blanch  VJ, Deal  CD, Castric  P,     ( 2002 )

Identification of the Pseudomonas aeruginosa 1244 pilin glycosylation site.

Infection and immunity 70 (6)
PMID : 12010970  :   DOI  :   10.1128/iai.70.6.2837-2845.2002     PMC  :   PMC128005    
Abstract >>
Previous work (P. Castric, F. J. Cassels, and R. W. Carlson, J. Biol. Chem. 276:26479-26485, 2001) has shown the Pseudomonas aeruginosa 1244 pilin glycan to be covalently bound to a serine residue. N-terminal sequencing of pilin fragments produced from endopeptidase treatment and identified by reaction with a glycan-specific monoclonal antibody indicated that the glycan was present between residue 75 and the pilin carboxy terminus. Further sequencing of these peptides revealed that serine residues 75, 81, 84, 105, 106, and 108 were not modified. Conversion of serine 148, but not serine 118, to alanine by site-directed mutagenesis, resulted in loss of the ability to carry out pilin glycosylation when tested in an in vivo system. These results showed the pilin glycan to be attached to residue 148, the carboxy-terminal amino acid. The carboxy-proximal portion of the pilin disulfide loop, which is adjacent to the pilin glycan, was found to be a major linear B-cell epitope, as determined by peptide epitope mapping analysis. Immunization of mice with pure pili produced antibodies that recognized the pilin glycan. These sera also reacted with P. aeruginosa 1244 lipopolysaccharide as measured by Western blotting and enzyme-linked immunosorbent assay.
KeywordMeSH Terms
37. Poirel  L, Gerome  P, De Champs  C, Stephanazzi  J, Naas  T, Nordmann  P,     ( 2002 )

Integron-located oxa-32 gene cassette encoding an extended-spectrum variant of OXA-2 beta-lactamase from Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 46 (2)
PMID : 11796380  :   DOI  :   10.1128/aac.46.2.566-569.2002     PMC  :   PMC127075    
Abstract >>
Pseudomonas aeruginosa clinical isolate CY-1, which was resistant to ceftazidime, harbored a conjugative ca. 250-kb plasmid that contained a class 1 integron with two gene cassettes encoding OXA-32, an OXA-2- type beta-lactamase, and the aminoglycoside acetyltransferase AAC(6')Ib(9). OXA-32 differed from OXA-2 by an Leu169Ile amino acid substitution (class D numbering). Site-directed mutagenesis established that Ile169 is responsible for resistance to ceftazidime but not to cefotaxime.
KeywordMeSH Terms
38. Partridge  SR, Brown  HJ, Hall  RM,     ( 2002 )

Characterization and movement of the class 1 integron known as Tn2521 and Tn1405.

Antimicrobial agents and chemotherapy 46 (5)
PMID : 11959558  :   DOI  :   10.1128/aac.46.5.1288-1294.2002     PMC  :   PMC127177    
Abstract >>
Two putative transposons, Tn2521 and Tn1405, carrying determinants for the PSE-4 beta-lactamase and for resistance to streptomycin, spectinomycin, and sulfonamides were previously isolated from the chromosome of Pseudomonas aeruginosa Dalgleish. Detailed mapping and determination of the complete sequence of Tn2521 revealed that it is a class 1 integron, here renamed In33, with a backbone structure identical to that of In4 from Tn1696. In33 contains two gene cassettes, blaP1 and aadA1, replacing the aacC1-orfE-aadA2-cmlA1 cassette array in In4. Although In33 does not include any transposition genes, movement of In33 (Tn2521) targeted to a single location in the IncP-1 plasmid R18-18 has been reported previously (M. I. Sinclair and B. W. Holloway, J. Bacteriol. 151:569-579, 1982). A 5-bp duplication of the target, which lies within the res site recognized by the ParA resolvase of R18-18, was present, indicating that the mechanism of movement was transposition. Together, these data indicate that class 1 integrons that are defective in self-transposition can move under appropriate circumstances. The Tn1405 isolate studied was found to represent only the cassette array of In33, which had replaced the cassette array in the recipient plasmid R388, probably by homologous recombination.
KeywordMeSH Terms
Recombination, Genetic
39. Drenkard  E, Ausubel  FM,     ( 2002 )

Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation.

Nature 416 (6882)
PMID : 11961556  :   DOI  :   10.1038/416740a    
Abstract >>
Colonization of the lungs of cystic fibrosis (CF) patients by the opportunistic bacterial pathogen Pseudomonas aeruginosa is the principal cause of mortality in CF populations. Pseudomonas aeruginosa infections generally persist despite the use of long-term antibiotic therapy. This has been explained by postulating that P. aeruginosa forms an antibiotic-resistant biofilm consisting of bacterial communities embedded in an exopolysaccharide matrix. Alternatively, it has been proposed that resistant P. aeruginosa variants may be selected in the CF respiratory tract by antimicrobial therapy itself. Here we report that both explanations are correct, and are interrelated. We found that antibiotic-resistant phenotypic variants of P. aeruginosa with enhanced ability to form biofilms arise at high frequency both in vitro and in the lungs of CF patients. We also identified a regulatory protein (PvrR) that controls the conversion between antibiotic-resistant and antibiotic-susceptible forms. Compounds that affect PvrR function could have an important role in the treatment of CF infections.
KeywordMeSH Terms
Drug Resistance, Bacterial
40. Folders  J, Algra  J, Roelofs  MS, van Loon  LC, Tommassen  J, Bitter  W,     ( 2001 )

Characterization of Pseudomonas aeruginosa chitinase, a gradually secreted protein.

Journal of bacteriology 183 (24)
PMID : 11717261  :   DOI  :   10.1128/JB.183.24.7044-7052.2001     PMC  :   PMC95551    
Abstract >>
The gram-negative bacterium Pseudomonas aeruginosa secretes many proteins into its extracellular environment via the type I, II, and III secretion systems. In this study, a gene, chiC, coding for an extracellular chitinolytic enzyme, was identified. The chiC gene encodes a polypeptide of 483 amino acid residues, without a typical N-terminal signal sequence. Nevertheless, an N-terminal segment of 11 residues was found to be cleaved off in the secreted protein. The protein shows sequence similarity to the secreted chitinases ChiC of Serratia marcescens, ChiA of Vibrio harveyi, and ChiD of Bacillus circulans and consists of an activity domain and a chitin-binding domain, which are separated by a fibronectin type III domain. ChiC was able to bind and degrade colloidal chitin and was active on the artificial substrates carboxymethyl-chitin-Remazol Brilliant Violet and p-nitrophenyl-beta-D-N,N',N"-triacetylchitotriose, but not on p-nitrophenyl-beta-D-N-acetylglucosamine, indicating that it is an endochitinase. Expression of the chiC gene appears to be regulated by the quorum-sensing system of P. aeruginosa, since this gene was not expressed in a lasIR vsmI mutant. After overnight growth, the majority of the ChiC produced was found intracellularly, whereas only small amounts were detected in the culture medium. However, after several days, the cellular pool of ChiC was largely depleted, and the protein was found in the culture medium. This release could not be ascribed to cell lysis. Since ChiC did not appear to be secreted via any of the known secretion systems, a novel secretion pathway seems to be involved.
KeywordMeSH Terms
41. Hickey  WJ, Sabat  G,     ( 2001 )

Integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular cloning for the identification and functional characterization of mobile ortho-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2.

Applied and environmental microbiology 67 (12)
PMID : 11722919  :   DOI  :   10.1128/AEM.67.12.5648-5655.2001     PMC  :   PMC93356    
Abstract >>
Protein mass spectrometry and molecular cloning techniques were used to identify and characterize mobile o-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2 and Pseudomonas huttiensis strain D1. Proteins induced in strains JB2 and D1 by growth on 2-chlorobenzoate (2-CBa) were extracted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two bands gave significant matches to OhbB and OhbA, which have been reported to be the alpha and beta subunits, respectively, of an ortho-1,2-halobenzoate dioxygenase of P. aeruginosa strain 142 (T. V. Tsoi, E. G. Plotnikova, J. R. Cole, W. F. Guerin, M. Bagdasarian, and J. M. Tiedje, Appl. Environ. Microbiol. 65:2151-2162, 1999). PCR and Southern hybridization experiments confirmed that ohbAB were present in strain JB2 and were transferred from strain JB2 to strain D1. While the sequences of ohbA from strains JB2, D1, and 142 were identical, the sequences of ohbB from strains JB2 and D1 were identical to each other but differed slightly from that of strain 142. PCR analyses and Southern hybridization analyses indicated that ohbAB were conserved in strains JB2 and D1 and in strain 142 but that the regions adjoining these genes were divergent. Expression of ohbAB in Escherichia coli resulted in conversion of o-chlorobenzoates to the corresponding (chloro)catechols with the following apparent affinity: 2-CBa approximately 2,5-dichlorobenzoate > 2,3,5-trichlorobenzoate > 2,4-dichlorobenzoate. The activity of OhbAB(JB2) appeared to differ from that reported for OhbAB(142) primarily in that a chlorine in the para position posed a greater impediment to catalysis with the former. Hybridization analysis of spontaneous 2-CBa(-) mutants of strains JB2 and D1 verified that ohbAB were lost along with the genes, suggesting that all of the genes may be contained in the same mobile element. Strains JB2 and 142 originated from California and Russia, respectively. Thus, ohbAB and/or the mobile element on which they are carried may have a global distribution.
KeywordMeSH Terms
Cloning, Molecular
42. Aubert  D, Poirel  L, Ali  AB, Goldstein  FW, Nordmann  P,     ( 2001 )

OXA-35 is an OXA-10-related beta-lactamase from Pseudomonas aeruginosa.

The Journal of antimicrobial chemotherapy 48 (5)
PMID : 11679562  :   DOI  :   10.1093/jac/48.5.717    
Abstract >>
Pseudomonas aeruginosa clinical isolate PA35 is resistant to amino- and ureido-penicillins, has intermediate susceptibility to cefsulodin, cefepime and aztreonam, and is susceptible to imipenem and ceftazidime. Cloning and sequencing revealed a new beta-lactamase variant, OXA-35, sharing 96% amino acid identity with OXA-10. OXA-35 displays a restricted-substrate hydrolysis profile with improved hydrolysis of amoxicillin and cloxacillin compared with OXA-10. OXA-35 differs from derivatives OXA-19 and OXA-28 by one amino acid substitution and may be a progenitor of these OXA-13-like extended-spectrum beta-lactamases.
KeywordMeSH Terms
43. Mavroidi  A, Tzelepi  E, Tsakris  A, Miriagou  V, Sofianou  D, Tzouvelekis  LS,     ( 2001 )

An integron-associated beta-lactamase (IBC-2) from Pseudomonas aeruginosa is a variant of the extended-spectrum beta-lactamase IBC-1.

The Journal of antimicrobial chemotherapy 48 (5)
PMID : 11679551  :   DOI  :   10.1093/jac/48.5.627    
Abstract >>
An extended-spectrum beta-lactamase, IBC-2, produced by a clinical strain of Pseudomonas aeruginosa, was characterized. bla(IBC-2) was found, as a gene cassette, to be the sole gene within the variable region of a class 1 integron probably located in the chromosome. IBC-2 is a variant of IBC-1 and GES-1, differing by one amino acid from each of these beta-lactamases. When expressed in Escherichia coli, IBC-2 was observed to confer resistance to ceftazidime and decreased susceptibility to other oxyimino-beta-lactams.
KeywordMeSH Terms
44. Frisk  A, Jyot  J, Arora  SK, Ramphal  R,     ( 2002 )

Identification and functional characterization of flgM, a gene encoding the anti-sigma 28 factor in Pseudomonas aeruginosa.

Journal of bacteriology 184 (6)
PMID : 11872701  :   DOI  :   10.1128/jb.184.6.1514-1521.2002     PMC  :   PMC134903    
Abstract >>
We describe here the functional characterization of the putative flgM gene of Pseudomonas aeruginosa. FlgM of P. aeruginosa is most similar to FlgM of Vibrio parahaemolyticus. A conserved region is present in the C-terminal half of the FlgM of P. aeruginosa and in FlgM homologues of other organisms that includes the sigma(28) binding domain. A role for the flgM gene of P. aeruginosa in motility was demonstrated by its inactivation. The beta-galactosidase activity of a transcriptional fusion of the fliC promoter to lacZ was upregulated in the flgM mutant, suggesting that the activity of FliA, the sigma factor that regulates fliC, was increased. Consistent with these results, an increased amount of flagellin was demonstrated in the flgM mutant of P. aeruginosa strain PAK by Western blot, suggesting that FlgM negatively regulates transcription of fliC by inhibiting the activity of FliA. Direct interaction of the P. aeruginosa FlgM with the alternative sigma factor sigma(28) was demonstrated by utilizing the yeast two-hybrid system. Three putative consensus sigma(54) recognition sites and one sigma(28) site were found in the flgM upstream region. However, analysis of the transcriptional fusion of the flgM promoter to lacZ in different mutant backgrounds showed that the flgM promoter was not entirely dependent on either sigma(28) or sigma(54). A transcript was detected by primer extension that was 8 bp downstream of the consensus sigma(28)-binding site. Thus, a system for the control of flagellin synthesis by FlgM exists in P. aeruginosa that is different from that in the enteric bacteria and seems to be most similar to that of V. cholerae where both sigma(28)-dependent and -independent mechanisms of transcription exist.
KeywordMeSH Terms
45. Lee  K, Lim  JB, Yum  JH, Yong  D, Chong  Y, Kim  JM, Livermore  DM,     ( 2002 )

bla(VIM-2) cassette-containing novel integrons in metallo-beta-lactamase-producing Pseudomonas aeruginosa and Pseudomonas putida isolates disseminated in a Korean hospital.

Antimicrobial agents and chemotherapy 46 (4)
PMID : 11897589  :   DOI  :   10.1128/aac.46.4.1053-1058.2002     PMC  :   PMC127086    
Abstract >>
We investigated the phenotypic and genetic properties of metallo-beta-lactamase-producing Pseudomonas isolates collected at a tertiary-care hospital in Korea since 1995. The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates reached 16% in 1997, when 9% of the resistant organisms were found to produce VIM-2 beta-lactamase, a class B enzyme previously found only in P. aeruginosa isolates from Europe. VIM-2-producing isolates of Pseudomonas putida were also detected. Resistance was transferable from both these species to P. aeruginosa PAO4089Rp by filter mating, although the resistance determinant could not be found on any detectable plasmid. Serotyping showed that many of the VIM-2-producing P. aeruginosa isolates belonged to serotypes O:11 and O:12, and pulsed-field gel electrophoresis of XbaI-digested genomic DNA revealed that many had identical profiles, whereas the P. putida isolates were diverse. Sequencing showed that the bla(VIM-2) genes resided as cassettes in class 1 integrons. In contrast to previous VIM-encoding integrons, the integron sequenced from a P. aeruginosa isolate had bla(VIM) located downstream of a variant of aacA4. bla(VIM) also lay in a class 1 integron in a representative P. putida strain, but the organization of this integron was different from that sequenced from the P. aeruginosa strain. In conclusion, the metallo-beta-lactamase produced by these imipenem-resistant Pseudomonas isolates was VIM-2, and the accumulation of producers reflected clonal dissemination as well as horizontal spread. Strict measures are required in order to control a further spread of resistance.
KeywordMeSH Terms
46. Pai  H, Jacoby  GA,     ( 2001 )

Sequences of the NPS-1 and TLE-1 beta-lactamase genes.

Antimicrobial agents and chemotherapy 45 (10)
PMID : 11557499  :   DOI  :   10.1128/AAC.45.10.2947-2948.2001     PMC  :   PMC90761    
Abstract >>
The NPS-1 and TLE-1 beta-lactamase genes were cloned and sequenced. NPS-1 differed from LCR-1 beta-lactamase in 8 of 260 amino acids. TLE-1 differed from TEM-1 by a single Asp(115)-->Gly substitution and has been renamed TEM-90.
KeywordMeSH Terms
Bacterial Proteins
47. Martínez  A, Soberón-Chávez  G,     ( 2001 )

Characterization of the lipA gene encoding the major lipase from Pseudomonas aeruginosa strain IGB83.

Applied microbiology and biotechnology 56 (5��6��)
PMID : 11601622  :  
Abstract >>
The lipases produced by Pseudomonas have a wide range of potential biotechnological applications. Pseudomonas aeruginosa IGB83 was isolated as a highly lipolytic strain which produced a thermotolerant and alkaline lipase. In the present work, we have characterized the P. aeruginosa IGB83 gene (lipA) encoding this enzyme. We describe the construction of a lipA mutant and report on the effect of two carbon sources on lipase expression.
KeywordMeSH Terms
48. Hertle  R, Mrsny  R, Fitzgerald  DJ,     ( 2001 )

Dual-function vaccine for Pseudomonas aeruginosa: characterization of chimeric exotoxin A-pilin protein.

Infection and immunity 69 (11)
PMID : 11598071  :   DOI  :   10.1128/IAI.69.11.6962-6969.2001     PMC  :   PMC100076    
Abstract >>
Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis patients. Strategies to prevent colonization by this bacterium and/or neutralize its virulence factors are clearly needed. Here we characterize a dual-function vaccine designed to generate antibodies to reduce bacterial adherence and to neutralize the cytotoxic activity of exotoxin A. To construct the vaccine, key sequences from type IV pilin were inserted into a vector encoding a nontoxic (active-site deletion) version of exotoxin A. The chimeric protein, termed PE64Delta553pil, was expressed in Escherichia coli, refolded to a near-native conformation, and then characterized by various biochemical and immunological assays. PE64Delta553pil bound specifically to asialo-GM1, and, when injected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell-killing activity of exotoxin A. Results support further evaluation of this chimeric protein as a vaccine to prevent Pseudomonas colonization in susceptible individuals.
KeywordMeSH Terms
ADP Ribose Transferases
Bacterial Toxins
Virulence Factors
49. Hickey  WJ, Sabat  G, Yuroff  AS, Arment  AR, Pérez-Lesher  J,     ( 2001 )

Cloning, nucleotide sequencing, and functional analysis of a novel, mobile cluster of biodegradation genes from Pseudomonas aeruginosa strain JB2.

Applied and environmental microbiology 67 (10)
PMID : 11571162  :   DOI  :   10.1128/aem.67.10.4603-4609.2001     PMC  :   PMC93209    
Abstract >>
We have identified in Pseudomonas aeruginosa strain JB2 a novel cluster of mobile genes encoding degradation of hydroxy- and halo-aromatic compounds. Nineteen open reading frames were located and, based on sequence similarities, were putatively identified as encoding a ring hydroxylating oxygenase (hybABCD), an ATP-binding cassette-type transporter, an extradiol ring-cleavage dioxygenase, transcriptional regulatory proteins, enzymes mediating chlorocatechol degradation, and transposition functions. Expression of hybABCD in Escherichia coli cells effected stoichiometric transformation of 2-hydroxybenzoate (salicylate) to 2,5-dihydroxybenzoate (gentisate). This activity was predicted from sequence similarity to functionally characterized genes, nagAaGHAb from Ralstonia sp. strain U2 (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522-2530, 1998), and is the second confirmed example of salicylate 5-hydroxylase activity effected by an oxygenase outside the flavoprotein group. Growth of strain JB2 or Pseudomonas huttiensis strain D1 (an organism that had acquired the 2-chlorobenzoate degradation phenotype from strain JB2) on benzoate yielded mutants that were unable to grow on salicylate or 2-chlorobenzoate and that had a deletion encompassing hybABCD and the region cloned downstream. The mutants' inability to grow on 2-chlorobenzoate suggested the loss of additional genes outside of, but contiguous with, the characterized region. Pulsed-field gel electrophoresis revealed a plasmid of >300 kb in strain D1, but no plasmids were detected in strain JB2. Hybridization analyses confirmed that the entire 26-kb region characterized here was acquired by strain D1 from strain JB2 and was located in the chromosome of both organisms. Further studies to delineate the element's boundaries and functional characteristics could provide new insights into the mechanisms underlying evolution of bacterial genomes in general and of catabolic pathways for anthropogenic pollutants in particular.
KeywordMeSH Terms
DNA Transposable Elements
Genes, Bacterial
50. Calhoun  DH, Bonner  CA, Gu  W, Xie  G, Jensen  RA,     ( 2001 )

The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa.

Genome biology 2 (8)
PMID : 11532214  :   DOI  :   10.1186/gb-2001-2-8-research0030     PMC  :   PMC55327    
Abstract >>
Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea. Many of these exist as domains that are fused with other aromatic-pathway catalytic domains. Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment. Whether or not this might be unique to E. herbicola was unknown. The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S. typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location. The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins. The increased size is due to a carboxy-terminal extension of unknown function. In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P. aeruginosa. Our analysis has detected a number of additional *aroQ genes. The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P. aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the hidden overflow pathway" to phenylalanine described previously."
KeywordMeSH Terms
51. Yorgey  P, Rahme  LG, Tan  MW, Ausubel  FM,     ( 2001 )

The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice.

Molecular microbiology 41 (5)
PMID : 11555287  :   DOI  :   10.1046/j.1365-2958.2001.02580.x    
Abstract >>
We are exploiting the broad host range of the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 to elucidate the molecular basis of bacterial virulence in plants, nematodes, insects and mice. In this report, we characterize the role that two PA14 gene products, MucD and AlgD, play in virulence. MucD is orthologous to the Escherichia coli periplasmic protease and chaperone DegP. DegP homologues are known virulence factors that play a protective role in stress responses in various species. AlgD is an enzyme involved in the biosynthesis of the exopolysaccharide alginate, which is hyperinduced in mucD mutants. A PA14 mucD mutant was significantly impaired in its ability to cause disease in Arabidopsis thaliana and mice and to kill the nematode Caenorhabditis elegans. Moreover, MucD was found to be required for the production of an extracellular toxin involved in C. elegans killing. In contrast, a PA14 algD mutant was not impaired in virulence in plants, nematodes or mice. A mucDalgD double mutant had the same phenotype as the mucD single mutant in the plant and nematode pathogenesis models. However, the mucDalgD double mutant was synergistically reduced in virulence in mice, suggesting that alginate can partially compensate for the loss of MucD function in mouse pathogenesis.
KeywordMeSH Terms
Serine Endopeptidases
52. Arora  SK, Bangera  M, Lory  S, Ramphal  R,     ( 2001 )

A genomic island in Pseudomonas aeruginosa carries the determinants of flagellin glycosylation.

Proceedings of the National Academy of Sciences of the United States of America 98 (16)
PMID : 11481492  :   DOI  :   10.1073/pnas.161249198     PMC  :   PMC55422    
Abstract >>
Protein glycosylation has been long recognized as an important posttranslational modification process in eukaryotic cells. Glycoproteins, predominantly secreted or surface localized, have also been identified in bacteria. We have identified a cluster of 14 genes, encoding the determinants of the flagellin glycosylation machinery in Pseudomonas aeruginosa PAK, which we called the flagellin glycosylation island. Flagellin glycosylation can be detected only in bacteria expressing the a-type flagellin sequence variants, and the survey of 30 P. aeruginosa isolates revealed coinheritance of the a-type flagellin genes with at least one of the flagellin glycosylation island genes. Expression of the b-type flagellin in PAK, an a-type strain carrying the glycosylation island, did not lead to glycosylation of the b-type flagellin of PAO1, suggesting that flagellins expressed by b-type bacteria not only lack the glycosylation island, they cannot serve as substrates for glycosylation. Providing the entire glycosylation island of PAK, including its a-type flagellin in a flagellin mutant of a b-type strain, results in glycosylation of the heterologous flagellin. These results suggest that some or all of the 14 genes on the glycosylation island are the genes that are missing from strain PAO1 to allow glycosylation of an appropriate flagellin. Inactivation of either one of the two flanking genes present on this island abolished flagellin glycosylation. Based on the limited homologies of these gene products with enzymes involved in glycosylation, we propose that the island encodes similar proteins involved in synthesis, activation, or polymerization of sugars that are necessary for flagellin glycosylation.
KeywordMeSH Terms
Genome, Bacterial
53. Poirel  L, Weldhagen  GF, Naas  T, De Champs  C, Dove  MG, Nordmann  P,     ( 2001 )

GES-2, a class A beta-lactamase from Pseudomonas aeruginosa with increased hydrolysis of imipenem.

Antimicrobial agents and chemotherapy 45 (9)
PMID : 11502535  :   DOI  :   10.1128/aac.45.9.2598-2603.2001     PMC  :   PMC90698    
Abstract >>
Pseudomonas aeruginosa GW-1 was isolated in 2000 in South Africa from blood cultures of a 38-year-old female who developed nosocomial pneumonia. This isolate harbored a self-transferable ca. 100-kb plasmid that conferred an expanded-spectrum cephalosporin resistance profile associated with an intermediate susceptibility to imipenem. A beta-lactamase gene, bla(GES-2), was cloned from whole-cell DNA of P. aeruginosa GW-1 and expressed in Escherichia coli. GES-2, with a pI value of 5.8, hydrolyzed expanded-spectrum cephalosporins, and its substrate profile was extended to include imipenem compared to that of GES-1, identified previously in Klebsiella pneumoniae. GES-2 activity was less inhibited by clavulanic acid, tazobactam and imipenem than GES-1. The GES-2 amino acid sequence differs from that of GES-1 by a glycine-to-asparagine substitution in position 170 located in the omega loop of Ambler class A enzymes. This amino acid change may explain the extension of the substrate profile of the plasmid-encoded beta-lactamase GES-2.
KeywordMeSH Terms
54. Pernot  L, Frénois  F, Rybkine  T, L'Hermite  G, Petrella  S, Delettré  J, Jarlier  V, Collatz  E, Sougakoff  W,     ( 2001 )

Crystal structures of the class D beta-lactamase OXA-13 in the native form and in complex with meropenem.

Journal of molecular biology 310 (4)
PMID : 11453693  :   DOI  :   10.1006/jmbi.2001.4805    
Abstract >>
The therapeutic problems posed by class D beta-lactamases, a family of serine enzymes that hydrolyse beta-lactam antibiotics following an acylation-deacylation mechanism, are increased by the very low level of sensitivity of these enzymes to beta-lactamase inhibitors. To gain structural and mechanistic insights to aid the design of new inhibitors, we have determined the crystal structure of OXA-13 from Pseudomonas aeruginosa in the apo form and in complex with the carbapenem meropenem. The native form consisted of a dimer displaying an overall organisation similar to that found in the closely related enzyme OXA-10. In the acyl-enzyme complex, the positioning of the antibiotic appeared to be ensured mainly by (i) the covalent acyl bond and (ii) a strong salt-bridge involving the carboxylate moiety of the drug. Comparison of the structures of OXA-13 in the apo form and in complex with meropenem revealed an unsuspected flexibility in the region of the essential serine 115 residue, with possible consequences for the catalytic properties of the enzyme. In the apo form, the Ser115 side-chain is oriented outside the active site, whereas the general base Lys70 adopts a conformation that seems to be incompatible with the activation of the catalytic water molecule required for the deacylation step. In the OXA-13:meropenem complex, a 3.5 A movement of the backbone of the 114-116 loop towards the side-chain of Lys70 was observed, which seems to be driven by a displacement of the neighbouring 91-104 loop and which results in the repositioning of the side-chain hydroxyl group of Ser115 toward the catalytic centre. Concomitantly, the side-chain of Lys70 is forced to curve in the direction of the deacylating water molecule, which is then strongly bound and activated by this residue. However, a distance of ca 5 A separates the catalytic water molecule from the acyl carbonyl group of meropenem, a structural feature that accounts for the inhibition of OXA-13 by this drug. Finally, the low level of penicillinase activity revealed by the kinetic analysis of OXA-13 could be related to the specific presence in position 73 of a serine residue located close to the general base Lys70, which results in a decrease of the number of hydrogen-bonding interactions stabilising the catalytic water molecule.
KeywordMeSH Terms
55. Yan  JJ, Hsueh  PR, Ko  WC, Luh  KT, Tsai  SH, Wu  HM, Wu  JJ,     ( 2001 )

Metallo-beta-lactamases in clinical Pseudomonas isolates in Taiwan and identification of VIM-3, a novel variant of the VIM-2 enzyme.

Antimicrobial agents and chemotherapy 45 (8)
PMID : 11451678  :   DOI  :   10.1128/AAC.45.8.2224-2228.2001     PMC  :   PMC90635    
Abstract >>
A total of 209 clinical isolates of Pseudomonas (193 Pseudomonas aeruginosa, 10 P. putida, 4 P. stutzeri, and 2 P. fluorescens isolates) with reduced susceptibilities to imipenem and/or ceftazidime were subjected to PCR assays with primers specific for bla(IMP-1), bla(IMP-2), bla(VIM-1), and bla(VIM-2) and sequence analysis to identify the metallo-beta-lactamases (MBLs) prevalent among these organisms in Taiwan; and 21 isolates gave positive results. Five isolates including two P. putida and three P. stutzeri isolates were found to carry bla(IMP-1), and six isolates including five P. putida and one P. stutzeri isolates harbored bla(VIM-2). The remaining 10 isolates were P. aeruginosa, and all were found to carry a novel variant of bla(VIM-2), designated bla(VIM-3). There are only two nucleotide differences between bla(VIM-2) and bla(VIM-3), leading to two amino acid alterations. Our findings indicate that VIM-2 and its variant have become the most prevalent metalloenzymes in Pseudomonas in Taiwan. Southern hybridization with the bla(VIM-2)-, bla(VIM-3)-, and bla(IMP-1)-specific probes revealed that only two VIM-2-producing P. putida isolates appeared to carry the MBL gene on plasmids. Pulsed-field gel electrophoresis showed that six VIM-3-producing P. aeruginosa isolates and two IMP-1-producing P. stutzeri isolates were genetically related, suggesting that the spread of these MBL genes in Taiwan could be due to clonal dissemination as well as genetic exchange between different clones.
KeywordMeSH Terms
56. Nishijyo  T, Haas  D, Itoh  Y,     ( 2001 )

The CbrA-CbrB two-component regulatory system controls the utilization of multiple carbon and nitrogen sources in Pseudomonas aeruginosa.

Molecular microbiology 40 (4)
PMID : 11401699  :   DOI  :   10.1046/j.1365-2958.2001.02435.x    
Abstract >>
A novel two-component system, CbrA-CbrB, was discovered in Pseudomonas aeruginosa; cbrA and cbrB mutants of strain PAO were found to be unable to use several amino acids (such as arginine, histidine and proline), polyamines and agmatine as sole carbon and nitrogen sources. These mutants were also unable to use, or used poorly, many other carbon sources, including mannitol, glucose, pyruvate and citrate. A 7 kb EcoRI fragment carrying the cbrA and cbrB genes was cloned and sequenced. The cbrA and cbrB genes encode a sensor/histidine kinase (Mr 108 379, 983 residues) and a cognate response regulator (Mr 52 254, 478 residues) respectively. The amino-terminal half (490 residues) of CbrA appears to be a sensor membrane domain, as predicted by 12 possible transmembrane helices, whereas the carboxy-terminal part shares homology with the histidine kinases of the NtrB family. The CbrB response regulator shows similarity to the NtrC family members. Complementation and primer extension experiments indicated that cbrA and cbrB are transcribed from separate promoters. In cbrA or cbrB mutants, as well as in the allelic argR9901 and argR9902 mutants, the aot-argR operon was not induced by arginine, indicating an essential role for this two-component system in the expression of the ArgR-dependent catabolic pathways, including the aruCFGDB operon specifying the major aerobic arginine catabolic pathway. The histidine catabolic enzyme histidase was not expressed in cbrAB mutants, even in the presence of histidine. In contrast, proline dehydrogenase, responsible for proline utilization (Pru), was expressed in a cbrB mutant at a level comparable with that of the wild-type strain. When succinate or other C4-dicarboxylates were added to proline medium at 1 mM, the cbrB mutant was restored to a Pru+ phenotype. Such a succinate-dependent Pru+ property was almost abolished by 20 mM ammonia. In conclusion, the CbrA-CbrB system controls the expression of several catabolic pathways and, perhaps together with the NtrB-NtrC system, appears to ensure the intracellular carbon: nitrogen balance in P. aeruginosa.
KeywordMeSH Terms
Amino Acid Transport Systems
Periplasmic Binding Proteins
57. Caspi  R, Pacek  M, Consiglieri  G, Helinski  DR, Toukdarian  A, Konieczny  I,     ( 2001 )

A broad host range replicon with different requirements for replication initiation in three bacterial species.

The EMBO journal 20 (12)
PMID : 11406602  :   DOI  :   10.1093/emboj/20.12.3262     PMC  :   PMC150194    
Abstract >>
Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.
KeywordMeSH Terms
DNA Replication
Escherichia coli Proteins
Replicon
58. Aubert  D, Poirel  L, Chevalier  J, Leotard  S, Pages  JM, Nordmann  P,     ( 2001 )

Oxacillinase-mediated resistance to cefepime and susceptibility to ceftazidime in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 45 (6)
PMID : 11353602  :   DOI  :   10.1128/AAC.45.6.1615-1620.2001     PMC  :   PMC90522    
Abstract >>
Pseudomonas aeruginosa clinical isolate SOF-1 was resistant to cefepime and susceptible to ceftazidime. This resistance phenotype was explained by the expression of OXA-31, which shared 98% amino acid identity with a class D beta-lactamase, OXA-1. The oxa-31 gene was located on a ca. 300-kb nonconjugative plasmid and on a class 1 integron. No additional efflux mechanism for cefepime was detected in P. aeruginosa SOF-1. Resistance to cefepime and susceptibility to ceftazidime in P. aeruginosa were conferred by OXA-1 as well.
KeywordMeSH Terms
59. Poirel  L, Rotimi  VO, Mokaddas  EM, Karim  A, Nordmann  P,     ( N/A )

VEB-1-like extended-spectrum beta-lactamases in Pseudomonas aeruginosa, Kuwait.

Emerging infectious diseases 7 (3)
PMID : 11384532  :   DOI  :   10.3201/eid0703.010322     PMC  :   PMC2631808    
Abstract >>
Two clinical Pseudomonas aeruginosa isolates from patients in intensive care units in Kuwait were resistant to expanded-spectrum cephalosporins and showed a synergistic effect between ceftazidime and clavulanic acid. This is the first report of extended-spectrum enzymes from nosocomial isolates from the Middle East.
KeywordMeSH Terms
60. Toney  JH, Hammond  GG, Fitzgerald  PM, Sharma  N, Balkovec  JM, Rouen  GP, Olson  SH, Hammond  ML, Greenlee  ML, Gao  YD,     ( 2001 )

Succinic acids as potent inhibitors of plasmid-borne IMP-1 metallo-beta-lactamase.

The Journal of biological chemistry 276 (34)
PMID : 11390410  :   DOI  :   10.1074/jbc.M104742200    
Abstract >>
IMP-1 metallo-beta-lactamase (class B) is a plasmid-borne zinc metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics, including carbapenems, rendering them ineffective. Because IMP-1 has been found in several clinically important carbapenem-resistant pathogens, there is a need for inhibitors of this enzyme that could protect broad spectrum antibiotics such as imipenem from hydrolysis and thus extend their utility. We have identified a series of 2,3-(S,S)-disubstituted succinic acids that are potent inhibitors of IMP-1. Determination of high resolution crystal structures and molecular modeling of succinic acid inhibitor complexes with IMP-1 has allowed an understanding of the potency, stereochemistry, and structure-activity relationships of these inhibitors.
KeywordMeSH Terms
Plasmids
61. Azzolina  BA, Yuan  X, Anderson  MS, El-Sherbeini  M,     ( 2001 )

The cell wall and cell division gene cluster in the Mra operon of Pseudomonas aeruginosa: cloning, production, and purification of active enzymes.

Protein expression and purification 21 (3)
PMID : 11281713  :   DOI  :   10.1006/prep.2001.1390    
Abstract >>
We have cloned the Pseudomonas aeruginosa cell wall biosynthesis and cell division gene cluster that corresponds to the mra operon in the 2-min region of the Escherichia coli chromosome. The organization of the two chromosomal regions in P. aeruginosa and E. coli is remarkably similar with the following gene order: pbp3/pbpB, murE, murF, mraY, murD, ftsW, murG, murC, ddlB, ftsQ, ftsA, ftsZ, and envA/LpxC. All of the above P. aeruginosa genes are transcribed from the same strand of DNA with very small, if any, intragenic regions, indicating that these genes may constitute a single operon. All five amino acid ligases, MurC, MurD, MurE, MurF, and DdlB, in addition to MurG and MraY were cloned in expression vectors. The four recombinant P. aeruginosa Mur ligases, MurC, MurD, MurE, and MurF were overproduced in E. coli and purified as active enzymes.
KeywordMeSH Terms
62. Partridge  SR, Brown  HJ, Stokes  HW, Hall  RM,     ( 2001 )

Transposons Tn1696 and Tn21 and their integrons In4 and In2 have independent origins.

Antimicrobial agents and chemotherapy 45 (4)
PMID : 11257044  :   DOI  :   10.1128/AAC.45.4.1263-1270.2001     PMC  :   PMC90453    
Abstract >>
The first 13.6 kb of the mercury and multidrug resistance transposon Tn1696, which includes the class 1 integron In4, has been sequenced. In4 is 8.33 kb long and contains the 5'-conserved segment (5'-CS) and 2.24 kb of the 3'-conserved segment (3'-CS) flanking four integrated cassettes. The 3'-CS region is followed by one full copy and an adjacent partial copy of the insertion sequence IS6100 flanked, in inverse orientation, by two short segments (123 and 152 bp) from the outer right-hand end of class 1 integrons. This structure is representative of a distinct group of class 1 integrons that differs from In2, found in Tn21, and other related class 1 integrons. In4 does not include transposition genes but is bounded by characteristic 25-bp inverted repeats and flanked by a direct duplication of 5 bp of the target sequence, indicating that it was inserted by a transpositional mechanism. In4 lies between the resII and resI sites of a backbone mercury resistance transposon which is >99.5% identical to Tn5036. Although Tn21 and Tn1696 are both classified as members of the Tn21 subfamily of the Tn3 transposon family, the backbone mercury resistance transposons are only 79 to 96% identical. Tn21 also contains a region of about 0.7 kb not found in Tn1696. The integrons In2 and In4 carrying the antibiotic resistance genes have been inserted at different locations into distinct ancestral mercury resistance transposons. Thus, Tn21 and Tn1696 have independent histories and origins. Other transposons (Tn1403 and Tn1412) that include a class 1 integron also have independent origins. In all except Tn21, the integron is located within the res region of the backbone transposon.
KeywordMeSH Terms
DNA Transposable Elements
Drug Resistance, Multiple
Evolution, Molecular
63. Ferguson  MW, Maxwell  JA, Vincent  TS, da Silva  J, Olson  JC,     ( 2001 )

Comparison of the exoS gene and protein expression in soil and clinical isolates of Pseudomonas aeruginosa.

Infection and immunity 69 (4)
PMID : 11254575  :   DOI  :   10.1128/IAI.69.4.2198-2210.2001     PMC  :   PMC98147    
Abstract >>
Exoenzyme S (ExoS) is translocated into eukaryotic cells by the type III secretory process and has been hypothesized to function in conjunction with other virulence factors in the pathogenesis of Pseudomonas aeruginosa. To gain further understanding of how ExoS might contribute to P. aeruginosa survival and virulence, ExoS expression and the structural gene sequence were determined in P. aeruginosa soil isolates and compared with ExoS of clinical isolates. Significantly higher levels of ExoS ADP-ribosyltransferase (ADPRT) activity were detected in culture supernatants of soil isolates compared to those of clinical isolates. The higher levels of ADPRT activity of soil isolates reflected both the increased production of ExoS and the production of ExoS having a higher specific activity. ExoS structural gene sequence comparisons found the gene to be highly conserved among soil and clinical isolates, with the greatest number of nonsynonymous substitutions occurring within the region of ExoS encoding GAP function. The lack of amino acid changes in the ADPRT region in association with a higher specific activity implies that other factors produced by P. aeruginosa or residues outside the ADPRT region are affecting ExoS ADPRT activity. The data are consistent with ExoS being integral to P. aeruginosa survival in the soil and suggest that, in the transition of P. aeruginosa from the soil to certain clinical settings, the loss of ExoS expression is favored.
KeywordMeSH Terms
Bacterial Toxins
Soil Microbiology
64. Keizer  DW, Slupsky  CM, Kalisiak  M, Campbell  AP, Crump  MP, Sastry  PA, Hazes  B, Irvin  RT, Sykes  BD,     ( 2001 )

Structure of a pilin monomer from Pseudomonas aeruginosa: implications for the assembly of pili.

The Journal of biological chemistry 276 (26)
PMID : 11294863  :   DOI  :   10.1074/jbc.M100659200    
Abstract >>
Type IV pilin monomers assemble to form fibers called pili that are required for a variety of bacterial functions. Pilin monomers oligomerize due to the interaction of part of their hydrophobic N-terminal alpha-helix. Engineering of a truncated pilin from Pseudomonas aeruginosa strain K122-4, where the first 28 residues are removed from the N terminus, yields a soluble, monomeric protein. This truncated pilin is shown to bind to its receptor and to decrease morbidity and mortality in mice upon administration 15 min before challenge with a heterologous strain of Pseudomonas. The structure of this truncated pilin reveals an alpha-helix at the N terminus that lies across a 4-stranded antiparallel beta-sheet. A model for a pilus is proposed that takes into account both electrostatic and hydrophobic interactions of pilin subunits as well as previously published x-ray fiber diffraction data. Our model indicates that DNA or RNA cannot pass through the center of the pilus, however, the possibility exists for small organic molecules to pass through indicating a potential mechanism for signal transduction.
KeywordMeSH Terms
65. Bartels  F, Fernández  S, Holtel  A, Timmis  KN, de Lorenzo  V,     ( 2001 )

The essential HupB and HupN proteins of Pseudomonas putida provide redundant and nonspecific DNA-bending functions.

The Journal of biological chemistry 276 (20)
PMID : 11278879  :   DOI  :   10.1074/jbc.M011295200    
Abstract >>
A protein mixture containing two major components able to catalyze a beta-recombination reaction requiring nonspecific DNA bending was obtained by fractionation of a Pseudomonas putida extract. N-terminal sequence analysis and genomic data base searches identified the major component as an analogue of HupB of Pseudomonas aeruginosa and Escherichia coli, encoding one HU protein variant. The minor component of the fraction, termed HupN, was divergent enough from HupB to predict a separate DNA-bending competence. The determinants of the two proteins were cloned and hyperexpressed, and the gene products were purified. Their activities were examined in vitro in beta-recombination assays and in vivo by complementation of the Hbsu function of Bacillus subtilis. HupB and HupN were equally efficient in all tests, suggesting that they are independent and functionally redundant DNA bending proteins. This was reflected in the maintenance of in vivo activity of the final sigma54 Ps promoter of the toluene degradation plasmid, TOL, which requires facilitated DNA bending, in DeltahupB or DeltahupN strains. However, hupB/hupN double mutants were not viable. It is suggested that the requirement for protein-facilitated DNA bending is met in P. putida by two independent proteins that ensure an adequate supply of an essential cellular activity.
KeywordMeSH Terms
66. Castric  P, Cassels  FJ, Carlson  RW,     ( 2001 )

Structural characterization of the Pseudomonas aeruginosa 1244 pilin glycan.

The Journal of biological chemistry 276 (28)
PMID : 11342554  :   DOI  :   10.1074/jbc.M102685200    
Abstract >>
An antigenic similarity between lipopolysaccharide (LPS) and glycosylated pilin of Pseudomonas aeruginosa 1244 was noted. We purified a glycan-containing molecule from proteolytically digested pili and showed it to be composed of three sugars and serine. This glycan competed with pure pili and LPS for reaction with an LPS-specific monoclonal antibody, which also inhibited twitching motility by P. aeruginosa bearing glycosylated pili. One-dimensional NMR analysis of the glycan indicated the sugars to be 5N beta OHC(4)7NfmPse, Xyl, and FucNAc. The complete proton assignments of these sugars as well as the serine residue were determined by COSY and TOCSY. Electrospray ionization mass spectrometry (MS) determined the mass of this molecule to be 771.5. The ROESY NMR spectrum, tandem MS/MS analysis, and methylation analysis provided information on linkage and the sequence of oligosaccharide components. These data indicated that the molecule had the following structure: alpha-5N beta OHC(4)7NFmPse-(2-->4)beta-Xyl-(1-->3)-beta-FucNAc-(1-->3)-beta-Ser.
KeywordMeSH Terms
67. Würtele  M, Renault  L, Barbieri  JT, Wittinghofer  A, Wolf  E,     ( 2001 )

Structure of the ExoS GTPase activating domain.

FEBS letters 491 (1��2��)
PMID : 11226412  :   DOI  :   10.1016/s0014-5793(01)02105-6    
Abstract >>
Pseudomonas aeruginosa is an opportunistic bacterial pathogen of great medical relevance. One of its major toxins, exoenzyme S (ExoS), is a dual function protein with a C-terminal Ras-ADP-ribosylation domain and an N-terminal GTPase activating protein (GAP) domain specific for Rho-family proteins. We report here the three-dimensional structure of the N-terminal domain of ExoS determined by X-ray crystallography to 2.4 A resolution. Its fold is all helical with a four helix bundle core capped by additional irregular helices. Loops that are known to interact with Rho-family proteins show very large mobility. Considering the importance of ExoS in Pseudomonas pathogenicity, this structure could be of interest for drug targeting.
KeywordMeSH Terms
Bacterial Toxins
68. Poirel  L, Girlich  D, Naas  T, Nordmann  P,     ( 2001 )

OXA-28, an extended-spectrum variant of OXA-10 beta-lactamase from Pseudomonas aeruginosa and its plasmid- and integron-located gene.

Antimicrobial agents and chemotherapy 45 (2)
PMID : 11158739  :   DOI  :   10.1128/AAC.45.2.447-453.2001     PMC  :   PMC90311    
Abstract >>
Pseudomonas aeruginosa ED-1, isolated from a pulmonary brush of a patient hospitalized in a suburb of Paris, France, was resistant to ceftazidime and of intermediate susceptibility to ureidopenicillins and to cefotaxime. Cloning and expression of the beta-lactamase gene content of this isolate in Escherichia coli DH10B identified a novel OXA-10 variant, OXA-28, with a pI value of 8.1 and a molecular mass of 29 kDa. It differed from OXA-10 by 10 amino acid changes and from OXA-13 and OXA-19 by 2 amino acid changes, including a glycine instead of tryptophan at position 164, which is likely involved in its resistance to ceftazidime. Like OXA-11, -14, -16, and -19 and as opposed to OXA-17, OXA-28 predominantly compromised ceftazidime and had only marginal effect on the MICs of aztreonam and cefotaxime in P. aeruginosa. Once expressed in E. coli, OXA-28 raised the MIC of ceftazidime to a much higher level than those of amoxicillin, cephalothin, and cefotaxime (128, 16, 8, and 4 microg/ml, respectively). OXA-28 beta-lactamase had a broad spectrum of activity, including ceftazidime. Its activity was partially antagonized by clavulanic acid (50% inhibitory concentration, 10 microM) and NaCl addition. The oxa28 gene cassette was inserted in the variable region of a class 1 integron, In57, immediately downstream of an amino 6'-N-acetyltransferase gene cassette, aac(6')Ib. The structures of the integrons carrying either oxa28, oxa13, or oxa19 gene cassettes were almost identical, suggesting that they may have derived from a common ancestor as a result of the common European origin of the P. aeruginosa isolates. In57 was located on a self-transferable plasmid of ca. 150 kb that was transferred from P. aeruginosa to P. aeruginosa.
KeywordMeSH Terms
69. Murata  T, Bognar  AL, Hayashi  T, Ohnishi  M, Nakayama  K, Terawaki  Y,     ( 2000 )

Molecular analysis of the folC gene of Pseudomonas aeruginosa.

Microbiology and immunology 44 (11)
PMID : 11145267  :   DOI  :   10.1111/j.1348-0421.2000.tb02578.x    
Abstract >>
We have cloned the Pseudomonas aeruginosa folC gene coding for folylpolyglutamate synthetase-dihydrofolate synthetase, which was located between the trpF and purF loci, and determined the nucleotide sequence of the folC gene and its flanking region. The deduced amino acid sequence of P. aeruginosa FolC was highly homologous to that of Escherichia coli FolC. The cloned gene complemented E. coli folC mutations and was found to encode both folylpolyglutamate synthetase and dihydrofolate synthetase activities. The gene organization around the folC gene in P. aeruginosa was completely conserved with that in E. coli; the accD gene was located upstream of the folC gene, and dedD, cvpA and purF genes followed the folC gene in this order. The gene arrangement and the result of the promoter activity assay suggested that the P. aeruginosa accD and folC genes were co-transcribed.
KeywordMeSH Terms
70. Würtele  M, Wolf  E, Pederson  KJ, Buchwald  G, Ahmadian  MR, Barbieri  JT, Wittinghofer  A,     ( 2001 )

How the Pseudomonas aeruginosa ExoS toxin downregulates Rac.

Nature structural biology 8 (1)
PMID : 11135665  :   DOI  :   10.1038/83007    
Abstract >>
Pseudomonas aeruginosa is an opportunistic bacterial pathogen. One of its major toxins, ExoS, is translocated into eukaryotic cells by a type III secretion pathway. ExoS is a dual function enzyme that affects two different Ras-related GTP binding proteins. The C-terminus inactivates Ras through ADP ribosylation, while the N-terminus inactivates Rho proteins through its GTPase activating protein (GAP) activity. Here we have determined the three-dimensional structure of a complex between Rac and the GAP domain of ExoS in the presence of GDP and AlF3. Composed of approximately 130 residues, this ExoS domain is the smallest GAP hitherto described. The GAP domain of ExoS is an all-helical protein with no obvious structural homology, and thus no recognizable evolutionary relationship, with the eukaryotic RhoGAP or RasGAP fold. Similar to other GAPs, ExoS downregulates Rac using an arginine finger to stabilize the transition state of the GTPase reaction, but the details of the ExoS-Rac interaction are unique. Considering the intrinsic resistance of P. aeruginosa to antibiotics, this might open up a new avenue towards blocking its pathogenicity.
KeywordMeSH Terms
Down-Regulation
71. Branny  P, Pearson  JP, Pesci  EC, Köhler  T, Iglewski  BH, Van Delden  C,     ( 2001 )

Inhibition of quorum sensing by a Pseudomonas aeruginosa dksA homologue.

Journal of bacteriology 183 (5)
PMID : 11160083  :   DOI  :   10.1128/JB.183.5.1531-1539.2001     PMC  :   PMC95037    
Abstract >>
The Pseudomonas aeruginosa las (lasR-lasI) and rhl (rhlR-rhlI) quorum-sensing systems regulate the expression of several virulence factors, including elastase and rhamnolipid. P. aeruginosa strain PR1-E4 is a lasR deletion mutant that contains a second, undefined mutation which allows production of elastase and rhamnolipid despite a nonfunctional las system. We have previously shown that this strain accomplishes this by increasing the expression of the autoinducer synthase gene rhlI. In this report, we show that the elastolytic phenotype of mutant PR1-E4 can be complemented with a P. aeruginosa homologue of the Escherichia coli dnaK mutation suppressor gene dksA. When supplied in trans on a multicopy plasmid, this gene completely suppressed elastase production by mutant PR1-E4. Cloning and Northern blot analysis revealed that dksA was neither mutated nor less transcribed in mutant PR1-E4. When overexpressed, dksA also reduced rhamnolipid production by both mutant PR1-E4 and the wild type, PAO1. Using Northern blot analysis and lacZ reporter fusions, we show that dksA inhibits rhlI, rhlAB, and lasB transcription. Exogenous N-butyryl-L-homoserine lactone overcame the reduced expression of rhlI and restored rhlAB and lasB expression, as well as elastase production. Our results suggest that the overproduction of the P. aeruginosa DksA homologue inhibits quorum-sensing-dependent virulence factor production by downregulating the transcription of the autoinducer synthase gene rhlI.
KeywordMeSH Terms
Escherichia coli Proteins
Gene Expression Regulation, Bacterial
Signal Transduction
72. Schwan  WR, Barker  L, Brody  LL,     ( 2000 )

Differences in sensitivity to PA-1806 among iron transport mutants of Pseudomonas aeruginosa compared to Escherichia coli.

Antimicrobial agents and chemotherapy 44 (11)
PMID : 11184229  :   DOI  :   10.1128/aac.44.11.3237-3238.2000     PMC  :   PMC101644    
Abstract >>
N/A
KeywordMeSH Terms
Escherichia coli Proteins
73. Duong  F, Bonnet  E, Géli  V, Lazdunski  A, Murgier  M, Filloux  A,     ( 2001 )

The AprX protein of Pseudomonas aeruginosa: a new substrate for the Apr type I secretion system.

Gene 262 (1��2��)
PMID : 11179678  :   DOI  :   10.1016/s0378-1119(00)00541-2    
Abstract >>
Protein secretion in Pseudomonas aeruginosa involves different mechanisms. The type II and type III secretory pathways control the extracellular release of a wide range of substrates. The type I secretion process, or ABC transporter, was believed to be exclusively involved in alkaline protease secretion. Recently, it was discovered that a P. aeruginosa heme binding protein, HasAp, is also secreted by a type I process. We present here the identification of a third putative type I-dependent protein of P. aeruginosa, AprX. The function of this protein has not yet been elucidated but very interestingly it appears to be linked to the apr cluster, and organized in one single operon together with the aprD, -E and -F genes.
KeywordMeSH Terms
ATP-Binding Cassette Transporters
Membrane Proteins
Membrane Transport Proteins
74. Pallecchi  L, Riccio  ML, Docquier  JD, Fontana  R, Rossolini  GM,     ( 2001 )

Molecular heterogeneity of bla(VIM-2)-containing integrons from Pseudomonas aeruginosa plasmids encoding the VIM-2 metallo-beta-lactamase.

FEMS microbiology letters 195 (2)
PMID : 11179643  :   DOI  :   10.1111/j.1574-6968.2001.tb10512.x    
Abstract >>
A bla(VIM-2) metallo-beta-lactamase determinant, identical to that previously identified in Pseudomonas aeruginosa COL-1 isolate from a French hospital, was detected on a 28-kb plasmid carried by a nosocomial isolate of P. aeruginosa from Verona, Italy. In this plasmid the bla(VIM-2) determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1 integrase gene and two gene cassettes. The first cassette carries an aacA4 aminoglycoside acetyl transferase determinant. The second cassette carries a bla(VIM-2) determinant followed by a partially deleted attC site. The structure of In72 was notably different from that of In56, the bla(VIM-2)-containing integron found in the COL-1 isolate, revealing the existence of molecular heterogeneity among bla(VIM-2)-containing integrons in clinical isolates of P. aeruginosa from Europe.
KeywordMeSH Terms
DNA Transposable Elements
Plasmids
Recombination, Genetic
75. Kerschen  EJ, Irani  VR, Hassett  DJ, Rowe  JJ,     ( 2001 )

snr-1 gene is required for nitrate reduction in Pseudomonas aeruginosa PAO1.

Journal of bacteriology 183 (6)
PMID : 11222615  :   DOI  :   10.1128/JB.183.6.2125-2131.2001     PMC  :   PMC95112    
Abstract >>
Pseudomonas aeruginosa is able to use nitrate for both assimilation and anaerobic respiration. One set of genes, designated snr (for "shared nitrate reduction"), have been recently cloned and partially characterized. In this study, we demonstrate that the snr-1 gene encodes a predicted 52.5-kDa protein that is 82% similar to a unique cytochrome c of Desulfomonile tiedjei DCB-1. Importantly, the Snr-1 protein sequence of P. aeruginosa differed from that of the cytochrome c of D. tiedjei primarily in the first 25 amino acids, which are required for membrane attachment in D. tiedjei. In P. aeruginosa, the Snr-1 protein hydropathy profile indicates that it is a soluble protein. An isogenic snr-1::Gm insertional mutant was unable to grow aerobically with nitrate as a sole nitrogen source or anaerobically with nitrate as an electron acceptor. Complementation of the snr-1::Gm mutant with the snr-1 gene restored the wild-type phenotypes. Interestingly, anaerobic growth rates were significantly higher in the snr-1 mutant harboring a multicopy plasmid containing snr-1. In contrast, aerobic growth rates of the restored mutant using nitrate as the sole nitrogen source were similar to those of the wild type. Transcriptional lacZ fusions demonstrated that snr-1 was not regulated by molybdate, oxygen, or nitrate.
KeywordMeSH Terms
76. Liang  X, Pham  XQ, Olson  MV, Lory  S,     ( 2001 )

Identification of a genomic island present in the majority of pathogenic isolates of Pseudomonas aeruginosa.

Journal of bacteriology 183 (3)
PMID : 11208781  :   DOI  :   10.1128/JB.183.3.843-853.2001     PMC  :   PMC94950    
Abstract >>
Pseudomonas aeruginosa, a ubiquitous gram-negative bacterium, is capable of colonizing a wide range of environmental niches and can also cause serious infections in humans. In order to understand the genetic makeup of pathogenic P. aeruginosa strains, a method of differential hybridization of arrayed libraries of cloned DNA fragments was developed. An M13 library of DNA from strain X24509, isolated from a patient with a urinary tract infection, was screened using a DNA probe from P. aeruginosa strain PAO1. The genome of PAO1 has been recently sequenced and can be used as a reference for comparisons of genetic organization in different strains. M13 clones that did not react with a DNA probe from PAO1 carried X24509-specific inserts. When a similar array hybridization analysis with DNA probes from different strains was used, a set of M13 clones which carried sequences present in the majority of human P. aeruginosa isolates from a wide range of clinical sources was identified. The inserts of these clones were used to identify cosmids encompassing a contiguous 48.9-kb region of the X24509 chromosome called PAGI-1 (for "P. aeruginosa genomic island 1"). PAGI-1 is incorporated in the X24509 chromosome at a locus that shows a deletion of a 6,729-bp region present in strain PAO1. Survey of the incidence of PAGI-1 revealed that this island is present in 85% of the strains from clinical sources. Approximately half of the PAGI-1-carrying strains show the same deletion as X24509, while the remaining strains contain both the PAGI-1 sequences and the 6,729-bp PAO1 segment. Sequence analysis of PAGI-1 revealed that it contains 51 predicted open reading frames. Several of these genes encoded products with predictable function based on their sequence similarities to known genes, including insertion sequences, determinants of regulatory proteins, a number of dehydrogenase gene homologs, and two for proteins of implicated in detoxification of reactive oxygen species. It is very likely that PAGI-1 was acquired by a large number of P. aeruginosa isolates through horizontal gene transfer. The selection for its maintenance may be the consequence of expression of any one of the genes of unknown function or the genes which allow P. aeruginosa to survive under the conditions that generate reactive oxygen species. Alternatively, one or both of the transcriptional regulators encoded in PAGI-1 may control the expression of genes in the P. aeruginosa chromosome, which provides a selective advantage for strains that have acquired this genomic island.
KeywordMeSH Terms
Oligonucleotide Array Sequence Analysis
77. Poirel  L, Lambert  T, Türkoglü  S, Ronco  E, Gaillard  J, Nordmann  P,     ( 2001 )

Characterization of Class 1 integrons from Pseudomonas aeruginosa that contain the bla(VIM-2) carbapenem-hydrolyzing beta-lactamase gene and of two novel aminoglycoside resistance gene cassettes.

Antimicrobial agents and chemotherapy 45 (2)
PMID : 11158753  :   DOI  :   10.1128/AAC.45.2.546-552.2001     PMC  :   PMC90325    
Abstract >>
Two clonally unrelated Pseudomonas aeruginosa clinical strains, RON-1 and RON-2, were isolated in 1997 and 1998 from patients hospitalized in a suburb of Paris, France. Both isolates expressed the class B carbapenem-hydrolyzing beta-lactamase VIM-2 previously identified in Marseilles in the French Riviera. In both isolates, the bla(VIM-2) cassette was part of a class 1 integron that also encoded aminoglycoside-modifying enzymes. In one case, two novel aminoglycoside resistance gene cassettes, aacA29a and aacA29b, were located at the 5' and 3' end of the bla(VIM-2) gene cassette, respectively. The aacA29a and aacA29b gene cassettes were fused upstream with a 101-bp part of the 5' end of the qacE cassette. The deduced amino acid sequence AAC(6')-29a protein shared 96% identity with AAC(6')-29b but only 34% identity with the aacA7-encoded AAC(6')-I1, the closest relative of the AAC(6')-I family enzymes. These aminoglycoside acetyltransferases had amino acid sequences much shorter (131 amino acids) than the other AAC(6')-I enzymes (144 to 153 amino acids). They conferred resistance to amikacin, isepamicin, kanamycin, and tobramycin but not to gentamicin, netilmicin, and sisomicin.
KeywordMeSH Terms
78. Reimmann  C, Patel  HM, Serino  L, Barone  M, Walsh  CT, Haas  D,     ( 2001 )

Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.

Journal of bacteriology 183 (3)
PMID : 11208777  :   DOI  :   10.1128/JB.183.3.813-820.2001     PMC  :   PMC94946    
Abstract >>
The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa.
KeywordMeSH Terms
79. Raivio  TL, Olson  JC, Gallant  CV,     ( 2000 )

Pseudomonas aeruginosa cystic fibrosis clinical isolates produce exotoxin A with altered ADP-ribosyltransferase activity and cytotoxicity.

Microbiology (Reading, England) 146 (Pt 8) (N/A)
PMID : 10931893  :   DOI  :   10.1099/00221287-146-8-1891    
Abstract >>
The role of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in the lung infections of cystic fibrosis (CF) patients is not well understood. Transcript-accumulation studies of bacterial populations in sputum reveal high levels of transcription of toxA, which encodes ETA, in some patients with CF. However, in general, tissue damage in the lungs of patients with CF does not seem to be consistent with a high level of expression of active ETA. To address this discrepancy the authors analysed the production and activity of ETA produced by a number of P. aeruginosa CF isolates. One CF isolate, strain 4384, transcribed toxA at levels similar to the hypertoxigenic strain PA103 but produced an ETA with reduced ADP-ribosyltransferase (ADPRT) activity. Complementation in trans of strain 4384 with the wild-type toxA and a mixed toxin experiment suggested the absence of inhibitory accessory factors within this strain. The toxA gene from strain 4384 was cloned and sequenced, revealing only three mutations in the gene, all within the enzymic domain. The first mutation changed Ser-410 to Asn. The second mutation was located within an alpha-helix, altering Ala-476 to Glu. The third mutation, Ser-515 to Gly, was found at the protein surface. To date, Ser-410, Ala-476 and Ser-515 have not been reported to play a role in the ADPRT activity of ETA. However, it may be the combination of these mutations that reduces the enzymic activity of ETA produced by strain 4384. Expression of 4384 toxA and wild-type toxA in an isogenic strain revealed that 4384 ETA had 10-fold less ADPRT activity than wild-type ETA. ETA purified from strain 4384 also demonstrated 10-fold less ADPRT activity as compared to wild-type ETA. Cytotoxicity assays of purified ETA from strain 4384 indicated that the cytotoxicity of 4384 ETA is not reduced; it may be slightly more toxic than wild-type ETA. Analysis of five other CF isolates revealed a similar reduction in ADPRT activity to that seen in strain 4384. Sequence analysis of the enzymic domain of toxA from the five CF strains identified a number of mutations that could account for the reduction in ADPRT activity. These results suggest that some CF isolates produce an ETA with reduced enzymic activity and this may partially explain the pathogenesis of chronic lung infections of CF due to P. aeruginosa.
KeywordMeSH Terms
ADP Ribose Transferases
Bacterial Toxins
Virulence Factors
80. Liebert  CA, Watson  AL, Summers  AO,     ( 2000 )

The quality of merC, a module of the mer mosaic.

Journal of molecular evolution 51 (6)
PMID : 11116334  :  
Abstract >>
We examined a region of high variability in the mosaic mercury resistance (mer) operon of natural bacterial isolates from the primate intestinal microbiota. The region between the merP and merA genes of nine mer loci was sequenced and either the merC, the merF, or no gene was present. Two novel merC genes were identified. Overall nucleotide diversity, pi (per 100 sites), of the merC gene was greater (49.63) than adjacent merP (35.82) and merA (32.58) genes. However, the consequences of this variability for the predicted structure of the MerC protein are limited and putative functional elements (metal-binding ligands and transmembrane domains) are strongly conserved. Comparison of codon usage of the merTP, merC, and merA genes suggests that several merC genes are not coeval with their flanking sequences. Although evidence of homologous recombination within the very variable merC genes is not apparent, the flanking regions have higher homologies than merC, and recombination appears to be driving their overall sequence identities higher. The synonymous codon usage bias (EN(C)) values suggest greater variability in expression of the merC gene than in flanking genes in six different bacterial hosts. We propose a model for the evolution of MerC as a host-dependent, adventitious module of the mer operon.
KeywordMeSH Terms
Bacterial Proteins
Cation Transport Proteins
81. Winzer  K, Falconer  C, Garber  NC, Diggle  SP, Camara  M, Williams  P,     ( 2000 )

The Pseudomonas aeruginosa lectins PA-IL and PA-IIL are controlled by quorum sensing and by RpoS.

Journal of bacteriology 182 (22)
PMID : 11053384  :   DOI  :   10.1128/jb.182.22.6401-6411.2000     PMC  :   PMC94786    
Abstract >>
In Pseudomonas aeruginosa, many exoproduct virulence determinants are regulated via a hierarchical quorum-sensing cascade involving the transcriptional regulators LasR and RhlR and their cognate activators, N-(3-oxododecanoyl)-L-homoserine lactone (3O-C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL). In this paper, we demonstrate that the cytotoxic lectins PA-IL and PA-IIL are regulated via quorum sensing. Using immunoblot analysis, the production of both lectins was found to be directly dependent on the rhl locus while, in a lasR mutant, the onset of lectin synthesis was delayed but not abolished. The PA-IL structural gene, lecA, was cloned and sequenced. Transcript analysis indicated a monocistronic organization with a transcriptional start site 70 bp upstream of the lecA translational start codon. A lux box-type element together with RpoS (sigma(S)) consensus sequences was identified upstream of the putative promoter region. In Escherichia coli, expression of a lecA::lux reporter fusion was activated by RhlR/C4-HSL, but not by LasR/3O-C12-HSL, confirming direct regulation by RhlR/C4-HSL. Similarly, in P. aeruginosa PAO1, the expression of a chromosomal lecA::lux fusion was enhanced but not advanced by the addition of exogenous C4-HSL but not 3O-C12-HSL. Furthermore, mutation of rpoS abolished lectin synthesis in P. aeruginosa, demonstrating that both RpoS and RhlR/C4-HSL are required. Although the C4-HSL-dependent expression of the lecA::lux reporter in E. coli could be inhibited by the presence of 3O-C12-HSL, this did not occur in P. aeruginosa. This suggests that, in the homologous genetic background, 3O-C12-HSL does not function as a posttranslational regulator of the RhlR/C4-HSL-dependent activation of lecA expression.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
82. Nakayama  K, Takashima  K, Ishihara  H, Shinomiya  T, Kageyama  M, Kanaya  S, Ohnishi  M, Murata  T, Mori  H, Hayashi  T,     ( 2000 )

The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage.

Molecular microbiology 38 (2)
PMID : 11069649  :   DOI  :   10.1046/j.1365-2958.2000.02135.x    
Abstract >>
Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.
KeywordMeSH Terms
Genes, Bacterial
Pyocins
83. Hager  P, Phibbs  P, Ochsner  U, Swanson  BL,     ( 2000 )

Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1.

Molecular microbiology 37 (3)
PMID : 10931350  :   DOI  :   10.1046/j.1365-2958.2000.02012.x    
Abstract >>
The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.
KeywordMeSH Terms
84. Kiewitz  C, Larbig  K, Klockgether  J, Weinel  C, Tümmler  B,     ( 2000 )

Monitoring genome evolution ex vivo: reversible chromosomal integration of a 106 kb plasmid at two tRNA(Lys) gene loci in sequential Pseudomonas aeruginosa airway isolates.

Microbiology (Reading, England) 146 (Pt 10) (N/A)
PMID : 11021913  :   DOI  :   10.1099/00221287-146-10-2365    
Abstract >>
The genome rearrangements in sequential Pseudomonas aeruginosa clone K isolates from the airways of a patient with cystic fibrosis were determined by an integrated approach of mapping, sequencing and bioinformatics. Restriction mapping uncovered an 8.9 kb deletion of PAO sequence between phnAB and oprL in clone K, and two 106 kb insertions either adjacent to this deletion or several hundred kilobases away, close to the pilA locus. These 106 kb blocks of extra DNA also co-existed as the circular plasmid pKLK106 in several clone K isolates and were found to be closely related to plasmid pKLC102 in P. aeruginosa clone C isolates. The breakpoints of the deletion in clone K and the attB-attP sequences for the reversible integration of the plasmid in clones C and K were located within the 3' end of the lysine tRNA structural genes (att site). pKLK106 sequentially recombined with either of the two tRNA(Lys) genes in clone K isolates. The att site of the pilA hypervariable region has been utilized by clone C to target its plasmid pKLC102 into the chromosome; the att site of the phnAB-oprL region has been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements. The use of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms used by P. aeruginosa to generate the mosaic genome structure of blocks of species-, clone- and strain-specific DNA. The example described here demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes.
KeywordMeSH Terms
Evolution, Molecular
Genome, Bacterial
85. Dean  CR,     ( 2000 )

The wbpM gene in Pseudomonas aeruginosa serogroup O17 resides on a cryptic copy of the serogroup O11 O antigen gene locus.

FEMS microbiology letters 187 (1)
PMID : 10828401  :   DOI  :   10.1111/j.1574-6968.2000.tb09137.x    
Abstract >>
The Pseudomonas aeruginosa serogroup O11 strain PA103 O antigen gene locus consists of 11 genes designated wzz, wzx, wbjA, wzy, wbjB-F, wbpL, and wbpM. The distribution of each of these genes amongst the 20 P. aeruginosa international antigenic typing system (IATS) serogroups was analyzed by Southern blot. As shown previously, wbpM was present in all 20 serogroups. The remaining O11 O antigen genes, with the exception of wzy, were present in the serogroup O17 strain IATSO17, despite the structural unrelatedness of the O11 and O17 O antigens. Sequencing revealed the presence of a cryptic serogroup O11 locus in the IATSO17 interrupted by two copies of a 1.1-kb insertion element. Introduction of plasmid pLPS2, containing the complete O11 O antigen locus from strain PA103, into IATSO17 resulted in production of both the O11 and O17 O antigens. The results of insertional inactivation of wbpM in IATSO17 are discussed.
KeywordMeSH Terms
86. Whitchurch  CB, Semmler  AB,     ( 2000 )

Identification of a novel gene, fimV, involved in twitching motility in Pseudomonas aeruginosa.

Microbiology (Reading, England) 146 (Pt 6) (N/A)
PMID : 10846211  :   DOI  :   10.1099/00221287-146-6-1321    
Abstract >>
Transposon mutagenesis was used to identify a new locus required for twitching motility in Pseudomonas aeruginosa. Four Tn5-B21 mutants which lacked twitching motility and a fifth which exhibited impaired motility were found to map to the same KPN:I restriction fragment at approximately 40 min on the P. aeruginosa genome. Cloning and sequencing studies showed that all five transposon insertions occurred within the same 2.8 kb ORF, which was termed fimV. The product of this gene has a putative peptidoglycan-binding domain, predicted transmembrane domains, a highly acidic C terminus and anomalous electrophoretic migration, indicating unusual primary or secondary structure. The P. aeruginosa genome also possesses a paralogue of fimV. Homologues of fimV were also found in the sequenced genomes of the other type-IV-fimbriated bacteria Neisseria gonorrhoeae, Neisseria meningitidis, Legionella pneumophila and Vibrio cholerae, but not in those of other bacteria which lack type IV fimbriae. A fimV homologue was also found in the genome sequence of Shewanella putrefaciens, along with many other homologues of type IV fimbrial genes, indicating that this bacterium is also likely to produce type IV fimbriae. Wild-type twitching motility was restored to fimV mutants by complementation in a dosage-dependent manner. Overexpression of fimV resulted in an unusual phenotype where the cells were massively elongated and migrated in large convoys at the periphery of the colony. It is suggested that FimV may be involved in remodelling of the peptidoglycan layer to enable assembly of the type IV fimbrial structure and machinery.
KeywordMeSH Terms
Genes, Bacterial
87. G Ordóñez  L, Campos-García  J,     ( 2000 )

The Pseudomonas aeruginosa hscA gene encodes Hsc66, a DnaK homologue.

Microbiology (Reading, England) 146 (Pt 6) (N/A)
PMID : 10846221  :   DOI  :   10.1099/00221287-146-6-1429    
Abstract >>
Under heat-stress conditions bacteria induce, among other heat-shock proteins, the Hsp70 molecular chaperone (DnaK), which is involved in protein stabilization. It has been shown in Escherichia coli that an Hsp70 homologue called Hsc66, which is widespread in bacteria, functions as a chaperone in vitro. This paper reports the isolation of a Pseudomonas aeruginosa W51D mutant (W51M22) by insertion of the mini-Tn5-Hg transposon, which was unable to grow on ethanol and other short-chain alcohols as sole source of carbon. The transposon insertion in this mutant was shown to be located in the hscA gene encoding Hsc66. The inability of mutant W51M22 to use ethanol was complemented by the E. coli hscBA-fdx operon. The authors characterized the transcriptional arrangement of hscA, showing that it forms part of an operon with the upstream hscB gene, and that it is also expressed from its own promoter. These results are compatible with the P. aeruginosa Hsc66 protein being a functional molecular chaperone involved in the stabilization, in the presence of ethanol, of some proteins required for bacterial growth on short-chain alcohols.
KeywordMeSH Terms
Escherichia coli Proteins
Genes, Bacterial
88. Naas  T, Nicolas  D, Collet  L, Poirel  L,     ( 2000 )

Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France.

Antimicrobial agents and chemotherapy 44 (4)
PMID : 10722487  :   DOI  :   10.1128/aac.44.4.891-897.2000     PMC  :   PMC89788    
Abstract >>
Pseudomonas aeruginosa COL-1 was identified in a blood culture of a 39-year-old-woman treated with imipenem in Marseilles, France, in 1996. This strain was resistant to beta-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefepime, ceftazidime, imipenem, and meropenem, but remained susceptible to the monobactam aztreonam. The carbapenem-hydrolyzing beta-lactamase gene of P. aeruginosa COL-1 was cloned, sequenced, and expressed in Escherichia coli DH10B. The deduced 266-amino-acid protein was an Ambler class B beta-lactamase, with amino acid identities of 32% with B-II from Bacillus cereus; 31% with IMP-1 from several gram-negative rods in Japan, including P. aeruginosa; 27% with CcrA from Bacteroides fragilis; 24% with BlaB from Chryseobacterium meningosepticum; 24% with IND-1 from Chryseobacterium indologenes; 21% with CphA-1 from Aeromonas hydrophila; and 11% with L-1 from Stenotrophomonas maltophilia. It was most closely related to VIM-1 beta-lactamase recently reported from Italian P. aeruginosa clinical isolates (90% amino acid identity). Purified VIM-2 beta-lactamase had a pI of 5.6, a relative molecular mass of 29.7 kDa, and a broad substrate hydrolysis range, including penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems, but not monobactams. As a metallo-beta-lactamase, its activity was zinc dependent and inhibited by EDTA (50% inhibitory concentration, 50 microM). VIM-2 conferred a resistance pattern to beta-lactams in E. coli DH10B that paralleled its in vitro hydrolytic properties, except for susceptibility to ureidopenicillins, carbapenems, and cefepime. bla(VIM-2) was located on a ca. 45-kb plasmid that in addition conferred resistance to sulfamides and that was not self-transmissible either from P. aeruginosa to E. coli or from E. coli to E. coli. bla(VIM-2) was the only gene cassette located within the variable region of a novel class 1 integron, In56, that was weakly related to the bla(VIM-1)-containing integron. VIM-2 is the second carbapenem-hydrolyzing metalloenzyme characterized from a P. aeruginosa isolate outside Japan.
KeywordMeSH Terms
89. Lafontaine  ER, Duan  K,     ( 2000 )

RegA, iron, and growth phase regulate expression of the Pseudomonas aeruginosa tol-oprL gene cluster.

Journal of bacteriology 182 (8)
PMID : 10735848  :   DOI  :   10.1128/jb.182.8.2077-2087.2000     PMC  :   PMC111254    
Abstract >>
The tol-oprL region in Pseudomonas aeruginosa appears to be involved in pyocin uptake and required for cell viability. The complete nucleotide sequences of the tolQRA and oprL genes as well as the incomplete sequences of tolB and orf2 have been previously reported. In addition, the sequence of a P. aeruginosa iron-regulated gene (pig6) has been described and found to share homology with an open reading frame located upstream of the Escherichia coli tolQRA genes (U. A. Ochsner and M. L. Vasil, Proc. Natl. Acad. Sci. USA 93:4409-4414, 1996). In this study, we cloned the remainder of the P. aeruginosa tol-oprL gene cluster and determined its nucleotide sequence. This cluster was found to consist of seven genes in the order orf1 tolQ tolR tolA tolB oprL orf2. Transcriptional analysis of this gene cluster was performed by detecting the presence of mRNAs spanning adjacent genes as well as by using a promoterless lacZ reporter gene fused to each of the seven genes contained in the tol-oprL locus. The results show that there are three major transcriptional units or operons in this region, orf1-tolQRA, tolB, and oprL-orf2, in contrast to the E. coli tol-pal region, where there are only two operons, orf1-tolQRA and tolB-pal-orf2. Analysis of gene expression indicated that the tol-oprL genes of P. aeruginosa are both iron and growth phase modulated. The first operon, orf1-tolQRA, is iron regulated throughout growth, but iron-regulated expression of tolB and oprL fusions occurs only in late log phase. The expression of the three operons was significantly less repressed by iron in fur mutants than in the wild-type strain, suggesting the involvement of Fur in the iron regulation of all three operons. RegA is a positive yet nonessential regulator of tol-oprL expression.
KeywordMeSH Terms
Bacterial Outer Membrane Proteins
Gene Expression Regulation, Bacterial
Proteoglycans
90. Fukui  T, Matsusaki  H, Taguchi  S, Tsuge  T,     ( 2000 )

Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis.

FEMS microbiology letters 184 (2)
PMID : 10713420  :   DOI  :   10.1111/j.1574-6968.2000.tb09013.x    
Abstract >>
Two Pseudomonas aeruginosa genes, termed phaJ1(Pa) and phaJ2(Pa), homologous to the Aeromonas caviae (R)-specific enoyl-CoA hydratase gene (phaJ(Ac)) were cloned using a PCR technique to investigate the monomer-supplying ability for polyhydroxyalkanoate (PHA) synthesis from beta-oxidation cycle. Two expression plasmids for phaJ1(Pa) and phaJ2(Pa) were constructed and introduced into Escherichia coli DH5alpha strain. The recombinants harboring phaJ1(Pa) or phaJ2(Pa) showed high (R)-specific enoyl-CoA hydratase activity with different substrate specificities, that is, specific for short chain-length enoyl-CoA or medium chain-length enoyl-CoA, respectively. In addition, co-expression of these two hydratase genes with PHA synthase gene in E. coli LS5218 resulted in the accumulation of PHA up to 14-29 wt% of cell dry weight from dodecanoate as a sole carbon source. It has been suggested that phaJ1(Pa) and phaJ2(Pa) products have the monomer-supplying ability for PHA synthesis from beta-oxidation cycle.
KeywordMeSH Terms
91. Simpson  DA,     ( 2000 )

RpmA is required for nonopsonic phagocytosis of Pseudomonas aeruginosa.

Infection and immunity 68 (5)
PMID : 10768936  :   DOI  :   10.1128/iai.68.5.2493-2502.2000     PMC  :   PMC97451    
Abstract >>
Pseudomonas aeruginosa causes severe respiratory tract infections in patients with cystic fibrosis (CF). We have been examining nonopsonic phagocytosis of P. aeruginosa by macrophages. To study the P. aeruginosa-macrophage interaction at the molecular level, we have constructed a transposon Tn5G bank in a clinical isolate of P. aeruginosa (strain 4020) and identified mutants resistant to nonopsonic phagocytosis. Phagocytosis-resistant mutants were enriched by passaging the transposon bank over 18 macrophage monolayers. Of 900 individual mutants isolated from this enriched pool in a nonopsonic phagocytosis assay, we identified 85 putative mutants that were resistant to phagocytosis. In this study, we have characterized one of these transposon mutants, P. aeruginosa 4020 H27A, which was poorly ingested. H27A possessed a Tn5G insertion in a gene encoding a protein with homology to the MotA proteins of several species of bacteria. We have called this gene rpmA for required for phagocytosis by macrophages. RpmA is one of two MotA paralogs in P. aeruginosa. This rpmA::Tn5G mutant was motile both on agar plates and in visual examination of wet mounts. The phagocytosis defect was partially complemented by providing the rpmA gene in trans and fully complemented when both rpmA and rpmB were provided. A rpmA null mutant was ingested by macrophages similar to the H27A transposon mutant. These data suggest that the rpmA and rpmB gene products are required for the efficient ingestion of P. aeruginosa by macrophages.
KeywordMeSH Terms
92. Lewis  C, Galleni  M, Frère  JM, Clarke  BP, Cheever  CA, Pearson  S, Rowling  P, Janson  CA, Concha  NO,     ( 2000 )

Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broad-spectrum inhibitor.

Biochemistry 39 (15)
PMID : 10757977  :   DOI  :   10.1021/bi992569m    
Abstract >>
Metallo beta-lactamase enzymes confer antibiotic resistance to bacteria by catalyzing the hydrolysis of beta-lactam antibiotics. This relatively new form of resistance is spreading unchallenged as there is a current lack of potent and selective inhibitors of metallo beta-lactamases. Reported here are the crystal structures of the native IMP-1 metallo beta-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor, 2-[5-(1-tetrazolylmethyl)thien-3-yl]-N-[2-(mercaptomethyl)-4 -(phenylb utyrylglycine)]. The structures were determined by molecular replacement, and refined to 3.1 A (native) and 2.0 A (complex) resolution. Binding of the inhibitor in the active site induces a conformational change that results in closing of the flap and transforms the active site groove into a tunnel-shaped cavity enclosing 83% of the solvent accessible surface area of the inhibitor. The inhibitor binds in the active site through interactions with residues that are conserved among metallo beta-lactamases; the inhibitor's carboxylate group interacts with Lys161, and the main chain amide nitrogen of Asn167. In the "oxyanion hole", the amide carbonyl oxygen of the inhibitor interacts through a water molecule with the side chain of Asn167, the inhibitor's thiolate bridges the two Zn(II) ions in the active site displacing the bridging water, and the phenylbutyryl side chain binds in a hydrophobic pocket (S1) at the base of the flap. The flap is displaced 2.9 A compared to the unbound structure, allowing Trp28 to interact edge-to-face with the inhibitor's thiophene ring. The similarities between this inhibitor and the beta-lactam substrates suggest a mode of substrate binding and the role of the conserved residues in the active site. It appears that the metallo beta-lactamases bind their substrates by establishing a subset of binding interactions near the catalytic center with conserved characteristic chemical groups of the beta-lactam substrates. These interactions are complemented by additional nonspecific binding between the more variable groups in the substrates and the flexible flap. This unique mode of binding of the mercaptocarboxylate inhibitor in the enzyme active site provides a binding model for metallo beta-lactamase inhibition with utility for future drug design.
KeywordMeSH Terms
beta-Lactamase Inhibitors
93. Lehoux  DE, Sanschagrin  F, Mahan  MJ, Handfield  M,     ( 2000 )

In vivo-induced genes in Pseudomonas aeruginosa.

Infection and immunity 68 (4)
PMID : 10722644  :   DOI  :   10.1128/iai.68.4.2359-2362.2000     PMC  :   PMC97428    
Abstract >>
In vivo expression technology was used for testing Pseudomonas aeruginosa in the rat lung model of chronic infection and in a mouse model of systemic infection. Three of the eight ivi proteins found showed sequence identity to known virulence factors involved in iron acquisition via an open reading frame (called pvdI) implicated in pyoverdine biosynthesis, membrane biogenesis (FtsY), and adhesion (Hag2).
KeywordMeSH Terms
Oligopeptides
94. Poirel  L, Naas  T,     ( 1999 )

Molecular characterisation of In51, a class 1 integron containing a novel aminoglycoside adenylyltransferase gene cassette, aadA6, in Pseudomonas aeruginosa.

Biochimica et biophysica acta 1489 (2��3��)
PMID : 10673049  :   DOI  :   10.1016/s0167-4781(99)00202-x    
Abstract >>
Polymerase chain reaction-amplification and subsequent sequencing of the variable region of a novel integron, In51, from Pseudomonas aeruginosa revealed the presence of a novel aminoglycoside adenylyltransferase gene, aadA6, together with an open reading frame of unknown function, orfD. AADA6 enzyme has only 75% amino acid identity with AADA1 and is able to confer high level resistance to streptomycin and spectinomycin in Escherichia coli.
KeywordMeSH Terms
95. Dasgupta  N, Arora  SK,     ( 2000 )

Identification of two distinct types of flagellar cap proteins, FliD, in Pseudomonas aeruginosa.

Infection and immunity 68 (3)
PMID : 10678962  :   DOI  :   10.1128/iai.68.3.1474-1479.2000     PMC  :   PMC97303    
Abstract >>
Binding of Pseudomonas aeruginosa strain PAK to mucin has been shown to be mediated by the flagellar cap protein, product of the fliD gene. Since the flagellar cap is very likely an exposed structure, the FliD polypeptide should be recognized by the host immune system, analogous to the recognition of dominant epitopes located in the exposed parts of the flagellin polypeptide within the assembled flagellum. In P. aeruginosa, a number of distinct flagellin variants are made, and these variable sequences presumably allow the newly infected P. aeruginosa to escape recognition by the antibody induced during a previous infection. Since similar mechanisms may direct the selection of FliD variants, we examined the extent of sequence heterogeneity among various FliD sequences among a selected group of P. aeruginosa. The results of PCR and nucleotide sequencing of the fliD region of eight different P. aeruginosa strains (laboratory strains PAK, PAO1, and PA103; clinical strains 1244, CS2, and CS32; cystic fibrosis strains CS29 and MDR) suggested that there were two distinct types of FliD in P. aeruginosa, which we named A type and B type. The results of Western blotting using the polyclonal antibodies raised against the purified FliD of A type (PAK) or B type (PAO1) further confirmed the existence of two distinct antigenic types of FliD proteins, with no cross-reactivity between the two serotypes. Further Western immunoblot analysis of the same strains using polyclonal FliC antibody showed that the strains with A-type FliD possessed a-type FliC and those with B-type FliD had b-type FliC. Similar Western blot analyses of 50 more P. aeruginosa strains obtained from varied sources revealed that all strains contained either A-type or B-type FliD, suggesting the existence of only two types of FliD in P. aeruginosa and indicating that fliC and fliD were coinherited. This limited diversity of FliC and FliD serotypes seems to be a unique feature of flagellar proteins. A chromosomal mutant having an insertion in the fliD gene of P. aeruginosa PAO1 was constructed. The motility defect of this mutant and a previously constructed PAK fliD mutant was better complemented with the fliD gene of the homologous types.
KeywordMeSH Terms
96. Johnson  Z, Ochsner  UA,     ( 2000 )

Genetics and regulation of two distinct haem-uptake systems, phu and has, in Pseudomonas aeruginosa.

Microbiology (Reading, England) 146 (Pt 1) (N/A)
PMID : 10658665  :   DOI  :   10.1099/00221287-146-1-185    
Abstract >>
A gene cluster similar to haem iron uptake loci of bacterial pathogens was identified in Pseudomonas aeruginosa. This phu locus ('Pseudomonas haem uptake') consisted of the phuR receptor gene and the phuSTUVW operon encoding a typical ABC transporter. Expression of phuR and phuSTUVW from mapped transcriptional-start sites occurred under iron-restricted growth conditions and was directly controlled by the Fur protein. Binding of Fur was demonstrated by DNase footprinting of two adjacent 'Fur boxes' that overlapped both the phuR and phuSTUVW promoters. Two tandem repeats of 154 bp were identified downstream of the phuSTUVW operon, each of which contained a strong Fur-dependent promoter driving expression of iron-regulated RNAs antisense to phuSTUVW. Mutant strains with deletions in phuR and phuSTUV showed greatly reduced growth with either haem or haemoglobin as the only iron source: the defects were complemented by plasmids harbouring the phuR or the phuSTUV genes, respectively. Deletions of phuW or of the tandem repeats had only minor effects on haem utilization. The remaining haem and haemoglobin uptake still observed in the deltaphuR or deltaphuSTUV deletion mutants was due to a second haem-acquisition system, has, which was also under the direct control of Fur. This second haem-receptor gene, hasR, was identified upstream of and in an operon with hasA, encoding a haem-binding extracellular protein. A deltahasR mutant also exhibited decreased utilization of haem and haemoglobin, and a deltaphuR deltahasR double mutant was virtually unable to take up either compound. Both the PhuR and HasR proteins were detected in the outer-membrane fraction of P. aeruginosa grown in low-iron media. Taken together, the evidence suggests that the phu and has loci encode two distinct systems required for the acquisition of haem and haemoglobin in P. aeruginosa.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
Sigma Factor
97. Zhao  Q,     ( 2000 )

A second tonB gene in Pseudomonas aeruginosa is linked to the exbB and exbD genes.

FEMS microbiology letters 184 (1)
PMID : 10689178  :   DOI  :   10.1111/j.1574-6968.2000.tb09002.x    
Abstract >>
The exbBD genes of Pseudomonas aeruginosa PAO were cloned by complementation of the growth defect of an Escherichia coli exbB tolQ double mutant on iron-restricted medium. Nucleotide sequence analysis confirmed that these genes are contiguous and preceded by a second tonB gene in this organism, which we have designated tonB2. lacZ promoter fusions confirmed that expression of the tonB2-exbB-exbD genes is increased under conditions of iron limitation. Deletions within any of these genes, in contrast to deletions in the first tonB gene, tonB1, did not adversely affect growth on iron-restricted medium. On the other hand, tonB1 tonB2 double mutants were more compromised as regards growth in an iron-restricted medium than a tonB1 deletion, indicating that TonB2 could partially replace TonB1 in its role in iron acquisition. TonB1 but not TonB2 deletion strains were also compromised as regards the utilization of hemin or hemoglobin as sole iron sources, indicating that heme transport requires TonB1.
KeywordMeSH Terms
Escherichia coli Proteins
98. Nájera  R, Camarena  L, Campos-García  J,     ( 2000 )

The pseudomonas aeruginosa motR gene involved in regulation of bacterial motility.

FEMS microbiology letters 184 (1)
PMID : 10689166  :   DOI  :   10.1111/j.1574-6968.2000.tb08990.x    
Abstract >>
A mini-Tn5-Hg insertion mutant derived from Pseudomonas aeruginosa W51D (W51M1) was isolated in which mini-Tn5 insertion disrupted the motR gene showing that it forms part of the cluster involved in bacterial motility and chemotaxis. Characterization of the W51M1 motility behavior, and also of a PAO1 motR::mini-Tn5-Hg mutant, suggests that the product of the motR gene is a negative regulator of bacterial motility which controls the number of flagella per cell.
KeywordMeSH Terms
99. Burrows  LL, Monteiro  MA, Matewish  MJ, Walsh  AG,     ( 2000 )

Lipopolysaccharide core phosphates are required for viability and intrinsic drug resistance in Pseudomonas aeruginosa.

Molecular microbiology 35 (4)
PMID : 10692150  :   DOI  :   10.1046/j.1365-2958.2000.01741.x    
Abstract >>
Pseudomonas aeruginosa is an opportunistic pathogen that is notorious for its intrinsic drug resistance. We have used chemical and genetic techniques to characterize three putative kinase genes that are involved in the addition of phosphate to the inner core region of P. aeruginosa lipopolysaccharide. The first gene is a waaP homologue, whereas the other two (wapP and wapQ) are unique to P. aeruginosa. Repeated attempts using a variety of membrane-stabilizing conditions to generate waaP:Gm (Gm, gentamicin) or wapP:Gm mutants were unsuccessful. We were able to generate a chromosomal waaP mutant that had a wild-type copy of either waaPPa or waaPEc in trans, but were unable to cure this plasmid-borne copy of the gene. These results are consistent with the fact that P. aeruginosa mutants lacking inner core heptose (Hep) or phosphate have never been isolated and demonstrate the requirement of Hep-linked phosphate for P. aeruginosa viability. A wapQ:Gm mutant was isolated and it had an unaltered minimum inhibitory concentration (MIC) for novobiocin and only a small decrease in the MIC for sodium dodecyl sulphate (SDS), suggesting that the loss of a phosphate group transferred by WapQ may only be having a small impact on outer-membrane permeability. Nuclear magnetic resonance and methylation linkage analysis showed that WaaPPa could add one phosphate to O4 of HepI in a Salmonella typhimurium waaP mutant. The expression of WaaPPa increased the outer-membrane integrity of these complemented mutants, as evidenced by 35-fold and 75-fold increases in the MIC for novobiocin and SDS respectively. The S. typhimurium waaP mutant transformed with both waaP and wapP had over 250-fold and 1000-fold increases, respectively, in these MICs. The inner core phosphates of P. aeruginosa appear to be playing a key role in the intrinsic drug resistance of this bacterium.
KeywordMeSH Terms
Drug Resistance, Microbial
100. Nguyen  LY, Shawar  RM, Warrener  P, Zhu  YQ, Coulter  SN, Hickey  MJ, Sherman  DR, Westbrock-Wadman  S,     ( 1999 )

Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability.

Antimicrobial agents and chemotherapy 43 (12)
PMID : 10582892  :   PMC  :   PMC89597    
Abstract >>
Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibiotics; however, in cystic fibrosis patients, more than 90% of aminoglycoside-resistant P. aeruginosa isolates are of the impermeability phenotype. The precise molecular mechanisms that produce aminoglycoside impermeability-type resistance are yet to be elucidated. A subtractive hybridization technique was used to reveal gene expression differences between PAO1 and isogenic, spontaneous aminoglycoside-resistant mutants of the impermeability phenotype. Among the many genes found to be up-regulated in these laboratory mutants were the amrAB genes encoding a recently discovered efflux system. The amrAB genes appear to be the same as the recently described mexXY genes; however, the resistance profile that we see in P. aeruginosa is very different from that described for Escherichia coli with mexXY. Direct evidence for AmrAB involvement in aminoglycoside resistance was provided by the deletion of amrB in the PAO1-derived laboratory mutant, which resulted in the restoration of aminoglycoside sensitivity to a level nearly identical to that of the parent strain. Furthermore, transcription of the amrAB genes was shown to be up-regulated in P. aeruginosa clinical isolates displaying the impermeability phenotype compared to a genotypically matched sensitive clinical isolate from the same patient. This suggests the possibility that AmrAB-mediated efflux is a clinically relevant mechanism of aminoglycoside resistance. Although it is unlikely that hyperexpression of AmrAB is the sole mechanism conferring the impermeability phenotype, we believe that the Amr efflux system can contribute to a complex interaction of molecular events resulting in the aminoglycoside impermeability-type resistance phenotype.
KeywordMeSH Terms
101. Ostrovsky  P, Martínez  A,     ( 1999 )

LipC, a second lipase of Pseudomonas aeruginosa, is LipB and Xcp dependent and is transcriptionally regulated by pilus biogenesis components.

Molecular microbiology 34 (2)
PMID : 10564475  :   DOI  :   10.1046/j.1365-2958.1999.01601.x    
Abstract >>
We have isolated cosmids that complement a Pseudomonas aeruginosa export-impaired mutant by increasing growth on lipid agar, a medium that requires lipase expression and export. These cosmids encode a previously unidentified lipase, LipC, which has high homology to the P. aeruginosa lipA gene product. Like LipA, LipC activity requires the chaperone activity of the lipB gene product and a functional xcp gene cluster for export. However, expression of LipC is barely detectable in a wild-type background. Transposon insertions that increase lipC promoter activity have been obtained that inactivate two pilus biogenesis genes, pilX and pilY1. This suggests that these proteins either directly or indirectly repress the expression of LipC and may be involved in transducing an extracellular signal that regulates this lipase.
KeywordMeSH Terms
Fimbriae Proteins
Gene Expression Regulation, Bacterial
Serine Endopeptidases
102. Arora  SK, Dasgupta  N,     ( 2000 )

fleN, a gene that regulates flagellar number in Pseudomonas aeruginosa.

Journal of bacteriology 182 (2)
PMID : 10629180  :   DOI  :   10.1128/jb.182.2.357-364.2000     PMC  :   PMC94283    
Abstract >>
The single polar flagellum of Pseudomonas aeruginosa plays an important role in the pathogenesis of infection by this organism. However, regulation of the assembly of this organelle has not been delineated. In analyzing the sequence available at the Pseudomonas genome database, an open reading frame (ORF), flanked by flagellar genes flhF and fliA, that coded for a protein (280 amino acids) with an ATP-binding motif at its N terminus was found. The ORF was inactivated by inserting a gentamicin cassette in P. aeruginosa PAK and PAO1. The resulting mutants were nonmotile on motility agar plates, but under a light microscope they exhibited random movement and tumbling behavior. Electron microscopic studies of the wild-type and mutant strains revealed that the mutants were multiflagellate, with three to six polar flagella per bacterium as rather than one as in the wild type, indicating that this ORF was involved in regulating the number of flagella and chemotactic motility in P. aeruginosa. The ORF was named fleN. An intact copy of fleN on a plasmid complemented the mutant by restoring motility and monoflagellate status. The beta-galactosidase activities of eight flagellar operon or gene promoters in the wild-type and fleN mutant strains revealed a direct correlation between six promoters that were upregulated in the fleN mutant (fliLMNOPQ, flgBCDE, fliEFG, fliDS orf126, fleSR, and fliC) and positive regulation by FleQ, an NtrC-like transcriptional regulator for flagellar genes. Based on these results, we propose a model where FleN influences FleQ activity (directly or indirectly) in regulating flagellar number in P. aeruginosa.
KeywordMeSH Terms
103. Springael  D, Römling  U, van der Lelie  D, Hassan  MT,     ( 1999 )

Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa.

Gene 238 (2)
PMID : 10570969  :   DOI  :   10.1016/s0378-1119(99)00349-2    
Abstract >>
Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA-DNA hybridization indicated strong conservation of czr in other environmental P. aeruginosa isolates and in the P. aeruginosa type strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
Multigene Family
104. Keating  TA, Quadri  LE,     ( 1999 )

Assembly of the Pseudomonas aeruginosa nonribosomal peptide siderophore pyochelin: In vitro reconstitution of aryl-4, 2-bisthiazoline synthetase activity from PchD, PchE, and PchF.

Biochemistry 38 (45)
PMID : 10555976  :   DOI  :   10.1021/bi991787c    
Abstract >>
Three Pseudomonas aeruginosa proteins involved in biogenesis of the nonribosomal peptide siderophore pyochelin, PchD, PchE, and PchF, have been expressed in and purified from Escherichia coli and are found to produce the tricyclic acid hydroxyphenyl-thiazolyl-thiazolinyl-carboxylic acid (HPTT-COOH), an advanced intermediate containing the aryl-4,2-bis-heterocyclic skeleton of the bithiazoline class of siderophores. The three proteins contain three adenylation domains, one specific for salicylate activation and two specific for cysteine activation, and three carrier protein domains (two in PchE and one in PchF) that undergo posttranslational priming with phosphopantetheine to enable covalent tethering of salicyl and cysteinyl moieties as acyl-S-enzyme intermediates. Two cyclization domains (Cy1 in PchE and Cy2 in PchF) create the two amide linkages in the elongating chains and the cyclodehydrations of acylcysteine moieties into thiazolinyl rings. The ninth domain, the most downstream domain in PchF, is the chain-terminating, acyl-S-enzyme thioester hydrolase that releases the HPTT-S-enzyme intermediate to the observed tandem bis-heterocyclic acid product. A PchF-thioesterase domain active site double mutant fails to turn over, but a monocyclic hydroxyphenyl-thiazolinyl-cysteine (HPT-Cys) product continues to be released from PchE, allowing assignment of the cascade of acyl-S-enzyme intermediates involved in initiation, elongation, and termination steps.
KeywordMeSH Terms
Thiazoles
105. Burrows  LL, Bélanger  M,     ( 1999 )

Functional analysis of genes responsible for the synthesis of the B-band O antigen of Pseudomonas aeruginosa serotype O6 lipopolysaccharide.

Microbiology (Reading, England) 145 (Pt 12) (N/A)
PMID : 10627048  :   DOI  :   10.1099/00221287-145-12-3505    
Abstract >>
This study reports the organization of the wbp gene cluster and characterization of a number of genes that are essential for B-band O antigen biosynthesis in the clinically prevalent Pseudomonas aeruginosa serotype 06. Twelve genes were identified that share homology with other LPS and polysaccharide biosynthetic genes. This cluster contains homologues of wzx (encoding the O antigen flippase/translocase) and wzz (which modulates O antigen chain length distribution) genes, typical of a wzy-dependent pathway. However, a complete wzy gene (encoding the O-polymerase) was not found within the cluster. Four biosynthetic genes, wbpO, wbpP, wbpV and wbpM, and four putative glycosyltransferase genes, wbpR, wbpT, wbpU and wbpL, were identified in the cluster. To characterize their roles in LPS biosynthesis, null mutants of wbpO, wbpP, wbpV, wbpL and wbpM were generated using a gene-replacement strategy. Mutations in each of these genes caused deficiency in B-band synthesis. The wbpL mutant was deficient in both A-band and B-band LPS. WbpL(O6) is a bi-functional enzyme which could initiate B-band synthesis through the addition of QuiNAc to undecaprenol phosphate, and A-band synthesis by transferring either a GalNAc or a GlcNAc residue. Another approach used to assign function to the wbp(O6) genes was by complementation analysis. Two genes from Salmonella typhi, wcdA and wcdB, responsible for the synthesis of a homopolymer of GalNAcA called Vi antigen were used in complementation experiments to verify the functions of wbpO and wbpP. wcdA and wcdB restored B-band synthesis in wbpO and wbpP mutants respectively, implying that wbpO and wbpP are involved in UDP-GalNAcA synthesis. Although wbpV has homology to wbpK of the serotype O5 B-band LPS synthesis cluster, complementation analysis using the respective null mutants showed that the genes are not interchangeable. A knockout mutation of wbpN (located downstream of wbpM) did not abrogate LPS synthesis in either 05 or 06; therefore, it has been renamed orf48.5. These results establish the organization of genes involved in P. aeruginosa B-band O antigen synthesis and provide the evidence to assign functions to a number of LPS biosynthetic genes.
KeywordMeSH Terms
Genes, Bacterial
106. Epp  SF, Köhler  T,     ( 1999 )

Characterization of MexT, the regulator of the MexE-MexF-OprN multidrug efflux system of Pseudomonas aeruginosa.

Journal of bacteriology 181 (20)
PMID : 10515918  :   PMC  :   PMC103763    
Abstract >>
We investigated the regulation of the MexEF-OprN multidrug efflux system of Pseudomonas aeruginosa, which is overexpressed in nfxC-type mutants and confers resistance to quinolones, chloramphenicol and trimethoprim. Sequencing of the DNA region upstream of the mexEF-oprN operon revealed the presence of an open reading frame (ORF) of 304 amino acids encoding a LysR-type transcriptional activator, termed MexT. By using T7-polymerase, a 34-kDa protein was expressed in Escherichia coli from a plasmid carrying the mexT gene. Expression of a mexE::lacZ fusion was 10-fold higher in nfxC-type mutants than in the wild-type strain; however, transcription of mexT as well as the mexT DNA region was unchanged. Located adjacent to mexT but transcribed in opposite direction, the beginning of an ORF termed qrh (quinone oxidoreductase homologue) was identified. Expression of a qrh::lacZ fusion was also found to be activated by MexT. Further, we present evidence for coregulation at the transcriptional and the posttranscriptional level between the MexEF-OprN efflux system and the OprD porin responsible for cross-resistance of nfxC-type mutants to carbapenem antibiotics.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
107. Brown  AL, Baynham  PJ,     ( 1999 )

Pseudomonas aeruginosa AlgZ, a ribbon-helix-helix DNA-binding protein, is essential for alginate synthesis and algD transcriptional activation.

Molecular microbiology 33 (5)
PMID : 10476040  :   DOI  :   10.1046/j.1365-2958.1999.01550.x    
Abstract >>
The Pseudomonas aeruginosa algD gene is the first gene of an operon encoding most of the enzymes necessary for biosynthesis of the exopolysaccharide alginate. Transcriptional activation of algD results in the high-level synthesis of alginate, an important P. aeruginosa virulence factor with antiphagocytic and adherence properties. Previously, we have identified a protein(s), AlgZ, expressed in mucoid P. aeruginosa CF isolates that specifically bound to sequences located 280 bp upstream of the algD promoter. Mutagenesis of the AlgZ DNA binding site and transcription assays were used to show that AlgZ was an activator of algD transcription. In the current study, the monomeric size of AlgZ was estimated to be between 6 kDa and 15 kDa by electroelution of a protein preparation from an SDS-PAGE gel and analysis of the fractions via protein staining and electrophoretic mobility shift assays. A biochemical enrichment procedure, resulting in a 130-fold enrichment for AlgZ, was devised, the protein identified and a partial amino-terminal sequence obtained. Using the P. aeruginosa Genome Project database, a complete sequence was obtained, and algZ was cloned and expressed in Escherichia coli. Expression of algZ was sufficient for the observed AlgZ DNA binding previously observed from extracts of P. aeruginosa. A protein database search revealed that AlgZ is homologous to the Mnt and Arc repressors of the ribbon-helix-helix family of DNA-binding proteins. An algZ deletion mutant was constructed in the mucoid CF isolate FRD1. The resulting strain was non-mucoid and exhibited no detectable algD transcription. As an indirect role in transcription would probably result in some residual algD transcription, these data suggest that AlgZ is an integral activator of algD and support the hypothesis that both AlgZ and the response regulator AlgR are involved in direct contact with RNA polymerase containing the alternative sigma factor, AlgT. The cloning of algZ is a crucial step in determining the mechanism of algD activation.
KeywordMeSH Terms
108. Köhler  T, Aires  JR,     ( 1999 )

Involvement of an active efflux system in the natural resistance of Pseudomonas aeruginosa to aminoglycosides.

Antimicrobial agents and chemotherapy 43 (11)
PMID : 10543738  :   PMC  :   PMC89534    
Abstract >>
A mutant, named 11B, hypersusceptible to aminoglycosides, tetracycline, and erythromycin was isolated after Tn501 insertion mutagenesis of Pseudomonas aeruginosa PAO1. Cloning and sequencing experiments showed that 11B was deficient in an, at that time, unknown active efflux system that contains homologs of MexAB. This locus also contained a putative regulatory gene, mexZ, transcribed divergently from the efflux operon. Introduction of a recombinant plasmid that carries the genes of the efflux system restored the resistance of 11B to parental levels, whereas overexpression of these genes strongly increased the MICs of substrate antibiotics for the PAO1 host. Antibiotic accumulation studies confirmed that this new system is an energy-dependent active efflux system that pumps out aminoglycosides. Furthermore, this system appeared to function with an outer membrane protein, OprM. While the present paper was being written and reviewed, genes with a sequence identical to our pump genes, mexXY of P. aeruginosa, have been reported to increase resistance to erythromycin, fluoroquinolones, and organic cations in Escherichia coli hosts, although efflux of aminoglycosides was not examined (Mine et al., Antimicrob. Agents Chemother. 43:415-417, 1999). Our study thus shows that the MexXY system plays an important role in the intrinsic resistance of P. aeruginosa to aminoglycosides. Although overexpression of MexXY increased the level of resistance to fluoroquinolones, disruption of the mexXY operon in P. aeruginosa had no detectable effect on susceptibility to these agents.
KeywordMeSH Terms
109. Poirel  L, Karim  A, Nordmann  P,     ( 1999 )

Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum beta-lactamase VEB-1 in Pseudomonas aeruginosa.

FEMS microbiology letters 176 (2)
PMID : 10427724  :   DOI  :   10.1111/j.1574-6968.1999.tb13691.x    
Abstract >>
A clinical isolate of Pseudomonas aeruginosa, JES, was resistant to extended-spectrum cephalosporins with a marked synergistic effect with clavulanic acid on a routine antibiogram. Preliminary PCR analysis revealed the presence of blaVEB-1, an integron-located gene encoding an extended-spectrum beta-lactamase previously identified in Escherichia coli MG-1. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1 containing integron along with some flanking sequence from P. aeruginosa JES was amplified and subsequently sequenced. In50 contains within its variable region, in addition to qacE delta 1 and sull genes commonly found in class 1 integrons, two gene cassettes, veb1 and aadB. In50 is peculiar since its attI1 site is interrupted by two novel insertion sequences, IS1999 and IS2000. P. aeruginosa JES and Escherichia coli MG-1 strains were isolated from patients previously hospitalized in south east Asian countries. The finding of blaVEB-1 in these strains and on different integrons underlines the interspecies spread of this integron-located extended-spectrum beta-lactamase gene.
KeywordMeSH Terms
110. Hoang  TT, Schweizer  HP,     ( 1999 )

Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD).

Journal of bacteriology 181 (17)
PMID : 10464226  :   PMC  :   PMC94061    
Abstract >>
A DNA fragment containing the Pseudomonas aeruginosa fabD (encoding malonyl-coenzyme A [CoA]:acyl carrier protein [ACP] transacylase), fabG (encoding beta-ketoacyl-ACP reductase), acpP (encoding ACP), and fabF (encoding beta-ketoacyl-ACP synthase II) genes was cloned and sequenced. This fab gene cluster is delimited by the plsX (encoding a poorly understood enzyme of phospholipid metabolism) and pabC (encoding 4-amino-4-deoxychorismate lyase) genes; the fabF and pabC genes seem to be translationally coupled. The fabH gene (encoding beta-ketoacyl-ACP synthase III), which in most gram-negative bacteria is located between plsX and fabD, is absent from this gene cluster. A chromosomal temperature-sensitive fabD mutant was obtained by site-directed mutagenesis that resulted in a W258Q change. A chromosomal fabF insertion mutant was generated, and the resulting mutant strain contained substantially reduced levels of cis-vaccenic acid. Multiple attempts aimed at disruption of the chromosomal fabG gene were unsuccessful. We purified FabD as a hexahistidine fusion protein (H6-FabD) and ACP in its native form via an ACP-intein-chitin binding domain fusion protein, using a novel expression and purification scheme that should be applicable to ACP from other bacteria. Matrix-assisted laser desorption-ionization spectroscopy, native polyacrylamide electrophoresis, and amino-terminal sequencing revealed that (i) most of the purified ACP was properly modified with its 4'-phosphopantetheine functional group, (ii) it was not acylated, and (iii) the amino-terminal methionine was removed. In an in vitro system, purified ACP functioned as acyl acceptor and H(6)-FabD exhibited malonyl-CoA:ACP transacylase activity.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
Multigene Family
111. Colmer  JA,     ( 1999 )

The Pseudomonas aeruginosa exotoxin A regulatory gene, ptxS: evidence for negative autoregulation.

Journal of bacteriology 181 (16)
PMID : 10438759  :   PMC  :   PMC93976    
Abstract >>
We have previously described a Pseudomonas aeruginosa gene, ptxR, which enhances exotoxin A production at the transcriptional level. We have also described another gene, ptxS, which is transcribed divergently from ptxR and interferes with the enhancement of exotoxin A synthesis by ptxR. However, the mechanisms through which ptxR and/or ptxS are regulated is not known. In this study, we attempted (by using the DNA gel shift assay) to determine if P. aeruginosa contains a potential regulatory protein that binds specifically to the ptxR or ptxS upstream region. In the initial analysis, different-sized gel shift bands were detected when a probe containing the ptxR-ptxS intergenic region was incubated with the lysate of P. aeruginosa PAO1. The strongest binding activity was detected with a smaller fragment that represents the ptxS upstream region. Additional deletion analysis localized the binding to a 52-bp fragment immediately upstream of ptxS. The gel shift band was not detected when the 52-bp fragment was incubated with the lysate of the ptxS isogenic mutant PAO1::ptxS. However, the binding band was regenerated when a plasmid carrying ptxS intact was introduced into PAO1::ptxS. In addition, the gel shift band was detected when the 52-bp fragment was incubated with a lysate of Escherichia coli in which ptxS was overexpressed from the T7 promoter. The effect of PtxS on ptxS expression was examined by using a ptxS-lacZ fusion plasmid. The level of beta-galactosidase activity produced by PAO1::ptxS carrying the fusion plasmid was four- to fivefold higher than that produced by PAO1 carrying the same plasmid. Using DNase I footprinting analysis, the binding region was specified to a 20-bp fragment. Within the fragment, a 14-bp palindromic sequence exists that may function as a PtxS binding site. These results suggest that PtxS autoregulates its synthesis by binding to a specific sequence within the ptxS upstream region.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
112. Schoofs  G, Hancock  RE, De Mot  R,     ( 1999 )

Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens.

Journal of bacteriology 181 (16)
PMID : 10438740  :   PMC  :   PMC93957    
Abstract >>
The gene encoding OprF, a major outer membrane protein in Pseudomonas species (formerly known as type 1 pseudomonads), was thought to be constitutively transcribed from a single sigma 70 promoter immediately upstream of the gene. We now report the identification of a novel putative ECF (extracytoplasmic function) sigma factor gene, sigX, located immediately upstream of oprF in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 and show that disruption of this gene significantly reduces OprF expression. In P. aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF. Comparison of sigX-disrupted and wild-type cell transcripts by primer extension indicated that monocistronic transcription of oprF occurs from two overlapping promoters, one that is SigX-dependent and resembles ECF sigma factor promoters in its minus-35 region and another promoter that is independent of SigX and is analogous to the sigma 70-type promoter previously reported. Complementation of the P. aeruginosa sigX-disrupted mutant with plasmid-encoded OprF did not resolve the phenotypes associated with this mutant, which included a markedly reduced logarithmic-phase growth rate in rich medium (compared to that in minimal medium), further reduction of the growth rate in a low-osmolarity environment, secretion of an unidentified pigment, and increased sensitivity to the antibiotic imipenem. This indicates that SigX is involved in the regulation of other genes in P. aeruginosa. Disruption of the sigX gene in P. fluorescens also had an effect on the logarithmic-phase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P. aeruginosa clinical isolates. Collectively, these data indicate that an ECF sigma factor plays a role in the regulation and expression of OprF and also affects other genes.
KeywordMeSH Terms
Bacterial Proteins
Gene Expression Regulation, Bacterial
113. Motley  ST,     ( 1999 )

Functional characterization of a serine/threonine protein kinase of Pseudomonas aeruginosa.

Infection and immunity 67 (10)
PMID : 10496921  :   PMC  :   PMC96896    
Abstract >>
Protein kinases play a key role in signal transduction pathways in both eukaryotic and prokaryotic cells. Using in vivo expression technology, we have identified several promoters in Pseudomonas aeruginosa which are preferentially activated during infection of neutropenic mice. One of these promoters directs the transcription of a gene encoding a putative protein kinase similar to the enzymes found in eukaryotic cells. The full characterization of this protein, termed PpkA, is presented in this communication. The ppkA gene encodes a 1,032-amino-acid polypeptide with an N-terminal catalytic domain showing all of the conserved residues of protein kinases with the substrate phosphorylation specificities for serine and threonine residues. The catalytic domain is linked to the rest of the protein by a short proline-rich segment. The enzymes showed anomalous migration behavior when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be attributed to autophosphorylation activity. The full-length enzyme was expressed as an oligohistidine fusion protein and was shown to phosphorylate several artificial protein substrates. Both autophosphorylation and phosphorylation of added substrates were strongly reduced by a single-amino-acid substitution in the catalytic domain of PpkA. Although PpkA appears to be differentially phosphorylated by autocatalysis, the levels of phosphorylation have minimal effect on its overall enzymatic activity. Our results, therefore, indicate the operation of a novel protein phosphorylation mechanism during transduction of signals in P. aeruginosa, and this pathway may be important in regulating the expression of virulence factors by this pathogen during certain phases of infection.
KeywordMeSH Terms
114. Goldberg  JB, Pier  GB, Hatano  K, Franklund  CV, Retief  JD, Coyne  MJ,     ( 1999 )

Characterization of the serogroup O11 O-antigen locus of Pseudomonas aeruginosa PA103.

Journal of bacteriology 181 (14)
PMID : 10400585  :   PMC  :   PMC93929    
Abstract >>
We previously cloned a genomic DNA fragment from the serogroup O11 Pseudomonas aeruginosa strain PA103 that contained all genes necessary for O-antigen synthesis and directed the expression of serogroup O11 antigen on recombinant Escherichia coli and Salmonella. To elucidate the pathway of serogroup O11 antigen synthesis, the nucleotide sequence of the biosynthetic genes was determined. Eleven open reading frames likely to be involved in serogroup O11 O-antigen biosynthesis were identified and are designated in order as wzzPaO111 (wzz from P. aeruginosa serogroup O11), wzxPaO11, wbjA, wzyPaO11, wbjB to wbjF, wbpLO11 and wbpMO11 (wbpL and wbpM from serogroup O11). Consistent with previous descriptions of O-antigen biosynthetic gene loci, the entire region with the exception of wbpMO11 has a markedly reduced G+C content relative to the chromosomal average. WzyPaO11 shows no significant similarity at the protein or DNA sequence level to any database sequence and is very hydrophobic, with 10 to 12 putative transmembrane domains, both typical characteristics of O-antigen polymerases. A nonpolar chromosomal insertion mutation in wzyPaO11 in P. aeruginosa PA103 confirmed the identity of this gene. There is striking similarity between WbjBCDE and Cap(5/8)EFGL, involved in type 5 and type 8 capsule biosynthesis in Staphylococcus aureus. There is nearly total identity between wbpMO11 and wbpMO5, previously shown by others to be present in all 20 P. aeruginosa serogroups. Using similarity searches, we have assigned functions to the proteins encoded by the PA103 O-antigen locus and present the potential steps in the pathway for the biosynthesis of P. aeruginosa serogroup O11 O antigen.
KeywordMeSH Terms
Genes, Bacterial
115. Posey  JE, Vasil  ML, Monaco  JJ, Hassett  DJ, Johnson  Z, Howell  ML, Ochsner  UA, Klotz  MG, Nanayakkara  VK,     ( 1999 )

Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa.

Journal of bacteriology 181 (12)
PMID : 10368148  :   PMC  :   PMC93851    
Abstract >>
We have cloned a 3.6-kb genomic DNA fragment from Pseudomonas aeruginosa harboring the rpoA, rplQ, katA, and bfrA genes. These loci are predicted to encode, respectively, (i) the alpha subunit of RNA polymerase; (ii) the L17 ribosomal protein; (iii) the major catalase, KatA; and (iv) one of two iron storage proteins called bacterioferritin A (BfrA; cytochrome b1 or b557). Our goal was to determine the contributions of KatA and BfrA to the resistance of P. aeruginosa to hydrogen peroxide (H2O2). When provided on a multicopy plasmid, the P. aeruginosa katA gene complemented a catalase-deficient strain of Escherichia coli. The katA gene was found to contain two translational start codons encoding a heteromultimer of approximately 160 to 170 kDa and having an apparent Km for H2O2 of 44.7 mM. Isogenic katA and bfrA mutants were hypersusceptible to H2O2, while a katA bfrA double mutant demonstrated the greatest sensitivity. The katA and katA bfrA mutants possessed no detectable catalase activity. Interestingly, a bfrA mutant expressed only approximately 47% the KatA activity of wild-type organisms, despite possessing wild-type katA transcription and translation. Plasmids harboring bfrA genes encoding BfrA altered at critical amino acids essential for ferroxidase activity could not restore wild-type catalase activity in the bfrA mutant. RNase protection assays revealed that katA and bfrA are on different transcripts, the levels of which are increased by both iron and H2O2. Mass spectrometry analysis of whole cells revealed no significant difference in total cellular iron levels in the bfrA, katA, and katA bfrA mutants relative to wild-type bacteria. Our results suggest that P. aeruginosa BfrA may be required as one source of iron for the heme prosthetic group of KatA and thus for protection against H2O2.
KeywordMeSH Terms
Bacterial Proteins
116. Vasil  ML, Meyer  JM, Ochsner  U, Stonehouse  M, Johnson  Z,     ( 1999 )

The pvc gene cluster of Pseudomonas aeruginosa: role in synthesis of the pyoverdine chromophore and regulation by PtxR and PvdS.

Journal of bacteriology 181 (13)
PMID : 10383985  :   PMC  :   PMC93907    
Abstract >>
A putative operon of four genes implicated in the synthesis of the chromophore moiety of the Pseudomonas aeruginosa siderophore pyoverdine, dubbed pvcABCD (where pvc stands for pyoverdine chromophore), was cloned and sequenced. Mutational inactivation of the pvc genes abrogated pyoverdine biosynthesis, consistent with their involvement in the biosynthesis of this siderophore. pvcABCD expression was negatively regulated by iron and positively regulated by both PvdS, the alternate sigma factor required for pyoverdine biosynthesis, and PtxR, a LysR family activator previously implicated in exotoxin A regulation.
KeywordMeSH Terms
Genes, Bacterial
Multigene Family
Oligopeptides
117. Fontana  R, Amicosante  G, Cornaglia  G, Mazzariol  A, Riccio  ML,     ( 1999 )

Cloning and characterization of blaVIM, a new integron-borne metallo-beta-lactamase gene from a Pseudomonas aeruginosa clinical isolate.

Antimicrobial agents and chemotherapy 43 (7)
PMID : 10390207  :   PMC  :   PMC89328    
Abstract >>
Production of a metallo-beta-lactamase activity was detected in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate (isolate VR-143/97) from an Italian inpatient at the Verona University Hospital (northern Italy). The metallo-beta-lactamase determinant was isolated from a genomic library of VR-143/97, constructed in an Escherichia coli plasmid vector, by screening for clones with reduced susceptibility to imipenem. Sequencing of the cloned gene revealed that it encoded a new class B beta-lactamase that was named VIM-1. At the sequence level VIM-1 was rather divergent from the other class B enzymes (16.4 to 38.7% identity), overall being more similar to members of subclass B1 including the beta-lactamase II of Bacillus cereus (Bc-II), the Bacteroides fragilis CcrA, the Chryseobacterium meningosepticum BlaB, and the cassette-encoded IMP-1 enzymes. Among these, VIM-1 showed the highest degree of similarity to Bc-II. Similarly to blaIMP, blaVIM was also found to be carried on a gene cassette inserted into a class 1 integron. The blaVIM-containing integron was located on the chromosome of P. aeruginosa VR-143/97, and the metallo-beta-lactamase-encoding determinant was not transferable to E. coli by conjugation. Expression of the integron-borne blaVIM gene in E. coli resulted in a significant decrease in susceptibility to a broad array of beta-lactams (ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems), revealing a very broad substrate specificity of the VIM-1 enzyme.
KeywordMeSH Terms
Bacterial Proteins
118. Yoshihara  E, Nakae  T, Nishino  T, Yamada  H, Gotoh  N, Tsujimoto  H,     ( 1999 )

Molecular cloning and characterization of the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa.

Microbiology and immunology 43 (3)
PMID : 10338201  :   DOI  :   10.1111/j.1348-0421.1999.tb02407.x    
Abstract >>
We cloned and characterized the oprQ gene coding for outer membrane protein OprE3 of Pseudomonas aeruginosa PAO1. The oprQ gene was composed of 1,275 base pairs including a sequence encoding for the signal sequence and a mature protein with a Mr of 44,602. Computer-aided alignment and hydropathy analyses of the predicted amino acid sequences suggested that OprE3 is a transmembrane protein homologous to outer membrane proteins of P. aeruginosa such as OprD2 (OprD) porin and OprE1 (OprE) porin. Susceptibility to several antibiotics of the strains lacking or overproducing OprE3 was indistinguishable from that of the wild-type strain, suggesting that OprE3 is unlikely involved in the diffusion of carbapenems and other beta-lactam antibiotics.
KeywordMeSH Terms
Bacterial Outer Membrane Proteins
119. Livermore  DM, Hall  LM, Duke  B, Gur  D,     ( 1999 )

OXA-17, a further extended-spectrum variant of OXA-10 beta-lactamase, isolated from Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 43 (6)
PMID : 10348753  :   PMC  :   PMC89279    
Abstract >>
Pseudomonas aeruginosa isolates 871 and 873 were isolated at Hacettepe University Hospital in Ankara and were highly resistant to ceftazidime (MIC, 128 microg/ml). Each produced three beta-lactamases, with pIs of 5.3, 6.1, and 7.9. The beta-lactamase with a pI of 5.3 was previously shown to be PER-1 enzyme. The antibiograms of the isolates were not entirely explained by production of PER-1 enzyme, insofar as ceftazidime resistance was incompletely reversed by clavulanate. The enzymes with pIs of 6.1 and 7.9 were therefore investigated. The enzyme with a pI of 6.1 proved to be a novel mutant of OXA-10, which we designated OXA-17, and had asparagine changed to serine at position 73 of the protein. When cloned into Escherichia coli XL1-blue, OXA-17 enzyme conferred greater resistance to cefotaxime, latamoxef, and cefepime than did OXA-10, but it had only a marginal (two- to fourfold) effect on the MIC of ceftazidime. This behavior contrasted with that of previous OXA-10 mutants, specifically OXA-11, -14, and -16, which predominately compromise ceftazidime. Extracted OXA-17 enzyme had relatively greater activity than OXA-10 against oxacillin, cloxacillin, and cefotaxime but, in terms of kcat/Km, it had lower catalytic efficiency against most beta-lactams. The enzyme with a pI of 7.9 was shown by gene sequencing to be OXA-2.
KeywordMeSH Terms
120. Tiedje  JM, Bagdasarian  M, Tsoi  TV, Guerin  WF, Cole  JR,     ( 1999 )

Cloning, expression, and nucleotide sequence of the Pseudomonas aeruginosa 142 ohb genes coding for oxygenolytic ortho dehalogenation of halobenzoates.

Applied and environmental microbiology 65 (5)
PMID : 10224014  :   PMC  :   PMC91311    
Abstract >>
We have cloned and characterized novel oxygenolytic ortho-dehalogenation (ohb) genes from 2-chlorobenzoate (2-CBA)- and 2,4-dichlorobenzoate (2,4-dCBA)-degrading Pseudomonas aeruginosa 142. Among 3,700 Escherichia coli recombinants, two clones, DH5alphaF'(pOD22) and DH5alphaF'(pOD33), converted 2-CBA to catechol and 2,4-dCBA and 2,5-dCBA to 4-chlorocatechol. A subclone of pOD33, plasmid pE43, containing the 3,687-bp minimized ohb DNA region conferred to P. putida PB2440 the ability to grow on 2-CBA as a sole carbon source. Strain PB2440(pE43) also oxidized but did not grow on 2,4-dCBA, 2,5-dCBA, or 2,6-dCBA. Terminal oxidoreductase ISPOHB structural genes ohbA and ohbB, which encode polypeptides with molecular masses of 20,253 Da (beta-ISP) and 48,243 Da (alpha-ISP), respectively, were identified; these proteins are in accord with the 22- and 48-kDa (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) polypeptides synthesized in E. coli and P. aeruginosa parental strain 142. The ortho-halobenzoate 1,2-dioxygenase activity was manifested in the absence of ferredoxin and reductase genes, suggesting that the ISPOHB utilized electron transfer components provided by the heterologous hosts. ISPOHB formed a new phylogenetic cluster that includes aromatic oxygenases featuring atypical structural-functional organization and is distant from the other members of the family of primary aromatic oxygenases. A putative IclR-type regulatory gene (ohbR) was located upstream of the ohbAB genes. An open reading frame (ohbC) of unknown function that overlaps lengthwise with ohbB but is transcribed in the opposite direction was found. The ohbC gene codes for a 48,969-Da polypeptide, in accord with the 49-kDa protein detected in E. coli. The ohb genes are flanked by an IS1396-like sequence containing a putative gene for a 39,715-Da transposase A (tnpA) at positions 4731 to 5747 and a putative gene for a 45,247-Da DNA topoisomerase I/III (top) at positions 346 to 1563. The ohb DNA region is bordered by 14-bp imperfect inverted repeats at positions 56 to 69 and 5984 to 5997.
KeywordMeSH Terms
Genes, Bacterial
121. Zago  A, Chugani  S,     ( 1999 )

Cloning and characterization of polyphosphate kinase and exopolyphosphatase genes from Pseudomonas aeruginosa 8830.

Applied and environmental microbiology 65 (5)
PMID : 10224002  :   PMC  :   PMC91299    
Abstract >>
Pseudomonas aeruginosa accumulates polyphosphates in response to nutrient limitations. To elucidate the function of polyphosphate in this microorganism, we have investigated polyphosphate metabolism by isolating from P. aeruginosa 8830 the genes encoding polyphosphate kinase (PPK) and exopolyphosphatase (PPX), which are involved in polyphosphate synthesis and degradation, respectively. The 690- and 506-amino-acid polypeptides encoded by the two genes have been expressed in Escherichia coli and purified, and their activities have been tested in vitro. Gene replacement was used to construct a PPK-negative strain of P. aeruginosa 8830. Low residual PPK activity in the ppk mutant suggests a possible alternative pathway of polyphosphate synthesis in this microorganism. Primer extension analysis indicated that ppk is transcribed from a sigmaE-dependent promoter, which could be responsive to environmental stresses. However, no coregulation between ppk and ppx promoters has been demonstrated in response to osmotic shock or oxidative stress.
KeywordMeSH Terms
Genes, Bacterial
122. Naas  T, Nordmann  P, Ronco  E, Poirel  L,     ( 1999 )

An SHV-derived extended-spectrum beta-lactamase in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 43 (5)
PMID : 10223953  :   PMC  :   PMC89260    
Abstract >>
A clinical isolate of Pseudomonas aeruginosa RP-1 produced the extended-spectrum beta-lactamase (ESBL) SHV-2a. Its gene was expressed from a composite promoter made of the -35 region derived from the left inverted repeat of IS26 and the -10 region from the blaSHV-2a promoter itself. The DNA sequences immediately surrounding blaSHV-2a were homologous to plasmid pMPA2a from Klebsiella pneumoniae KpZU-3, while further away and 3' to the blaSHV-2a gene, a sequence corresponding to the left end of Tn1721 was detected, thus indicating a likely enterobacterial origin of this ESBL gene.
KeywordMeSH Terms
Genes, Bacterial
123. Foglino  M, Méjean  V, Clepet  C,     ( 1999 )

ThrH, a homoserine kinase isozyme with in vivo phosphoserine phosphatase activity in Pseudomonas aeruginosa.

Microbiology (Reading, England) 145 (Pt 4) (N/A)
PMID : 10220164  :   DOI  :   10.1099/13500872-145-4-845    
Abstract >>
Homoserine kinase, the product of the thrB gene, catalyses an obligatory step of threonine biosynthesis. In Pseudomonas aeruginosa, unlike Escherichia coli, inactivation of the previously identified thrB gene does not result in threonine auxotrophy. A new gene, named thrH, was isolated that, when expressed in E. coli thrB mutant strains, results in complementation of the mutant phenotype. In P. aeruginosa, threonine auxotrophy is observed only when both thrB and thrH are simultaneously inactivated. Thus, thrH encodes a protein with an in vivo homoserine-kinase-like activity. Surprisingly, thrH overexpression allows complementation of serine auxotrophy of E. coli and P. aeruginosa serB mutants. These mutants are affected in the phosphoserine phosphatase protein, an enzyme involved in serine biosynthesis. Comparison analysis revealed sequence homology between ThrH and the SerB proteins from different organisms. This could explain the in vivo phosphoserine phosphatase activity of ThrH when overproduced. ThrH differs from the protein encoded by the serB gene which was identified in P. aeruginosa. Thus, two SerB-like proteins co-exist in P. aeruginosa, a situation also found in Mycobacterium tuberculosis.
KeywordMeSH Terms
Bacterial Proteins
124. Goldberg  JB,     ( 1999 )

Cloning of the glutamyl-tRNA synthetase (gltX) gene from Pseudomonas aeruginosa.

Journal of bacteriology 181 (11)
PMID : 10348873  :   PMC  :   PMC93828    
Abstract >>
The glutamyl-tRNA synthetase (gltX) gene from Pseudomonas aeruginosa was identified. A plasmid containing a 2.3-kb insert complemented the temperature-sensitive gltX mutation of Escherichia coli JP1449, and GltX activity was demonstrated. The inferred amino acid sequence of this gene showed 50.6% identity with GltX from Rhizobium meliloti.
KeywordMeSH Terms
125. Akasaka  T, Sato  K, Tanaka  M,     ( 1999 )

Cloning, expression, and enzymatic characterization of Pseudomonas aeruginosa topoisomerase IV.

Antimicrobial agents and chemotherapy 43 (3)
PMID : 10049263  :   PMC  :   PMC89156    
Abstract >>
The topoisomerase IV subunit A gene, parC homolog, has been cloned and sequenced from Pseudomonas aeruginosa PAO1, with cDNA encoding the N-terminal region of Escherichia coli parC used as a probe. The homolog and its upstream gene were presumed to be parC and parE through sequence homology with the parC and parE genes of other organisms. The deduced amino acid sequence of ParC and ParE showed 33 and 32% identity with that of the P. aeruginosa DNA gyrase subunits, GyrA and GyrB, respectively, and 69 and 75% identity with that of E. coli ParC and ParE, respectively. The putative ParC and ParE proteins were overexpressed and separately purified by use of a fusion system with a maltose-binding protein, and their enzymatic properties were examined. The reconstituted enzyme had ATP-dependent decatenation activity, which is the main catalytic activity of bacterial topoisomerase IV, and relaxing activities but had no supercoiling activity. So, the cloned genes were identified as P. aeruginosa topoisomerase IV genes. The inhibitory effects of quinolones on the activities of topoisomerase IV and DNA gyrase were compared. The 50% inhibitory concentrations of quinolones for the decatenation activity of topoisomerase IV were from five to eight times higher than those for the supercoiling activities of P. aeruginosa DNA gyrase. These results confirmed that topoisomerase IV is less sensitive to fluoroquinolones than is DNA gyrase and may be a secondary target of new quinolones in wild-type P. aeruginosa.
KeywordMeSH Terms
126. Hallin  S, Lindgren  PE,     ( 1999 )

PCR detection of genes encoding nitrite reductase in denitrifying bacteria.

Applied and environmental microbiology 65 (4)
PMID : 10103263  :   PMC  :   PMC91233    
Abstract >>
Using consensus regions in gene sequences encoding the two forms of nitrite reductase (Nir), a key enzyme in the denitrification pathway, we designed two sets of PCR primers to amplify cd1- and Cu-nir. The primers were evaluated by screening defined denitrifying strains, denitrifying isolates from wastewater treatment plants, and extracts from activated sludge. Sequence relationships of nir genes were also established. The cd1 primers were designed to amplify a 778 to 799-bp region of cd1-nir in the six published sequences. Likewise, the Cu primers amplified a 473-bp region in seven of the eight published Cu-nir sequences. Together, the two sets of PCR primers amplified nir genes in nine species within four genera, as well as in four of the seven sludge isolates. The primers did not amplify genes of nondenitrifying strains. The Cu primers amplified the expected fragment in all 13 sludge samples, but cd1-nir fragments were only obtained in five samples. PCR products of the expected sizes were verified as nir genes after hybridization to DNA probes, except in one case. The sequenced nir fragments were related to other nir sequences, demonstrating that the primers amplified the correct gene. The selected primer sites for Cu-nir were conserved, while broad-range primers targeting conserved regions of cd1-nir seem to be difficult to find. We also report on the existence of Cu-nir in Paracoccus denitrificans Pd1222.
KeywordMeSH Terms
Genes, Bacterial
127. Schobert  M, Görisch  H,     ( 1999 )

Cytochrome c550 is an essential component of the quinoprotein ethanol oxidation system in Pseudomonas aeruginosa: cloning and sequencing of the genes encoding cytochrome c550 and an adjacent acetaldehyde dehydrogenase.

Microbiology (Reading, England) 145 (Pt 2) (N/A)
PMID : 10075429  :   DOI  :   10.1099/13500872-145-2-471    
Abstract >>
Pseudomonas aeruginosa ATCC 17933 grown aerobically on ethanol produces a soluble cytochrome c550 together with a quinoprotein ethanol dehydrogenase. A 3.2 kb genomic DNA fragment containing the gene encoding cytochrome c550 was cloned and sequenced. Two other complete and two truncated ORFs were also identified. A truncated ORF encoding the quinoprotein ethanol dehydrogenase (exaA) was found upstream of the cytochrome c550 gene (exaB) and in reverse orientation. An ORF encoding a NAD(+)-dependent acetaldehyde dehydrogenase (exaC) was located downstream of the cytochrome c550 gene and in the same orientation. Another ORF showed similarity to the pqqA gene and a truncated ORF similarity to the pqqB gene, both involved in the biosynthesis of the prosthetic group PQQ. The organization of these genes was found to be different from the well-studied methanol oxidation system in methylotrophic bacteria. The deduced amino acid sequence of cytochrome c550 from P. aeruginosa showed some similarity to cytochrome c6 of the alga Chlamydomonas reinhardtii and the haem domain of quinohaemoprotein alcohol dehydrogenases of acetic acid bacteria, but no similarity to the soluble cytochrome cL of the quinoprotein methanol oxidation system of methylotrophs could be detected. A mutant of P. aeruginosa with an interrupted cytochrome c550 gene was unable to grow on ethanol, which proves that cytochrome c550 is an essential component of the ethanol oxidation system in this organism.
KeywordMeSH Terms
128. Berger  B, Haas  D,     ( 1999 )

Target joining of duplicated insertion sequence IS21 is assisted by IstB protein in vitro.

Journal of bacteriology 181 (7)
PMID : 10094711  :   PMC  :   PMC93646    
Abstract >>
Tandemly repeated insertion sequence IS21, located on a suicide plasmid, promoted replicon fusion with bacteriophage lambda in vitro in the presence of ATP. This reaction was catalyzed in a cell extract containing the 45-kDa IstA protein (cointegrase) and the 30-kDa IstB helper protein of IS21 after both proteins had been overproduced in Escherichia coli. Without IstB, replicon fusion was inefficient and did not produce the 4-bp target duplications typical of IS21.
KeywordMeSH Terms
DNA Transposable Elements
Tandem Repeat Sequences
129. Tribuddharat  C,     ( 1999 )

Integron-mediated rifampin resistance in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 43 (4)
PMID : 10103210  :   PMC  :   PMC89236    
Abstract >>
A new rifampin resistance gene, arr-2, has been found in Pseudomonas aeruginosa. The ARR-2 protein shows 54% amino acid identity to the rifampin ADP-ribosylating transferase encoded by the arr gene from Mycobacterium smegmatis. This arr-2 gene is located on a gene cassette within a class I integron.
KeywordMeSH Terms
130. Frère  JM, Amicosante  G, Thamm  I, Laraki  N, Riccio  ML,     ( 1999 )

Structure of In31, a blaIMP-containing Pseudomonas aeruginosa integron phyletically related to In5, which carries an unusual array of gene cassettes.

Antimicrobial agents and chemotherapy 43 (4)
PMID : 10103196  :   PMC  :   PMC89222    
Abstract >>
The location and environment of the acquired blaIMP gene, which encodes the IMP-1 metallo-beta-lactamase, were investigated in a Japanese Pseudomonas aeruginosa clinical isolate (isolate 101/1477) that produced the enzyme. In this isolate, blaIMP was carried on a 36-kb plasmid, and similar to the identical alleles found in Serratia marcescens and Klebsiella pneumoniae clinical isolates, it was located on a mobile gene cassette inserted into an integron. The entire structure of this integron, named In31, was determined. In31 is a class 1 element belonging to the same group of defective transposon derivatives that originated from Tn402-like ancestors such as In0, In2, and In5. The general structure of In31 appeared to be most closely related to that of In5 from pSCH884, suggesting a recent common phylogeny for these two elements. In In31, the blaIMP cassette is the first of an array of five gene cassettes that also includes an aacA4 cassette and three original cassettes that have never been described in other integrons. The novel cassettes carry, respectively, (i) a new chloramphenicol acetyltransferase-encoding allele of the catB family, (ii) a qac allele encoding a new member of the small multidrug resistance family of proteins, and (iii) an open reading frame encoding a protein of unknown function. All the resistance genes carried on cassettes inserted in In31 were found to be functional in decreasing the in vitro susceptibilities of host strains to the corresponding antimicrobial agents.
KeywordMeSH Terms
Bacterial Proteins
131. de Kievit  T, Iglewski  BH, Passador  L,     ( 1999 )

RsaL, a novel repressor of virulence gene expression in Pseudomonas aeruginosa.

Journal of bacteriology 181 (7)
PMID : 10094696  :   PMC  :   PMC93631    
Abstract >>
As components of a Pseudomonas aeruginosa quorum-sensing system, LasR and PAI-1 globally regulate expression of multiple virulence determinants, as well as the second P. aeruginosa quorum-sensing system. To date, no information exists on negative regulation of the quorum-sensing cascade in P. aeruginosa. Here we describe a novel gene, rsaL, which is located downstream from lasR and transcribed antisense relative to lasR. In P. aeruginosa, overexpression of rsaL results in reduced lasB expression and decreased elastase activity. With the use of a six-His protein fusion system, we demonstrate that rsaL encodes an 11-kDa protein. Direct quantitation of PAI-1 levels in cultures and studies utilizing Escherichia coli lambda lysogens carrying lacZ transcriptional fusions reveal that RsaL specifically represses transcription of the PAI-1 autoinducer synthase gene, lasI. RsaL's repressive effect on lasI and the associated decrease in elastase activity have important implications for the expression of all LasR-PAI-1-dependent virulence genes and the overall pathogenicity of P. aeruginosa.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
132. Tan  MW, Ausubel  FM, Tompkins  RG,     ( 1999 )

Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors.

Proceedings of the National Academy of Sciences of the United States of America 96 (5)
PMID : 10051655  :   DOI  :   10.1073/pnas.96.5.2408     PMC  :   PMC26797    
Abstract >>
We reported recently that the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 kills Caenorhabditis elegans and that many P. aeruginosa virulence factors (genes) required for maximum virulence in mouse pathogenicity are also required for maximum killing of C. elegans. Here we report that among eight P. aeruginosa PA14 TnphoA mutants isolated that exhibited reduced killing of C. elegans, at least five also exhibited reduced virulence in mice. Three of the TnphoA mutants corresponded to the known virulence-related genes lasR, gacA, and lemA. Three of the mutants corresponded to known genes (aefA from Escherichia coli, pstP from Azotobacter vinelandii, and mtrR from Neisseria gonorrhoeae) that had not been shown previously to play a role in pathogenesis, and two of the mutants contained TnphoA inserted into novel sequences. These data indicate that the killing of C. elegans by P. aeruginosa can be exploited to identify novel P. aeruginosa virulence factors important for mammalian pathogenesis.
KeywordMeSH Terms
133. Bellingham  NF, Morgan  JA, Saunders  JR, Hart  CA,     ( 1999 )

Comparison of flagellin genes from clinical and environmental Pseudomonas aeruginosa isolates.

Applied and environmental microbiology 65 (3)
PMID : 10049879  :   PMC  :   PMC91160    
Abstract >>
Pseudomonas aeruginosa, an important opportunistic pathogen, was isolated from environmental samples and compared to clinically derived strains. While P. aeruginosa was isolated readily from an experimental mushroom-growing unit, it was found only rarely in other environmental samples. A flagellin gene PCR-restriction fragment length polymorphism analysis of the isolates revealed that environmental and clinical P. aeruginosa strains are not readily distinguishable. The variation in the central regions of the flagellin genes of seven of the isolates was investigated further. The strains used included two strains with type a genes (998 bp), four strains with type b genes (1,258 bp), and one strain, K979, with a novel flagellin gene (2,199 bp). The route by which flagellin gene variation has occurred in P. aeruginosa is discussed.
KeywordMeSH Terms
Environmental Microbiology
134. Schmidt-Larbig  K, Wüest  T,     ( 1999 )

A novel reduced flavin mononucleotide-dependent methanesulfonate sulfonatase encoded by the sulfur-regulated msu operon of Pseudomonas aeruginosa.

Journal of bacteriology 181 (5)
PMID : 10049377  :   PMC  :   PMC93535    
Abstract >>
When Pseudomonas aeruginosa is grown with organosulfur compounds as sulfur sources, it synthesizes a set of proteins whose synthesis is repressed in the presence of sulfate, cysteine, or thiocyanate (so-called sulfate starvation-induced proteins). The gene encoding one of these proteins, PA13, was isolated from a cosmid library of P. aeruginosa PAO1 and sequenced. It encoded a 381-amino-acid protein that was related to several reduced flavin mononucleotide (FMNH2)-dependent monooxygenases, and it was the second in an operon of three genes, which we have named msuEDC. The MsuD protein catalyzed the desulfonation of alkanesulfonates, requiring oxygen and FMNH2 for the reaction, and showed highest activity with methanesulfonate. MsuE was an NADH-dependent flavin mononucleotide (FMN) reductase, which provided reduced FMN for the MsuD enzyme. Expression of the msu operon was analyzed with a transcriptional msuD::xylE fusion and was found to be repressed in the presence of sulfate, sulfite, sulfide, or cysteine and derepressed during growth with methionine or alkanesulfonates. Growth with methanesulfonate required an intact cysB gene, and the msu operon is therefore part of the cys regulon, since sulfite utilization was found to be CysB independent in this species. Measurements of msuD::xylE expression in cysN and cysI genetic backgrounds showed that sulfate, sulfite, and sulfide or cysteine play independent roles in negatively regulating msu expression, and sulfonate utilization therefore appears to be tightly regulated.
KeywordMeSH Terms
Bacterial Proteins
Operon
135. Nordmann  P, Chaibi  EB, Labia  R, Naas  T,     ( 1999 )

Molecular and biochemical characterization of VEB-1, a novel class A extended-spectrum beta-lactamase encoded by an Escherichia coli integron gene.

Antimicrobial agents and chemotherapy 43 (3)
PMID : 10049269  :   PMC  :   PMC89162    
Abstract >>
A clinical isolate, Escherichia coli MG-1, isolated from a 4-month-old Vietnamese orphan child, produced a beta-lactamase conferring resistance to extended-spectrum cephalosporins and aztreonam. In a disk diffusion test, a typical synergistic effect between ceftazidime or aztreonam and clavulanic acid was observed along with an unusual synergy between cefoxitin and cefuroxime. The gene for VEB-1 (Vietnamese extended-spectrum beta-lactamase) was cloned and expressed in E. coli JM109. The recombinant plasmid pRLT1 produced a beta-lactamase with a pI of 5.35 and conferred high-level resistance to extended-spectrum (or oxyimino) cephalosporins and to aztreonam. Vmax values for extended-spectrum cephalosporins were uncommonly high, while the affinity of the enzyme for ceftazidime and aztreonam was relatively low. blaVEB-1 showed significant homology at the DNA level with only blaPER-1 and blaPER-2. Analysis of the deduced protein sequence showed that VEB-1 is a class A penicillinase having very low levels of homology with any other known beta-lactamases. The highest percentage of amino acid identity was 38% with PER-1 or PER-2, two uncommon class A extended-spectrum enzymes. Exploration of the genetic environment of blaVEB-1 revealed the presence of gene cassette features, i.e., (i) a 59-base element associated with blaVEB-1; (ii) a second 59-base element just upstream of blaVEB-1, likely belonging to the aacA1-orfG gene cassette; (iii) two core sites (GTTRRRY) on both sides of blaVEB-1; and (iv) a second antibiotic resistance gene 3' of blaVEB-1, aadB. blaVEB-1 may therefore be the first class A extended-spectrum beta-lactamase that is part of a gene cassette, which itself is likely to be located on a class 1 integron, as sulfamide resistance may indicate. Furthermore, blaVEB-1 is encoded on a large (> 100-kb) transferable plasmid found in a Klebsiella pneumoniae MG-2 isolated at the same time from the same patient, indicating a horizontal gene transfer.
KeywordMeSH Terms
136. Li  Y, Xia  H, Bai  F, Xu  H, Yang  L, Yao  H, Zhang  L, Zhang  X, Bai  Y, Saris  PE, Tolker-Nielsen  T, Qiao  M,     ( 2007 )

Identification of a new gene PA5017 involved in flagella-mediated motility, chemotaxis and biofilm formation in Pseudomonas aeruginosa.

FEMS microbiology letters 272 (2)
PMID : 17521365  :   DOI  :   10.1111/j.1574-6968.2007.00753.x    
Abstract >>
Flagella-mediated motility is recognized as one of the major factors contributing to virulence in Pseudomonas aeruginosa. During a screening of a mini-Mu transposon mutant library of P. aeruginosa PA68, a mutant partially deficient in swimming and swarming motility was identified in a new locus that encodes a predicted protein of unknown function annotated PA5017 in the P. aeruginosa PAO1 genome sequence. Chemotaxis plate assay indicated that inactivation of the PA5017 gene led to a decreased chemotactic response. Complementation of the PA5017 mutant with the wild-type PA5017 gene restored normal motility and chemotaxis phenotype. A promoter-lacZ reporter activity assay of the cheYZAB operon from chemotaxis gene cluster 1 showed that there was almost a twofold difference in expression levels of the wild-type PA68 and the PA5017 mutant. This suggested that the PA5017 affected expression of the cheYZAB operon negatively. Further study showed that inactivation of the PA5017 gene in PA68 led to increased biofilm formation in a static system and to the formation of a heterogeneous biofilm in a flow-chamber system. These results suggested that PA5017 possibly affected flagellum-dependent motility and in turn biofilm formation via the chemotaxis signal transduction pathway.
KeywordMeSH Terms
Biofilms
Genes, Bacterial
137. Maniati  M, Ikonomidis  A, Mantzana  P, Daponte  A, Maniatis  AN, Pournaras  S,     ( 2007 )

A highly carbapenem-resistant Pseudomonas aeruginosa isolate with a novel blaVIM-4/blaP1b integron overexpresses two efflux pumps and lacks OprD.

The Journal of antimicrobial chemotherapy 60 (1)
PMID : 17483142  :   DOI  :   10.1093/jac/dkm126    
Abstract >>
A Pseudomonas aeruginosa clinical isolate that exhibited high-level carbapenem resistance and produced metallo-beta-lactamase (MBL) was recovered from a Greek patient. This study was conducted to determine the underlying mechanisms that conferred the carbapenem resistance phenotype. MICs were determined by Etest and Etest MBL. PCR assays were performed for identification of bla(VIM-type), other antibiotic resistance and efflux pump genes and mapping of class 1 integrons. Expression of efflux pump genes was quantified by real-time PCR. Nucleotide sequencing was used to determine the bla(VIM) allele. The location of the MBL allele was investigated by mating experiments, plasmid analysis and hybridization studies. The isolate was highly carbapenem-resistant (MICs of imipenem and meropenem were 512 and 128 mg/L, respectively) and multidrug-resistant. It harboured the beta-lactamase genes bla(VIM-4) and bla(P1b) in a novel class 1 integron named InV4P1, and a second integron with aac(6')-Ib and bla(OXA-35) gene cassettes. The isolate was deficient in porin OprD and overexpressed efflux pumps MexAB-OprM and MexXY-OprM. Conjugation experiments failed to detect transferable MBL determinants, plasmids were not visualized and bla(VIM) was detected by PCR in the chromosomal band. Multiple carbapenem resistance mechanisms are demonstrated to coexist in a single P. aeruginosa isolate and might confer the high-level carbapenem resistance.
KeywordMeSH Terms
138. Tseng  SP, Hsueh  PR, Tsai  JC, Teng  LJ,     ( 2007 )

Tn6001, a transposon-like element containing the blaVIM-3-harboring integron In450.

Antimicrobial agents and chemotherapy 51 (11)
PMID : 17846142  :   DOI  :   10.1128/AAC.00542-07     PMC  :   PMC2151417    
Abstract >>
We describe the structure of a transposon-like element named Tn6001, which contains a bla(VIM-3)-harboring integron In450, which was derived from a multidrug-resistant Pseudomonas aeruginosa clinical isolate in Taiwan. The transposon backbone structure is most closely related to those of Tn1404* and Tn1403. Tn6001 was inserted into the chromosome of the clinical isolate.
KeywordMeSH Terms
139. Schue  M, Glendinning  KJ, Hobman  JL, Brown  NL,     ( 2008 )

Evidence for direct interactions between the mercuric ion transporter (MerT) and mercuric reductase (MerA) from the Tn501 mer operon.

Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine 21 (2)
PMID : 17457514  :   DOI  :   10.1007/s10534-007-9097-4    
Abstract >>
Mercuric ion resistance in bacteria requires transport of mercuric ions (Hg(2+)) into the cytoplasmic compartment where they are reduced to the less toxic metallic mercury (Hg(0)) by mercuric reductase (MR). The long-established model for the resistance mechanism predicts interactions between the inner membrane mercuric ion transporter, MerT, and the N-terminal domain of cytoplasmic MR, but attempts to demonstrate this interaction have thus far been unsuccessful. A recently developed bacterial two-hybrid protein interaction detection system was used to show that the N-terminal region of MR interacts with the cytoplasmic face of MerT. We also show that the cysteine residues on the cytoplasmic face of the MerT protein are required for maximal mercuric ion transport but not for the interaction with mercuric reductase.
KeywordMeSH Terms
Operon
140. Evans  JC, Segal  H,     ( 2007 )

A novel insertion sequence, ISPA26, in oprD of Pseudomonas aeruginosa is associated with carbapenem resistance.

Antimicrobial agents and chemotherapy 51 (10)
PMID : 17682099  :   DOI  :   10.1128/AAC.00837-07     PMC  :   PMC2043271    
Abstract >>
N/A
KeywordMeSH Terms
141. Qiu  D, Eisinger  VM, Rowen  DW, Yu  HD,     ( 2007 )

Regulated proteolysis controls mucoid conversion in Pseudomonas aeruginosa.

Proceedings of the National Academy of Sciences of the United States of America 104 (19)
PMID : 17470813  :   DOI  :   10.1073/pnas.0702660104     PMC  :   PMC1876579    
Abstract >>
Overproduction of the exopolysaccharide alginate causes mucoid conversion in Pseudomonas aeruginosa and is a poor prognosticator in cystic fibrosis. The ECF sigma factor AlgU and its cognate anti-sigma factor MucA are two principal regulators of alginate production. Here, we report the identification of three positive regulators of alginate biosynthesis: PA4033 (designated mucE), PA3649 (designated mucP), and algW. MucE, a small protein (9.5 kDa), was identified as part of a global mariner transposon screen for new regulators of alginate production. A transposon located in its promoter caused the overexpression of MucE and mucoid conversion in P. aeruginosa strains PAO1 and PA14. Accumulation of MucE in the envelope resulted in increased AlgU activity and reduced MucA levels. Three critical amino acid residues at the C terminus of MucE (WVF) were required for mucoid conversion via two predicted proteases AlgW (DegS) and MucP (RseP/YaeL). Moreover, as in Escherichia coli, the PDZ domain of AlgW was required for signal transduction. These results suggest that AlgU is regulated similarly to E. coli sigma(E) except that the amino acid triad signals from MucE and other envelope proteins that activate AlgW are slightly different from those activating DegS.
KeywordMeSH Terms
142. Ohlasova  D, Kmet  V, Niks  M,     ( 2007 )

First report of the carbapenem-resistant Pseudomonas aeruginosa producing IMP-7 metallo-beta-lactamase in Slovakia.

International journal of antimicrobial agents 30 (4)
PMID : 17709230  :   DOI  :   10.1016/j.ijantimicag.2007.06.008    
Abstract >>
N/A
KeywordMeSH Terms
143. Bai  F, Li  Y, Xu  H, Xia  H, Yin  T, Yao  H, Zhang  L, Zhang  X, Bai  Y, Jin  S, Qiao  M,     ( 2007 )

Identification and functional characterization of pfm, a novel gene involved in swimming motility of Pseudomonas aeruginosa.

Gene 401 (1��2��)
PMID : 17714889  :   DOI  :   10.1016/j.gene.2007.06.019    
Abstract >>
Pseudomonas aeruginosa, an important opportunistic pathogen, has a single polar flagellum which is an important virulence and colonization factor by providing swimming motility. This paper describes the functional characterization of a novel gene pfm (PA2950) of P. aeruginosa. The pfm encodes a protein that is similar to a number of short-chain alcohol dehydrogenases of other bacterial species. Mutation of this gene results in a defect in swimming motility which can be completed back to that of wild type by a plasmid containing the pfm. Interestingly, the pfm mutant possesses an intact flagellum which does not rotate, thus giving rise to a non-motile phenotype. The pfm gene is encoded on an operon together with two upstream genes which code for electron transfer flavoprotein (ETF). Yeast two-hybrid tests indicated that the PFM interacts with the ETF, suggesting that the putative dehydrogenase (PFM) is involved in energy metabolism that is critical for the rotation of flagellum in P. aeruginosa.
KeywordMeSH Terms
Genes, Bacterial
144. Vu-Thien  H, Corbineau  G, Hormigos  K, Fauroux  B, Corvol  H, Clément  A, Vergnaud  G, Pourcel  C,     ( 2007 )

Multiple-locus variable-number tandem-repeat analysis for longitudinal survey of sources of Pseudomonas aeruginosa infection in cystic fibrosis patients.

Journal of clinical microbiology 45 (10)
PMID : 17699654  :   DOI  :   10.1128/JCM.00702-07     PMC  :   PMC2045346    
Abstract >>
In order to identify the source of infection by Pseudomonas aeruginosa in patients with cystic fibrosis (CF), systematic genotyping of isolates is necessary. Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) was used to survey the sources of P. aeruginosa infections in a French (Paris, France) pediatric CF center. Between January 2004 and December 2006, 108 patients ages 2 to 21 years who were regularly monitored at the center provided sputum for culture. P. aeruginosa was detected in 46 children, 17 of whom had primary colonization. A total of 163 isolates were recovered. MLVA was improved from a previously published method by the addition of new, informative, and easily typeable markers. Upon genotyping with 15 VNTRs, a total of 39 lineages composed of indistinguishable or closely related isolates, were observed. One of them corresponds to "clone C," which is widely distributed in Europe, and another corresponds to reference strain PA14. Six patients were colonized with two different strains, and the remaining 40 patients were colonized with a single strain. Strains from seven lineages were shared by at least two and up to four patients among a total of 20 patients. The study demonstrates that MLVA is an efficient, easy, and rapid molecular method for epidemiological surveillance for P. aeruginosa infection. The resulting data and strain genetic profiles can be queried on http://bacterial-genotyping.igmors.u-psud.fr.
KeywordMeSH Terms
Minisatellite Repeats
145. Gupta  A, Ray  S, Kapoor  S, Khare  SK,     ( 2008 )

Solvent-stable Pseudomonas aeruginosa PseA protease gene: identification, molecular characterization, phylogenetic and bioinformatic analysis to study reasons for solvent stability.

Journal of molecular microbiology and biotechnology 15 (4)
PMID : 17715461  :   DOI  :   10.1159/000107488    
Abstract >>
We have previously isolated a solvent-stable protease from a novel solvent-tolerant strain of Pseudomonas aeruginosa (PseA). Here we report cloning and characterization of the gene coding for this solvent-tolerant protease. A homology search of the N-terminal amino acid sequence of the purified PseA protease revealed an exact match to a P. aeruginosa PST-01 protease gene, lasB. The c-DNA sequence of the PST-01 protease was used to design primers for the amplification of a 1,494-bp open reading frame encoding a 53.6-kDa, 498-amino-acid PseA LasB polypeptide. The deduced PseA LasB protein contained a 23-residue signal peptide (2.6 kDa) followed by a propeptide of 174 residues and a 33-kDa mature product of 301 residues. A phylogenetic analysis placed PseA lasB closest to the known zinc metalloproteases from P. aeruginosa. This gene was also found to contain a conserved HEXXH zinc-binding motif, characteristic of all zinc metallopeptidases. The 3D structure analysis of PseA protease revealed the presence of 7 alpha-helices (36% of the sequence). The molecule was found to have two disulfide bonds (between Cys-227 and Cys-255 and between Cys-467 and Cys-494) and had a number of hydrophobic clusters at the protein surface. These hydrophobic patches (21% of the sequence) and disulfide bonds may possibly be responsible for the solvent-stable nature of the enzyme.
KeywordMeSH Terms
146. Cipriano  R, da Fonseca  EL, das Graças Azevedo Soares  M, Léda Moura  MC, Vieira  VV, Vicente  AC,     ( 2007 )

Recurrent infections caused by Pseudomonas aeruginosa strains in a unique patient.

American journal of infection control 35 (1)
PMID : 17276795  :   DOI  :   10.1016/j.ajic.2006.08.014    
Abstract >>
N/A
KeywordMeSH Terms
Infection Control
147. Zhao  GS, Xia  TH, Fischer  RS, Jensen  RA,     ( 1992 )

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The Journal of biological chemistry 267 (4)
PMID : 1733946  :  
Abstract >>
The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.
KeywordMeSH Terms
148. Stokes  HW, Elbourne  LD, Hall  RM,     ( 2007 )

Tn1403, a multiple-antibiotic resistance transposon made up of three distinct transposons.

Antimicrobial agents and chemotherapy 51 (5)
PMID : 17261631  :   DOI  :   10.1128/AAC.01279-06     PMC  :   PMC1855573    
Abstract >>
Transposon Tn1403 from a clinical Pseudomonas strain is composed of three transposons, including Tn5393c. A related transposon Tn1404* from a plant-associated Pseudomonas strain lacks Tn5393 but includes a transposon carrying the tet(C) tetracycline resistance determinant. These compound transposons illustrate the role of preexisting transposons in generating clusters of antibiotic resistance genes.
KeywordMeSH Terms
DNA Transposable Elements
149. Ogino  H, Katou  Y, Akagi  R, Mimitsuka  T, Hiroshima  S, Gemba  Y, Doukyu  N, Yasuda  M, Ishimi  K, Ishikawa  H,     ( 2007 )

Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03.

Extremophiles : life under extreme conditions 11 (6)
PMID : 17657406  :   DOI  :   10.1007/s00792-007-0101-2    
Abstract >>
Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively activated by lipase-specific foldase.
KeywordMeSH Terms
Cloning, Molecular
DNA, Bacterial
150. Kaluzny  K, Abeyrathne  PD, Lam  JS,     ( 2007 )

Coexistence of two distinct versions of O-antigen polymerase, Wzy-alpha and Wzy-beta, in Pseudomonas aeruginosa serogroup O2 and their contributions to cell surface diversity.

Journal of bacteriology 189 (1��11��)
PMID : 17384183  :   DOI  :   10.1128/JB.00237-07     PMC  :   PMC1913395    
Abstract >>
Assembly of B-band lipopolysaccharide (LPS) in Pseudomonas aeruginosa follows a Wzy-dependent pathway, requiring the O-antigen polymerase Wzy and other proteins. The peptide sequences of the wzy(alpha) product from strains of serotypes O2, O5, and O16 are identical, but the O units in O5 are alpha-glycosidically linked, while those in O2 and O16 are beta-linked. We hypothesized that a derivative of the D3 bacteriophage wzy(beta) is present in the chromosomes of O2 and O16 and that this gene is responsible for the beta-linkage. By a combination of PCR and primer walking, wzy(beta) genes of both serotypes have been amplified and cloned. They are identical but share only 87.42% sequence identity with their xenolog in D3. A chromosomal knockout mutant of O16 wzy(beta) was made, and it produces semirough LPS devoid of B-band O antigen. The cloned wzy(beta) is capable of complementing the O16 wzy(beta) mutant, as well as cross-complementing a wzy(alpha) knockout mutant. However, in the latter case, the restored O antigen was beta-linked. Using reverse transcription-PCR, we showed that wzy(alpha) was transcribed in O2 and O16 strains and was functional, since both of these genes could complement the wzy(alpha) mutant of O5. With the coexistence of wzy(alpha) and wzy(beta) in O2 and O16 and the B-band O polysaccharides in these being beta-linked, we hypothesized that iap, an inhibitor of the alpha-polymerase gene, must be present in these serotypes. Indeed, through PCR, TOPO-cloning, and nucleotide-sequencing results, we verified the presence of iap in both O2 and O16 serotypes.
KeywordMeSH Terms
151. Haines  AS, Jones  K, Batt  SM, Kosheleva  IA, Thomas  CM,     ( 2007 )

Sequence of plasmid pBS228 and reconstruction of the IncP-1alpha phylogeny.

Plasmid 58 (1)
PMID : 17320955  :   DOI  :   10.1016/j.plasmid.2007.01.001    
Abstract >>
The antibiotic resistance plasmid pBS228 has been completely sequenced, and revealed to be descended from a plasmid virtually identical to the Birmingham IncP-1alpha plasmid RK2/RP4/RP1. However, it has three additional transposon insertions, one of which is responsible for the extra antibiotic resistances conferred. Loss of kanamycin resistance, which is characteristic of most IncP-1alpha plasmids, is the result of this insertion. A second transposon causes inactivation of the mating pair formation apparatus, rendering the plasmid non-self-transmissible. Comparison with the published data for other IncP-1alpha plasmids gives insight into the recent evolutionary history of this group as well as the acquisition and transmission of one of the first ampicillin resistance transposons discovered.
KeywordMeSH Terms
Evolution, Molecular
Phylogeny
Sequence Analysis, DNA
152. da Fonseca  EL, Vieira  VV, Cipriano  R, Vicente  AC,     ( 2007 )

Emergence of blaGES-5 in clinical colistin-only-sensitive (COS) Pseudomonas aeruginosa strain in Brazil.

The Journal of antimicrobial chemotherapy 59 (3)
PMID : 17284538  :   DOI  :   10.1093/jac/dkl517    
Abstract >>
N/A
KeywordMeSH Terms
153. Gu  B, Tong  M, Zhao  W, Liu  G, Ning  M, Pan  S, Zhao  W,     ( 2007 )

Prevalence and characterization of class I integrons among Pseudomonas aeruginosa and Acinetobacter baumannii isolates from patients in Nanjing, China.

Journal of clinical microbiology 45 (1)
PMID : 17122024  :   DOI  :   10.1128/JCM.01318-06     PMC  :   PMC1828976    
Abstract >>
Class I integrons were detected in 40.8% (40/98) of Pseudomonas aeruginosa strains and 52.8% (56/106) of Acinetobacter baumannii strains in the Nanjing area of China, including several cassette arrays not previously reported.
KeywordMeSH Terms
154. Doi  Y, de Oliveira Garcia  D, Adams  J, Paterson  DL,     ( 2007 )

Coproduction of novel 16S rRNA methylase RmtD and metallo-beta-lactamase SPM-1 in a panresistant Pseudomonas aeruginosa isolate from Brazil.

Antimicrobial agents and chemotherapy 51 (3)
PMID : 17158944  :   DOI  :   10.1128/AAC.01345-06     PMC  :   PMC1803107    
Abstract >>
Serious infections with Pseudomonas aeruginosa are frequently treated with the combination of a beta-lactam antimicrobial and an aminoglycoside. P. aeruginosa strain PA0905 was isolated in 2005 from an inpatient in Brazil. It showed a panresistant phenotype that included resistance to beta-lactams, aminoglycosides, and fluoroquinolones. The beta-lactam resistance was conferred by the production of the metallo-beta-lactamase SPM-1. No inhibitory zone was observed when a disk diffusion test was performed with the semisynthetic aminoglycoside arbekacin, raising suspicion of 16S rRNA methylase production. A cloning experiment subsequently revealed the presence of a novel 16S rRNA methylase, RmtD, which accounted for the high-level resistance to all 4,6-disubstituted deoxystreptamine aminoglycosides, such as amikacin, tobramycin, and gentamicin. RmtD shared a moderate degree of identity with RmtA, another 16S rRNA methylase that was initially reported to occur in P. aeruginosa in Japan in 2003. This is the first identification of aminoglycoside resistance mediated by a 16S rRNA methylase in South America. This is also the first report to document coproduction of a metallo-beta-lactamase and a 16S rRNA methylase, a combination that would severely compromise therapeutic options for the infected patients.
KeywordMeSH Terms
155. Monzingo  AF, Ozburn  A, Xia  S, Meyer  RJ, Robertus  JD,     ( 2007 )

The structure of the minimal relaxase domain of MobA at 2.1 A resolution.

Journal of molecular biology 366 (1��1��)
PMID : 17157875  :   DOI  :   10.1016/j.jmb.2006.11.031     PMC  :   PMC1894915    
Abstract >>
The plasmid R1162 encodes proteins that enable its conjugative mobilization between bacterial cells. It can transfer between many different species and is one of the most promiscuous of the mobilizable plasmids. The plasmid-encoded protein MobA, which has both nicking and priming activities on single-stranded DNA, is essential for mobilization. The nicking, or relaxase, activity has been localized to the 186 residue N-terminal domain, called minMobA. We present here the 2.1 A X-ray structure of minMobA. The fold is similar to that seen for two other relaxases, TraI and TrwC. The similarity in fold, and action, suggests these enzymes are evolutionary homologs, despite the lack of any significant amino acid similarity. MinMobA has a well- defined target DNA called oriT. The active site metal is observed near Tyr25, which is known to form a phosphotyrosine adduct with the substrate. A model of the oriT substrate complexed with minMobA has been made, based on observed substrate binding to TrwC and TraI. The model is consistent with observations of substrate base specificity, and provides a rationalization for elements of the likely enzyme mechanism.
KeywordMeSH Terms
156. Yang  X, Lee  WH, Sobott  F, Papagrigoriou  E, Robinson  CV, Grossmann  JG, Sundström  M, Doyle  DA, Elkins  JM,     ( 2006 )

Structural basis for protein-protein interactions in the 14-3-3 protein family.

Proceedings of the National Academy of Sciences of the United States of America 103 (46)
PMID : 17085597  :   DOI  :   10.1073/pnas.0605779103     PMC  :   PMC1859916    
Abstract >>
The seven members of the human 14-3-3 protein family regulate a diverse range of cell signaling pathways by formation of protein-protein complexes with signaling proteins that contain phosphorylated Ser/Thr residues within specific sequence motifs. Previously, crystal structures of three 14-3-3 isoforms (zeta, sigma, and tau) have been reported, with structural data for two isoforms deposited in the Protein Data Bank (zeta and sigma). In this study, we provide structural detail for five 14-3-3 isoforms bound to ligands, providing structural coverage for all isoforms of a human protein family. A comparative structural analysis of the seven 14-3-3 proteins revealed specificity determinants for binding of phosphopeptides in a specific orientation, target domain interaction surfaces and flexible adaptation of 14-3-3 proteins through domain movements. Specifically, the structures of the beta isoform in its apo and peptide bound forms showed that its binding site can exhibit structural flexibility to facilitate binding of its protein and peptide partners. In addition, the complex of 14-3-3 beta with the exoenzyme S peptide displayed a secondary structural element in the 14-3-3 peptide binding groove. These results show that the 14-3-3 proteins are adaptable structures in which internal flexibility is likely to facilitate recognition and binding of their interaction partners.
KeywordMeSH Terms
157. Wang  J, Bo  R, Xu  L, Mi  Z, Wang  C,     ( 2006 )

A CARB-like beta-lactamase gene from a multiple-drug-resistant Pseudomonas aeruginosa clinical isolate in China.

Journal of medical microbiology 55 (Pt 11)
PMID : 17030927  :   DOI  :   10.1099/jmm.0.46758-0    
Abstract >>
N/A
KeywordMeSH Terms
158. Lagatolla  C, Edalucci  E, Dolzani  L, Riccio  ML, De Luca  F, Medessi  E, Rossolini  GM, Tonin  EA,     ( 2006 )

Molecular evolution of metallo-beta-lactamase-producing Pseudomonas aeruginosa in a nosocomial setting of high-level endemicity.

Journal of clinical microbiology 44 (7)
PMID : 16825348  :   DOI  :   10.1128/JCM.00258-06     PMC  :   PMC1489503    
Abstract >>
An outbreak of multidrug-resistant Pseudomonas aeruginosa strains producing VIM-type metallo-beta-lactamases (MBLs) has occurred in an Italian hospital since 2000 (C. Lagatolla, E. A. Tonin, C. Monti-Bragadin, L. Dolzani, F. Gombac, C. Bearzi, E. Edalucci, F. Gionechetti, and G. M. Rossolini, Emerg. Infect. Dis. 10:535-538, 2004). In this work, using molecular methods, we characterized 128 carbapenem-resistant isolates (including 98 VIM-positive isolates) collected from that hospital from 2000 to 2002 to investigate the dynamics of the dissemination of MBL producers in the clinical setting. Genotyping by random amplification of polymorphic DNA and pulsed-field gel electrophoresis showed that most VIM-positive isolates belonged to two different clonal lineages, producing either a VIM-1- or a VIM-2-like MBL, whose ancestors were detected for the first time in the hospital in 1999, suggesting that clonal expansion played a predominant role in the dissemination of these isolates. The 86 clonally related isolates carrying a blaVIM-1-like gene on an In70-like integron were clearly related to a VIM-1-positive P. aeruginosa clone circulating in various Italian hospitals since the late 1990s. VIM-negative P. aeruginosa strains related to the VIM-1-positive clone were detected during the same period, suggesting that the latter strain was derived from a clonal lineage already circulating in the hospital. In the VIM-2-like positive clone, the MBL gene was carried by an unusual class 1 integron, named In71, lacking the 3' conserved sequence region typical of sul1-associated integrons. A different class 1 integron with an original structure carrying a blaVIM-2 determinant, named In74, was detected in a sporadic isolate. A retrospective investigation did not reveal the presence of strains related to any of the VIM-producing isolates earlier than 1997.
KeywordMeSH Terms
Evolution, Molecular
159. Yan  JJ, Hsueh  PR, Lu  JJ, Chang  FY, Ko  WC, Wu  JJ,     ( 2006 )

Characterization of acquired beta-lactamases and their genetic support in multidrug-resistant Pseudomonas aeruginosa isolates in Taiwan: the prevalence of unusual integrons.

The Journal of antimicrobial chemotherapy 58 (3)
PMID : 16816399  :   DOI  :   10.1093/jac/dkl266    
Abstract >>
The present study was conducted to investigate acquired beta-lactamases and their genetic support in 26 Pseudomonas aeruginosa isolates that were resistant to nearly all antipseudomonal drugs from six medical centres in Taiwan. Acquired beta-lactamases and their genetic support were determined by PCR-based strategies. Four and 16 of the 26 isolates were found to produce VIM-2 and VIM-3 metallo-beta-lactamases (MBLs), respectively, and 1, 1 and 2 isolates produced OXA-17, OXA-10 and PSE-1, respectively. These bla genes are all in class 1 integrons that are probably chromosomally located. The bla(VIM-3)-containing integron, with a deletion between int1 and the bla(VIM-3) structural gene, has six gene cassettes, bla(VIM-3), a probable fosfomycin resistance determinant, aacA4, aacA4, aadB and aacA4. The bla(VIM-2)-containing integron, without detectable 5'-conserved segment, contains four genes cassettes (aacA7-bla(VIM-2)-dhfr-aacA5) and is ended by tniC. The bla(OXA-10)-containing integron includes a catB3 cassette and a fused gene cassette, which is made up of bla(OXA-17) and a novel streptomycin-spectinomycin gene, designated aadA15. The bla(OXA-17)-containing integron has three gene cassettes (aacA4-catB2-bla(OXA-17)) but the 59-base element of the bla(OXA-17) cassette is interrupted by a putative transposase gene. The bla(PSE-1)-containing integron has three gene cassettes, aacA4, an aadA3-related gene designated aadA3b and bla(PSE-1). PFGE revealed genetic diversity among the multidrug-resistant isolates from different hospitals. This study demonstrated the high prevalence of VIM-type MBLs and the presence of unusual bla-encoding integrons in multidrug-resistant P. aeruginosa isolates in Taiwan. The spread of bla(VIM-2)-related genes by horizontal transfer might have occurred.
KeywordMeSH Terms
Genes, Bacterial
Pseudomonas aeruginosa
160. Libisch  B, Muzslay  M, Gacs  M, Minárovits  J, Knausz  M, Watine  J, Ternák  G, Kenéz  E, Kustos  I, Rókusz  L, Széles  K, Balogh  B, Füzi  M,     ( 2006 )

Molecular epidemiology of VIM-4 metallo-beta-lactamase-producing Pseudomonas sp. isolates in Hungary.

Antimicrobial agents and chemotherapy 50 (12)
PMID : 17000739  :   DOI  :   10.1128/AAC.00300-06     PMC  :   PMC1693993    
Abstract >>
VIM metallo-beta-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonizing 19 patients from seven hospitals in Hungary were characterized between October 2003 and November 2005. Macrorestriction analysis revealed the involvement of hospitals from three different towns in northwest Hungary in an outbreak caused by VIM-4-producing P. aeruginosa.
KeywordMeSH Terms
Molecular Epidemiology
161. Sun  GX, Zhong  JJ,     ( 2006 )

Mechanism of augmentation of organotin decomposition by ferripyochelin: formation of hydroxyl radical and organotin-pyochelin-iron ternary complex.

Applied and environmental microbiology 72 (11)
PMID : 16997992  :   DOI  :   10.1128/AEM.01477-06     PMC  :   PMC1636177    
Abstract >>
Pyochelin (PCH), a kind of siderophore secreted by Pseudomonas aeruginosa, was recently found to have triphenyltin (TPT)-decomposing capacity. In this work, significant augmentation of TPT decomposition by ferripyochelin (FePCH), the chelating compound of PCH with iron, was demonstrated in Tris-HCl buffer (pH 8.0). The generation of hydroxyl radical (HO.) in the presence of FePCH was observed. Inhibition of HO. generation by adding catalase and HO. scavengers (methanol and dimethyl sulfoxide) decreased TPT decomposition, while an increase in HO. formation in the presence of H(2)O(2) enhanced its decomposition. Our findings indicated that HO. generated in the reaction system was responsible for the enhanced TPT decomposition by FePCH versus PCH. The existence of the TPT-pyochelin-iron ternary complex was demonstrated by electron spray ionization-mass spectrometry, tandem mass spectrometry, and (1)H nuclear magnetic resonance. On the basis of the above results, HO. produced in the presence of FePCH was deduced to be in close proximity to TPT and has more opportunity to attack the Sn-C bond, which resulted in the enhanced organotin decomposition. The information obtained may have considerable environmental significance.
KeywordMeSH Terms
162. Song  JK, Ahn  HJ, Kim  HS, Song  BK,     ( 2006 )

Molecular cloning and expression of perhydrolase genes from Pseudomonas aeruginosa and Burkholderia cepacia in Escherichia coli.

Biotechnology letters 28 (12)
PMID : 16786268  :   DOI  :   10.1007/s10529-006-9016-8    
Abstract >>
Two bacterial perhydrolase genes, perPA and perBC, were cloned from Pseudomonas aeruginosa and Burkholderia cepacia, respectively, using PCR amplification with primers designed to be specific for conserved amino acid sequences of the already-known perhydrolases. The amino acid sequence of PerPA was identical to a putative perhydrolase of P. aeruginosa PAO1 genome sequences, whereas PerBC of B. cepacia was a novel bacterial perhydrolase showing similarity of less than 80% with all other existing perhydrolases. Most importantly, the perPA gene was expressed as a soluble intracellular form to an extent of more than 50% of the total protein content in Escherichia coli. Two perhydrolase enzymes were confirmed to exhibit the halogenation activity towards Phenol Red and monochlorodimedone. These results suggested that we successfully obtained the newly identified members of the bacterial perhydrolase family, expanding the pool of available perhydrolases.
KeywordMeSH Terms
163. Whitchurch  CB, Hobbs  M, Livingston  SP, Krishnapillai  V, Mattick  JS,     ( 1991 )

Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria.

Gene 101 (1)
PMID : 1676385  :   DOI  :   10.1016/0378-1119(91)90221-v    
Abstract >>
Type-4 fimbriae (pili) are associated with a phenomenon known as twitching motility, which appears to be involved with bacterial translocation across solid surfaces. Pseudomonas aeruginosa mutants which produce fimbriae, but which have lost the twitching motility function, display altered colony morphology and resistance to fimbrial-specific bacteriophage. We have used phenotypic complementation of such mutants to isolate a region of DNA involved in twitching motility. This region was physically mapped to a SpeI fragment around 20 min on the P. aeruginosa PAO chromosome, remote from the major fimbrial locus (around 75 min) where the structural subunit-encoding gene (fimA/pilA) and ancillary genes required for fimbrial assembly (pilB, C and D) are found. A gene, pilT, within the twitching motility region is predicted to encode a 344-amino acid protein which has strong homology to a variety of other bacterial proteins. These include the P. aeruginosa PilB protein, the ComG ORF-1 protein from the Bacillus subtilis comG operon (necessary for competence), the PulE protein from the Klebsiella oxytoca (formerly K. pneumoniae) pulC-O operon (involved in pullulanase export), and the VirB-11 protein from the virB operon (involved in virulence) which is located on the Agrobacterium tumefaciens Ti plasmid. We have also identified other sets of homologies between P. aeruginosa fimbrial assembly (Pil) proteins and B. subtilis Com and K. oxytoca Pul proteins, which suggest that these are all related members of a specialised protein export pathway which is widespread in the eubacteria.
KeywordMeSH Terms
Fimbriae, Bacterial
Genes, Bacterial
164. Sun  GX, Zhou  WQ, Zhong  JJ,     ( 2006 )

Organotin decomposition by pyochelin, secreted by Pseudomonas aeruginosa even in an iron-sufficient environment.

Applied and environmental microbiology 72 (9)
PMID : 16957273  :   DOI  :   10.1128/AEM.00957-06     PMC  :   PMC1563630    
Abstract >>
A triphenyltin (TPT)-decomposing strain, Pseudomonas aeruginosa CGMCC 1.860, was screened out. It secreted an unknown TPT-decomposing factor into the medium, later shown to be pyochelin, even in the presence of 100 muM iron. To our knowledge, this is the first report of organotin decomposition by pyochelin.
KeywordMeSH Terms
165. Heylen  K, Gevers  D, Vanparys  B, Wittebolle  L, Geets  J, Boon  N, De Vos  P,     ( 2006 )

The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers.

Environmental microbiology 8 (11)
PMID : 17014499  :   DOI  :   10.1111/j.1462-2920.2006.01081.x    
Abstract >>
Gene sequence analysis of nirS and nirK, both encoding nitrite reductases, was performed on cultivated denitrifiers to assess their incidence in different bacterial taxa and their taxonomical value. Almost half of the 227 investigated denitrifying strains did not render an nir amplicon with any of five previously described primers. NirK and nirS were found to be prevalent in Alphaproteobacteria and Betaproteobacteria, respectively, nirK was detected in the Firmicutes and Bacteroidetes and nirS and nirK with equal frequency in the Gammaproteobacteria. These observations deviated from the hitherto reported incidence of nir genes in bacterial taxa. NirS gene phylogeny was congruent with the 16S rRNA gene phylogeny on family or genus level, although some strains did group within clusters of other bacterial classes. Phylogenetic nirK gene sequence analysis was incongruent with the 16S rRNA gene phylogeny. NirK sequences were also found to be significantly more similar to nirK sequences from the same habitat than to nirK sequences retrieved from highly related taxa. This study supports the hypothesis that horizontal gene transfer events of denitrification genes have occurred and underlines that denitrification genes should not be linked with organism diversity of denitrifiers in cultivation-independent studies.
KeywordMeSH Terms
166. Giske  CG, Libisch  B, Colinon  C, Scoulica  E, Pagani  L, Füzi  M, Kronvall  G, Rossolini  GM,     ( 2006 )

Establishing clonal relationships between VIM-1-like metallo-beta-lactamase-producing Pseudomonas aeruginosa strains from four European countries by multilocus sequence typing.

Journal of clinical microbiology 44 (12)
PMID : 17021059  :   DOI  :   10.1128/JCM.00817-06     PMC  :   PMC1698408    
Abstract >>
Ten multidrug-resistant Pseudomonas aeruginosa strains producing VIM-1-like acquired metallo-beta-lactamases (MBLs), isolated from four European countries (Greece, Hungary, Italy, and Sweden), were analyzed for genetic relatedness by several methodologies, including fliC sequence analysis, macrorestriction profiling of genomic DNA by pulsed-field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD), and multilocus sequence typing (MLST). The four approaches yielded consistent results overall but showed different resolution powers in establishing relatedness between isolates (PFGE>RAPD>MLST>fliC typing) and could usefully complement each other to address issues in the molecular epidemiology of P. aeruginosa strains producing acquired MBLs. In particular, the recently developed MLST approach was useful in revealing clonal relatedness between isolates when this was not readily apparent using RAPD and PFGE, and it suggested a common ancestry for some of the VIM-1-like MBL-positive P. aeruginosa strains currently spreading in Europe. The MBL producers belonged in three clonal complexes/burst groups (BGs). Of these, one corresponded to the previously described BG4 and included serotype O12 strains from Hungary and Sweden, while the other two were novel and included serotype O11 or nonserotypable strains from Greece, Sweden, and/or Italy. Comparison of the integrons carrying blaVIM-1-like cassettes of various isolates revealed a remarkable structural heterogeneity, suggesting the possibility that multiple independent events of acquisition of different blaVIM-containing integrons had occurred in members of the same clonal lineage, although a contribution of integrase-mediated cassette shuffling or other recombination mechanisms during the evolution of similar strains could also have played a role in determining this variability.
KeywordMeSH Terms
167. Ohama  M, Hiramatsu  K, Miyajima  Y, Kishi  K, Nasu  M, Kadota  J,     ( 2006 )

Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice.

FEMS immunology and medical microbiology 47 (1)
PMID : 16706793  :   DOI  :   10.1111/j.1574-695X.2006.00069.x    
Abstract >>
We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.
KeywordMeSH Terms
168. Kulasekara  BR, Kulasekara  HD, Wolfgang  MC, Stevens  L, Frank  DW, Lory  S,     ( 2006 )

Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa.

Journal of bacteriology 188 (11)
PMID : 16707695  :   DOI  :   10.1128/JB.02000-05     PMC  :   PMC1482899    
Abstract >>
ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNA(Lys) gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of beta- and gamma-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNA(Lys)-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNA(Lys) gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.
KeywordMeSH Terms
Evolution, Molecular
169. Hanson  ND, Hossain  A, Buck  L, Moland  ES, Thomson  KS,     ( 2006 )

First occurrence of a Pseudomonas aeruginosa isolate in the United States producing an IMP metallo-beta-lactamase, IMP-18.

Antimicrobial agents and chemotherapy 50 (1��6��)
PMID : 16723605  :   DOI  :   10.1128/AAC.01440-05     PMC  :   PMC1479107    
Abstract >>
N/A
KeywordMeSH Terms
170. Verma  A, Schirm  M, Arora  SK, Thibault  P, Logan  SM, Ramphal  R,     ( 2006 )

Glycosylation of b-Type flagellin of Pseudomonas aeruginosa: structural and genetic basis.

Journal of bacteriology 188 (12)
PMID : 16740946  :   DOI  :   10.1128/JB.01642-05     PMC  :   PMC1482937    
Abstract >>
The flagellin of Pseudomonas aeruginosa can be classified into two major types-a-type or b-type-which can be distinguished on the basis of molecular weight and reactivity with type-specific antisera. Flagellin from the a-type strain PAK was shown to be glycosylated with a heterogeneous O-linked glycan attached to Thr189 and Ser260. Here we show that b-type flagellin from strain PAO1 is also posttranslationally modified with an excess mass of up to 700 Da, which cannot be explained through phosphorylation. Two serine residues at positions 191 and 195 were found to be modified. Each site had a deoxyhexose to which is linked a unique modification of 209 Da containing a phosphate moiety. In comparison to strain PAK, which has an extensive flagellar glycosylation island of 14 genes in its genome, the equivalent locus in PAO1 comprises of only four genes. PCR analysis and sequence information suggested that there are few or no polymorphisms among the islands of the b-type strains. Mutations were made in each of the genes, PA1088 to PA1091, and the flagellin from these isogenic mutants was examined by mass spectrometry to determine whether they were involved in posttranslational modification of the type-b flagellin. While mutation of PA1088, PA1089, and PA1090 genes altered the composition of the flagellin glycan, only unmodified flagellin was produced by the PA1091 mutant strain. There were no changes in motility or lipopolysaccharide banding in the mutants, implying a role that is limited to glycosylation.
KeywordMeSH Terms
Protein Processing, Post-Translational
171. Orlik-Eisel  G, Lutz  F, Henschen  A, Eisel  U, Struckmeier  M, Kräuter  J, Niemann  H,     ( 1990 )

The cytotoxin of Pseudomonas aeruginosa: cytotoxicity requires proteolytic activation.

Archives of microbiology 153 (6)
PMID : 1695085  :   DOI  :   10.1007/bf00245265    
Abstract >>
The primary structure of a cytotoxin from Pseudomonas aeruginosa was determined by sequencing of the structural gene. The cytotoxin (31,700 Mr) lacks an N-terminal signal sequence for bacterial secretion but contains a pentapeptide consensus sequence commonly found in prokaryotic proteins which function in a TonB-dependent manner. The cytotoxin gene has a [G + C]-content of 53.8% which is considerably lower than generally observed for genes from Pseudomonas aeruginosa. The cytotoxin gene was exclusively detected in strain 158 but not in three other clinical isolates, as determined by Southern and Northern hybridization. The latter technique revealed that the toxin is translated from monocistronic mRNA. The promoter of the cytotoxin is inactive in Escherichia coli. Upon site-directed modification of the 5'-noncoding region by the polymerase chain reaction the gene was expressed under control of the trc-promoter. The gene product obtained in Escherichia coli was nontoxic. Toxicity was induced by subsequent treatment with trypsin. [35S]methionine-labeled cytotoxin with high specific radioactivity was obtained by in vitro transcription/translation. Like [125I] labeled material from Pseudomonas aeruginosa this polypeptide bound to membrane preparations from Ehrlich ascites cells, as evidenced by sedimentation through a sucrose gradient at neutral pH.
KeywordMeSH Terms
172. Stokes  HW, Hall  RM,     ( 1991 )

Sequence analysis of the inducible chloramphenicol resistance determinant in the Tn1696 integron suggests regulation by translational attenuation.

Plasmid 26 (1)
PMID : 1658833  :  
Abstract >>
The sequence of the Tn1696 determinant for inducible nonenzymatic chloramphenicol resistance has been determined. The cml region, the fourth insert of the Tn1696 integron, is 1547 bases and includes a 59-base element at the 3' end, as is typical of integron inserts. One gene, designated cmlA and predicting a polypeptide of 44.2 kDa, is encoded in the insert. However, the cmlA region shows one feature not previously found in an integron insert. A promoter is located within the cmlA insert, and translational attenuation signals related to those of the inducible cat and ermC genes found in gram-positive organisms are also present. The regulatory region includes a leader peptide of nine amino acids, a ribosome stall sequence related to those preceding cat genes, and two alternative pairs of stem-loop structures which either sequester or disclose the ribosome binding site and start codon preceding the cmlA gene.
KeywordMeSH Terms
DNA Transposable Elements
Gene Expression Regulation, Bacterial
Plasmids
Protein Biosynthesis
173. Wang  C, Cai  P, Chang  D, Mi  Z,     ( 2006 )

A Pseudomonas aeruginosa isolate producing the GES-5 extended-spectrum beta-lactamase.

The Journal of antimicrobial chemotherapy 57 (6)
PMID : 16617065  :   DOI  :   10.1093/jac/dkl116    
Abstract >>
N/A
KeywordMeSH Terms
174. Naas  T, Aubert  D, Lambert  T, Nordmann  P,     ( 2006 )

Complex genetic structures with repeated elements, a sul-type class 1 integron, and the blaVEB extended-spectrum beta-lactamase gene.

Antimicrobial agents and chemotherapy 50 (5)
PMID : 16641445  :   DOI  :   10.1128/AAC.50.5.1745-1752.2006     PMC  :   PMC1472224    
Abstract >>
Two clinical isolates of Pseudomonas aeruginosa, TL-1 and TL-2, were isolated from a patient transferred from Bangladesh and hospitalized for osteomyelitis in Paris, France. P. aeruginosa TL-1 expressed the extended-spectrum beta-lactamase VEB-1a and was susceptible only to imipenem and colistin, while P. aeruginosa TL-2 expressed only the naturally occurring bla(AmpC) gene at a basal level and exhibited a wild-type beta-lactam resistance phenotype. In TL-1, the typical 5'-end conserved sequence (5'-CS) region of class 1 integrons usually present upstream of the bla(VEB-1a) gene was replaced by a truncated 3'-CS and a 135-bp repeated element (Re). Downstream of the bla(VEB-1a) gene, an insertion sequence, ISPa31 disrupted by ISPa30, and an orf513 sequence, belonging to a common region (conserved region 1 [CR1]) immediately upstream of the aphA-6 gene, were present. Further downstream, a second truncated 3'-CS region in direct repeat belonged to In51, an integron containing two gene cassettes (aadA6 and the OrfD cassette). Thus, the overall structure corresponded to a sul-type class 1 integron termed In121. Genetic analyses revealed that both isolates were clonally related and differed by a ca. 100-kb fragment that contained In121. Both isolates contained another integron, In122, that carried three gene cassettes: aadB, dfrA1, and the OrfX cassette. This work identifies for the first time the spread of Re-associated bla(VEB) genes located on a sul-type integron. It also reports for the first time a CR1 element in P. aeruginosa that is associated with an aminoglycoside resistance aphA-6 gene that is expressed from a composite promoter.
KeywordMeSH Terms
Genes, Bacterial
175. Rodrigues  JL, Kachel  CA, Aiello  MR, Quensen  JF, Maltseva  OV, Tsoi  TV, Tiedje  JM,     ( 2006 )

Degradation of aroclor 1242 dechlorination products in sediments by Burkholderia xenovorans LB400(ohb) and Rhodococcus sp. strain RHA1(fcb).

Applied and environmental microbiology 72 (4)
PMID : 16597946  :   DOI  :   10.1128/AEM.72.4.2476-2482.2006     PMC  :   PMC1449002    
Abstract >>
Burkholderia xenovorans strain LB400, which possesses the biphenyl pathway, was engineered to contain the oxygenolytic ortho dehalogenation (ohb) operon, allowing it to grow on 2-chlorobenzoate and to completely mineralize 2-chlorobiphenyl. A two-stage anaerobic/aerobic biotreatment process for Aroclor 1242-contaminated sediment was simulated, and the degradation activities and genetic stabilities of LB400(ohb) and the previously constructed strain RHA1(fcb), capable of growth on 4-chlorobenzoate, were monitored during the aerobic phase. The population dynamics of both strains were also followed by selective plating and real-time PCR, with comparable results; populations of both recombinants increased in the contaminated sediment. Inoculation at different cell densities (10(4) or 10(6) cells g(-1) sediment) did not affect the extent of polychlorinated biphenyl (PCB) biodegradation. After 30 days, PCB removal rates for high and low inoculation densities were 57% and 54%, respectively, during the aerobic phase.
KeywordMeSH Terms
176. Frank  DW, Iglewski  BH,     ( 1991 )

Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa.

Journal of bacteriology 173 (20)
PMID : 1655713  :   DOI  :   10.1128/jb.173.20.6460-6468.1991     PMC  :   PMC208981    
Abstract >>
Exoenzyme S is an ADP-ribosyltransferase enzyme distinct from exotoxin A that is synthesized and secreted by Pseudomonas aeruginosa. Yields of exoenzyme S are variable and depend on strain and growth conditions. Since certain medium additives are required for exoenzyme S production, its regulation may be influenced by environmental stimuli. In this study, we have cloned a region that complements the exoenzyme S-deficient phenotype of strain 388 exs1::Tn1, a chromosomal Tn1 insertional mutation. A large clone (28 kb) was shown to restore both synthesis and secretory functions to the mutant strain. Subcloning and Tn501 mutagenesis experiments localized the region required for exoenzyme S synthesis to a 3.2-kb fragment. Nucleotide sequence analysis demonstrated several open reading frames. Comparison of the N-terminal amino acid sequence of purified exoenzyme S with predicted amino acid sequences of all open reading frames indicated that the structural gene was not encoded within the sequenced region. Homology studies suggested that the region encoded three regulatory genes, exsC, exsB, and exsA. ExsA was homologous to the AraC family of transcriptional activator proteins, with extensive homology being found with one member of this family, VirF of Yersinia enterocolitica. VirF and ExsA both contain carboxy-terminal domains with the helix-turn-helix motif of DNA-binding proteins. The ExsA gene product appeared to be required for induction of exoenzyme S synthesis above a low basal level. Expression of ExsA was demonstrated by cloning the region under the control of the T7 promoter. Gene replacement experiments suggested that the expression of ExsC affects the final yield of exoenzyme S.
KeywordMeSH Terms
ADP Ribose Transferases
Bacterial Toxins
177. Caetano  T, Ferreira  S, Mondego  AP, Correia  A, Mendo  S,     ( 2007 )

In99, an In100-related integron, its occurrence and prevalence in clinical Pseudomonas aeruginosa strains from a central region of Portugal.

Epidemiology and infection 135 (3)
PMID : 16870030  :   DOI  :   10.1017/S095026880600700X     PMC  :   PMC2870590    
Abstract >>
In99, a possible ancestor of In100, is a class 1 integron associated with carbenicillinase (blaPSE) and aminoglycoside resistance genes [aac(6')-Ib and aadA2]. In99 was present in 8 of 81 clinical isolates of Pseudomonas aeruginosa from unrelated patients collected in different years. The strains fell into two clonal groups and exhibited resistance to beta-lactams and aminoglycosides.
KeywordMeSH Terms
Integrons
178. Lamont  IL, Martin  LW, Sims  T, Scott  A, Wallace  M,     ( 2006 )

Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa.

Journal of bacteriology 188 (8)
PMID : 16585778  :   DOI  :   10.1128/JB.188.8.3149-3152.2006     PMC  :   PMC1446982    
Abstract >>
Strains of Pseudomonas aeruginosa secrete one of three pyoverdine siderophores (types I to III). We have characterized a gene, pvdY(II) (for the pvdY gene present in type II P. aeruginosa strains), that is only present in strains that make type II pyoverdine. A mutation in pvdY(II) prevented pyoverdine synthesis. Bioinformatic, genetic, and biochemical approaches indicate that the PvdYII enzyme catalyzes acetylation of hydroxyornithine. Expression of pvdY(II) is repressed by the presence of iron and upregulated by the presence of type II pyoverdine. Characterization of pvdY(II) provides insights into the molecular basis for production of different pyoverdines by different strains of P. aeruginosa.
KeywordMeSH Terms
Genes, Bacterial
179. Kresse  AU, Blöcker  H, Römling  U,     ( 2006 )

ISPa20 advances the individual evolution of Pseudomonas aeruginosa clone C subclone C13 strains isolated from cystic fibrosis patients by insertional mutagenesis and genomic rearrangements.

Archives of microbiology 185 (4)
PMID : 16474952  :   DOI  :   10.1007/s00203-006-0089-5    
Abstract >>
Pseudomonas aeruginosa clone C strains, which chronically colonize the lungs of cystic fibrosis patients reorganize their genome structure. In this study, a novel member of the IS3 subfamily of IS elements, ISPa20, was detected which was specific for clone C subclone C13 strains. ISPa20, which was present in high copy number, mediated events of genomic reorganization. ISPa20 was inserted into P. aeruginosa backbone genes leading to adaptation to the cystic fibrosis lung habitat and into DNA acquired through horizontal gene transfer. Further on, large chromosomal inversions were mediated by ISPa20. In contrast to strains of other subclonal linages high rates of genomic rearrangements of subclone C13 strains were observed in vitro. The acquisition of mobile elements by P. aeruginosa clone C strains in the lungs of cystic fibrosis patients supports the chronic colonization by insertional mutagenesis and chromosome restructuring leading to microevolution within clone C that reflects macroevolution observed on the species level.
KeywordMeSH Terms
Chromosome Inversion
DNA Transposable Elements
Evolution, Molecular
Mutagenesis, Insertional
180. Vliegenthart  JS, Ketelaar-van Gaalen  PA, van de Klundert  JA,     ( 1991 )

Nucleotide sequence of the aacC3 gene, a gentamicin resistance determinant encoding aminoglycoside-(3)-N-acetyltransferase III expressed in Pseudomonas aeruginosa but not in Escherichia coli.

Antimicrobial agents and chemotherapy 35 (5)
PMID : 1649572  :   DOI  :   10.1128/aac.35.5.892     PMC  :   PMC245125    
Abstract >>
A chromosomal gentamicin resistance determinant from Pseudomonas aeruginosa was cloned on a 2.4-kb fragment in the broad-host-range vector pLAFR3. Substrate profiles for eight aminoglycosides at three concentrations showed that resistance was due to aminoglycoside-(3)-N-acetyltransferase III. This enzyme was produced in Pseudomonas strains but not in an Escherichia coli strain bearing the aacC3 gene. Nucleotide sequencing revealed two contiguous open reading frames (ORFs) preceded by a potential promoter and a ribosome-binding site. ORF-1 was 642 bp in length and encoded a protein of unknown function with a molecular mass of 23.9 kDa. ORF-2 was 813 bp in length and encoded a protein of 29.6 kDa. From deletion mutagenesis, in vitro transcription-translation data, and protein analysis of bacterial lysates, it was inferred that this 29.6-kDa protein represents the aminoglycoside-(3)-N-acetyltransferase III enzyme. A polymerase chain reaction with two specific intragenic 20-mer primers was developed to detect the aacC3 gene. A BstEII restriction site in the amplified DNA region was used to demonstrate the specificity of the reaction. Tests of 23 reference strains, which produced 12 different aminoglycoside-modifying enzymes, confirmed the specificities of the primers. The gene proved to be absent from a collection of 50 gentamicin-resistant P. aeruginosa strains selected at random in The Netherlands.
KeywordMeSH Terms
181. Murphy  TA, Catto  LE, Halford  SE, Hadfield  AT, Minor  W, Walsh  TR, Spencer  J,     ( 2006 )

Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-beta-lactamases.

Journal of molecular biology 357 (3)
PMID : 16460758  :   DOI  :   10.1016/j.jmb.2006.01.003    
Abstract >>
Metallo-beta-lactamases (mbetals) confer broad-spectrum resistance to beta-lactam antibiotics upon host bacteria and escape the action of existing beta-lactamase inhibitors. SPM-1 is a recently discovered mbetal that is distinguished from related enzymes by possession of a substantial central insertion and by sequence variation at positions that maintain active site structure. Biochemical data show SPM-1 to contain two Zn2+ sites of differing affinities, a phenomenon that is well documented amongst mbetals but for which a structural explanation has proved elusive. Here, we report the crystal structure of SPM-1 to 1.9 A resolution. The structure reveals SPM-1 to lack a mobile loop implicated in substrate binding by related mbetals and to accommodate the central insertion in an extended helical interdomain region. Deleting this had marginal effect upon binding and hydrolysis of a range of beta-lactams. These data suggest that the interactions of SPM-1 with substrates differ from those employed by other mbetals. SPM-1 as crystallised contains a single Zn2+. Both the active site hydrogen-bonding network and main-chain geometry at Asp120, a key component of the binding site for the second zinc ion, differ significantly from previous mbetal structures. We propose that variable interactions made by the Asp120 carbonyl group modulate affinity for a second Zn2+ equivalent in mbetals of the B1 subfamily. We further predict that SPM-1 possesses the capacity to evolve variants of enhanced catalytic activity by point mutations altering geometry and hydrogen bonding in the vicinity of the second Zn2+ site.
KeywordMeSH Terms
182. Bissonnette  L, Champetier  S, Buisson  JP, Roy  PH,     ( 1991 )

Characterization of the nonenzymatic chloramphenicol resistance (cmlA) gene of the In4 integron of Tn1696: similarity of the product to transmembrane transport proteins.

Journal of bacteriology 173 (14)
PMID : 1648560  :   DOI  :   10.1128/jb.173.14.4493-4502.1991     PMC  :   PMC208113    
Abstract >>
Integrons constitute a novel family of DNA elements which evolved by site-specific integration of discrete units between two conserved segments. On the In4 integron of Tn1696, a precisely inserted gene cassette of 1,549 bp conferring nonenzymatic chloramphenicol resistance (cmlA) is present between the streptomycin-spectinomycin resistance (aadA2) gene cassette and the 3'-conserved segment of the integron. In this study, we present the nucleotide sequence of the cmlA gene cassette of Tn1696, show its similarity to bacterial efflux systems and other transport proteins, and present evidence for alterations that its expression exerts on bacterial membranes. The cmlA gene cassette apparently carries its own promoter(s), a situation that has not heretofore been observed in the integrons of multiresistance plasmids and transposons of gram-negative bacteria. One or more of these promoters were shown to be functionally active in expressing a cat marker gene from promoter-probe vectors. The putative CmlA polypeptide appears to provoke a reduction of the content of the major porins OmpA and OmpC.
KeywordMeSH Terms
DNA Transposable Elements
Genes, Bacterial
183. Goussard  S, Courvalin  P,     ( 1991 )

Sequence of the genes blaT-1B and blaT-2.

Gene 102 (1)
PMID : 1650734  :   DOI  :   10.1016/0378-1119(91)90540-r    
Abstract >>
We have completed the nucleotide sequence of the genes blaT-1B from transposon Tn2, and blaT-2 from Tn1, which encode the penicillinases TEM-1 and TEM-2, respectively.
KeywordMeSH Terms
184. Lachapelle  J, Dufresne  J, Levesque  RC,     ( 1991 )

Characterization of the blaCARB-3 gene encoding the carbenicillinase-3 beta-lactamase of Pseudomonas aeruginosa.

Gene 102 (1)
PMID : 1650733  :   DOI  :   10.1016/0378-1119(91)90530-o    
Abstract >>
We have isolated the blaCARB-3 structural gene encoding the CARB-3 carbenicillinase of Pseudomonas aeruginosa strain Cilote, tested the specificity of blaCARB-3 DNA probes and determined the nucleotide sequence of blaCARB-3. Three restriction fragment probes internal or delimiting the blaCARB-3 structural gene were hybridized with purified plasmid DNA coding for 18 other beta-lactamases (Blas). Under high-stringency conditions, only blaPSE-1, blaPSE-4, and blaCARB-4 sequences cross-hybridized with blaCARB-3. Sequencing of blaCARB-3 identified the structural gene which encodes a polypeptide product of 268 amino acids with a calculated estimated Mr of 29,246 for the mature form of the protein. Homology studies and computer analysis of primary structures confirmed that CARB-3 is a class-A Bla. The CARB-3 carbenicillinase differs from PSE-4 at two positions: Phe (PSE-4) instead of Leu188 (CARB-3), and Glu (PSE-4) instead of Ala266 (CARB-3), which changes the isoelectric value from (PSE-4) 5.4 to 5.75 (CARB-3). The possible effects of these two mutations were examined by comparisons on the 2 A crystal structure of the Staphylococcus aureus penicillinase, and they were shown to be silent substitutions causing no changes in the phenotype. The nucleic acid hybridization studies and sequence data confirmed that carbenicillinase-encoding bla genes are closely related and that blaCARB-3 is a variant of blaPSE-4.
KeywordMeSH Terms
Genes, Bacterial
185. Fiett  J, Baraniak  A, Mrówka  A, Fleischer  M, Drulis-Kawa  Z, Naumiuk  ?, Samet  A, Hryniewicz  W, Gniadkowski  M,     ( 2006 )

Molecular epidemiology of acquired-metallo-beta-lactamase-producing bacteria in Poland.

Antimicrobial agents and chemotherapy 50 (3)
PMID : 16495246  :   DOI  :   10.1128/AAC.50.3.880-886.2006     PMC  :   PMC1426447    
Abstract >>
We have analyzed 40 metallo-beta-lactamase (MBL)-producing isolates of Pseudomonas aeruginosa (n = 38), Pseudomonas putida (n = 1), and Acinetobacter genospecies 3 (n = 1) from 17 hospitals in 12 cities in Poland that were identified in 2000 to 2004. Pulsed field gel electrophoresis typing classified the P. aeruginosa isolates into eight types, with two types differentiated further into subtypes. Each of the types was specific either to a given center or to several hospitals of the same or neighboring geographic area. Almost all of the organisms produced beta-lactamase VIM-2; the only exceptions were several P. aeruginosa isolates from two centers which expressed VIM-4. The bla(VIM) genes resided exclusively within class 1 integrons, and these were located in either chromosomal or plasmid DNA. PCR-restriction fragment length polymorphism study of the variable regions of the integrons, followed by DNA sequencing, revealed the presence of eight different, mostly novel gene cassette arrays, six of which contained bla(VIM-2) and two of which contained bla(VIM-4). The occurrence of the integron variants correlated well with the geographic distribution of the MBL-producing organisms, and this suggested that their emergence in particular parts of the country had been likely due to a number of independent events. The following regional dissemination of MBL producers could be attributed to various phenomena, including their clonal spread, horizontal transmission of resistance determinants, or both. All of the data collected in this study revealed that even at this early stage of detection, the epidemiological situation concerning MBL producers in Poland has already been complex and very dynamic.
KeywordMeSH Terms
186. Iwaya  A, Nakagawa  S, Iwakura  N, Taneike  I, Kurihara  M, Kuwano  T, Gondaira  F, Endo  M, Hatakeyama  K, Yamamoto  T,     ( 2005 )

Rapid and quantitative detection of blood Serratia marcescens by a real-time PCR assay: its clinical application and evaluation in a mouse infection model.

FEMS microbiology letters 248 (2)
PMID : 15964718  :   DOI  :   10.1016/j.femsle.2005.05.041    
Abstract >>
Large-scale nosocomial outbreaks of Serratia marcescens septicaemia in Japan have had a fatality rate of 20-60% within 48 h. As a countermeasure, a real-time PCR assay was constructed for the rapid diagnosis of S. marcescens septicaemia. This assay indeed detected S. marcescens in clinical blood specimens (at ca. 10(2)CFU ml(-1)), at a frequency of 0.5% in suspected cases of septicaemia. In mice, the assay provided estimates of blood S. marcescens levels at various infectious stages: namely, 10(7) to 10(8)CFU ml(-1) at a fatal stage (resulting in 100% death), 10(4)-10(5)CFU ml(-1) at a moderately fatal stage (resulting in 50% or more death), and <10(3)CFU ml(-1) at a mild stage (resulting in 100% survival), consistent with actual CFU measurements. Blood bacterial levels could be an important clinical marker that reflects the severity of septicaemia. The simultaneous detection of S. marcescens and the carbapenem resistance gene was also demonstrated.
KeywordMeSH Terms
187. Hendrickx  B, Junca  H, Vosahlova  J, Lindner  A, Rüegg  I, Bucheli-Witschel  M, Faber  F, Egli  T, Mau  M, Schlömann  M, Brennerova  M, Brenner  V, Pieper  DH, Top  EM, Dejonghe  W, Bastiaens  L, Springael  D,     ( 2006 )

Alternative primer sets for PCR detection of genotypes involved in bacterial aerobic BTEX degradation: distribution of the genes in BTEX degrading isolates and in subsurface soils of a BTEX contaminated industrial site.

Journal of microbiological methods 64 (2)
PMID : 15949858  :   DOI  :   10.1016/j.mimet.2005.04.018    
Abstract >>
Eight new primer sets were designed for PCR detection of (i) mono-oxygenase and dioxygenase gene sequences involved in initial attack of bacterial aerobic BTEX degradation and of (ii) catechol 2,3-dioxygenase gene sequences responsible for meta-cleavage of the aromatic ring. The new primer sets allowed detection of the corresponding genotypes in soil with a detection limit of 10(3)-10(4) or 10(5)-10(6) gene copies g(-1) soil, assuming one copy of the gene per cell. The primer sets were used in PCR to assess the distribution of the catabolic genes in BTEX degrading bacterial strains and DNA extracts isolated from soils sampled from different locations and depths (vadose, capillary fringe and saturated zone) within a BTEX contaminated site. In both soil DNA and the isolates, tmoA-, xylM- and xylE1-like genes were the most frequently recovered BTEX catabolic genes. xylM and xylE1 were only recovered from material from the contaminated samples while tmoA was detected in material from both the contaminated and non-contaminated samples. The isolates, mainly obtained from the contaminated locations, belonged to the Actinobacteria or Proteobacteria (mainly Pseudomonas). The ability to degrade benzene was the most common BTEX degradation phenotype among them and its distribution was largely congruent with the distribution of the tmoA-like genotype. The presence of tmoA and xylM genes in phylogenetically distant strains indicated the occurrence of horizontal transfer of BTEX catabolic genes in the aquifer. Overall, these results show spatial variation in the composition of the BTEX degradation genes and hence in the type of BTEX degradation activity and pathway, at the examined site. They indicate that bacteria carrying specific pathways and primarily carrying tmoA/xylM/xylE1 genotypes, are being selected upon BTEX contamination.
KeywordMeSH Terms
Soil Microbiology
188. Haines  AS, Jones  K, Cheung  M, Thomas  CM,     ( 2005 )

The IncP-6 plasmid Rms149 consists of a small mobilizable backbone with multiple large insertions.

Journal of bacteriology 187 (14)
PMID : 15995187  :   DOI  :   10.1128/JB.187.14.4728-4738.2005     PMC  :   PMC1169491    
Abstract >>
Plasmid Rms149, the archetype of Pseudomonas plasmid incompatibility group IncP-6, was identified in Pseudomonas aeruginosa as an agent conferring resistance to streptomycin, sulfanilamide, gentamicin, and carbenicillin in 1975. It has been classed as a broad-host-range plasmid due to its ability to replicate in both Escherichia coli (where it is designated IncG) and Pseudomonas species, although both species are gamma-proteobacteria. To provide reference information on this Inc group, we have determined the complete sequence of Rms149 and found that, although the genome comprises 57,121 bp, it is essentially a small mobilizable plasmid carrying multiple mobile elements, which make up 79% (>45 kb) of its genome. A replicon has been identified which encodes a single polypeptide with moderate identity to other replication proteins. The region encoding this protein can replicate in Pseudomonas putida and E. coli. This sequence is directly downstream of a putative partitioning region highly similar to that of pRA2. A functional IncQ-type mobilization region is also present. Thus, the backbone appears to be a novel combination of modules already identified in other plasmid systems. Analysis of the segments that fall outside this core of stable inheritance and transfer functions show that this plasmid has been subject to multiple insertion events and that the plasmid appears to carry a considerable load of DNA that no longer should be phenotypically advantageous. The plasmid therefore functions not just as a vehicle for spread of selective traits but also as a store for DNA that is not currently under selection.
KeywordMeSH Terms
DNA Transposable Elements
189. Ait Tayeb  L, Ageron  E, Grimont  F, Grimont  PA,     ( N/A )

Molecular phylogeny of the genus Pseudomonas based on rpoB sequences and application for the identification of isolates.

Research in microbiology 156 (5��6��)
PMID : 15950132  :   DOI  :   10.1016/j.resmic.2005.02.009    
Abstract >>
Phylogenetic relationships within the genus Pseudomonas were examined by comparing partial (about 1000 nucleotides) rpoB gene sequences. A total of 186 strains belonging to 75 species of Pseudomonas sensu stricto and related species were studied. The phylogenetic resolution of the rpoB tree was approximately three times higher than that of the rrs tree. Ribogroups published earlier correlated well with rpoB sequence clusters. The rpoB sequence database generated by this study was used for identification. A total of 89 isolates (79.5%) were identified to a named species, while 16 isolates (14.3%) corresponded to unnamed species, and 7 isolates (6.2%) had uncertain affiliation. rpoB sequencing is now being used for routine identification of Pseudomonas isolates in our laboratory.
KeywordMeSH Terms
Phylogeny
190. Llanes  C, Neuwirth  C, El Garch  F, Hocquet  D, Plésiat  P,     ( 2006 )

Genetic analysis of a multiresistant strain of Pseudomonas aeruginosa producing PER-1 beta-lactamase.

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 12 (3)
PMID : 16451415  :   DOI  :   10.1111/j.1469-0691.2005.01333.x    
Abstract >>
A multiresistant strain of Pseudomonas aeruginosa, PA2345, belonging to serotype O:1, was isolated at the Teaching Hospital of Besan?on, France. Resistance to beta-lactams, including third-generation cephalosporins, depended upon a chromosomally-located composite transposon carrying the bla(PER-1) gene encoding extended-spectrum beta-lactamase PER-1. PA2345 was unrelated genotypically to two previous PER-1-producing isolates of P. aeruginosa. Sequence analysis of the transposon in PA2345 revealed the presence of two insertion sequences (ISPa23 and ISPa24) with very different predicted transposases (TnpA1, TnpA2), which were both bordered by closely related 16-bp inverted repeats. High resistance of PA2345 to aminoglycosides was caused, in part, by a chromosomal class-I integron containing gene cassettes aadB, encoding an ANT(2'') enzyme, and aadA11, encoding a new ANT(3'') enzyme with 281 amino-acids that conferred elevated resistance to streptomycin and spectinomycin. Stable overproduction of efflux system MexXY contributed to resistance to amikacin, while mutations in the quinolone resistance-determining regions of gyrA and parC accounted for the high resistance of PA2345 to fluoroquinolones. The study indicates that multidrug resistance in P. aeruginosa might arise from sequential acquisition of a variety of mechanisms provided by both horizontal gene transfers and mutations in chromosomal genes.
KeywordMeSH Terms
Genome, Bacterial
191. Mima  T, Sekiya  H, Mizushima  T, Kuroda  T, Tsuchiya  T,     ( 2005 )

Gene cloning and properties of the RND-type multidrug efflux pumps MexPQ-OpmE and MexMN-OprM from Pseudomonas aeruginosa.

Microbiology and immunology 49 (11)
PMID : 16301811  :   DOI  :   10.1111/j.1348-0421.2005.tb03696.x    
Abstract >>
We cloned two operons for putative RND-type multidrug efflux pumps from Pseudomonas aeruginosa by a PCR method. We designated the genes in one operon mexPQ(-opmE) and in another operon mexMN. Introduction of the mexPQ-opmE into drug hypersensitive cells resulted in elevated MICs of macrolides, fluoroquinolones and some other drugs. Introduction of the mexMN into the hypersensitive cells possessing oprM, but not into cells not possessing oprM, resulted in elevated MICs of chloramphenicol and thiamphenicol. Thus, we conclude that MexPQ-OpmE and MexMN-OprM are functional multidrug efflux pumps when expressed in P. aeruginosa.
KeywordMeSH Terms
Genes, Bacterial
192. Poirel  L, Cabanne  L, Vahaboglu  H, Nordmann  P,     ( 2005 )

Genetic environment and expression of the extended-spectrum beta-lactamase blaPER-1 gene in gram-negative bacteria.

Antimicrobial agents and chemotherapy 49 (5)
PMID : 15855485  :   DOI  :   10.1128/AAC.49.5.1708-1713.2005     PMC  :   PMC1087670    
Abstract >>
The genetic location of the gene coding for the expanded-spectrum beta-lactamase PER-1 was analyzed in a series of gram-negative isolates. It was identified as part of a composite transposon bracketed by two novel insertion elements, ISPa12 and ISPa13, belonging to the IS4 family that possess transposases that share 63% amino acid identity and that are chromosomally located in Pseudomonas aeruginosa, Providencia stuartii, and Acinetobacter baumannii. On the contrary, the bla(PER-1) gene was identified just downstream of an ISPa12 element but not within a composite transposon when it was located on a plasmid in Salmonella enterica serovar Typhimurium and A. baumannii isolates. In both cases, expression of the bla(PER-1) gene was driven by promoter sequences located in ISPa12.
KeywordMeSH Terms
193. Giuliani  F, Docquier  JD, Riccio  ML, Pagani  L, Rossolini  GM,     ( 2005 )

OXA-46, a new class D beta-lactamase of narrow substrate specificity encoded by a blaVIM-1-containing integron from a Pseudomonas aeruginosa clinical isolate.

Antimicrobial agents and chemotherapy 49 (5)
PMID : 15855521  :   DOI  :   10.1128/AAC.49.5.1973-1980.2005     PMC  :   PMC1087641    
Abstract >>
A novel OXA-type enzyme, named OXA-46, was found to be encoded by a gene cassette inserted into a class 1 integron from a multidrug-resistant Pseudomonas aeruginosa clinical isolate. The variable region of the integron also contained a bla(VIM-1) metallo-beta-lactamase cassette and a duplicated aacA4 aminoglycoside acetyltransferase cassette. OXA-46 belongs to the OXA-2 lineage of class D beta-lactamases. It exhibits 78% sequence identity with OXA-2 and the highest similarity (around 92% identity) with another OXA-type enzyme detected in clinical isolates of Burkholderia cepacia and in unidentified bacteria from a wastewater plant. Expression of bla(OXA-46) in Escherichia coli decreased susceptibility to penicillins and narrow-spectrum cephalosporins but not to extended-spectrum cephalosporins, cefsulodin, aztreonam, or carbapenems. The enzyme was overproduced in E. coli and purified by two anion-exchange chromatography steps (approximate yield, 6 mg/liter). OXA-46 was made of a 28.5-kDa polypeptide and exhibited an alkaline pI (7.8). In its native form OXA-46 appeared to be dimeric, and the oligomerization state was not affected by EDTA. Kinetic analysis of OXA-46 revealed a specificity for narrow-spectrum substrates, including oxacillin, other penicillins (but not temocillin), and narrow-spectrum cephalosporins. The enzyme apparently did not interact with temocillin, oxyimino-cephalosporins, or aztreonam. OXA-46 was inactivated by tazobactam and carbapenems and, although less efficiently, also by clavulanic acid. Enzyme activity was not affected either by EDTA or by divalent cations and exhibited low susceptibility to NaCl. These findings underscore the functional and structural diversity that can be encountered among class D beta-lactamases.
KeywordMeSH Terms
194. Lee  J, Boyapati  G, Song  K, Rhee  S, Kim  C,     ( 2000 )

Cloning and sequence analysis of the estA gene encoding enzyme for producing (R)-beta-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001.

Journal of bioscience and bioengineering 90 (6)
PMID : 16232934  :  
Abstract >>
The estA gene encoding the enzyme that catalyzes the production of (R)-beta-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A + T and C + G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70-100 amino acids upstream of the G-X-S-X-G consensus sequence.
KeywordMeSH Terms
195. Xu  H, Lin  W, Xia  H, Xu  S, Li  Y, Yao  H, Bai  F, Zhang  X, Bai  Y, Saris  P, Qiao  M,     ( 2005 )

Influence of ptsP gene on pyocyanin production in Pseudomonas aeruginosa.

FEMS microbiology letters 253 (1)
PMID : 16239083  :   DOI  :   10.1016/j.femsle.2005.09.027    
Abstract >>
A pyocyanin overproducer with insertional inactivation of ptsP gene was isolated from a mini-Mu insertion library in Pseudomonas aeruginosa PA68. The mutation was complemented by a functional ptsP gene in trans. The pyocyanin-overproducing phenotype was also found in a ptsP mutant constructed by gene replacement in the P. aeruginosa PAO1 strain. Reporter plasmids with P(qscR)-lacZ, P(lasI)-lacZ and P(rhlI)-lacZ were constructed and the beta-galactosidase activity in the ptsP mutant/wild-type background was measured. The results showed that lack of Enzyme I(Ntr) (EI(Ntr), encoded by ptsP) decreased transcription from the P(qscR) promoter and increased the activity of the P(lasI) and P(rhlI) promoters. Normally, QscR represses the quorum-sensing LasR-LasI and RhlR-RhlI systems involved in pyocyanin regulation. Our results showed that the ptsP gene has an important role in the regulation of pyocyanin production and that two quorum-sensing systems and their repressor QscR are involved in this regulation.
KeywordMeSH Terms
Genes, Bacterial
196. Lewis  DA, Jones  A, Parkhill  J, Speert  DP, Govan  JR, Lipuma  JJ, Lory  S, Webb  AK, Mahenthiralingam  E,     ( 2005 )

Identification of DNA markers for a transmissible Pseudomonas aeruginosa cystic fibrosis strain.

American journal of respiratory cell and molecular biology 33 (1)
PMID : 15834046  :   DOI  :   10.1165/rcmb.2004-0352OC    
Abstract >>
A number of transmissible Pseudomonas aeruginosa strains have been identified which potentially constitute an emerging threat to patients with cystic fibrosis (CF). We sought to identify DNA markers that were specific to a transmissible P. aeruginosa CF clone and evaluate these probes on a large collection of genotypically distinct P. aeruginosa strains. Using subtractive DNA hybridization, in combination with analysis using the P. aeruginosa PAO1 genome chip, DNA markers specific for or absent from the Manchester transmissible CF strain (MA) were identified. Five subtractive DNA hybridization markers (MA15, MA18, MA21, MA22, and MA30) were found to be specific to strain MA and were located within a novel 13,318-bp genomic island, designated the MA island. The MA island encoded 18 genes and consisted of two bacteriophage-like regions; one region encoded the MA-specific subtractive hybridization markers, while the other bacteriophage-like region contained a Vibrio cholera-like toxin gene. Probes MA15, MA18, MA21, MA22, and MA30 were all found to be specific to strain MA when a collection of 141 P. aeruginosa strains was examined by hybridization with each DNA marker. In contrast, a previously isolated DNA marker for the Liverpool transmissible CF strain, PS21, was not found to be specific, detecting two additional strain types in the collection screened. Both the Manchester and Liverpool strain types were not encountered in CF populations outside the United Kingdom. The MA genomic island and Vibrio cholera-like toxin gene within it constitute novel genetic factors associated with a transmissible P. aeruginosa strain and their role in pathogenesis remains to be determined.
KeywordMeSH Terms
Oligonucleotide Probes
197. Juan  C, Maciá  MD, Gutiérrez  O, Vidal  C, Pérez  JL, Oliver  A,     ( 2005 )

Molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in Pseudomonas aeruginosa clinical strains.

Antimicrobial agents and chemotherapy 49 (11)
PMID : 16251318  :   DOI  :   10.1128/AAC.49.11.4733-4738.2005     PMC  :   PMC1280133    
Abstract >>
The molecular mechanisms of beta-lactam resistance mediated by AmpC hyperproduction in natural strains of Pseudomonas aeruginosa were investigated in a collection of 10 isogenic, ceftazidime-susceptible and -resistant pairs of isolates, each sequentially recovered from a different intensive care unit patient treated with beta-lactams. All 10 ceftazidime-resistant mutants hyper-produced AmpC (beta-lactamase activities were 12- to 657-fold higher than those of the parent strains), but none of them harbored mutations in ampR or the ampC-ampR intergenic region. On the other hand, six of them harbored inactivating mutations in ampD: four contained frameshift mutations, one had a C-->T mutation, creating a premature stop codon, and finally, one had a large deletion, including the complete ampDE region. Complementation studies revealed that only three of the six ampD mutants could be fully trans-complemented with either ampD- or ampDE-harboring plasmids, whereas one of them could be trans-complemented only with ampDE and two of them (including the mutant with the deletion of the ampDE region and one with an ampD frameshift mutation leading to an ampDE-fused open reading frame) could not be fully trans-complemented with any of the plasmids. Finally, one of the four mutants with no mutations in ampD could be trans-complemented, but only with ampDE. Although the inactivation of AmpD is found to be the most frequent mechanism of AmpC hyperproduction in clinical strains, our findings suggest that for certain types of mutations, AmpE plays an indirect role in resistance and that there are other unknown genes involved in AmpC hyperproduction, with at least one of them apparently located close to the ampDE operon.
KeywordMeSH Terms
beta-Lactam Resistance
198. Mukherjee  S, Chakraborty  R,     ( 2006 )

Incidence of class 1 integrons in multiple antibiotic-resistant Gram-negative copiotrophic bacteria from the River Torsa in India.

Research in microbiology 157 (3)
PMID : 16239097  :   DOI  :   10.1016/j.resmic.2005.08.003    
Abstract >>
The presence of class 1 integrons in multiple-antibiotic-resistant (MAR) Gram-negative copiotrophic bacteria from the River Torsa in India was detected using a polymerase chain reaction (PCR)-based screening method. Among 100 isolates that were resistant to at least five of the twelve antibiotics tested, 40 carried class 1 integrons, with inserted DNA regions of 0.7-3.2 kb. Carriage of integrons in strains of higher MAR index was found to be statistically significant. DNA sequencing was used to identify the genetic content of the integron-variable regions. In addition to the identification of gene cassettes dfrA1, dfrA5, dfrA7, dfrA17 and a variant of dfrA12 for trimethoprim, aac(6')-Ib for amikacin and tobramycin and aadA1 and aadA6 for streptomycin and spectinomycin resistance, a novel ORF predicted from a sequence of Morganella sp. TR 90 bearing homology with the Vibrio cholerae dfrA1 gene cassette was characterized. To our knowledge, this is the first report of the incidence and abundance of class 1 integrons in copiotrophic river water bacteria from India.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Integrons
199. Noda  K, Watanabe  K, Maruhashi  K,     ( 2003 )

Isolation of the Pseudomonas aeruginosa gene affecting uptake of dibenzothiophene in n-tetradecane.

Journal of bioscience and bioengineering 95 (5)
PMID : 16233447  :  
Abstract >>
The dsz desulfurization gene cluster from Rhodococcus erythropolis KA2-5-1 was transferred into the chromosomes of Pseudomonas aeruginosa strain NCIMB9571 using a transposon vector. All of the recombinant strains completely desulfurized 1 mM dibenzothiophene (DBT) in n-tetradecane (n-TD) except one, named strain PARMI. Strain PARMI was unable to desulfurize DBT in n-TD, but was able to desulfurize it in water. The n-alkane utilization ability, the biosurfactant production and the fatty acid composition of cells in strain PARMI were the same level as those of the other recombinants. The transposon insertion area of strain PARMI was analyzed by transposon tagging. We cloned three possible open reading frames (ORFs), designated hcuA, hcuB and hcuC, from the genomic DNA of P. aeruginosa NCIMB9571 using the transposon insertion area of strain PARMI as a DNA probe. Examination of the sequence revealed the transposon was inserted into hcuA. The full length of the hcuABC genes transformed into strain PARMI achieved 87% recovery of the desulfurization activity of DBT in n-TD, but partial hcuABC genes achieved only 0-12%. These results indicate that DBT desulfurization in the oil phase by recombinant P. aeruginosa strain NCIMB9571 requires the full length of the hcuABC gene cluster. The hcuABC gene cluster relates to DBT uptake from the oil phase to inside of the cell, and the uptake ability is independent of the n-alkane utilization ability, the biosurfactant production and the fatty acid composition of cells.
KeywordMeSH Terms
200. Vidal-Mas  J, Busquets  M, Manresa  A,     ( 2005 )

Cloning and expression of a lipoxygenase from Pseudomonas aeruginosa 42A2.

Antonie van Leeuwenhoek 87 (3)
PMID : 15803390  :   DOI  :   10.1007/s10482-004-4021-1    
Abstract >>
In order to produce (S) 10-monohydroxy-8E-octadecenoic acid (MHOD) from oleic acid, a full-length probable lipoxygenase cDNA from Pseudomonas aeruginosa 42A2 was cloned and expressed in Escherichia coli BL21(DE3). The recombinant protein was purified by affinity chromatography to electrophoretic homogeneity and specifically stained. Its molecular mass was 70 kDa. The activity of the rec-LOX with oleic acid was about 30% of that of the preferred substrate, linoleic acid (100%). Bacterial LOX forms a new subfamily in the lipoxygenase phylogenetic tree.
KeywordMeSH Terms
201. Saeed  HM, Abdel-Fattah  YR, Berekaa  MM, Gohar  YM, Elbaz  MA,     ( 2004 )

Identification, cloning and expression of Pseudomonas aeruginosa Ps-x putative urate oxidase gene in Escherichia coli.

Polish journal of microbiology 53 (4)
PMID : 15790071  :  
Abstract >>
In a previous study we reported for the first time the isolation and characterization ofurate oxidase enzyme from Pseudomonas aeruginosa. In this work we isolated and cloned a 1.350 kilobase DNA fragment that encode a putative urate oxidase gene from the genomic library of P. aeruginosa Ps-x. The nucleotide sequence of the cloned DNA insert revealed an open reading frame that encodes a protein of a molecular weight of 54.0 kDa. The cloned DNA fragment showed an uricolytic activity when expressed in E. coli DH5alpha. Surprisingly, the nucleotide sequence of the cloned gene showed more than 99% identity to the gene encoding hypothetical protein of P. aeruginosa PAO1. Moreover, the sequence of the cloned gene was closely similar to the corresponding uricase gene of Cellulomonas flavigena (44% similarity), but showed lower similarity values to that of Bacillus sp. BT-90 (24% similarity), Candida utilis (24% similarity). Interestingly, the isolated uricase gene showed closer similarity to uricase from yeast-like symbiotic fungi Beauveria bassiana (35%), Tolypocladium inflatum (29%), Paecilomyces tenuipes (27%) and Cerataphis fransseni (24%).
KeywordMeSH Terms
Cloning, Molecular
Urate Oxidase
202. Bröms  JE, Edqvist  PJ, Carlsson  KE, Forsberg  A, Francis  MS,     ( 2005 )

Mapping of a YscY binding domain within the LcrH chaperone that is required for regulation of Yersinia type III secretion.

Journal of bacteriology 187 (22)
PMID : 16267298  :   DOI  :   10.1128/JB.187.22.7738-7752.2005     PMC  :   PMC1280294    
Abstract >>
Type III secretion systems are used by many animal and plant interacting bacteria to colonize their host. These systems are often composed of at least 40 genes, making their temporal and spatial regulation very complex. Some type III chaperones of the translocator class are important regulatory molecules, such as the LcrH chaperone of Yersinia pseudotuberculosis. In contrast, the highly homologous PcrH chaperone has no regulatory effect in native Pseudomonas aeruginosa or when produced in Yersinia. In this study, we used LcrH-PcrH chaperone hybrids to identify a discrete region in the N terminus of LcrH that is necessary for YscY binding and regulatory control of the Yersinia type III secretion machinery. PcrH was unable to bind YscY and the homologue Pcr4 of P. aeruginosa. YscY and Pcr4 were both essential for type III secretion and reciprocally bound to both substrates YscX of Yersinia and Pcr3 of P. aeruginosa. Still, Pcr4 was unable to complement a DeltayscY null mutant defective for type III secretion and yop-regulatory control in Yersinia, despite the ability of YscY to function in P. aeruginosa. Taken together, we conclude that the cross-talk between the LcrH and YscY components represents a strategic regulatory pathway specific to Yersinia type III secretion.
KeywordMeSH Terms
Protein Interaction Mapping
Protein Structure, Tertiary
203. Naas  T, Nordmann  P, Vedel  G, Poyart  C,     ( 2005 )

Plasmid-mediated carbapenem-hydrolyzing beta-lactamase KPC in a Klebsiella pneumoniae isolate from France.

Antimicrobial agents and chemotherapy 49 (10)
PMID : 16189140  :   DOI  :   10.1128/AAC.49.10.4423-4424.2005     PMC  :   PMC1251528    
Abstract >>
N/A
KeywordMeSH Terms
204. Marquart  ME, Caballero  AR, Chomnawang  M, Thibodeaux  BA, Twining  SS, O'Callaghan  RJ,     ( 2005 )

Identification of a novel secreted protease from Pseudomonas aeruginosa that causes corneal erosions.

Investigative ophthalmology & visual science 46 (10)
PMID : 16186360  :   DOI  :   10.1167/iovs.04-1483    
Abstract >>
The purpose of this study was to identify a new Pseudomonas protease and determine its possible role in keratitis. Concentrated culture supernatants of the Pseudomonas aeruginosa strains PA103 and ATCC 19660 were analyzed by zymography. P. aeruginosa small protease (PASP) was purified from strain PA103, and modified elastase B (LasB) was purified from strain ATCC 19660. SDS-PAGE and Western blot analysis were performed on purified PASP and modified LasB. PASP was further analyzed by mass spectrometry and amino-terminal sequencing. The Pasp gene was cloned and expressed, affinity-purified in denatured form from inclusion bodies, and refolded by removal of the denaturant. Purified recombinant PASP was analyzed by zymography for protease activity. PASP and heat-inactivated PASP were injected into rabbit corneas, and the corneas were monitored for erosions caused by protease activity. Each strain produced a protease with a molecular mass of 80 kDa on zymograms. LasB antiserum identified the ATCC 19660 protease as modified LasB. Mass spectrometry defined the PA103 protease as having a molecular mass of 18.5 kDa. Amino-terminal sequencing and analysis of the P. aeruginosa genome sequence determined that the PA103 Pasp gene sequence was >99% identical with the PA0423 sequence of strain PAO1. Recombinant PASP was proteolytic, with a zymogram mass of 50 kDa. PASP purified from PA103 produced extensive corneal epithelial erosions, whereas heat-inactivated PASP produced no erosions. PASP is a protease that has not been previously identified. It causes corneal epithelial erosions, indicating its likely activity as a virulence-promoting factor in Pseudomonas keratitis.
KeywordMeSH Terms
205. Viana Vieira  V, Lourenço da Fonseca  E, Paulo Vincente  AC,     ( 2005 )

Metallo-beta-lactamases produced by carbapenem-resistant Pseudomonas aeruginosa in Brazil.

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 11 (11)
PMID : 16216114  :   DOI  :   10.1111/j.1469-0691.2005.01249.x    
Abstract >>
N/A
KeywordMeSH Terms
beta-Lactam Resistance
206. Sekiguchi  J, Asagi  T, Miyoshi-Akiyama  T, Fujino  T, Kobayashi  I, Morita  K, Kikuchi  Y, Kuratsuji  T, Kirikae  T,     ( 2005 )

Multidrug-resistant Pseudomonas aeruginosa strain that caused an outbreak in a neurosurgery ward and its aac(6')-Iae gene cassette encoding a novel aminoglycoside acetyltransferase.

Antimicrobial agents and chemotherapy 49 (9)
PMID : 16127047  :   DOI  :   10.1128/AAC.49.9.3734-3742.2005     PMC  :   PMC1195402    
Abstract >>
We characterized multidrug-resistant Pseudomonas aeruginosa strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a P. aeruginosa strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, beta-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of P. aeruginosa, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of bla(IMP-1), a novel aminoglycoside resistance gene, and the aadA1 gene. The aminoglycoside resistance gene, designated aac(6')-Iae, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6')-Iq. Recombinant AAC(6')-Iae protein showed aminoglycoside 6'-N-acetyltransferase activity by thin-layer chromatography. Escherichia coli expressing exogenous aac(6')-Iae showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of gyrA and parC at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that P. aeruginosa IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the aac(6')-I family and mutations in drug resistance genes.
KeywordMeSH Terms
207. Poirel  L, Brinas  L, Verlinde  A, Ide  L, Nordmann  P,     ( 2005 )

BEL-1, a novel clavulanic acid-inhibited extended-spectrum beta-lactamase, and the class 1 integron In120 in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 49 (9)
PMID : 16127048  :   DOI  :   10.1128/AAC.49.9.3743-3748.2005     PMC  :   PMC1195426    
Abstract >>
Screening by a double-disk synergy test identified a Pseudomonas aeruginosa isolate that produced a clavulanic acid-inhibited expanded-spectrum beta-lactamase (ESBL). Cloning and sequencing identified a novel ESBL, BEL-1, weakly related to other Ambler class A ESBLs. beta-Lactamase BEL-1 hydrolyzed significantly most expanded-spectrum cephalosporins and aztreonam, and its activity was inhibited by clavulanic acid, tazobactam, cefoxitin, moxalactam, and imipenem. This chromosome-encoded ESBL gene was embedded in a class 1 integron containing three other gene cassettes. In addition, this integron was bracketed by Tn1404 transposon sequences at its right end and by P. aeruginosa-specific sequences at its left end.
KeywordMeSH Terms
208. Chen  Y-, Liu  H, Zhu  L-, Jin  Y-,     ( N/A )

[Cloning and characterization of a chromosome-encoded catechol 2,3-dioxygenase gene from Pseudomonas aeruginosa ZD 4-3].

Mikrobiologiia 73 (6)
PMID : 15688939  :  
Abstract >>
Catechol 2,3-dioxygenase (C23O), one of extradiol-type dioxygenases cleaving the aromatic C-C bond at the meta-position of dihydroxylated aromatic substrates, catalyzes the conversion of catechol to 2-hydroxymuconic semialdehyde. Based on curing experiment, PCR identification, and Southern hybridization, the gene responsible for C23O was localized on a 3.5-kb EcoRI/BamHI fragment and cloned from P. aeruginosa ZD 4-3 able to degrade both single and bicyclic compounds via the meta-cleavage pathway. A complete nucleotide sequence analysis of the C23O revealed that it had one ORF, which showed a strong amino acid sequence similarity to the known C23Os of mesophilic gram-negative bacteria. The alignment analysis indicated that distinct difference existed between the C23O in this study and the 2,3-dihydroxybiphenyl dioxygenases cleaving bicyclic aromatic compounds. The heterogenous expression of the pheB gene in Escherichia coli BL21(DE3) demonstrated that this C23O possessed a meta-cleavage activity.
KeywordMeSH Terms
209. Kong  KF, Jayawardena  SR, Del Puerto  A, Wiehlmann  L, Laabs  U, Tümmler  B, Mathee  K,     ( 2005 )

Characterization of poxB, a chromosomal-encoded Pseudomonas aeruginosa oxacillinase.

Gene 358 (N/A)
PMID : 16120476  :   DOI  :   10.1016/j.gene.2005.05.027    
Abstract >>
Pseudomonas aeruginosa is the major pathogen associated with morbidity and mortality of patients with cystic fibrosis. One of the reasons for the failure of beta-lactam antibiotic regimens appears to be mediated by de-regulation of the ampC gene, encoding the chromosomal Ambler's Class C beta-lactamase. Currently, the AmpC is the only known chromosomal beta-lactamase whose expression is regulated by a transcriptional regulator, AmpR. We generated an ampC mutation in the prototypic P. aeruginosa strain PAO1. The mutation in ampC did not abolish the beta-lactamase activity entirely suggesting the expression of yet another unreported beta-lactamase. Our genomic analysis revealed the presence of an open reading frame encoding a protein with high homology to the Class D beta-lactamases, commonly known as oxacillinases. The gene was named poxB for Pseudomonas oxacillinase. Cloning and expression of poxB in Escherichia coli conferred beta-lactam resistance to the host. We detected the presence of poxB both in clinical and environmental isolates. Our studies show that P. aeruginosa possesses two beta-lactamases, AmpC and PoxB, which contribute to its resistance against a wide spectrum of beta-lactam antibiotics.
KeywordMeSH Terms
210. Whitchurch  CB, Beatson  SA, Comolli  JC, Jakobsen  T, Sargent  JL, Bertrand  JJ, West  J, Klausen  M, Waite  LL, Kang  PJ, Tolker-Nielsen  T, Mattick  JS, Engel  JN,     ( 2005 )

Pseudomonas aeruginosa fimL regulates multiple virulence functions by intersecting with Vfr-modulated pathways.

Molecular microbiology 55 (5)
PMID : 15720546  :   DOI  :   10.1111/j.1365-2958.2005.04479.x     PMC  :   PMC1266277    
Abstract >>
Virulence of Pseudomonas aeruginosa involves the co-ordinate expression of a range of factors including type IV pili (tfp), the type III secretion system (TTSS) and quorum sensing. Tfp are required for twitching motility, efficient biofilm formation, and for adhesion and type III secretion (TTS)-mediated damage to mammalian cells. We describe a novel gene (fimL) that is required for tfp biogenesis and function, for TTS and for normal biofilm development in P. aeruginosa. The predicted product of fimL is homologous to the N-terminal domain of ChpA, except that its putative histidine and threonine phosphotransfer sites have been replaced with glutamine. fimL mutants resemble vfr mutants in many aspects including increased autolysis, reduced levels of surface-assembled tfp and diminished production of type III secreted effectors. Expression of vfr in trans can complement fimL mutants. vfr transcription and production is reduced in fimL mutants whereas cAMP levels are unaffected. Deletion and insertion mutants of fimL frequently revert to wild-type phenotypes suggesting that an extragenic suppressor mutation is able to overcome the loss of fimL. vfr transcription and production, as well as cAMP levels, are elevated in these revertants, while Pseudomonas quinolone signal (PQS) production is reduced. These results suggest that the site(s) of spontaneous mutation is in a gene(s) which lies upstream of vfr transcription, cAMP, production, and PQS synthesis. Our studies indicate that Vfr and FimL are components of intersecting pathways that control twitching motility, TTSS and autolysis in P. aeruginosa.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
211. Smith  EE, Sims  EH, Spencer  DH, Kaul  R, Olson  MV,     ( 2005 )

Evidence for diversifying selection at the pyoverdine locus of Pseudomonas aeruginosa.

Journal of bacteriology 187 (6)
PMID : 15743962  :   DOI  :   10.1128/JB.187.6.2138-2147.2005     PMC  :   PMC1064051    
Abstract >>
Pyoverdine is the primary siderophore of the gram-negative bacterium Pseudomonas aeruginosa. The pyoverdine region was recently identified as the most divergent locus alignable between strains in the P. aeruginosa genome. Here we report the nucleotide sequence and analysis of more than 50 kb in the pyoverdine region from nine strains of P. aeruginosa. There are three divergent sequence types in the pyoverdine region, which correspond to the three structural types of pyoverdine. The pyoverdine outer membrane receptor fpvA may be driving diversity at the locus: it is the most divergent alignable gene in the region, is the only gene that showed substantial intratype variation that did not appear to be generated by recombination, and shows evidence of positive selection. The hypothetical membrane protein PA2403 also shows evidence of positive selection; residues on one side of the membrane after protein folding are under positive selection. R', previously identified as a type IV strain, is clearly derived from a type III strain via a 3.4-kb deletion which removes one amino acid from the pyoverdine side chain peptide. This deletion represents a natural modification of the product of a nonribosomal peptide synthetase enzyme, whose consequences are predictive from the DNA sequence. There is also linkage disequilibrium between the pyoverdine region and pvdY, a pyoverdine gene separated by 30 kb from the pyoverdine region. The pyoverdine region shows evidence of horizontal transfer; we propose that some alleles in the region were introduced from other soil bacteria and have been subsequently maintained by diversifying selection.
KeywordMeSH Terms
Genetic Variation
212. Pagani  L, Colinon  C, Migliavacca  R, Labonia  M, Docquier  JD, Nucleo  E, Spalla  M, Li Bergoli  M, Rossolini  GM,     ( 2005 )

Nosocomial outbreak caused by multidrug-resistant Pseudomonas aeruginosa producing IMP-13 metallo-beta-lactamase.

Journal of clinical microbiology 43 (8)
PMID : 16081918  :   DOI  :   10.1128/JCM.43.8.3824-3828.2005     PMC  :   PMC1233900    
Abstract >>
An outbreak of Pseudomonas aeruginosa showing a multidrug-resistant (MDR) phenotype (including carbapenems, ceftazidime, cefepime, gentamicin, tobramycin, and fluoroquinolones) was observed, during a 5-month period, in a general intensive care unit of a large tertiary care and clinical research hospital in southern Italy. The outbreak involved 15 patients, with a total of 87 isolates, mostly from lower respiratory tract specimens. Analysis of isolates involved in the outbreak revealed production of metallo-beta-lactamase (MBL) activity, and genotyping by pulsed-field gel electrophoresis of genomic DNA digested by SpeI revealed clonal relatedness among isolates. Molecular analysis of the MBL determinant showed the presence of a bla(IMP-13) gene carried on a gene cassette inserted in a class 1 integron which also contained an aacA4 aminoglycoside resistance cassette encoding an AAC(6')-Ib enzyme. The bla(IMP-13)-containing integron and its genetic environment appeared to be similar to those found in P. aeruginosa isolates producing IMP-13 from a hospital in Rome. The bla(IMP-13) gene was not transferable by conjugation and was apparently carried on the chromosome. The outbreak was coincidental with a shortage of nursing personnel, and resolution was apparently associated with reinstatement of nursing personnel and reinforcement of general infection control practices within the intensive care unit. To our best knowledge this is the first description of a nosocomial outbreak of relatively large size caused by an IMP-producing gram-negative pathogen in Europe.
KeywordMeSH Terms
Disease Outbreaks
213. Poirel  L, Brinas  L, Fortineau  N, Nordmann  P,     ( 2005 )

Integron-encoded GES-type extended-spectrum beta-lactamase with increased activity toward aztreonam in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 49 (8)
PMID : 16048994  :   DOI  :   10.1128/AAC.49.8.3593-3597.2005     PMC  :   PMC1196234    
Abstract >>
A Pseudomonas aeruginosa strain expresses an extended-spectrum beta-lactamase, GES-9, which differs from GES-1 by a Gly243Ser substitution, is inhibited by clavulanic acid and imipenem, and hydrolyzes aztreonam. The bla(GES-9) gene was located inside a class 1 integron structure containing two copies of a novel insertion sequence belonging to the IS1111 family.
KeywordMeSH Terms
beta-Lactam Resistance
Integrons
214. Lolans  K, Queenan  AM, Bush  K, Sahud  A, Quinn  JP,     ( 2005 )

First nosocomial outbreak of Pseudomonas aeruginosa producing an integron-borne metallo-beta-lactamase (VIM-2) in the United States.

Antimicrobial agents and chemotherapy 49 (8)
PMID : 16048978  :   DOI  :   10.1128/AAC.49.8.3538-3540.2005     PMC  :   PMC1196250    
Abstract >>
Carbapenemases are rare in the United States. This is the first report of a United States nosocomial outbreak of pan-resistant Pseudomonas aeruginosa infections due to an integron-borne metallo-beta-lactamase, VIM-2. This emergence of carbapenemases on mobile genetic elements in the United States warrants surveillance.
KeywordMeSH Terms
beta-Lactam Resistance
Disease Outbreaks
Integrons
215. Vernez  I, Hauser  P, Bernasconi  MV, Blanc  DS,     ( 2005 )

Population genetic analysis of Pseudomonas aeruginosa using multilocus sequence typing.

FEMS immunology and medical microbiology 43 (1)
PMID : 15607633  :   DOI  :   10.1016/j.femsim.2004.06.024    
Abstract >>
To study the population genetic structure of Pseudomonas aeruginosa, we developed a multilocus sequence typing scheme. The sequences of internal fragments of seven housekeeping genes were obtained for 34 P. aeruginosa isolates from patients hospitalized in five different European cities. Twenty-six different allelic profiles were identified. The mean allelic diversity was 0.854 (range: 0.606-0.978), which was about six times greater than the results obtained with the multilocus enzyme electrophoresis method. Linkage disequilibrium was measured with the index of association. An index of 1.95+/-0.24 was calculated when all the strains were considered. This index was 1.76+/-0.27 when only one strain per sequence type was considered. Both results were different from 0, indicating linkage among loci, which means that the population structure of our set of P. aeruginosa isolates is clonal. The clonal structure of the population was also suggested by the congruence of the topology of the different trees obtained from the seven housekeeping genes. These results are in contrast to previous studies, finding a non clonal population structure. Since a small number of isolates was analyzed in this study, there might be a bias of selection which includes the possibility that they belong to widely disseminated epidemic clones. Another possibility is that recombination did not occurred homogeneously throughout the genome of P. aeruginosa, so that part of it has a clonal structure, while the remaining part of the genome is more frequently subject to recombination.
KeywordMeSH Terms
Bacterial Typing Techniques
216. Pasteran  F, Faccone  D, Petroni  A, Rapoport  M, Galas  M, Vázquez  M, Procopio  A,     ( 2005 )

Novel variant (bla(VIM-11)) of the metallo-{beta}-lactamase bla(VIM) family in a GES-1 extended-spectrum-{beta}-lactamase-producing Pseudomonas aeruginosa clinical isolate in Argentina.

Antimicrobial agents and chemotherapy 49 (1)
PMID : 15616342  :   DOI  :   10.1128/AAC.49.1.474-475.2005     PMC  :   PMC538874    
Abstract >>
N/A
KeywordMeSH Terms
Drug Resistance, Bacterial
217. Quinteira  S, Sousa  JC, Peixe  L,     ( 2005 )

Characterization of In100, a new integron carrying a metallo-{beta}-lactamase and a carbenicillinase, from Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 49 (1)
PMID : 15616334  :   DOI  :   10.1128/AAC.49.1.451-453.2005     PMC  :   PMC538865    
Abstract >>
In100, a new integron carrying a carbapenemase gene (bla(VIM-2)) associated with a carbenicillinase (blaP1b) and aminoglycoside resistance genes (aacA4 and aadA2), was detected in a Pseudomonas aeruginosa clinical isolate. The particular gene cassette organization of In100 seems to reflect the evolution of antibiotic usage in therapeutics.
KeywordMeSH Terms
218. Riccio  ML, Pallecchi  L, Docquier  JD, Cresti  S, Catania  MR, Pagani  L, Lagatolla  C, Cornaglia  G, Fontana  R, Rossolini  GM,     ( 2005 )

Clonal relatedness and conserved integron structures in epidemiologically unrelated Pseudomonas aeruginosa strains producing the VIM-1 metallo-{beta}-lactamase from different Italian hospitals.

Antimicrobial agents and chemotherapy 49 (1)
PMID : 15616282  :   DOI  :   10.1128/AAC.49.1.104-110.2005     PMC  :   PMC538861    
Abstract >>
Three epidemiologically independent Pseudomonas aeruginosa isolates, representative of the first VIM-1 metallo-beta-lactamase producers detected at three different hospitals in northern Italy, were investigated to determine their genomic relatedness and to compare the structures of the genetic supports for the VIM-1 determinants. The three isolates, all of serotype O11, appeared to be clonally related according to the results of genotyping by macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. Investigation of the genetic support for the bla(VIM-1) determinant revealed that it was carried on identical or almost identical integrons (named In70.2 and In70.3) located within a conserved genomic context. The integrons were structurally related to In70 and In110, two plasmid-borne bla(VIM-1)-containing integrons from Achromobacter xylosoxidans and Pseudomonas putida isolates, respectively, from the same geographic area (northern Italy) and were found to be inserted close to the res site of a Tn5051-like transposon, different from any of those described previously, that was apparently carried on the bacterial chromosome. The present findings suggest that the three VIM-1-producing isolates are members of the same clonal complex which have been spreading in hospitals in northern Italy since the late 1990s and point to a common ancestry of their bla(VIM-1)-containing integrons.
KeywordMeSH Terms
Conserved Sequence
Hospitals, University
219. Toleman  MA, Biedenbach  D, Bennett  DM, Jones  RN, Walsh  TR,     ( 2005 )

Italian metallo-beta-lactamases: a national problem? Report from the SENTRY Antimicrobial Surveillance Programme.

The Journal of antimicrobial chemotherapy 55 (1)
PMID : 15574470  :   DOI  :   10.1093/jac/dkh512    
Abstract >>
As part of the SENTRY Antimicrobial Surveillance Programme, 383 non-replicative randomly collected Pseudomonas aeruginosa isolates were collected during 1999-2002. These strains originated from three geographically distinct hospitals within Italy: Genoa (Northern Italy); Rome and Catania (Sicily), and were further studied to identify the prevalence of metallo-beta-lactamase (MbetaL) alleles across Italy and to determine their genetic details. Multidrug-resistant (MDR) strains were identified by MIC analysis followed by genotyping and PCR-based strategies. Initial MIC analysis identified 31 MDR isolates that displayed an Etest MbetaL-positive phenotype. Of these, 25 produced either the MbetaL VIM-1 or IMP-13 as detected by PCR and sequencing. VIM-1-producing isolates were found at all sites, whereas IMP-13-producing isolates were only found in Rome. MbetaL-producing isolates were found at all Italian SENTRY sites and together amounted to 6.5% of all P. aeruginosa isolates. Genetic analysis indicated that many strains contained multiple integrons and identified two novel MbetaL integrons, one from the site in Genoa and one from Sicily. Integrons identical in structure and sequence to In70, the first identified and characterized bla(VIM)-containing integron from Verona, were found in isolates with distinct ribotypes at the Roman and Sicilian sites indicating that this integron has recently disseminated across Italy. All 25 MbetaL-producing isolates were genetically linked in that all isolates contained Tn5051 sequences and all harboured the insertion sequence IsPa7 which may be involved in the mobilization of these resistance alleles. Taken together, these results indicate that Italy has a nationwide problem of MDR P. aeruginosa produced by mobile MbetaL genes.
KeywordMeSH Terms
Drug Resistance, Bacterial
Sentinel Surveillance
220. Wang  CX, Mi  ZH,     ( 2004 )

IMP-1 metallo-beta-lactamase-producing Pseudomonas aeruginosa in a university hospital in the People's Republic of China.

The Journal of antimicrobial chemotherapy 54 (6)
PMID : 15537691  :   DOI  :   10.1093/jac/dkh498    
Abstract >>
N/A
KeywordMeSH Terms
beta-Lactam Resistance
Hospitals, University
221. Crespo  MP, Woodford  N, Sinclair  A, Kaufmann  ME, Turton  J, Glover  J, Velez  JD, Castañeda  CR, Recalde  M, Livermore  DM,     ( 2004 )

Outbreak of carbapenem-resistant Pseudomonas aeruginosa producing VIM-8, a novel metallo-beta-lactamase, in a tertiary care center in Cali, Colombia.

Journal of clinical microbiology 42 (11)
PMID : 15528701  :   DOI  :   10.1128/JCM.42.11.5094-5101.2004     PMC  :   PMC525211    
Abstract >>
The prevalence of imipenem resistance among Pseudomonas aeruginosa isolates at a 195-bed tertiary care medical center in Cali, Colombia, rose from 2% in 1996 to 28% in 1997 and to over 40% in 2003. Many isolates showed high-level multiresistance, and phenotypic characterization suggested the spread of a predominant strain with minor variants. Sixty-six resistant isolates collected between February 1999 and July 2003 from hospitalized patients (n = 54) and environmental samples (n = 12) were subjected to a fuller analysis. Genetic fingerprints were compared by pulsed-field gel electrophoresis (PFGE) of SpeI-digested genomic DNA, and bla(IMP) and bla(VIM) genes were sought by PCR. PFGE and serotyping indicated that 52 of the 66 isolates belonged to a single strain, with 82% similarity; the PFGE pattern for this organism was designated pattern A. Two further pairs of isolates represented single strains; the remaining nine isolates were unique, and in the case of one isolate, no satisfactory PFGE profile could be obtained. The pattern A isolates were mostly of serotype O12 and were highly resistant to imipenem (MICs, 32 to >256 microg/ml), with this resistance decreased eightfold or more in the presence of EDTA. They yielded amplicons with bla(VIM)-specific primers, and sequencing of DNA from a representative isolate revealed bla(VIM-8), a novel allele with three polymorphisms compared with the sequence of bla(VIM-2). Two of these nucleotide changes were silent, but the third determined a Thr139Ala substitution. Only 4 of 13 resistant isolates (2 clinical isolates and 2 environmental isolates) assigned to other PFGE types carried bla(VIM) alleles, whereas the others were less multiresistant and mostly had lower levels of imipenem resistance (MICs, < or =32 microg/ml) which was not significantly reduced by EDTA. No bla(IMP) alleles were detected. During 2003, when the environmental study was undertaken, serotype O12 isolates with bla(VIM) were recovered from sinks and stethoscopes in the most-affected units, although not from the hands of staff; the problem declined once these reservoirs were disinfected and hygienic precautions were reinforced.
KeywordMeSH Terms
beta-Lactam Resistance
Disease Outbreaks
222. Mendes  RE, Toleman  MA, Ribeiro  J, Sader  HS, Jones  RN, Walsh  TR,     ( 2004 )

Integron carrying a novel metallo-beta-lactamase gene, blaIMP-16, and a fused form of aminoglycoside-resistant gene aac(6')-30/aac(6')-Ib': report from the SENTRY Antimicrobial Surveillance Program.

Antimicrobial agents and chemotherapy 48 (12)
PMID : 15561846  :   DOI  :   10.1128/AAC.48.12.4693-4702.2004     PMC  :   PMC529210    
Abstract >>
Since January 2002 Pseudomonas sp. strains resistant to carbapenems and ceftazidime have been routinely screened as part of the SENTRY Antimicrobial Surveillance Program for metallo-beta-lactamase production, and their resistance determinants have been analyzed. Pseudomonas aeruginosa index strain 101-4704, which harbors a novel bla(IMP) variant, bla(IMP-16), was isolated in April 2002 from a 60-year-old man in Brasilia, Brazil. bla(IMP-16) was found on the chromosome of the P. aeruginosa index strain, and the deduced amino acid sequence (IMP-16) showed the greatest identities to IMP-11 (90.3%) and IMP-8 (89.5%). Sequence analysis revealed that bla(IMP-16) was associated with a class 1 integron, which also encoded aminoglycoside-modifying enzymes. Downstream of bla(IMP-16) resided an open reading frame, which consisted of a new aminoglycoside-modifying gene, namely, aac(6')-30, which was fused with aac(6')-Ib'. The amino acid sequence of the aac(6')-30 putative protein showed the most identity (52.7%) to the sequence of AAC(6')-29b described previously. The fourth gene cassette constituted aadA1. The steady-state kinetics of IMP-16 demonstrated that the enzyme preferred cephalosporins and carbapenems to penicillins. The main functional difference observed among the kinetic values for IMP-16 compared to those for other IMPs was a lack of cefoxitin hydrolysis and a lower kcat/Km value for imipenem (0.36 microM(-1) . s(-1)). This report further emphasizes the spread of metallo-beta-lactamase genes and their close association with various aminoglycoside resistance genes.
KeywordMeSH Terms
223. Koh  TH, Wang  GC, Sng  LH,     ( 2004 )

Clonal spread of IMP-1-producing Pseudomonas aeruginosa in two hospitals in Singapore.

Journal of clinical microbiology 42 (11)
PMID : 15528748  :   DOI  :   10.1128/JCM.42.11.5378-5380.2004     PMC  :   PMC525177    
Abstract >>
Thirty-six isolates of carbapenem-resistant Pseudomonas aeruginosa were studied. Pulsed-field gel electrophoresis revealed the presence of two clones. One clone carried a bla(IMP-1) gene identical to that first described in Japan. The other clone carried a bla(IMP-1) variant containing four silent mutations. One isolate with a unique pulsed-field gel electrophoresis pattern contained bla(IMP-7).
KeywordMeSH Terms
beta-Lactam Resistance
Hospitals
224. Ogino  H, Mimitsuka  T, Muto  T, Matsumura  M, Yasuda  M, Ishimi  K, Ishikawa  H,     ( 2004 )

Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03.

Journal of molecular microbiology and biotechnology 7 (4)
PMID : 15383719  :   DOI  :   10.1159/000079830    
Abstract >>
A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.
KeywordMeSH Terms
225. Parsons  JF, Calabrese  K, Eisenstein  E, Ladner  JE,     ( 2004 )

Structure of the phenazine biosynthesis enzyme PhzG.

Acta crystallographica. Section D, Biological crystallography 60 (Pt 11)
PMID : 15502343  :   DOI  :   10.1107/S0907444904022474    
Abstract >>
PhzG is a flavin-dependent oxidase that is believed to play a role in phenazine antibiotic synthesis in various bacteria, including Pseudomonas. Phenazines are chorismic acid derivatives that provide the producing organisms, including the opportunistic pathogen P. aeruginosa, with a competitive growth advantage. Here, the crystal structures of PhzG from both P. aeruginosa and P. fluorescens solved in an unliganded state at 1.9 and 1.8 A resolution, respectively, are described. Although the specific reaction in phenazine biosynthesis catalyzed by PhzG is unknown, the structural data indicates that PhzG is closely related to pyridoxine-5'-phosphate oxidase, the Escherichia coli pdxH gene product, which catalyzes the final step in pyridoxal-5'-phosphate (PLP) biosynthesis. A previous proposal suggested that the physiological substrate of PhzG to be 2,3-dihydro-3-hydroxyanthranilic acid (DHHA), a phenazine precursor produced by the sequential actions of the PhzE and PhzD enzymes on chorismate, and that two DHHA molecules dimerized in another enzyme-catalyzed reaction to yield phenazine-1-carboxylate. However, it was not possible to demonstrate any in vitro activity upon incubation of PhzG and DHHA. Interestingly, analysis of the in vitro activities of PhzG in combination with PhzF suggests that PhzF acts on DHHA and that PhzG then reacts with a non-aromatic tricyclic phenazine precusor to catalyze an oxidation/aromatization reaction that yields phenazine-1-carboxylate. It is proposed that phzG arose by duplication of pdxH and that the subtle differences seen between the structures of PhzG and PdxH correlate with the loss of the ability of PhzG to catalyze PLP formation. Sequence alignments and superimpositions of the active sites of PhzG and PdxH reveal that the residues that form a positively charged pocket around the phosphate of PLP in the PdxH-PLP complex are not conserved in PhzG, consistent with the inability of phosphorylated compounds to serve as substrates for PhzG.
KeywordMeSH Terms
226. Castanheira  M, Toleman  MA, Jones  RN, Schmidt  FJ, Walsh  TR,     ( 2004 )

Molecular characterization of a beta-lactamase gene, blaGIM-1, encoding a new subclass of metallo-beta-lactamase.

Antimicrobial agents and chemotherapy 48 (12)
PMID : 15561840  :   DOI  :   10.1128/AAC.48.12.4654-4661.2004     PMC  :   PMC529189    
Abstract >>
As part of the SENTRY Antimicrobial Surveillance Program in 2002, five multidrug-resistant Pseudomonas aeruginosa clinical isolates were detected with metallo-beta-lactamase (MbetaL) activity. The isolates were recovered from different patients in a medical center located in Dusseldorf, Germany. The resistant determinant was isolated amplifying the region between the integrase and the aacA4 gene cassette. Sequencing revealed a novel MbetaL gene, designated bla(GIM-1). Additional analysis showed that GIM-1, comprising 250 amino acids and with a pI value of 5.4, differs in its primary sequence from that described for IMP, VIM, and SPM-1 enzymes by 39 to 43%, 28 to 31%, and 28%, respectively. The enzyme possesses unique amino acids within the major consensus sequence (HXHXD) of the MbetaL family. Kinetics analysis revealed that GIM-1 has no clear preference for any substrate and did not hydrolyze azlocillin, aztreonam, and the serine-beta-lactamase inhibitors. bla(GIM-1) was found on a 22-kb nontransferable plasmid. The new MbetaL gene was embedded in the first position of a 6-kb class 1 integron, In77, with distinct features, including an aacA4 cassette downstream of the MbetaL gene that appeared to be truncated with bla(GIM-1). The aacA4 was followed by an aadA1 gene cassette that was interrupted by a copy of the IS1394. This integron also carried an oxacillinase gene, bla(OXA-2), before the 3'-CS region. GIM-1 appears to be a unique MbetaL, which is located in a distinct integron structure, and represents the fourth subclass of mobile MbetaL enzymes to be characterized.
KeywordMeSH Terms
227. Wang  G, Skipper  HD,     ( 2004 )

Identification of denitrifying rhizobacteria from bentgrass and bermudagrass golf greens.

Journal of applied microbiology 97 (4)
PMID : 15357733  :   DOI  :   10.1111/j.1365-2672.2004.02368.x    
Abstract >>
As high rates of nitrogen fertilization are used in turfgrass management, there is a great potential for nitrogen loss. Research on identification of denitrifiers in turfgrass has been limited. Therefore, the aim was to identify denitrifier species and genes from turfgrass roots. Rhizobacteria were isolated from roots of bentgrass and bermudagrass in sand-based United States Golf Association (USGA) golf greens and used for denitrification biochemical analysis. Seventeen per cent (34 isolates) were identified as denitrifiers, 47% were classified as nitrate-reducers and 36% were nondenitrifiers. Identification of species of the denitrifiers was performed by chromatography fatty acid methyl ester (GC-FAME) and16S rDNA analyses. Bacillus and Pseudomonas were the major turfgrass denitrifiers. The two methods showed a 60% agreement at the genus level. Nitrite reductase genes nirK and nirS were detected in 74 and 15% of the denitrifiers, respectively, but not in nondenitrifiers. The nosZ gene encoding nitrous oxide reductase was detected in all the denitrifiers, but also in some nondenitrifiers. To our knowledge, this is the first report for identification of denitrifiers and denitrification-related genes associated with turfgrass roots. These results provide valuable data for future denitrification studies that seek to improve turfgrass nitrogen management for maximum efficiency.
KeywordMeSH Terms
Fertilizers
228. Libisch  B, Gacs  M, Csiszár  K, Muzslay  M, Rókusz  L, Füzi  M,     ( 2004 )

Isolation of an integron-borne blaVIM-4 type metallo-beta-lactamase gene from a carbapenem-resistant Pseudomonas aeruginosa clinical isolate in Hungary.

Antimicrobial agents and chemotherapy 48 (9)
PMID : 15328131  :   DOI  :   10.1128/AAC.48.9.3576-3578.2004     PMC  :   PMC514723    
Abstract >>
The first integron-borne metallo-beta-lactamase gene was isolated in Hungary. The bla(VIM-4) gene is located on a class 1 integron that also carries a novel bla(OXA)-like gene. The integron is harbored by a serotype O12 Pseudomonas aeruginosa strain and shows high structural similarity to integrons isolated in Greece and Poland.
KeywordMeSH Terms
229. Aubert  D, Girlich  D, Naas  T, Nagarajan  S, Nordmann  P,     ( 2004 )

Functional and structural characterization of the genetic environment of an extended-spectrum beta-lactamase blaVEB gene from a Pseudomonas aeruginosa isolate obtained in India.

Antimicrobial agents and chemotherapy 48 (9)
PMID : 15328086  :   DOI  :   10.1128/aac.48.9.3284-3290.2004     PMC  :   PMC514732    
Abstract >>
A Pseudomonas aeruginosa clinical strain isolated from a patient hospitalized in a New Delhi, India, hospital was resistant to expanded-spectrum cephalosporins, imipenem, and aztreonam. A bla(VEB-1)-like gene named bla(VEB-1a), which codes for the extended-spectrum beta-lactamase VEB-1a, was identified. The genetic environment of bla(VEB-1a) was peculiar: (i) no 5' conserved sequence (5'-CS) region was present upstream of the beta-lactamase gene, whereas bla(VEB-1)-like genes are usually associated with class 1 integrons; (ii) bla(VEB-1a) was inserted between two truncated 3'-CS regions in a direct repeat; and (iii) four 135-bp repeated DNA sequences (repeated elements) were located on each side of the bla(VEB-1a) gene. Expression of the bla(VEB-1a) gene was driven by a strong promoter located in one of these repeated sequences. In addition, cloning of the beta-lactamase content of this P. aeruginosa isolate followed by expression in Escherichia coli identified the naturally occurring AmpC beta-lactamase and a gene encoding an OXA-2-like beta-lactamase located in a class 1 integron, In78, in which an insertion sequence, ISpa7, was inserted within its 5'-CS region.
KeywordMeSH Terms
230. Shan  Z, Xu  H, Shi  X, Yu  Y, Yao  H, Zhang  X, Bai  Y, Gao  C, Saris  PE, Qiao  M,     ( 2004 )

Identification of two new genes involved in twitching motility in Pseudomonas aeruginosa.

Microbiology (Reading, England) 150 (Pt 8)
PMID : 15289561  :   DOI  :   10.1099/mic.0.27131-0    
Abstract >>
Mu transposition complexes were used for transposon mutagenesis of Pseudomonas aeruginosa strain PA68. Mu DNA transposition complexes were assembled with MuA transposase and an artificial mini-Mu transposon in vitro, and introduced into Pseudomonas aeruginosa by electroporation. Eight mutants deficient in twitching motility were isolated. Southern blotting confirmed that the insertions had occurred as single events. DNA sequencing of the region flanking the insertion in the twitching-motility mutants revealed that the mini-Mu transposon had inserted into six different genes, PAO171, PA1822, PAO413, PA4959, PA4551 and PA5040. Four of these have previously been proven to be needed for twitching motility, whereas the PA1822 and PA0171 genes have not previously been shown to be required for twitching motility. The twitching-motility defect in the PA1822 mutant was partially complemented by providing the PA1822 gene in trans, and the defect in the PA0171 mutant was fully complemented when PA0171 was provided. A PA0171 mutant and a PA1822 mutant were constructed by gene replacement in the P. aeruginosa PAO1 strain. These mutants were deficient in twitching motility, showing that both the PA1822 and the PA0171 gene are involved in twitching motility.
KeywordMeSH Terms
Genes, Bacterial
231. Hill  JE, Penny  SL, Crowell  KG, Goh  SH, Hemmingsen  SM,     ( 2004 )

cpnDB: a chaperonin sequence database.

Genome research 14 (8)
PMID : 15289485  :   DOI  :   10.1101/gr.2649204     PMC  :   PMC509277    
Abstract >>
Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.
KeywordMeSH Terms
232. Cladera  AM, Bennasar  A, Barceló  M, Lalucat  J, García-Valdés  E,     ( 2004 )

Comparative genetic diversity of Pseudomonas stutzeri genomovars, clonal structure, and phylogeny of the species.

Journal of bacteriology 186 (16)
PMID : 15292125  :   DOI  :   10.1128/JB.186.16.5239-5248.2004     PMC  :   PMC490930    
Abstract >>
A combined phylogenetic and multilocus DNA sequence analysis of 26 Pseudomonas stutzeri strains distributed within the 9 genomovars of the species has been performed. Type strains of the two most closely related species (P. balearica, former genomovar 6, and P. mendocina), together with P. aeruginosa, as the type species of the genus, have been included in the study. The extremely high genetic diversity and the clonal structure of the species were confirmed by the sequence analysis. Clustering of strains in the consensus phylogeny inferred from the analysis of seven nucleotide sequences (16S ribosomal DNA, internally transcribed spacer region 1, gyrB, rpoD, nosZ, catA, and nahH) confirmed the monophyletic origin of the genomovars within the Pseudomonas branch and is in good agreement with earlier DNA-DNA similarity analysis, indicating that the selected genes are representative of the whole genome in members of the species.
KeywordMeSH Terms
Genetic Variation
233. Yamane  K, Doi  Y, Yokoyama  K, Yagi  T, Kurokawa  H, Shibata  N, Shibayama  K, Kato  H, Arakawa  Y,     ( 2004 )

Genetic environments of the rmtA gene in Pseudomonas aeruginosa clinical isolates.

Antimicrobial agents and chemotherapy 48 (6)
PMID : 15155201  :   DOI  :   10.1128/AAC.48.6.2069-2074.2004     PMC  :   PMC415585    
Abstract >>
Nine Pseudomonas aeruginosa strains showing very high levels of resistance to various aminoglycosides have been isolated from clinical specimens in seven separate Japanese hospitals in five prefectures since 1997. These strains harbor the newly identified 16S rRNA methylase gene (rmtA). When an rmtA gene probe was hybridized with genomic DNAs of the nine strains digested with EcoRI, two distinct patterns were observed. The 11.1- and 15.8-kb regions containing the rmtA genes of strains AR-2 and AR-11, respectively, were sequenced and compared. In strain AR-2, a transposase gene-like sequence (sequence 1) and a probable tRNA ribosyltransferase gene (orfA) were located upstream of rmtA, and a Na(+)/H(+) antiporter gene-like sequence (sequence 2) was identified downstream of rmtA. This 6.2-kbp insert (the rmtA locus) was flanked by 262-bp kappagamma elements. Part of the orfQ gene adjacent to an inverted repeat was found outside of the rmtA locus. In strain AR-11, the rmtA gene and sequence 2 were found, but the 5' end of the orfA gene was truncated and replaced with IS6100. An orfQ-orfI region was present on each side of the rmtA gene in strain AR-11. The G+C content of the rmtA gene was about 55%, and since the newly identified rmtA gene may well be mediated by some mobile genetic elements such as Tn5041, further dissemination of the rmtA gene could become an actual clinical problem in the near future.
KeywordMeSH Terms
234. Girlich  D, Naas  T, Nordmann  P,     ( 2004 )

Biochemical characterization of the naturally occurring oxacillinase OXA-50 of Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 48 (6)
PMID : 15155197  :   DOI  :   10.1128/AAC.48.6.2043-2048.2004     PMC  :   PMC415580    
Abstract >>
The bla(OXA-50) gene (formerly known as the PA5514 gene) is an oxacillinase gene identified in silico in the genome of Pseudomonas aeruginosa PAO1. By using a mutant strain of P. aeruginosa PAO1 that had an inactivated bla(AmpC) cephalosporinase gene, the bla(OXA-50) gene was shown to be expressed constitutively in P. aeruginosa. This beta-lactamase gene was cloned onto a multicopy plasmid and expressed in P. aeruginosa and Escherichia coli. It conferred decreased susceptibility to ampicillin and ticarcillin and, interestingly, to moxalactam and meropenem in P. aeruginosa but not in E. coli. Overexpression and purification enabled us to determine the molecular mass (25 kDa), the pI value (8.6), and the hydrolysis spectrum of the OXA-50 beta-lactamase. It is a narrow-spectrum oxacillinase that uncommonly hydrolyzes imipenem, although at a low level. Very similar oxacillinase genes were identified in all P. aeruginosa isolates from various geographical origins tested. The weak variability of the nucleotide sequence of this gene (0 to 2%) corresponded to that found for the naturally occurring bla(AmpC) cephalosporinase gene of P. aeruginosa. The study indicated that P. aeruginosa harbors two naturally encoded beta-lactamase genes, one of which encodes an inducible cephalosporinase and the other of which encodes a constitutively expressed oxacillinase.
KeywordMeSH Terms
235. Hashim  S, Kwon  DH, Abdelal  A, Lu  CD,     ( 2004 )

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa.

Journal of bacteriology 186 (12)
PMID : 15175298  :   DOI  :   10.1128/JB.186.12.3848-3854.2004     PMC  :   PMC419967    
Abstract >>
The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
236. Friedman  J, Lad  L, Li  H, Wilks  A, Poulos  TL,     ( 2004 )

Structural basis for novel delta-regioselective heme oxygenation in the opportunistic pathogen Pseudomonas aeruginosa.

Biochemistry 43 (18)
PMID : 15122889  :   DOI  :   10.1021/bi049687g    
Abstract >>
The Gram-negative bacterium Pseudomonas aeruginosa contains a heme oxygenase (pa-HO) that primarily oxygenates the delta-meso heme carbon [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. This differs from other previously characterized heme oxygenases, which display regioselectivity for the alpha-meso heme carbon. Here we report the crystal structure of pa-HO at 1.60 A resolution and compare it to the 1.50 A structure of nm-HO from Neisseria meningitidis [Schuller, D. J., Zhu, W., Stojiljkovic, I., Wilks, A., and Poulos, T. L. (2001) Biochemistry 40, 11552-11558]. The crystal structure of pa-HO maintains the same overall fold as other bacterial and mammalian heme oxygenases, including a conserved network of hydrogen-bonded solvent molecules important for dioxygen activation. The novel delta-regioselectivity of heme oxygenation observed by pa-HO is due to the heme being rotated by approximately 100 degrees, which places the delta-meso heme carbon in the same position as the alpha-meso heme carbon in other heme oxygenases. The main interaction in pa-HO that stabilizes the unique heme orientation is a salt bridge between Lys132 and the heme 7-propionate, as well as hydrophobic contacts involving Leu29, Val33, and Phe189 with the heme methyl and vinyl groups.
KeywordMeSH Terms
237. Ohnishi  K, Okuta  A, Ju  J, Hamada  T, Misono  H, Harayama  S,     ( 2004 )

Molecular breeding of 2,3-dihydroxybiphenyl 1,2-dioxygenase for enhanced resistance to 3-chlorocatechol.

Journal of biochemistry 135 (3)
PMID : 15113829  :   DOI  :   10.1093/jb/mvh037    
Abstract >>
3-Chlorobiphenyl is known to be mineralized by biphenyl-utilizing bacteria to 3-chlorobenzoate, which is further metabolized to 3-chlorocatechol. An extradiol dioxygenase, 2,3-dihydroxybiphenyl 1,2-dioxygenase (DHB12O; EC 1.13.11.39), which is encoded by the bphC gene, catalyzes the third step of the upper pathway of 3-chlorobiphenyl degradation. In this study, two full-length bphCs and nine partial fragments of bphCs fused to the 3' end of bphC in Pseudomonas pseudoalcaligenes KF707 were cloned from different biphenyl-utilizing soil bacteria and expressed in Escherichia coli. The enzyme activities of the expressed DHB12Os were inhibited to varying degrees by 3-chlorocatechol, and the E. coli cells overexpressing DHB12O could not grow or grew very slowly in the presence of 3-chlorocatechol. These sensitivities of enzyme activity and cell growth to 3-chlorocatechol were well correlated, and this phenomenon was employed in screening chimeric BphCs formed by family shuffling of bphC genes isolated from Comamonas testosteroni KF704 and C. testosteroni KF712. The resultant DHB12Os were more resistant by a factor of two to 3-chlorocatechol than one of the best parents, KF707 DHB12O.
KeywordMeSH Terms
DNA Shuffling
238. Kus  JV, Tullis  E, Cvitkovitch  DG, Burrows  LL,     ( 2004 )

Significant differences in type IV pilin allele distribution among Pseudomonas aeruginosa isolates from cystic fibrosis (CF) versus non-CF patients.

Microbiology (Reading, England) 150 (Pt 5)
PMID : 15133094  :   DOI  :   10.1099/mic.0.26822-0    
Abstract >>
Type IV pili (TFP) are important colonization factors of the opportunistic pathogen Pseudomonas aeruginosa, involved in biofilm formation and attachment to host cells. This study undertook a comprehensive analysis of TFP alleles in more than 290 environmental, clinical, rectal and cystic fibrosis (CF) isolates of P. aeruginosa. Based on the results, a new system of nomenclature is proposed, in which P. aeruginosa TFP are divided into five distinct phylogenetic groups. Each pilin allele is stringently associated with characteristic, distinct accessory genes that allow the identification of the allele by specific PCR. The invariant association of the pilin and accessory genes implies horizontal transfer of the entire locus. Analysis of pilin allele distribution among isolates from various sources revealed a striking bias in the prevalence of isolates with group I pilin genes from CF compared with non-CF human sources (P<0.0001), suggesting this particular pilin type, which can be post-translationally modified by glycosylation via the action of TfpO (PilO), may confer a colonization or persistence advantage in the CF host. This allele was also predominant in paediatric CF isolates (29 of 43; 67.4 %), showing that this bias is apparent early in colonization. Group I pilins were also the most common type found in environmental isolates tested. To the authors' knowledge, this is the first example of a P. aeruginosa virulence factor allele that is strongly associated with CF isolates.
KeywordMeSH Terms
239. Schirm  M, Arora  SK, Verma  A, Vinogradov  E, Thibault  P, Ramphal  R, Logan  SM,     ( 2004 )

Structural and genetic characterization of glycosylation of type a flagellin in Pseudomonas aeruginosa.

Journal of bacteriology 186 (9)
PMID : 15090491  :   DOI  :   10.1128/jb.186.9.2523-2531.2004     PMC  :   PMC387798    
Abstract >>
Type a flagellins from two strains of Pseudomonas aeruginosa, strains PAK and JJ692, were found to be glycosylated with unique glycan structures. In both cases, two sites of O-linked glycosylation were identified on each monomer, and these sites were localized to the central, surface-exposed domain of the monomer in the assembled filament. The PAK flagellin was modified with a heterogeneous glycan comprising up to 11 monosaccharide units that were O linked through a rhamnose residue to the protein backbone. The flagellin of JJ692 was less complex and had a single rhamnose substitution at each site. The role of the glycosylation island gene cluster in the production of each of these glycosyl moieties was investigated. These studies revealed that the orfA and orfN genes were required for attachment of the heterologous glycan and the proximal rhamnose residue, respectively.
KeywordMeSH Terms
240. Hilario  E, Buckley  TR, Young  JM,     ( 2004 )

Improved resolution on the phylogenetic relationships among Pseudomonas by the combined analysis of atp D, car A, rec A and 16S rDNA.

Antonie van Leeuwenhoek 86 (1)
PMID : 15103237  :   DOI  :   10.1023/B:ANTO.0000024910.57117.16    
Abstract >>
A study of representatives of the bacterial genus Pseudomonas, analysing a combined data set of four molecular sequences with completely different properties and evolutionary constraints, is reported. The best evolutionary model was obtained with a hierarchical hypothesis testing program to describe each data set and the combined data set is presented and analysed under the likelihood criterion. The resolution among Pseudomonas taxa based on the combined data set analysis of the different lineages increased due to a synergistic effect of the individual data sets. The unresolved fluorescens lineage, as well as other weakly supported lineages in the single data set trees, should be revised in detail at the biochemical and molecular level. The taxonomic status of biovars of P. putida is discussed.
KeywordMeSH Terms
241. Pagani  L, Mantengoli  E, Migliavacca  R, Nucleo  E, Pollini  S, Spalla  M, Daturi  R, Romero  E, Rossolini  GM,     ( 2004 )

Multifocal detection of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase in Northern Italy.

Journal of clinical microbiology 42 (6)
PMID : 15184430  :   DOI  :   10.1128/JCM.42.6.2523-2529.2004     PMC  :   PMC427849    
Abstract >>
Forty-four nonreplicate clinical isolates of Pseudomonas aeruginosa that were resistant to extended-spectrum cephalosporins (ceftazidime and cefepime) and aztreonam, that putatively produced an acquired extended- spectrum beta-lactamase (ESBL), according to the results of a double-disk synergy test, and that had been involved in nosocomial outbreaks were obtained from six different hospitals in northern Italy and screened for the presence of bla(PER) ESBL determinants. Twenty isolates, associated with nine independent outbreaks that occurred in five hospitals in the Milan area and its surroundings during 1995-2000, were found to carry an acquired bla(PER-1) gene. PER-1 producers representative of the nine outbreaks exhibited a multidrug resistance (MDR) phenotype, including resistance to extended-spectrum cephalosporins, aztreonam, meropenem, aminoglycosides, and in most cases, imipenem and ciprofloxacin. An analysis of macrorestriction profiles of their genomic DNAs by pulsed-field gel electrophoresis revealed an overall clonal diversity of the PER-1 producers, although interhospital clonal spread was also observed. The bla(PER-1) gene was not transferable and appeared to be chromosomally located. An analysis of the EcoRI and EcoRV restriction fragment length polymorphisms of the bla(PER-1) locus revealed identical patterns for all isolates, and the characterization of a 1.9-kb region containing bla(PER-1) revealed a conserved structure in representatives of the various clonal lineages. The present findings indicate that MDR P. aeruginosa clones producing the PER-1 ESBL are endemic to this area of northern Italy, where they have been circulating since the mid-1990s and have been associated with several nosocomial outbreaks.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
242. Morales  G, Wiehlmann  L, Gudowius  P, van Delden  C, Tümmler  B, Martínez  JL, Rojo  F,     ( 2004 )

Structure of Pseudomonas aeruginosa populations analyzed by single nucleotide polymorphism and pulsed-field gel electrophoresis genotyping.

Journal of bacteriology 186 (13)
PMID : 15205425  :   DOI  :   10.1128/JB.186.13.4228-4237.2004     PMC  :   PMC421620    
Abstract >>
Pseudomonas aeruginosa has a wide ecological distribution that includes natural habitats and clinical settings. To analyze the population structure and distribution of P. aeruginosa, a collection of 111 isolates of diverse habitats and geographical origin, most of which contained a genome with a different SpeI macrorestriction profile, was typed by restriction fragment length polymorphism based on 14 single nucleotide polymorphisms (SNPs) located at seven conserved loci of the core genome (oriC, oprL, fliC, alkB2, citS, oprI, and ampC). The combination of these SNPs plus the type of fliC present (a or b) allowed the assignment of a genetic fingerprint to each strain, thus providing a simple tool for the discrimination of P. aeruginosa strains. Thirteen of the 91 identified SNP genotypes were found in two or more strains. In several cases, strains sharing their SNP genotype had different SpeI macrorestriction profiles. The highly virulent CHA strain shared its SNP genotype with other strains that had different SpeI genotypes and which had been isolated from nonclinical habitats. The reference strain PAO1 also shared its SNP genotype with other strains that had different SpeI genotypes. The P. aeruginosa chromosome contains a conserved core genome and variable amounts of accessory DNA segments (genomic islands and islets) that can be horizontally transferred among strains. The fact that some SNP genotypes were overrepresented in the P. aeruginosa population studied and that several strains sharing an SNP genotype had different SpeI macrorestriction profiles supports the idea that changes occur at a higher rate in the accessory DNA segments than in the conserved core genome.
KeywordMeSH Terms
Electrophoresis, Gel, Pulsed-Field
Polymorphism, Single Nucleotide
243. Bahar  G, Mazzariol  A, Koncan  R, Mert  A, Fontana  R, Rossolini  GM, Cornaglia  G,     ( 2004 )

Detection of VIM-5 metallo-beta-lactamase in a Pseudomonas aeruginosa clinical isolate from Turkey.

The Journal of antimicrobial chemotherapy 54 (1)
PMID : 15190017  :   DOI  :   10.1093/jac/dkh321    
Abstract >>
N/A
KeywordMeSH Terms
Bacterial Proteins
244. Ishiyama  N, Creuzenet  C, Lam  JS, Berghuis  AM,     ( 2004 )

Crystal structure of WbpP, a genuine UDP-N-acetylglucosamine 4-epimerase from Pseudomonas aeruginosa: substrate specificity in udp-hexose 4-epimerases.

The Journal of biological chemistry 279 (21)
PMID : 15016816  :   DOI  :   10.1074/jbc.M401642200    
Abstract >>
The O antigen of lipopolysaccharide in Gram-negative bacteria plays a critical role in bacterium-host interactions, and for pathogenic bacteria it is a major virulence factor. In Pseudomonas aeruginosa serotype O6 one of the initial steps in O-antigen biosynthesis is catalyzed by a saccharide epimerase, WbpP. WbpP is a member of the UDP-hexose 4-epimerase family of enzymes and exists as a homo-dimer. This enzyme preferentially catalyzes the conversion between UDP-GlcNAc and UDPGalNAc above UDP-Glc and UDP-Gal, using NAD(+) as a cofactor. The crystal structures of WbpP in complex with cofactor and either UDP-Glc or UDP-GalNAc were determined at 2.5 and 2.1 A, respectively, which represents the first structural studies of a genuine UDP-GlcNAc 4-epimerase. These structures in combination with complementary mutagenesis studies suggest that the basis for the differential substrate specificity of WbpP is a consequence of the presence of a pliable solvent network in the active site. This information allows for a comprehensive analysis of the relationship between sequence and substrate specificity for UDP-hexose 4-epimerases and enables the formulation of consensus sequences that predict substrate specificity of UDP-hexose 4-epimerases yet to be biochemically characterized. Furthermore, the examination indicates that as little as one residue can dictate substrate specificity. Nonetheless, phylogenetic analysis suggests that this substrate specificity is an evolutionary and highly conserved property within UDP-hexose 4-epimerases.
KeywordMeSH Terms
245. Wolter  DJ, Hanson  ND, Lister  PD,     ( 2004 )

Insertional inactivation of oprD in clinical isolates of Pseudomonas aeruginosa leading to carbapenem resistance.

FEMS microbiology letters 236 (1)
PMID : 15212803  :   DOI  :   10.1016/j.femsle.2004.05.039    
Abstract >>
Recently, a Texas, USA hospital isolated seven Pseudomonas aeruginosa strains displaying dual resistance to fluoroquinolones and imipenem. These isolates were resistant to the fluoroquinolones through overexpression of the MexXY efflux pump and/or QRDR mutations and resistant to imipenem through downregulation of oprD transcription. The purpose of this study was to evaluate the molecular events responsible for decreased transcriptional expression of oprD in these strains. Expression of oprD could only be detected in two of the strains, but expression was very low as indicated by the high number of RT-PCR cycles required to amplify the product. PCR was performed to amplify the oprD gene using primers upstream of the promoter and downstream of the structural gene. Amplified products were sequenced, and sequences were compared to wild-type P. aeruginosa strain PAO1. Two isolates provided PCR products of the predicted size of 1586 bp, but sequencing revealed a single base change within the structural gene resulting in a premature stop codon. The other five isolates provided PCR products that were 1.3-1.6 kb larger than expected, suggesting the presence of large inserts. Sequence analysis indicated these inserts were novel insertion sequence elements transposed into different locations within oprD. In summary, loss of OprD in all seven isolates was associated with mutations or insertions within oprD. Although the point mutations that resulted in premature stop codons would explain the loss of the OprD protein in two isolates. This observation does not explain the observed decrease in transcriptional expression. This is the first report of carbapenem resistance occurring through insertional inactivation of the oprD gene by IS elements.
KeywordMeSH Terms
246. Mendes  RE, Castanheira  M, Garcia  P, Guzman  M, Toleman  MA, Walsh  TR, Jones  RN, N/A  N/A,     ( 2004 )

First isolation of bla(VIM-2) in Latin America: report from the SENTRY Antimicrobial Surveillance Program.

Antimicrobial agents and chemotherapy 48 (4)
PMID : 15047562  :   DOI  :   10.1128/aac.48.4.1433-1434.2004     PMC  :   PMC375339    
Abstract >>
N/A
KeywordMeSH Terms
247. Poirel  L, Magalhaes  M, Lopes  M, Nordmann  P,     ( 2004 )

Molecular analysis of metallo-beta-lactamase gene bla(SPM-1)-surrounding sequences from disseminated Pseudomonas aeruginosa isolates in Recife, Brazil.

Antimicrobial agents and chemotherapy 48 (4)
PMID : 15047554  :   DOI  :   10.1128/aac.48.4.1406-1409.2004     PMC  :   PMC375305    
Abstract >>
The spread of clonally related carbapenem-resistant Pseudomonas aeruginosa producing the metallo-beta-lactamase SPM-1 was found in Recife, Brazil. Upstream of bla(SPM-1), a novel common region (CR4) was identified, comprising an open reading frame, orf495, whose product shares significant identity with putative recombinases, such as Orf513. CR4 may be responsible for mobilization and expression of bla(SPM-1).
KeywordMeSH Terms
248. Busquets  M, Deroncelé  V, Vidal-Mas  J, Rodríguez  E, Guerrero  A, Manresa  A,     ( 2004 )

Isolation and characterization of a lipoxygenase from Pseudomonas 42A2 responsible for the biotransformation of oleic acid into (S)-(E)-10-hydroxy-8-octadecenoic acid.

Antonie van Leeuwenhoek 85 (2)
PMID : 15028873  :   DOI  :   10.1023/B:ANTO.0000020152.15440.65    
Abstract >>
The isolation of a new lipoxygenase-like (LOX-like) enzyme from Pseudomonas 42A2 and its characterization is described. The enzyme, located in the periplasm of the cell, which contained 0.55 mol of Fe2+ per mol of protein, is monomeric and has a molecular mass of 45 kDa. In the presence of oxygen, the enzyme converts oleic acid into (E)-10-hydroperoxy-8-octadecenoic acid (HPOD), which decomposes to the corresponding (E)-10-hydroxy-8-octadecenoic acid (HOD). The absolute configuration of this acid was determined as S on the basis of exciton-coupled CD data, and specific rotation and NMR analysis of the corresponding p -bromobenzoate derivative. The reaction in vivo leads to the dihydroxy derivative (E)-7,10-dihydroxy-8-octadecenoic acid (DHOD), so that the three hydroxy-fatty acids can be isolated from the culture medium. The activity of the enzyme was optimal between 25 and 30 degrees C and 44% of its activity still remained at 55 degrees C. Its optimal pH is 8.5-9; and the presence of magnesium ions increased LOX activity by 1.5. The activity of the LOX is highest in unsaturated fatty acids containing double bonds in position 9 (oleic, linoleic and linolenic acids), linoleic acid being preferred (100% activity) over linolenic (60.4%) and oleic acids (46%). However, kinetic studies showed that the affinity of the enzyme is similar for the three substrates.
KeywordMeSH Terms
249. Arora  SK, Wolfgang  MC, Lory  S, Ramphal  R,     ( 2004 )

Sequence polymorphism in the glycosylation island and flagellins of Pseudomonas aeruginosa.

Journal of bacteriology 186 (7)
PMID : 15028697  :   DOI  :   10.1128/jb.186.7.2115-2122.2004     PMC  :   PMC374406    
Abstract >>
A genomic island consisting of 14 open reading frames, orfA to orfN was previously identified in Pseudomonas aeruginosa strain PAK and shown to be essential for glycosylation of flagellin. DNA microarray hybridization analysis of a number of P. aeruginosa strains from diverse origins showed that this island is polymorphic. PCR and sequence analysis confirmed that many P. aeruginosa strains carry an abbreviated version of the island (short island) in which orfD, -E and -H are polymorphic and orfI, -J, -K, -L, and -M are absent. To ascertain whether there was a relationship between the inheritance of the short island and specific flagellin sequence variants, complete or partial nucleotide sequences of flagellin genes from 24 a-type P. aeruginosa strains were determined. Two distinct flagellin subtypes, designated A1 and A2, were apparent. Strains with the complete 14-gene island (long island) were almost exclusively of the A1 type, whereas strains carrying the short island were associated with both A1- and A2-type flagellins. These findings indicate that P. aeruginosa possesses a relatively low number of distinct flagellin types and probably has the capacity to further diversify this antigenic surface protein by glycosylation.
KeywordMeSH Terms
Amino Acid Sequence
Polymorphism, Genetic
250. Patzer  J, Toleman  MA, Deshpande  LM, Kami?ska  W, Dzierzanowska  D, Bennett  PM, Jones  RN, Walsh  TR,     ( 2004 )

Pseudomonas aeruginosa strains harbouring an unusual blaVIM-4 gene cassette isolated from hospitalized children in Poland (1998-2001).

The Journal of antimicrobial chemotherapy 53 (3)
PMID : 14749341  :   DOI  :   10.1093/jac/dkh095    
Abstract >>
During 1997-2001, 151 isolates of imipenem-resistant Pseudomonas aeruginosa were obtained from clinical specimens taken from children hospitalized in Warsaw, Poland. These strains were investigated further to determine the mechanism of resistance. The strains were analysed by a combination of genotyping and PCR-based strategies. Eleven of these strains were found to contain the metallo-beta-lactamase (M beta L) gene bla(VIM-4). The first strain appeared in 1998, and P. aeruginosa strains harbouring this M beta L have become endemic in this hospital since then. All P. aeruginosa strains belonged to serotype O:6, and PFGE analysis revealed four different patterns and three sub-types. All 11 M beta L-producing strains contained an identical class 1 integron with the usual 5' and 3' conserved sequences. The integron included two resistance cassettes, aacA4 in the first position and the bla(VIM-4) cassette in the second position. The bla(VIM-4) gene included an unusual direct repeat of 169 bp of the 3' portion of the bla(VIM-4) gene. An unusual bla(VIM-4) M beta L has become endemic in P. aeruginosa isolates infecting Polish children hospitalized on surgical wards. The formation of this unusual bla(VIM-4) gene cassette could be explained by a mechanism involving deletion of a segment of an ancestral tandem repeat of bla(VIM-4) via slipped strand replication, mediated by a combination of polymerase and integrase.
KeywordMeSH Terms
251. Couture  F, Lachapelle  J, Levesque  RC,     ( 1992 )

Phylogeny of LCR-1 and OXA-5 with class A and class D beta-lactamases.

Molecular microbiology 6 (12)
PMID : 1495394  :   DOI  :   10.1111/j.1365-2958.1992.tb00894.x    
Abstract >>
The nucleotide sequences of blaLCR-1 and blaOXA-5 beta-lactamase genes have been determined. Polypeptide products of 260 and 267 amino acids with estimated molecular masses of 27 120 Da and 27,387 Da were obtained for the mature form of LCR-1 and OXA-5 proteins. A progressive alignment was used to evaluate the extent of identity between LCR-1 and OXA-5 with 29 other beta-lactamase amino acid sequences. The data showed that both belong to class D. We identified amino acids conserved in 24 positions for class A beta-lactamases and in 28 positions for five class D enzymes. The structural similarities between class A and class D beta-lactamases are more extensive than indicated by earlier biochemical studies with overall 16% identity between both classes. From the alignment, dendograms were constructed with a distance-matrix and parsimony methods which defined three major groups of proteins subdivided into clusters giving insight on beta-lactamase phylogeny and evolution.
KeywordMeSH Terms
Bacterial Proteins
Phylogeny
252. He  J, Baldini  RL, Déziel  E, Saucier  M, Zhang  Q, Liberati  NT, Lee  D, Urbach  J, Goodman  HM, Rahme  LG,     ( 2004 )

The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes.

Proceedings of the National Academy of Sciences of the United States of America 101 (8)
PMID : 14983043  :   DOI  :   10.1073/pnas.0304622101     PMC  :   PMC356984    
Abstract >>
The ubiquitous bacterium Pseudomonas aeruginosa is the quintessential opportunistic pathogen. Certain isolates infect a broad range of host organisms, from plants to humans. The pathogenic promiscuity of particular variants may reflect an increased virulence gene repertoire beyond the core P. aeruginosa genome. We have identified and characterized two P. aeruginosa pathogenicity islands (PAPI-1 and PAPI-2) in the genome of PA14, a highly virulent clinical isolate. The 108-kb PAPI-1 and 11-kb PAPI-2, which are absent from the less virulent reference strain PAO1, exhibit highly modular structures, revealing their complex derivations from a wide array of bacterial species and mobile elements. Most of the genes within these islands that are homologous to known genes occur in other human and plant bacterial pathogens. For example, PAPI-1 carries a complete gene cluster predicted to encode a type IV group B pilus, a well known adhesin absent from strain PAO1. However, >80% of the PAPI-1 DNA sequence is unique, and 75 of its 115 predicted ORF products are unrelated to any known proteins or functional domains. Significantly, many PAPI-1 ORFs also occur in several P. aeruginosa cystic fibrosis isolates. Twenty-three PAPI ORFs were mutated, and 19 were found to be necessary for full plant or animal virulence, with 11 required for both. The large set of "extra" virulence functions encoded by both PAPIs may contribute to the increased promiscuity of highly virulent P. aeruginosa strains, by directing additional pathogenic functions.
KeywordMeSH Terms
253. Head  NE, Yu  H,     ( 2004 )

Cross-sectional analysis of clinical and environmental isolates of Pseudomonas aeruginosa: biofilm formation, virulence, and genome diversity.

Infection and immunity 72 (1)
PMID : 14688090  :   DOI  :   10.1128/iai.72.1.133-144.2004     PMC  :   PMC343948    
Abstract >>
Chronic lung infections with Pseudomonas aeruginosa biofilms are associated with refractory and fatal pneumonia in cystic fibrosis (CF). In this study, a group of genomically diverse P. aeruginosa isolates were compared with the reference strain PAO1 to assess the roles of motility, twitching, growth rate, and overproduction of a capsular polysaccharide (alginate) in biofilm formation. In an in vitro biofilm assay system, P. aeruginosa displayed strain-specific biofilm formation that was not solely dependent on these parameters. Compared with non-CF isolates, CF isolates expressed two opposing growth modes: reduced planktonic growth versus efficient biofilm formation. Planktonic cells of CF isolates showed elevated sensitivity to hydrogen peroxide, a reactive oxygen intermediate, and decreased lung colonization in an aerosol infection mouse model. Despite having identical genomic profiles, CF sequential isolates produced different amounts of biofilm. While P. aeruginosa isolates exhibited genomic diversity, the genome size of these isolates was estimated to be 0.4 to 19% (27 to 1,184 kb) larger than that of PAO1. To identify these extra genetic materials, random amplification of polymorphic DNA was coupled with PAO1-subtractive hybridization. Three loci were found within the genomes of two CF isolates encoding one novel homolog involved in retaining a Shigella virulence plasmid (mvpTA) and two divergent genes that function in removing negative supercoiling (topA) and biosynthesis of pyoverdine (PA2402). Together, P. aeruginosa biodiversity could provide one cause for the variation of morbidity and mortality in CF. P. aeruginosa may possess undefined biofilm adhesins that are important to the development of an antibiofilm therapeutic target.
KeywordMeSH Terms
Genetic Variation
Pseudomonas aeruginosa
254. Ogino  H, Hiroshima  S, Hirose  S, Yasuda  M, Ishimi  K, Ishikawa  H,     ( 2004 )

Cloning, expression and characterization of a lipase gene (lip3) from Pseudomonas aeruginosa LST-03.

Molecular genetics and genomics : MGG 271 (2)
PMID : 14740297  :   DOI  :   10.1007/s00438-003-0970-8    
Abstract >>
A lipase gene (lip3) was cloned from the Pseudomonas aeruginosa strain LST-03 (which tolerates organic solvents) and expressed in Escherichia coli. The cloned sequence includes an ORF consisting of 945 nucleotides, encoding a protein of 315 amino acids (Lip3 lipase, 34.8 kDa). The predicted Lip3 lipase belongs to the class of serine hydrolases; the catalytic triad consists of the residues Ser-137, Asp-258, and His-286. The gene cloned in the present study does not encode the LST-03 lipase, a previously isolated solvent-stable lipase secreted by P. aeruginosa LST-03, because the N-terminal amino acid sequence of the Lip3 lipase differs from that of the LST-03 lipase. Although the effects of pH on the activity and stability of the Lip3 lipase, and the temperature optimum of the enzyme, were similar to those of the LST-03 lipase, the relative activity of the Lip3 lipase at lower temperatures (0-35 degrees C) was higher than that of the LST-03 lipase. In the absence of organic solvents, the half-life of the Lip3 lipase was similar to that of the LST-03 lipase. However, in the presence of most of the organic solvents tested in this study (the exceptions were ethylene glycol and glycerol), the stability of the Lip3 lipase was lower than that of the LST-03 lipase.
KeywordMeSH Terms
Gene Expression
255. Toleman  MA, Rolston  K, Jones  RN, Walsh  TR,     ( 2004 )

blaVIM-7, an evolutionarily distinct metallo-beta-lactamase gene in a Pseudomonas aeruginosa isolate from the United States.

Antimicrobial agents and chemotherapy 48 (1)
PMID : 14693560  :   DOI  :   10.1128/aac.48.1.329-332.2004     PMC  :   PMC310168    
Abstract >>
As part of the CANCER Antimicrobial Surveillance Program in North America, a Pseudomonas aeruginosa isolate, strain 07-406, was shown to possess a metallo-beta-lactamase, designated VIM-7. bla(VIM-7) is located on a 24-kb plasmid which can be readily transferred into Enterobacteriaceae and other pseudomonads. This is the first report of a mobile metallo-beta-lactamase gene, bla(VIM-7), being detected within the United States.
KeywordMeSH Terms
256. Moskowitz  SM, Ernst  RK, Miller  SI,     ( 2004 )

PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A.

Journal of bacteriology 186 (2)
PMID : 14702327  :   DOI  :   10.1128/jb.186.2.575-579.2004     PMC  :   PMC305751    
Abstract >>
Spontaneous polymyxin-resistant mutants of Pseudomonas aeruginosa were isolated. The mutations responsible for this phenotype were mapped to a two-component signal transduction system similar to PmrAB of Salmonella enterica serovar Typhimurium. Lipid A of these mutants contained aminoarabinose, an inducible modification that is associated with polymyxin resistance. Thus, P. aeruginosa possesses a mechanism that induces resistance to cationic antimicrobial peptides in response to environmental conditions.
KeywordMeSH Terms
Bacterial Proteins
257. Klockgether  J, Reva  O, Larbig  K, Tümmler  B,     ( 2004 )

Sequence analysis of the mobile genome island pKLC102 of Pseudomonas aeruginosa C.

Journal of bacteriology 186 (2)
PMID : 14702321  :   DOI  :   10.1128/jb.186.2.518-534.2004     PMC  :   PMC305764    
Abstract >>
The Pseudomonas aeruginosa plasmid pKLC102 coexists as a plasmid and a genome island in clone C strains. Whereas the related plasmid pKLK106 reversibly recombines with P. aeruginosa clone K chromosomes at one of the two tRNA(Lys) genes, pKLC102 is incorporated into the tRNA(Lys) gene only close to the pilA locus. Targeting of the other tRNA(Lys) copy in the chromosome is blocked by a 23,395-bp mosaic of truncated PAO open reading frames, transposons, and pKLC102 homologs. Annotation and phylogenetic analysis of the large 103,532-bp pKLC102 sequence revealed that pKLC102 is a hybrid of plasmid and phage origin. The plasmid lineage conferred oriV and genes for replication, partitioning, and conjugation, including a pil cluster encoding type IV thin sex pili and an 8,524-bp chvB glucan synthetase gene that is known to be a major determinant for host tropism and virulence. The phage lineage conferred integrase, att, and a syntenic set of conserved hypothetical genes also observed in the tRNA(Gly)-associated genome islands of P. aeruginosa clone C chromosomes. In subgroup C isolates from patients with cystic fibrosis, pKLC102 was irreversibly fixed into the chromosome by the insertion of the large 23,061-bp class I transposon TNCP23, which is a composite of plasmid, integron, and IS6100 elements. Intramolecular transposition of a copy of IS6100 led to chromosomal inversions and disruption of plasmid synteny. The case of pKLC102 in P. aeruginosa clone C documents the intraclonal evolution of a genome island from a mobile ancestor via a reversibly integrated state to irreversible incorporation and dissipation in the chromosome.
KeywordMeSH Terms
Genome, Bacterial
Plasmids
258. Yatsuyanagi  J, Saito  S, Harata  S, Suzuki  N, Ito  Y, Amano  K, Enomoto  K,     ( 2004 )

Class 1 integron containing metallo-beta-lactamase gene blaVIM-2 in Pseudomonas aeruginosa clinical strains isolated in Japan.

Antimicrobial agents and chemotherapy 48 (2)
PMID : 14742222  :   DOI  :   10.1128/aac.48.2.626-628.2004     PMC  :   PMC321541    
Abstract >>
Four bla(VIM-2) gene-harboring Pseudomonas aeruginosa strains were identified. These strains possessed a class 1 integron harboring ORF1, bla(VIM-2), and aacA4 gene cassettes. The transposon-mediated horizontal spread of the bla(VIM-2) gene among these strains was suggested, which increases the threat that the bla(VIM-2) gene will disseminate among diverse genera of bacteria.
KeywordMeSH Terms
259. Yokoyama  K, Doi  Y, Yamane  K, Kurokawa  H, Shibata  N, Shibayama  K, Yagi  T, Kato  H, Arakawa  Y,     ( 2003 )

Acquisition of 16S rRNA methylase gene in Pseudomonas aeruginosa.

Lancet (London, England) 362 (9399)
PMID : 14667745  :   DOI  :   10.1016/S0140-6736(03)14959-8    
Abstract >>
Bacteria develop resistance to aminoglycosides by producing aminoglycoside-modifying enzymes such as acetyltransferase, phosphorylase, and adenyltransferase. These enzymes, however, cannot confer consistent resistance to various aminoglycosides because of their substrate specificity. Notwithstanding, a Pseudomonas aeruginosa strain AR-2 showing high-level resistance (minimum inhibitory concentration >1024 mg/L) to various aminoglycosides was isolated clinically. We aimed to clone and characterise the genetic determinant of this resistance. We used conventional methods for DNA manipulation, susceptibility testing, and gene analyses to clone and characterise the genetic determinant of the resistance seen. PCR detection of the gene was also done on a stock of P aeruginosa strains that were isolated clinically since 1997. An aminoglycoside-resistance gene, designated rmtA, was identified in P aeruginosa AR-2. The Escherichia coli transformant and transconjugant harbouring the rmtA gene showed very high-level resistance to various aminoglycosides, including amikacin, tobramycin, isepamicin, arbekacin, kanamycin, and gentamicin. The 756-bp nucleotide rmtA gene encoded a protein, RmtA. This protein showed considerable similarity to the 16S rRNA methylases of aminoglycoside-producing actinomycetes, which protect bacterial 16S rRNA from intrinsic aminoglycosides by methylation. Incorporation of radiolabelled methyl groups into the 30S ribosome was detected in the presence of RmtA. Of 1113 clinically isolated P aeruginosa strains, nine carried the rmtA gene, as shown by PCR analyses. Our findings strongly suggest intergeneric lateral gene transfer of 16S rRNA methylase gene from some aminoglycoside-producing microorganisms to P aeruginosa. Further dissemination of the rmtA gene in nosocomial bacteria could be a matter of concern in the future.
KeywordMeSH Terms
260. Shibata  N, Doi  Y, Yamane  K, Yagi  T, Kurokawa  H, Shibayama  K, Kato  H, Kai  K, Arakawa  Y,     ( 2003 )

PCR typing of genetic determinants for metallo-beta-lactamases and integrases carried by gram-negative bacteria isolated in Japan, with focus on the class 3 integron.

Journal of clinical microbiology 41 (12)
PMID : 14662918  :   DOI  :   10.1128/jcm.41.12.5407-5413.2003     PMC  :   PMC309014    
Abstract >>
From January 2001 to December 2002, 587 strains of gram-negative bacterial isolates demonstrating resistance to ceftazidime and a combination of sulbactam and cefoperazone were subjected to a disk diffusion screening test using sodium mercaptoacetic acid; 431 strains (73.4%) appeared to produce metallo-beta-lactamase (MBL). Of these 431 strains, 357 were found by PCR to carry genes for IMP-1 type MBL (bla(IMP-1)), while only 7 and 67 strains carried the IMP-2 gene (bla(IMP-2)) and the VIM-2 gene (bla(VIM-2)), respectively. Neither VIM-1 nor SPM-1 type MBL genes were found among the strains tested. Of 431 strains, 427 carried the intI1 gene, and 4 strains carrying both the intI1 and intI3 genes were reidentified as Pseudomonas putida harboring bla(IMP-1). Of these four P. putida strains, three strains and one strain, respectively, were separately isolated from two hospitals located in the same prefecture, and the three strains showed very similar pulsed-field gel electrophoresis patterns. Of 357 bla(IMP-1) carriers, 116, 53, 51, 47, and 30 strains were identified as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, P. putida/fluorescens, Serratia marcescens, and Acinetobacter baumannii, respectively. Four strains carrying bla(IMP-2) were reidentified as P. putida. Sixty-three P. aeruginosa strains and four P. putida strains carried bla(VIM-2). Of 427 intI1-positive strains, 180, 53, 51, 47, and 35 were identified as P. aeruginosa, A. xylosoxidans, P. putida/fluorescens, S. marcescens, and A. baumannii, respectively. In the present study, it was confirmed that strains carrying bla(IMP-1) with a class 1 integron are the most prevalent type in Japan, although several intI3 carriers have also been identified sporadically in this country.
KeywordMeSH Terms
261. de Souza  JT, Mazzola  M, Raaijmakers  JM,     ( 2003 )

Conservation of the response regulator gene gacA in Pseudomonas species.

Environmental microbiology 5 (12)
PMID : 14641577  :  
Abstract >>
The response regulator gene gacA influences the production of several secondary metabolites in both pathogenic and beneficial Pseudomonas spp. In this study, we developed primers and a probe for the gacA gene of Pseudomonas species and sequenced a 425 bp fragment of gacA from ten Pseudomonas strains isolated from different plant-associated environments. Polymerase chain reaction analysis and Southern hybridization showed that gacA is highly conserved within the genus Pseudomonas: multiple strains of different Pseudomonas species all responded positively to the probe, whereas no response was obtained from 18 other strains representing 14 species that belong to eight different genera of Gram-negative bacteria other than Pseudomonas. Furthermore, from a total of approximately 550 indigenous bacterial isolates obtained from the rhizosphere of wheat, all isolates that hybridized with the gacA probe were classified as Pseudomonas spp. by group-specific primers. Isolates that did not respond with the gacA probe and primers were identified as bacterial genera other than Pseudomonas, including Stenotrophomonas, Cryseomonas and Comamonas spp. These results indicate that gacA can be used as a complementary genetic marker for detection of Pseudomonas spp. in environmental samples. Phylogenetic relationships inferred from the newly sequenced gacA fragments and the sequences of gacA homologues present in the databases, showed six distinct clusters that correspond to the following bacterial families: Pseudomonaceae, Enterobacteriaceae, Alteromonadaceae, Vibrionaceae, Burkholderia and Xanthomonas. Within the Pseudomonadaceae and Enterobacteriaceae, polymorphisms within gacA and its homologues allowed identification of six and five subclusters respectively. Comparison of the gacA gene and GacA protein-based trees with the tree inferred from 16S rDNA sequences yielded a similar overall clustering. These results suggest that gacA and its homologues may provide complementary markers for phylogenetic studies of Pseudomonas spp. and Gram-negative bacteria other than Pseudomonas.
KeywordMeSH Terms
Conserved Sequence
Genes, Regulator
262. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

The tmRNA website: reductive evolution of tmRNA in plastids and other endosymbionts.

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
263. Pritchard  AE, Vasil  ML,     ( 1990 )

Possible insertion sequences in a mosaic genome organization upstream of the exotoxin A gene in Pseudomonas aeruginosa.

Journal of bacteriology 172 (4)
PMID : 2156808  :   DOI  :   10.1128/jb.172.4.2020-2028.1990     PMC  :   PMC208700    
Abstract >>
Nucleotide sequence and Southern hybridization data revealed a mosaic genome organization in a region that extends several thousand base pairs upstream of the exotoxin A (toxA) gene in Pseudomonas aeruginosa. An interstrain comparison of DNA in this region showed a pattern of alternating segments of homologous and nonhomologous sequences. Two nonhomologous elements, approximately 1 kilobase pair upstream of the gene in strains PA103 and Ps388, were characterized in more detail. The sequence elements, denoted IS-PA-1 and IS-PA-2 for the different strains, are about 1,000 and 785 base pairs long, respectively, and have 5-base-pair direct repeats at their boundaries, consistent with their being DNA insertion sequences. The distribution of these elements in 34 different strains was determined. IS-PA-1 was found in a single copy upstream of toxA in half of the strains and was found in two copies in four of the strains. Some strains contained neither element, and one strain carried both. The genome of another strain, WR5, which lacks toxA, was shown to contain a 350-base-pair region that was highly homologous to DNA sequences located just upstream of toxA in other strains. The WR5 genome lacked several kilobase pairs of DNA that was found both upstream and downstream of this homologous region in the other strains.
KeywordMeSH Terms
ADP Ribose Transferases
Bacterial Toxins
DNA Transposable Elements
Genes, Bacterial
Virulence Factors
264. Drieux  L, Bourgeois-Nicolaos  N, Cremniter  J, Lawrence  C, Jarlier  V, Doucet-Populaire  F, Sougakoff  W,     ( 2011 )

Accumulation of carbapenemase-producing Gram-negative bacteria in a single patient linked to the acquisition of multiple carbapenemase producers and to the in vivo transfer of a plasmid encoding VIM-1.

International journal of antimicrobial agents 38 (2)
PMID : 21570257  :   DOI  :   10.1016/j.ijantimicag.2011.03.017    
Abstract >>
N/A
KeywordMeSH Terms
Gene Transfer, Horizontal
Plasmids
265. Ruiz-Martínez  L, López-Jiménez  L, Fusté  E, Vinuesa  T, Martínez  JP, Viñas  M,     ( 2011 )

Class 1 integrons in environmental and clinical isolates of Pseudomonas aeruginosa.

International journal of antimicrobial agents 38 (5)
PMID : 21873033  :   DOI  :   10.1016/j.ijantimicag.2011.06.016    
Abstract >>
The aims of this study were to ascertain the presence and spread of class 1 integrons amongst environmental and clinical isolates of Pseudomonas aeruginosa and to characterise their variable regions. A total of 76 isolates (56 clinical and 20 environmental) were studied. The presence of plasmids was explored, and polymerase chain reaction (PCR) was used for integron detection. All amplicons were sequenced. PCR detected class 1 integrons in 26 of the 56 clinical isolates; environmental isolates were integron-free. No plasmids were found, thus all the integrons found are possibly on the chromosome. Most isolates presented one amplicon, except PA110514 and PA116136, which showed two PCR products each. Variable regions revealed that 18 strains carried only one gene involved in aminoglycoside resistance, whereas in 3 strains gene cassettes were not found. The most prevalent cassettes amongst isolates were those encoding aminoglycoside adenyltransferase B (aadB). Several of the strains had acquired the same or a highly similar cassette array as those detected in geographically distant P. aeruginosa. This finding suggests that contact with bacterial reservoirs contributes to the evolution of this pathogen towards multiresistance. Empty structures found may represent a reservoir increasing the capacity to adapt to the environment. However, these integrons are not retained when the selective pressure disappears. It is hypothesised that integrons containing gene cassettes are crucial vehicles for the rapid horizontal transfer of resistance. If this is so, reduced use of antibiotics may lead to a significant decrease in the carriage of integrons amongst P. aeruginosa strains.
KeywordMeSH Terms
Water Microbiology
266. Bodilis  J, Nsigue Meilo  S, Cornelis  P, De Vos  P, Barray  S,     ( 2011 )

A long-branch attraction artifact reveals an adaptive radiation in pseudomonas.

Molecular biology and evolution 28 (10)
PMID : 21504889  :   DOI  :   10.1093/molbev/msr099    
Abstract >>
A significant proportion of protein-encoding gene phylogenies in bacteria is inconsistent with the species phylogeny. It was usually argued that such inconsistencies resulted from lateral transfers. Here, by further studying the phylogeny of the oprF gene encoding the major surface protein in the bacterial Pseudomonas genus, we found that the incongruent tree topology observed results from a long-branch attraction (LBA) artifact and not from lateral transfers. LBA in the oprF phylogeny could be explained by the faster evolution in a lineage adapted to the rhizosphere, highlighting an unexpected adaptive radiation. We argue that analysis of such artifacts in other inconsistent bacterial phylogenies could be a valuable tool in molecular ecology to highlight cryptic adaptive radiations in microorganisms.
KeywordMeSH Terms
267. Essar  DW, Eberly  L, Hadero  A, Crawford  IP,     ( 1990 )

Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications.

Journal of bacteriology 172 (2)
PMID : 2153661  :   DOI  :   10.1128/jb.172.2.884-900.1990     PMC  :   PMC208517    
Abstract >>
Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.
KeywordMeSH Terms
Biological Evolution
Genes, Bacterial
268. Edrington  TC, Kintz  E, Goldberg  JB, Tamm  LK,     ( 2011 )

Structural basis for the interaction of lipopolysaccharide with outer membrane protein H (OprH) from Pseudomonas aeruginosa.

The Journal of biological chemistry 286 (45)
PMID : 21865172  :   DOI  :   10.1074/jbc.M111.280933     PMC  :   PMC3234746    
Abstract >>
Pseudomonas aeruginosa is a major nosocomial pathogen that infects cystic fibrosis and immunocompromised patients. The impermeability of the P. aeruginosa outer membrane contributes substantially to the notorious antibiotic resistance of this human pathogen. This impermeability is partially imparted by the outer membrane protein H (OprH). Here we have solved the structure of OprH in a lipid environment by solution NMR. The structure reveals an eight-stranded �]-barrel protein with four extracellular loops of unequal size. Fast time-scale dynamics measurements show that the extracellular loops are disordered and unstructured. It was previously suggested that the function of OprH is to provide increased stability to the outer membranes of P. aeruginosa by directly interacting with lipopolysaccharide (LPS) molecules. Using in vivo and in vitro biochemical assays, we show that OprH indeed interacts with LPS in P. aeruginosa outer membranes. Based upon NMR chemical shift perturbations observed upon the addition of LPS to OprH in lipid micelles, we conclude that the interaction is predominantly electrostatic and localized to charged regions near both rims of the barrel, but also through two conspicuous tyrosines in the middle of the bilayer. These results provide the first molecular structure of OprH and offer evidence for multiple interactions between OprH and LPS that likely contribute to the antibiotic resistance of P. aeruginosa.
KeywordMeSH Terms
269. Mazzariol  A, Mammina  C, Koncan  R, Di Gaetano  V, Di Carlo  P, Cipolla  D, Corsello  G, Cornaglia  G,     ( 2011 )

A novel VIM-type metallo-beta-lactamase (VIM-14) in a Pseudomonas aeruginosa clinical isolate from a neonatal intensive care unit.

Clinical microbiology and infection : the official publication of the European Society of Clinical Microbiology and Infectious Diseases 17 (5)
PMID : 21521413  :   DOI  :   10.1111/j.1469-0691.2010.03424.x    
Abstract >>
A Pseudomonas aeruginosa highly resistant to carbapenems was isolated in a neonatal intensive care unit in Palermo, Italy. The strain was found to carry a novel VIM-type enzyme, classified as VIM-14. The novel enzyme differs from VIM-4 in a G31S mutation. VIM-14 was harboured in a class 1 integron with a new organization. The integron carried the genes aac7, blaVIM-14, blaOXA-20 and aac4 in that order.
KeywordMeSH Terms
Integrons
Intensive Care Units, Neonatal
270. Fontes  LC, Neves  PR, Oliveira  S, Silva  KC, Hachich  EM, Sato  MI, Lincopan  N,     ( 2011 )

Isolation of Pseudomonas aeruginosa coproducing metallo-�]-lactamase SPM-1 and 16S rRNA methylase RmtD1 in an urban river.

Antimicrobial agents and chemotherapy 55 (6)
PMID : 21464240  :   DOI  :   10.1128/AAC.00138-11     PMC  :   PMC3101456    
Abstract >>
N/A
KeywordMeSH Terms
271. Cervantes  C, Ohtake  H, Chu  L, Misra  TK, Silver  S,     ( 1990 )

Cloning, nucleotide sequence, and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505.

Journal of bacteriology 172 (1)
PMID : 2152903  :   DOI  :   10.1128/jb.172.1.287-291.1990     PMC  :   PMC208430    
Abstract >>
The chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505 was cloned into broad-host-range vector pSUP104. The hybrid plasmid containing an 11.1-kilobase insert conferred chromate resistance and reduced uptake of chromate in P. aeruginosa PAO1. Resistance to chromate was not expressed in Escherichia coli. Contiguous 1.6- and 6.3-kilobase HindIII fragments from this plasmid hybridized to pUM505 but not to P. aeruginosa chromosomal DNA and only weakly to chromate resistance plasmids pLHB1 and pMG6. Further subcloning produced a plasmid with an insert of 2,145 base pairs, which was sequenced. Analysis of deletions revealed that a single open reading frame was sufficient to determine chromate resistance. This open reading frame encodes a highly hydrophobic polypeptide, ChrA, of 416 amino acid residues that appeared to be expressed in E. coli under control of the T7 promoter. No significant homology was found between ChrA and proteins in the amino acid sequence libraries, but 29% amino acid identity was found with the ChrA amino acid sequence for another chromate resistance determinant sequenced in this laboratory from an Alcaligenes eutrophus plasmid (A. Nies, D. Nies, and S. Silver, submitted for publication).
KeywordMeSH Terms
Cloning, Molecular
Plasmids
272. Cuzon  G, Naas  T, Villegas  MV, Correa  A, Quinn  JP, Nordmann  P,     ( 2011 )

Wide dissemination of Pseudomonas aeruginosa producing beta-lactamase blaKPC-2 gene in Colombia.

Antimicrobial agents and chemotherapy 55 (11)
PMID : 21844315  :   DOI  :   10.1128/AAC.00297-11     PMC  :   PMC3195029    
Abstract >>
Ten bla(KPC-2)-harboring Pseudomonas aeruginosa isolates from hospitals located in five different Colombian cities have been characterized. Isolates were multidrug resistant, belonged to five different pulsotypes, and possessed naturally chromosome-encoded bla(AmpC) and bla(OXA-50) genes and the acquired bla(KPC-2) gene. In most cases, the bla(KPC-2) genes were carried by plasmids of different sizes and were associated with Tn4401b or a new structure containing only part of the Tn4401 sequence. This study revealed that several clones of P. aeruginosa producing bla(KPC-2) are disseminating in Colombia.
KeywordMeSH Terms
273. Lee  SH, Kim  JH, Mishra  D, Ni  YY, Rhee  YH,     ( 2011 )

Production of medium-chain-length polyhydroxyalkanoates by activated sludge enriched under periodic feeding with nonanoic acid.

Bioresource technology 102 (10)
PMID : 21463934  :   DOI  :   10.1016/j.biortech.2011.03.025    
Abstract >>
The potential use of activated sludge for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) was investigated. The enrichment of bacterial populations capable of producing MCL-PHAs was achieved by periodic feeding with nonanoic acid in a sequencing batch reactor (SBR). Denaturing gradient gel electrophoresis analysis revealed Pseudomonas aeruginosa strains to be predominant in the bacterial community during the SBR process. The composition of PHA synthesized by the enriched biomass from nonanoic acid consisted of a large concentration (>89 mol%) of MCL monomer units and a small amount of short-chain-length monomer units. Under fed-batch fermentation with continuous feeding of nonanoic acid at a flow rate of 0.225 g/L/h and a C/N ratio of 40, a maximum PHA content of 48.6% dry cell weight and a conversion yield (Y(p/s)) of 0.94 g/g were achieved. These results indicate that MCL-PHA production by activated sludge is a promising alternative to typical pure culture approaches.
KeywordMeSH Terms
Sewage
274. El Garch  F, Bogaerts  P, Bebrone  C, Galleni  M, Glupczynski  Y,     ( 2011 )

OXA-198, an acquired carbapenem-hydrolyzing class D beta-lactamase from Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 55 (10)
PMID : 21788473  :   DOI  :   10.1128/AAC.00522-11     PMC  :   PMC3186976    
Abstract >>
A carbapenem-resistant Pseudomonas aeruginosa strain (PA41437) susceptible to expanded-spectrum cephalosporins was recovered from several consecutive lower-respiratory-tract specimens of a patient who developed a ventilator-associated pneumonia while hospitalized in an intensive care unit. Cloning experiments identified OXA-198, a new class D �]-lactamase which was weakly related (less than 45% amino acid identity) to other class D �]-lactamases. Expression in Escherichia coli TOP10 and in P. aeruginosa PAO1 led to transformants that were resistant to ticarcillin and showed reduced susceptibility to carbapenems and cefepime. The bla(OXA-198) gene was harbored by a class 1 integron carried by a ca. 46-kb nontypeable plasmid. This study describes a novel class D �]-lactamase involved in carbapenem resistance in P. aeruginosa.
KeywordMeSH Terms
275. Khosravi  Y, Tay  ST, Vadivelu  J,     ( 2011 )

Analysis of integrons and associated gene cassettes of metallo-�]-lactamase-positive Pseudomonas aeruginosa in Malaysia.

Journal of medical microbiology 60 (Pt 7)
PMID : 21436370  :   DOI  :   10.1099/jmm.0.029868-0    
Abstract >>
In this study, 90 non-replicate imipenem-resistant Pseudomonas aeruginosa (IRPA) Malaysian isolates collected between October 2005 and March 2008 were subjected to a screening test for detection of the integron and the gene cassette. Class 1 integrons were detected in 54 IRPA clinical isolates, whilst three isolates contained class 2 integrons. Analysis of the gene cassettes associated with the class 1 integrons showed the detection of accC1 in isolates carrying bla(IMP-7) and aacA7 in isolates carrying bla(VIM-2). aadA6 was detected in two isolates carrying bla(IMP-4). Using random amplification of polymorphic DNA analysis, 14 PCR fingerprint patterns were generated from the 32 isolates carrying metallo-�]-lactamase (MBL) genes (35.5 %), whilst 20 patterns were generated from the 58 non-MBL gene isolates (64.4 %). Based on the differences in the fingerprinting patterns, two clusters (A and B) were identified among the MBL-producing isolates. Cluster A comprised 18 isolates (56 %) carrying the bla(VIM) gene, whereas cluster B comprised 14 (44 %) isolates carrying the bla(IMP) gene. The non-MBL isolates were divided into clusters C and D. Cluster C comprised 22 non-MBL isolates harbouring class 1 integrons, whilst cluster D consisted of three isolates carrying class 2 integrons. These findings suggest that the class 1 integron is widespread among P. aeruginosa isolated in Malaysia and that characterization of cassette arrays of integrons will be a useful epidemiological tool to study the evolution of multidrug resistance and the dissemination of antibiotic resistance genes.
KeywordMeSH Terms
276. Pinyon  JL, Hall  RM,     ( 2011 )

Evolution of IncP-1�\ plasmids by acquisition of antibiotic and mercuric ion resistance transposons.

Microbial drug resistance (Larchmont, N.Y.) 17 (3)
PMID : 21476866  :   DOI  :   10.1089/mdr.2010.0196    
Abstract >>
The aim of this study was to examine the relationship between the IncP-1�\ plasmid RP1 (representing RP4, RK2, R68, and R18) and two plasmids, R1033 and R934, that are known to be related to RP1. The region containing most of the antibiotic resistance genes in R1033 and R934 was mapped using polymerase chain reaction and sequenced. Both plasmids contained a copy of the class II transposon Tn1, but it was located at different sites between the position of Tn1 and the tet(A) determinant in RP1. Thus, Tn1 and the ampicillin resistance gene contained in it were acquired by the IncP1-�\ backbone on three separate occasions. In R1033, Tn1696 is located nearby, between Tn1 and the tet(A) determinant, and an IS186 insertion sequence was also found in this region. In R934, a defective class II transposon carrying a partial mercuric ion resistance (mer) module was found within the Tn1. These findings show that R1033 is not derived directly from RP1/RP4, refining the evolutionary pathways previously predicted for the IncP-1�\ plasmid family.
KeywordMeSH Terms
DNA Transposable Elements
277. Romão  C, Miranda  CA, Silva  J, Mandetta Clementino  M, de Filippis  I, Asensi  M,     ( 2011 )

Presence of qacE�G1 gene and susceptibility to a hospital biocide in clinical isolates of Pseudomonas aeruginosa resistant to antibiotics.

Current microbiology 63 (1)
PMID : 21479930  :   DOI  :   10.1007/s00284-011-9934-0    
Abstract >>
Biocides play an important role in healthcare-associated infection control by either minimizing or preventing microorganism dissemination. This study evaluated the susceptibility of Pseudomonas aeruginosa clinical isolates to a quaternary ammonium (QAC) disinfectant and antibiotics, and verified the presence of qacE�G1, a determinant of resistance to QAC. The disinfectant test was the Association of Official Analytical Chemists Use-Dilution Test, and polymerase chain reaction was used to examine for qacE�G1. The qacE�G1 gene was detected in 48% of the isolates. Eighty-eight percent of the multiresistant isolates carried qacE�G1 gene, while 35% of the non-multiresistant isolates was positive to this gene, and multiresistance well correlated with its presence. Among isolates tested for the disinfectant, 46% showed a reduced susceptibility to the disinfectant. qacE�G1 gene was present in 70% of the susceptible isolates to the biocide, whereas 90% of the less susceptible strains harbored this gene. Reduced susceptibility to the disinfectant was independent of presence of qacE�G1 suggesting that it does not play an important role in biocide resistance in P. aeruginosa. As far as we know, it is the first report confirming this fact and testing with disinfectant at its in-use concentration. The evidence of less susceptible strains than the reference bacterium used in disinfectant testing, and the high percentage of qacE�G1 gene detected are of special concern and suggests continued investigation in laboratory and in situ, not only in healthcare settings, but also in all areas of biocide usage, including different micro-organisms and biocides.
KeywordMeSH Terms
Drug Resistance, Bacterial
278. Rojo-Bezares  B, Estepa  V, de Toro  M, Undabeitia  E, Olarte  I, Torres  C, Sáenz  Y,     ( 2011 )

A novel class 1 integron array carrying blaVIM-2 genes and a new insertion sequence in a Pseudomonas aeruginosa strain isolated from a Spanish hospital.

Journal of medical microbiology 60 (Pt 7)
PMID : 21436368  :   DOI  :   10.1099/jmm.0.030973-0    
Abstract >>
N/A
KeywordMeSH Terms
279. Ramírez-Díaz  MI, Díaz-Magaña  A, Meza-Carmen  V, Johnstone  L, Cervantes  C, Rensing  C,     ( 2011 )

Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes.

Plasmid 66 (1)
PMID : 21421005  :   DOI  :   10.1016/j.plasmid.2011.03.002    
Abstract >>
We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.
KeywordMeSH Terms
Base Sequence
Operon
280. Totten  PA, Lory  S,     ( 1990 )

Characterization of the type a flagellin gene from Pseudomonas aeruginosa PAK.

Journal of bacteriology 172 (12)
PMID : 2123866  :   DOI  :   10.1128/jb.172.12.7188-7199.1990     PMC  :   PMC210844    
Abstract >>
Flagella in procaryotes are complex structures requiring the coordinate expression of over 50 genes, including flagellin, the major repeating structural protein. We have previously shown that a functional RpoN gene product is required for expression of flagellin in Pseudomonas aeruginosa PAK (P. A. Totten and S. Lory, J. Bacteriol. 172:389-396, 1990) and have now cloned, sequenced, and determined the transcriptional start site of the structural gene for this flagellin. The clones containing this gene produced a protein that reacted on Western immunoblots with polyclonal and four different monoclonal antibodies to purified flagella. However, this flagellin protein in Escherichia coli was slightly smaller (41 kDa) than flagellin protein produced in P. aeruginosa PAK (45 kDa), indicating degradation in E. coli or modification in P. aeruginosa. Comparison of the deduced amino acid sequence of this gene with the amino acid sequences of other flagellins revealed a conservation in the N- and C-terminal domains, suggesting conservation of secretion or assembly signals between these organisms. The sequence 5' of the structural gene contained potential RpoN-specific promoters as well as a promoter sequence recognized by RpoF (sigma 28), the alternative sigma factor required for expression of flagellin genes in E. coli (and Bacillus subtilis). Deletion analysis of the promoter region as well as transcriptional start site mapping implicated the RpoF, and not the RpoN, consensus sequences as the functional promoter for the flagellin gene. Models for the involvement of both RpoN and RpoF in the expression of flagellin in P. aeruginosa are presented.
KeywordMeSH Terms
Genes, Bacterial
281. Bailey  JK, Pinyon  JL, Anantham  S, Hall  RM,     ( 2011 )

Distribution of the blaTEM gene and blaTEM-containing transposons in commensal Escherichia coli.

The Journal of antimicrobial chemotherapy 66 (4)
PMID : 21393132  :   DOI  :   10.1093/jac/dkq529    
Abstract >>
The context of antibiotic resistance genes can provide valuable information about the epidemiology of mobile genetic elements. This study examined the distribution of the closely related blaTEM transposons Tn1, Tn2 and Tn3, or blaTEM-containing fragments of them, in ampicillin-resistant human commensal Escherichia coli isolates. A PCR mapping protocol was used to detect different segments of the transposons or to link partial copies to the insertion sequence IS26. Restriction digestion of one amplicon was used to assign transposons to Tn1, Tn2 or Tn3 groups and sequencing validated this approach. Restriction digestion and sequencing were used to determine how much of the transposon remained when blaTEM was linked to IS26. Sequences were compared with those in GenBank. Of 25 ampicillin-resistant E. coli strains recovered from the faecal flora of healthy humans that carried the blaTEM gene, 15 carried a complete copy of Tn2 or a Tn2 variant; one was interrupted by IS4. A further isolate carried Tn3. Tn2 was also most abundant in sequences available in GenBank. Two isolates carried Tn2 and an IS26-blaTEM fragment. The remaining 10 isolates carried only the blaTEM end of the transposon and 9 of these partial copies were flanked by IS26 at varying distances upstream of blaTEM. One configuration corresponded to that in Tn6029B and the complete transposon was shown to be present. Tn1, Tn2 and Tn3 can be simply and rapidly identified. Tn2 appears to be the most widely distributed. However, the blaTEM-containing end associated with an IS26 is also widely distributed.
KeywordMeSH Terms
DNA Transposable Elements
282. Tian  GB, Adams-Haduch  JM, Bogdanovich  T, Wang  HN, Doi  Y,     ( 2011 )

PME-1, an extended-spectrum �]-lactamase identified in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 55 (6)
PMID : 21402845  :   DOI  :   10.1128/AAC.01660-10     PMC  :   PMC3101390    
Abstract >>
A novel extended-spectrum �]-lactamase (ESBL) was identified in a Pseudomonas aeruginosa clinical isolate obtained from a patient admitted to a hospital in Pennsylvania in 2008. The patient had a prolonged hospitalization in a hospital in Dubai, United Arab Emirates, before being transferred to the United States. The novel ESBL, designated PME-1 (Pseudomonas aeruginosa ESBL 1), is a molecular class A, Bush-Jacoby-Medeiros group 2be enzyme and shared 50, 43, and 41% amino acid identity with the L2 �]-lactamase of Stenotrophomonas maltophilia, CTX-M-9, and KPC-2, respectively. PME-1 conferred clinically relevant resistance to ceftazidime, cefotaxime, cefepime, and aztreonam in P. aeruginosa PAO1 but not to carbapenems. Purified PME-1 showed good hydrolytic activity against ceftazidime, cefotaxime, and aztreonam, while activity against carbapenems and cefepime could not be measured. PME-1 was inhibited well by �]-lactamase inhibitors, including clavulanic acid, sulbactam, and tazobactam. The bla(PME-1) gene was carried by an approximately 9-kb plasmid and flanked by tandem ISCR24 elements.
KeywordMeSH Terms
283. Tuan  NN, Hsieh  HC, Lin  YW, Huang  SL,     ( 2011 )

Analysis of bacterial degradation pathways for long-chain alkylphenols involving phenol hydroxylase, alkylphenol monooxygenase and catechol dioxygenase genes.

Bioresource technology 102 (5)
PMID : 21227686  :   DOI  :   10.1016/j.biortech.2010.12.067    
Abstract >>
Eighteen 4-t-octylphenol-degrading bacteria were isolated and screened for the presence of degradative genes by polymerase chain reaction method using four designed primer sets. The primer sets were designed to amplify specific fragments from multicomponent phenol hydroxylase, single component monooxygenase, catechol 1,2-dioxygenase and catechol 2,3-dioxygenase genes. Seventeen of the 18 isolates exhibited the presence of a 232 bp amplicon that shared 61-92% identity to known multicomponent phenol hydroxylase gene sequences from short and/or medium-chain alkylphenol-degrading strains. Twelve of the 18 isolates were positive for a 324 bp region that exhibited 78-95% identity to the closest published catechol 1,2-dioxygenase gene sequences. The two strains, Pseudomonas putida TX2 and Pseudomonas sp. TX1, contained catechol 1,2-dioxygenase genes also have catechol 2,3-dioxygenase genes. Our result revealed that most of the isolated bacteria are able to degrade long-chain alkylphenols via multicomponent phenol hydroxylase and the ortho-cleavage pathway.
KeywordMeSH Terms
Environmental Microbiology
284. Okuda  K, Morihara  K, Atsumi  Y, Takeuchi  H, Kawamoto  S, Kawasaki  H, Suzuki  K, Fukushima  J,     ( 1990 )

Complete nucleotide sequence of the structural gene for alkaline proteinase from Pseudomonas aeruginosa IFO 3455.

Infection and immunity 58 (12)
PMID : 2123832  :   PMC  :   PMC313780    
Abstract >>
The DNA-encoding alkaline proteinase (AP) of Pseudomonas aeruginosa IFO 3455 was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, the gene-incorporated bacteria expressed high levels of both AP activity and AP antigens. The amino acid sequence deduced from the nucleotide sequence revealed that the mature AP consists of 467 amino acids with a relative molecular weight of 49,507. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified AP reported previously. The amino acid sequence analysis revealed that both the N-terminal side sequence of the purified AP and several internal lysyl peptide fragments were identical to the deduced amino acid sequences. The percent homology of amino acid sequences between AP and Serratia protease was about 55%. The zinc ligands and an active site of the AP were predicted by comparing the structure of the enzyme with of Serratia protease, thermolysin, Bacillus subtilis neutral protease, and Pseudomonas elastase.
KeywordMeSH Terms
Genes, Bacterial
285. Hoitink  CW, Woudt  LP, Turenhout  JC, van de Kamp  M, Canters  GW,     ( 1990 )

Isolation and sequencing of the Alcaligenes denitrificans azurin-encoding gene: comparison with the genes encoding blue copper proteins from Pseudomonas aeruginosa and Alcaligenes faecalis.

Gene 90 (1)
PMID : 2116366  :   DOI  :   10.1016/0378-1119(90)90434-s    
Abstract >>
The gene (azu) encoding azurin from Alcaligenes denitrificans has been cloned and sequenced. The gene codes for a pre-protein with a 19-aa signal peptide. Comparison with the sequences coding for the blue copper proteins from Pseudomonas aeruginosa and Alcaligenes faecalis reveals the presence of ntrA and fnr boxes in front of all three genes, instead of a regular [-10, -35]-promoter. In P. aeruginosa, the azu gene is terminated by a bidirectional terminator and flanked by open reading frames on the opposite strand.
KeywordMeSH Terms
Genes, Bacterial
286. Lassaux  P, Traoré  DA, Loisel  E, Favier  A, Docquier  JD, Sohier  JS, Laurent  C, Bebrone  C, Frère  JM, Ferrer  JL, Galleni  M,     ( 2011 )

Biochemical and structural characterization of the subclass B1 metallo-�]-lactamase VIM-4.

Antimicrobial agents and chemotherapy 55 (3)
PMID : 21149620  :   DOI  :   10.1128/AAC.01486-09     PMC  :   PMC3067066    
Abstract >>
The metallo-�]-lactamase VIM-4, mainly found in Pseudomonas aeruginosa or Acinetobacter baumannii, was produced in Escherichia coli and characterized by biochemical and X-ray techniques. A detailed kinetic study performed in the presence of Zn?+ at concentrations ranging from 0.4 to 100 �gM showed that VIM-4 exhibits a kinetic profile similar to the profiles of VIM-2 and VIM-1. However, VIM-4 is more active than VIM-1 against benzylpenicillin, cephalothin, nitrocefin, and imipenem and is less active than VIM-2 against ampicillin and meropenem. The crystal structure of the dizinc form of VIM-4 was solved at 1.9 ?. The sole difference between VIM-4 and VIM-1 is found at residue 228, which is Ser in VIM-1 and Arg in VIM-4. This substitution has a major impact on the VIM-4 catalytic efficiency compared to that of VIM-1. In contrast, the differences between VIM-2 and VIM-4 seem to be due to a different position of the flapping loop and two substitutions in loop 2. Study of the thermal stability and the activity of the holo- and apo-VIM-4 enzymes revealed that Zn?+ ions have a pronounced stabilizing effect on the enzyme and are necessary for preserving the structure.
KeywordMeSH Terms
287. Han  S, Zaniewski  RP, Marr  ES, Lacey  BM, Tomaras  AP, Evdokimov  A, Miller  JR, Shanmugasundaram  V,     ( 2010 )

Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa.

Proceedings of the National Academy of Sciences of the United States of America 107 (51)
PMID : 21135211  :   DOI  :   10.1073/pnas.1013092107     PMC  :   PMC3009787    
Abstract >>
Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by �]-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed �]-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.
KeywordMeSH Terms
Models, Molecular
288. Hirakawa  Y, Sasaki  H, Kawamoto  E, Ishikawa  H, Matsumoto  T, Aoyama  N, Kawasumi  K, Amao  H,     ( 2010 )

Prevalence and analysis of Pseudomonas aeruginosa in chinchillas.

BMC veterinary research 6 (N/A)
PMID : 21083906  :   DOI  :   10.1186/1746-6148-6-52     PMC  :   PMC2994850    
Abstract >>
Chinchillas (Chinchilla laniger) are popular as pets and are often used as laboratory animals for various studies. Pseudomonas aeruginosa is a major infectious agent that causes otitis media, pneumonia, septicaemia enteritis, and sudden death in chinchillas. This bacterium is also a leading cause of nosocomial infections in humans. To prevent propagation of P. aeruginosa infection among humans and animals, detailed characteristics of the isolates, including antibiotic susceptibility and genetic features, are needed. In this study, we surveyed P. aeruginosa distribution in chinchillas bred as pets or laboratory animals. We also characterized the isolates from these chinchillas by testing for antibiotic susceptibility and by gene analysis. P. aeruginosa was isolated from 41.8% of the 67 chinchillas included in the study. Slide agglutination and pulsed-field gel electrophoresis discriminated 5 serotypes and 7 unique patterns, respectively. For the antibiotic susceptibility test, 40.9% of isolates were susceptible to gentamicin, 77.3% to ciprofloxacin, 77.3% to imipenem, and 72.7% to ceftazidime. DNA analyses confirmed that none of the isolates contained the gene encoding extended-spectrum �]-lactamases; however, 2 of the total 23 isolates were found to have a gene similar to the pilL gene that has been identified in the pathogenicity island of a clinical isolate of P. aeruginosa. P. aeruginosa is widely spread in chinchillas, including strains with reduced susceptibility to the antibiotics and highly virulent strains. The periodic monitoring should be performed to help prevent the propagation of this pathogen and reduce the risk of infection from chinchillas to humans.
KeywordMeSH Terms
Chinchilla
289. Hrabák  J, Cervená  D, Izdebski  R, Duljasz  W, Gniadkowski  M, Fridrichová  M, Urbásková  P, Zemlicková  H,     ( 2011 )

Regional spread of Pseudomonas aeruginosa ST357 producing IMP-7 metallo-�]-lactamase in Central Europe.

Journal of clinical microbiology 49 (1)
PMID : 20980582  :   DOI  :   10.1128/JCM.00684-10     PMC  :   PMC3020450    
Abstract >>
N/A
KeywordMeSH Terms
290. Jin  Y, Yang  H, Qiao  M, Jin  S,     ( 2011 )

MexT regulates the type III secretion system through MexS and PtrC in Pseudomonas aeruginosa.

Journal of bacteriology 193 (2)
PMID : 21075931  :   DOI  :   10.1128/JB.01079-10     PMC  :   PMC3019812    
Abstract >>
The type III secretion system (T3SS) is the most important virulence factor in Pseudomonas aeruginosa, and its expression level varies in different isolates. We studied the molecular basis for such differences in two laboratory strains, PAK and PAO1. A chromosomal clone library from the high-T3SS-producer strain PAK was introduced into the low-producer strain PAO1, and we found that a mexS gene from PAK confers high T3SS expression in the PAO1 background. Further tests demonstrated that both mexS and its neighboring mexT gene are required for the repression of the T3SS in PAO1, while the PAK genome encodes a defective MexS, accounting for the derepression of the T3SS in PAK and the dominant negative effect when it is introduced into PAO1. MexS is a probable oxidoreductase whose expression is dependent on MexT, a LysR-type transcriptional regulator. Various genetic data support the idea that MexS modulates the transcriptional regulator function of MexT. In searching for the MexT-dependent repressor of the T3SS, a small gene product of PA2486 (ptrC) was found effective in suppressing the T3SS upon overexpression. However, deletion of ptrC in the PAO1 background did not result in derepression of the T3SS, indicating the presence of another repressor for the T3SS. Interestingly, overexpression of functional mexS alone was sufficient to repress T3SS even in the absence of MexT, suggesting that MexS is another mediator of MexT-dependent T3SS repression. Overexpression of mexS alone had no effect on the well-known MexT-dependent genes, including those encoding MexEF efflux pump, elastase, and pyocyanin, indicating alternative regulatory mechanisms. A model has been proposed for the MexS/MexT-mediated regulation of the T3SS, the MexEF efflux pump, and the production of elastase and pyocyanin.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
291. Hamood  AN, Iglewski  BH,     ( 1990 )

Expression of the Pseudomonas aeruginosa toxA positive regulatory gene (regA) in Escherichia coli.

Journal of bacteriology 172 (2)
PMID : 2105298  :   DOI  :   10.1128/jb.172.2.589-594.1990     PMC  :   PMC208481    
Abstract >>
The regA gene is a positive regulatory gene that regulates toxin A production in Pseudomonas aeruginosa at the transcriptional level. The product of the regA gene was examined in Escherichia coli with the expression vector pT7-7. A 1.3-kilobase AvaI-HindIII fragment containing the regA gene was cloned into the pT7-7 vector. A recombinant plasmid (pAH1) encoded a 29-kilodalton protein. The molecular weight of this protein correlated closely with the predicted molecular weight of the RegA protein. Production of the RegA protein in E. coli required both an E. coli promoter and an E. coli ribosome-binding site. Two in-frame deletion derivatives in which certain regions of the regA gene were expressed from the T7 promoter encoded 26- and 18-kilodalton fusion proteins, respectively. The RegA protein and the two fusion proteins were localized to the inner membrane of E. coli. Neither RegA protein nor the two fusion proteins showed DNA-binding activity to the 410-base-pair fragment containing the upstream region of toxA when synthesized in E. coli.
KeywordMeSH Terms
ADP Ribose Transferases
Genes, Bacterial
Genes, Regulator
Virulence Factors
292. Liu  W, Liu  X, Liao  J, Zhang  Y, Liang  X,     ( 2010 )

Identification of blaOXA-128 and blaOXA-129, two novel OXA-type extended-spectrum-�]-lactamases in Pseudomonas aeruginosa, in Hunan Province, China.

Journal of basic microbiology 50 Suppl 1 (N/A)
PMID : 20967789  :   DOI  :   10.1002/jobm.201000181    
Abstract >>
We collected 97 non-repetitive carbapenemases-sensitive clinical isolates of Pseudomonas aeruginosa in Human Province, China, during the period of October 2006 to January 2007. From these isolates, we identified two novel oxacillin-hydrolysing (OXA) type extended-spectrum-�]-lactamases (ESBLs): bla OXA-128 and bla OXA-129, which contain the mutations of I89V from bla OXA-56 and K134N from bla OXA-10, respectively. Clinical isolates containing either bla OXA-128 or bla OXA-129 show resistance to cephamycin-class antibiotics but sensitive to carbapenem-class antibiotics. The occurrence of novel OXA-type lactamases suggests a regional prevalent pattern of ESBLs Pseudomonas aeruginosa in this area.
KeywordMeSH Terms
293. Uemura  S, Yokota  S, Mizuno  H, Sakawaki  E, Sawamoto  K, Maekawa  K, Tanno  K, Mori  K, Asai  Y, Fujii  N,     ( 2010 )

Acquisition of a transposon encoding extended-spectrum beta-lactamase SHV-12 by Pseudomonas aeruginosa isolates during the clinical course of a burn patient.

Antimicrobial agents and chemotherapy 54 (9)
PMID : 20566763  :   DOI  :   10.1128/AAC.00110-10     PMC  :   PMC2934948    
Abstract >>
Three of seven clonally related Pseudomonas aeruginosa strains isolated from a burn patient produced the extended-spectrum beta-lactamase (ESBL) SHV-12. Its gene was flanked by two IS26 elements with a large transposon (>24 kb). The transposon also contained at least five IS26 elements and a gene encoding the amikacin resistance determinant aminoglycoside 6'-N-acetyltransferase type Ib [aac(6')-Ib]. It was inserted into the gene PA5317 in the P. aeruginosa chromosome.
KeywordMeSH Terms
294. Garza-Ramos  JU, Sanchez-Martinez  G, Barajas  JM, Suarez  S, Sanchez-Perez  A, Rojas-Moreno  T, Carrillo-Quiroz  B, Silva-Sanchez  J,     ( 2010 )

Variability of the bla(IMP-15)-containing integrons, highly related to In95, on an endemic clone of Pseudomonas aeruginosa in Mexico.

Microbial drug resistance (Larchmont, N.Y.) 16 (3)
PMID : 20617927  :   DOI  :   10.1089/mdr.2010.0017    
Abstract >>
The acquisition of �]-lactamases, such as class B metallo-�]-lactamases, in Pseudomonas aeruginosa is detrimental to antimicrobial therapy in hospitalized patients. In Mexico, metallo-�]-lactamase IMP-15 has been found to be encoded on the In95 class 1 integron in a major clone of P. aeruginosa. In this work, we describe the variability of this class 1 integron in an epidemic clone of carbapenem-resistant P. aeruginosa clinical isolates highly related to isolates previously described in Mexico.
KeywordMeSH Terms
Endemic Diseases
Genetic Variation
295. Chen  L, Wang  W, Sun  W, Surette  M, Duan  K,     ( 2010 )

Characterization of a cryptic plasmid from Pseudomonas sp. and utilization of its temperature-sensitive derivatives for genetic manipulation.

Plasmid 64 (2)
PMID : 20566404  :   DOI  :   10.1016/j.plasmid.2010.05.003    
Abstract >>
A new cryptic plasmid, named pMM101, in an environmental Pseudomonas sp. has been isolated, and its 2140-bp DNA sequence has been determined and analyzed. One open reading frame (Rep(MM)) similar to the rep gene of the rolling-circle replicon (RCR) group VIII has been allocated in the replication region where a putative double-strand origin dso and single-strand origin sso could also be identified. After in vitro mutagenesis by error-prone PCR and introduced into Pseudomonas aeruginosa PAO1, a mutant plasmid which was stably maintained at 30 degrees C but not at 42 degrees C was isolated and analyzed. It had two nucleotide substitutions in the putative sso and dso regions. Replacing one of the two sites with wild type sequence resulted in similar instability at 42 degrees C, indicating either mutation could result in the temperature-sensitive trait. Using the mutant replicon, a Pseudomonas (temperature-sensitive)-Escherichia coli shuttle vector, pUM109, has been constructed and used successfully to disrupt P. aeruginosa genes. This temperature-sensitive vector provides an alternative tool for genetic manipulations in this industrially as well as medically important bacterium.
KeywordMeSH Terms
296. Hotta  Y, Teramoto  K, Sato  H, Yoshikawa  H, Hosoda  A, Tamura  H,     ( 2010 )

Classification of genus Pseudomonas by MALDI-TOF MS based on ribosomal protein coding in S10-spc-alpha operon at strain level.

Journal of proteome research 9 (12)
PMID : 20945934  :   DOI  :   10.1021/pr100868d    
Abstract >>
We have proposed a rapid phylogenetic classification at the strain level by MALDI-TOF MS using ribosomal protein matching profiling. In this study, the S10-spc-alpha operon, encoding half of the ribosomal subunit proteins and highly conserved in eubacterial genomes, was selected for construction of the ribosomal protein database as biomarkers for bacterial identification by MALDI-TOF MS analysis to establish a more reliable phylogenetic classification. Our method revealed that the 14 reliable and reproducible ribosomal subunit proteins with less than m/z 15,000, except for L14, coded in the S10-spc-alpha operon were significantly useful biomarkers for bacterial classification at species and strain levels by MALDI-TOF MS analysis of genus Pseudomonas strains. The obtained phylogenetic tree was consisted with that based on genetic sequence (gyrB). Since S10-spc-alpha operons of genus Pseudomonas strains were sequenced using specific primers designed based on nucleotide sequences of genome-sequenced strains, the ribosomal subunit proteins encoded in S10-spc-alpha operon were suitable biomarkers for construction and correction of the database. MALDI-TOF MS analysis using these 14 selected ribosomal proteins is a rapid, efficient, and versatile bacterial identification method with the validation procedure for the obtained results.
KeywordMeSH Terms
297. Gurung  M, Moon  DC, Tamang  MD, Kim  J, Lee  YC, Seol  SY, Cho  DT, Lee  JC,     ( 2010 )

Emergence of 16S rRNA methylase gene armA and cocarriage of bla(IMP-1) in Pseudomonas aeruginosa isolates from South Korea.

Diagnostic microbiology and infectious disease 68 (4)
PMID : 20926221  :   DOI  :   10.1016/j.diagmicrobio.2010.07.021    
Abstract >>
Of the 100 multidrug-resistant Pseudomonas aeruginosa isolates from a Korean hospital, 14 isolates that were resistant to all aminoglycosides tested carried 16S rRNA methylase gene armA. Fourteen armA-positive isolates were classified into 8 pulsotypes. Seven armA-positive isolates cocarried bla(IMP-1). This study is the first report of occurrence of armA in P. aeruginosa.
KeywordMeSH Terms
298. Khosravi  Y, Tee Tay  S, Vadivelu  J,     ( 2010 )

Metallo-beta-lactamase-producing imipenem-resistant Pseudomonas aeruginosa clinical isolates in a university teaching hospital in Malaysia: detection of IMP-7 and first identification of IMP-4, VIM-2, and VIM-11.

Diagnostic microbiology and infectious disease 67 (3)
PMID : 20462725  :   DOI  :   10.1016/j.diagmicrobio.2010.02.010    
Abstract >>
Ninety (n = 90) imipenem-resistant Pseudomonas aeruginosa (IRPA) clinical isolates collected randomly during 2005 to 2008 from University Malaya Medical Center were assessed for the presence of different variants of metallo-beta-lactamase (MBL) genes. Polymerase chain reaction (PCR) assay detected 32 (n = 32) MBL gene PCR-positive isolates with the presence of bla(IMP) gene in 14 (n = 14) and bla(VIM) in 18 (n = 18) isolates. Four allelic variants, bla(IMP-7) (12 isolates), bla(IMP-4) (2 isolates), bla(VIM-2) (17 isolates), and bla(VIM-11) (1 isolate), of MBL genes were identified. This study is the first report of detection of bla(IMP-4), bla(VIM-2), and bla(VIM-11) MBL genes from IRPA clinical isolates in Malaysia.
KeywordMeSH Terms
beta-Lactam Resistance
299. Lassaux  P, Hamel  M, Gulea  M, Delbrück  H, Mercuri  PS, Horsfall  L, Dehareng  D, Kupper  M, Frère  JM, Hoffmann  K, Galleni  M, Bebrone  C,     ( 2010 )

Mercaptophosphonate compounds as broad-spectrum inhibitors of the metallo-beta-lactamases.

Journal of medicinal chemistry 53 (13)
PMID : 20527888  :   DOI  :   10.1021/jm100213c    
Abstract >>
Although commercialized inhibitors of active site serine beta-lactamases are currently used in coadministration with antibiotic therapy, no clinically useful inhibitors of metallo-beta-lactamases (MBLs) have yet been discovered. In this paper, we investigated the inhibitory effect of mercaptophosphonate derivatives against the three subclasses of MBLs (B1, B2, and B3). All 14 tested mercaptophosphonates, with the exception of 1a, behaved as competitive inhibitors for the three subclasses. Apart from 13 and 21, all the mercaptophosphonates tested exhibit a good inhibitory effect on the subclass B2 MBL CphA with low inhibition constants (K(i) < 15 muM). Interestingly, compound 18 turned out to be a potent broad spectrum MBL inhibitor. The crystallographic structures of the CphA-10a and CphA-18 complexes indicated that the sulfur atom of 10a and the phosphonato group of 18 interact with the Zn(2+) ion, respectively. Molecular modeling studies of the interactions between compounds 10a and 18 and the VIM-4 (B1), CphA (B2), and FEZ-1 (B3) enzymes brought to light different binding modes depending on the enzyme and the inhibitor, consistent with the crystallographic structures.
KeywordMeSH Terms
beta-Lactamase Inhibitors
300. Corich  L, Dolzani  L, Tonin  EA, Vitali  LA, Lagatolla  C,     ( 2010 )

Metallo-�]-lactamase expression confers an advantage to Pseudomonas aeruginosa isolates compared with other �]-lactam resistance mechanisms, favoring the prevalence of metallo-�]-lactamase producers in a clinical environment.

Microbial drug resistance (Larchmont, N.Y.) 16 (3)
PMID : 20735174  :   DOI  :   10.1089/mdr.2010.0016    
Abstract >>
The Pseudomonas aeruginosa isolate TS-832035 was responsible for an outbreak that occurred in an Italian hospital between 1999 and 2002. It exhibited a high-level resistance to carbapenems due to the contemporary presence of two independent mechanisms: the production of a carbapenemase, coded by a bla(VIM-1) determinant carried by the chromosomal class 1 integron In70.2 (containing also the aacA4, aphA15, and aadA1 genes in its cassette array), and the lack of the OprD porin. We compared TS-832035 with a strictly related isolate, TS-103, whose resistance to carbapenems was due to the lack of the OprD porin only, as it did not carry In70.2. We evaluated their growth kinetics, in both separate cultures and competition assays, under permissive conditions. These experiments highlighted a significant in vitro fitness cost associated with the integron. On the contrary, none of the resistance determinants other than the bla(VIM-1) seemed to confer a real selective advantage to its host. Comparison of these results with the in vivo behavior, showing that the In70.2-carrying isolates largely prevailed over the In70.2-lacking ones, besides the detection of similar integrons in other Italian clinical isolates, evidenced the need to investigate accurately the causes of their large distribution, as possible soft spots could exist in the ability of their hosts to adapt to the hospital settings.
KeywordMeSH Terms
beta-Lactam Resistance
301. Koh  TH, Khoo  CT, Tan  TT, Arshad  MA, Ang  LP, Lau  LJ, Hsu  LY, Ooi  EE,     ( 2010 )

Multilocus sequence types of carbapenem-resistant Pseudomonas aeruginosa in Singapore carrying metallo-beta-lactamase genes, including the novel bla(IMP-26) gene.

Journal of clinical microbiology 48 (7)
PMID : 20463166  :   DOI  :   10.1128/JCM.01905-09     PMC  :   PMC2897496    
Abstract >>
Nine imipenem-resistant Pseudomonas aeruginosa isolates were found to contain a variety of metallo-beta-lactamase genes, including bla(IMP-1), bla(IMP-7), bla(VIM-2), bla(VIM-6), and the novel bla(IMP-26). Multilocus sequence typing showed a diversity of sequence types. Comparison with isolates from an earlier study showed that the epidemic clones from 2000 have not become established.
KeywordMeSH Terms
Pseudomonas aeruginosa
302. Sharma  R, Gupta  R,     ( 2010 )

Extracellular expression of keratinase Ker P from Pseudomonas aeruginosa in E. coli.

Biotechnology letters 32 (12)
PMID : 20676920  :   DOI  :   10.1007/s10529-010-0361-2    
Abstract >>
Keratinase from Pseudomonas aeruginosa KS-1 was expressed constitutively as an extracellular protein in Escherichia coli with high specific activity of 3.7 kU/mg. It was purified fourfold as a 33 kDa monomeric protein by Q-Sepharose ion exchange chromatography with a recovery of 95%. It is a serine protease with optimal activity at pH 9 and 50�XC. It was stable from pH 4 to 12 for 1 h with a t(1/2) of 12 min at 70�XC. It hydrolyzed haemoglobin > fibrin > feather keratin > azo-casein > casein > meat protein > gelatin. Among synthetic substrates, it efficiently hydrolyzed N-Suc-ala-ala-pro-phe-pNA, N-Suc-ala-ala-ala-pNA, N-Suc-ala-ala-pro-leu-pNA and also plasmin substrate, D: -Val-Leu-Lys-pNA.
KeywordMeSH Terms
Gene Expression
303. Alguel  Y, Lu  D, Quade  N, Sauter  S, Zhang  X,     ( 2010 )

Crystal structure of MexZ, a key repressor responsible for antibiotic resistance in Pseudomonas aeruginosa.

Journal of structural biology 172 (3)
PMID : 20691272  :   DOI  :   10.1016/j.jsb.2010.07.012    
Abstract >>
Pseudomonas aeruginosa is responsible for around 10% of all hospital-acquired infections and the single most important pathogen of cystic fibrosis lungs. P. aeruginosa has high intrinsic and acquired antibiotic resistance, due to the extrusion of antibiotics by multidrug efflux pumps. The gene regulator MexZ controls the expression of mexXY, the efflux pump responsible for resistance to many drugs that are used for treating CF patients. MexZ is shown to be the most frequently mutated gene in P. aeruginosa isolated from CF patient lungs, confirming its importance in multidrug resistance. Here we present the crystal structure of MexZ at 2.9?. Combining the structural information with biochemical data on key mutants identified, we provide an explanation for the structural and functional consequences of these mutants. This work provides a framework for further characterisation of MexZ in order to fully understand its regulation and induction.
KeywordMeSH Terms
304. Smyth  TJ, Perfumo  A, Marchant  R, Banat  IM, Chen  M, Thomas  RK, Penfold  J, Stevenson  PS, Parry  NJ,     ( 2010 )

Directed microbial biosynthesis of deuterated biosurfactants and potential future application to other bioactive molecules.

Applied microbiology and biotechnology 87 (4)
PMID : 20405122  :   DOI  :   10.1007/s00253-010-2592-5    
Abstract >>
Deuterated rhamnolipids were produced using strain AD7 of Pseudomonas aeruginosa, which was progressively adapted to increasing levels of deuterium in D(2)O and carbon substrates. Fourteen different deuterated rhamnolipid structures, including structural isomers, were produced which is similar to normal protonated structures. There were two main products monorhamnolipid Rha-C(10)-C(10) and dirhamnolipid Rha(2)-C(10)-C(10). The levels of deuteration varied from 16% with 25% D(2)O + h-glycerol to 90% with 100% D(2)O + d-glycerol. When d-tetradecane was used with H(2)O, virtually all the deuterium appeared in the lipid chains while using h-tetradecane + D(2)O led to the majority of deuterium in the sugars. The adaptation to growth in deuterium appeared to be metabolic since no genetic changes could be found in the key rhamnolipid biosynthetic genes, the rhamnosyl transferases RhlB and RhlC. Deuterated sophorolipids were similarly produced using Candida bombicola and Candida apicola although in this case, no adaptation process was necessary. Up to 40 different sophorolipids were produced by these yeasts. However, unlike the rhamnolipids, use of D(2)O did not lead to any deuteration of the lipid chains, but direct incorporation into the lipid was achieved using d-isostearic acid. The results from these experiments show the feasibility of producing deuterated bioactive compounds from microorganisms coupled with the possibility of manipulating the pattern of labelling through judicious use of different deuterated substrates.
KeywordMeSH Terms
305. Giltner  CL, Rana  N, Lunardo  MN, Hussain  AQ, Burrows  LL,     ( 2011 )

Evolutionary and functional diversity of the Pseudomonas type IVa pilin island.

Environmental microbiology 13 (1)
PMID : 20738375  :   DOI  :   10.1111/j.1462-2920.2010.02327.x    
Abstract >>
In Pseudomonas aeruginosa, most proteins involved in type IVa pilus (T4aP) biogenesis are highly conserved except for the major pilin PilA and the minor pilins involved in pilus assembly. Here we show that each of the five major pilin alleles is associated with a specific set of minor pilins, and unrelated strains with the same major pilin type have identical minor pilin genes. The sequences of the minor pilin genes of strains with group III and V pilins are identical, suggesting that these groups diverged recently through further evolution of the major pilin cluster. Both gene clusters are localized on a single 'pilin island' containing putative tRNA recombinational hotspots, and a similar organization of pilin genes was identified in other Pseudomonas species. To address the biological significance of group-specific differences, cross-complementation studies using group II (PAO1) and group III (PA14) minor pilins were performed. Heterologous minor pilins complemented twitching motility to various extents except in the case of PilX, which was non-functional in non-native backgrounds. A recombinant PA14 strain expressing the PAO1 minor pilins regained motility only upon co-introduction of the PA14 pilX gene. Comparison of PilX and PilQ secretin sequences from group II, III and V genomes revealed discrete regions of sequence that co-varied between groups. Our data suggest that changes in PilX sequence have led to compensatory changes in the PilQ secretin monomer such that heterologous PilX proteins are no longer able to promote opening of the secretin to allow pili to appear on the cell surface.
KeywordMeSH Terms
Multigene Family
306. Wang  J, Zhou  JY, Qu  TT, Shen  P, Wei  ZQ, Yu  YS, Li  LJ,     ( 2010 )

Molecular epidemiology and mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from Chinese hospitals.

International journal of antimicrobial agents 35 (5)
PMID : 20185276  :   DOI  :   10.1016/j.ijantimicag.2009.12.014    
Abstract >>
We investigated the molecular epidemiology and carbapenem resistance mechanisms of 258 non-duplicate carbapenem-resistant clinical isolates of Pseudomonas aeruginosa collected from 2006 to 2007 at 28 hospitals in China. Up to 88% of the carbapenem-resistant isolates were multidrug-resistant. Pulsed-field gel electrophoresis (PFGE) revealed that levels of intrahospital and interhospital dissemination of clones were low. To assess the mechanisms leading to resistance, all 258 carbapenem-resistant isolates were analysed for expression of the chromosomal beta-lactamase (AmpC), the porin important for entry of carbapenems (OprD) and an efflux system (MexAB-OprM) known to extrude some beta-lactams. Carbapenem resistance was driven mainly by mutational inactivation of OprD, accompanied or not by hyperexpression of AmpC or MexAB-OprM. Metallo-beta-lactamase genes were detected in 22 carbapenem-resistant isolates in China, belonging to eight pulsotypes. The bla(OXA-50) gene was detected among all of the carbapenem-resistant isolates, whereas the bla(GES-5) gene was detected in only one carbapenem-resistant isolate.
KeywordMeSH Terms
Bacterial Typing Techniques
beta-Lactam Resistance
307. Cholley  P, Hocquet  D, Alauzet  C, Cravoisy-Popovic  A, Talon  D, Aissa  N, Plésiat  P, Bertrand  X,     ( 2010 )

Hospital outbreak of Pseudomonas aeruginosa producing extended-spectrum oxacillinase OXA-19.

Journal of medical microbiology 59 (Pt 7)
PMID : 20299501  :   DOI  :   10.1099/jmm.0.019364-0    
Abstract >>
N/A
KeywordMeSH Terms
Disease Outbreaks
308. Wang  W, Wang  L, Shao  Z,     ( 2010 )

Diversity and abundance of oil-degrading bacteria and alkane hydroxylase (alkB) genes in the subtropical seawater of Xiamen Island.

Microbial ecology 60 (2)
PMID : 20683589  :   DOI  :   10.1007/s00248-010-9724-4    
Abstract >>
In this report, the diversity of oil-degrading bacteria and alkB gene was surveyed in the seawater around Xiamen Island. Forty-four isolates unique in 16S rRNA sequence were obtained after enrichment with crude oil. Most of the obtained isolates exhibited growth with diesel oil and crude oil. alkB genes were positively detected in 16 isolates by degenerate polymerase chain reaction (PCR). And for the first time, alkB genes were found in bacteria of Gallaecimonas, Castellaniella, Paracoccus, and Leucobacter. Additional 29 alkB sequences were retrieved from genomic DNA of the oil-degrading communities. Phylogenetic analysis showed that the obtained alkB genes formed five groups, most of which exhibited 60-80% similarity at the amino acid level with sequences retrieved from the GenBank database. Furthermore, the abundance of alkB genes in seawater was examined by real-time PCR. The results showed that alkB genes of each group in situ ranged from about 3 �� 10(3) to 3 �� 10(5) copies L(-1), with the homologs of Alcanivorax and Pseudomonas being the most predominant. Bacteria of Alcanivorax, Acinetobacter, and Pseudomonas are important oil degraders in this area; while those frequently reported in other area, like Oleiphilus spp., Oleispira spp., and Thalassolituus spp. were not found in our report. These results indicate that bacteria and genes involved in oil degradation are quite diverse, and may have restriction in geographic distribution in some species.
KeywordMeSH Terms
Biodiversity
309. Docquier  JD, Benvenuti  M, Calderone  V, Giuliani  F, Kapetis  D, De Luca  F, Rossolini  GM, Mangani  S,     ( 2010 )

Crystal structure of the narrow-spectrum OXA-46 class D beta-lactamase: relationship between active-site lysine carbamylation and inhibition by polycarboxylates.

Antimicrobial agents and chemotherapy 54 (5)
PMID : 20145076  :   DOI  :   10.1128/AAC.01517-09     PMC  :   PMC2863608    
Abstract >>
Class D beta-lactamases represent a heterogeneous group of active-site serine beta-lactamases that show an extraordinary panel of functional features and substrate profiles, thus representing relevant models for biochemical and structural studies. OXA-46 is a narrow-spectrum enzyme belonging to the OXA-2 subgroup which was found in a Pseudomonas aeruginosa clinical isolate from northern Italy. In this work, we obtained the three-dimensional structure of OXA-46, which shows the overall fold of active serine beta-lactamases and a dimeric quaternary structure. Significant differences with currently available structures of class D beta-lactamases were found in the loops located close to the active site, which differ in length and conformation. Interestingly, the three subunits present in the asymmetric unit showed some structural heterogeneity, only one of which presented a carbamylated lysine recognized as an important functional feature of class D enzymes. The carbamylation state of residue Lys75 appeared to be associated with different shapes and dimensions of the active site. Moreover, a tartrate molecule from the crystallization buffer was found in the active site of the noncarbamylated subunits, which interacts with catalytically relevant residues. The OXA-46 crystal asymmetric units thus interestingly present the structures of the free carbamylated active site and of the ligand-bound uncarbamylated active site, offering the structural basis for investigating the potential of new scaffolds of beta-lactamase inhibitors.
KeywordMeSH Terms
310. Deligianni  E, Pattison  S, Berrar  D, Ternan  NG, Haylock  RW, Moore  JE, Elborn  SJ, Dooley  JS,     ( 2010 )

Pseudomonas aeruginosa cystic fibrosis isolates of similar RAPD genotype exhibit diversity in biofilm forming ability in vitro.

BMC microbiology 10 (N/A)
PMID : 20141637  :   DOI  :   10.1186/1471-2180-10-38     PMC  :   PMC2841157    
Abstract >>
Pseudomonas aeruginosa is considered to grow in a biofilm in cystic fibrosis (CF) chronic lung infections. Bacterial cell motility is one of the main factors that have been connected with P. aeruginosa adherence to both biotic and abiotic surfaces. In this investigation, we employed molecular and microscopic methods to determine the presence or absence of motility in P. aeruginosa CF isolates, and statistically correlated this with their biofilm forming ability in vitro. Our investigations revealed a wide diversity in the production, architecture and control of biofilm formation. Of 96 isolates, 49% possessed swimming motility, 27% twitching and 52% swarming motility, while 47% were non-motile. Microtitre plate assays for biofilm formation showed a range of biofilm formation ability from biofilm deficient phenotypes to those that formed very thick biofilms. A comparison of the motility and adherence properties of individual strains demonstrated that the presence of swimming and twitching motility positively affected biofilm biomass. Crucially, however, motility was not an absolute requirement for biofilm formation, as 30 non-motile isolates actually formed thick biofilms, and three motile isolates that had both flagella and type IV pili attached only weakly. In addition, CLSM analysis showed that biofilm-forming strains of P. aeruginosa were in fact capable of entrapping non-biofilm forming strains, such that these 'non-biofilm forming' cells could be observed as part of the mature biofilm architecture. Clinical isolates that do not produce biofilms in the laboratory must have the ability to survive in the patient lung. We propose that a synergy exists between isolates in vivo, which allows non biofilm-forming" isolates to be incorporated into the biofilm. Therefore, there is the potential for strains that are apparently non-biofilm forming in vitro to participate in biofilm-mediated pathogenesis in the CF lung."
KeywordMeSH Terms
Biofilms
311. Glupczynski  Y, Bogaerts  P, Deplano  A, Berhin  C, Huang  TD, Van Eldere  J, Rodriguez-Villalobos  H,     ( 2010 )

Detection and characterization of class A extended-spectrum-beta-lactamase-producing Pseudomonas aeruginosa isolates in Belgian hospitals.

The Journal of antimicrobial chemotherapy 65 (5)
PMID : 20200037  :   DOI  :   10.1093/jac/dkq048    
Abstract >>
To investigate the presence of extended-spectrum beta-lactamases (ESBLs) among Pseudomonas aeruginosa clinical isolates referred to two Belgian reference laboratories. Antibiograms were analysed for P. aeruginosa isolates referred between 2004 and 2008. Isolates resistant to ceftazidime (MIC > 8 mg/L) and with a positive double-disc synergy test between ceftazidime and clavulanate were serotyped and screened for the presence of ESBL-encoding genes. Genes encoding metallo-beta-lactamases (bla(MBL)) were sought by PCR in ESBL-producing isolates with positive imipenem/EDTA synergy tests. PFGE of SpeI-digested genomic DNA was used to compare isolates and selected strains were characterized by multilocus sequence typing. Forty-eight (2.2%) of 2150 P. aeruginosa isolates were confirmed as class A ESBL-producing isolates by molecular testing. bla(BEL) and bla(PER) alleles were detected, respectively, in 39 and 10 P. aeruginosa isolates originating from 16 hospitals (two isolates were simultaneously positive for BEL and PER). Fifteen of the isolates were found to co-produce ESBLs and VIM carbapenemases. These strains were pan-resistant and remained susceptible only to colistin (MICs
KeywordMeSH Terms
312. Coyne  S, Courvalin  P, Galimand  M,     ( 2010 )

Acquisition of multidrug resistance transposon Tn6061 and IS6100-mediated large chromosomal inversions in Pseudomonas aeruginosa clinical isolates.

Microbiology (Reading, England) 156 (Pt 5)
PMID : 20110294  :   DOI  :   10.1099/mic.0.033639-0    
Abstract >>
Pseudomonas aeruginosa is a major human opportunistic pathogen, especially for patients in intensive care units or with cystic fibrosis. Multidrug resistance is a common feature of this species. In a previous study we detected the ant(4')-IIb gene in six multiresistant clinical isolates of P. aeruginosa, and determination of the environment of the gene led to characterization of Tn6061. This 26 586 bp element, a member of the Tn3 family of transposons, carried 10 genes conferring resistance to six drug classes. The ant(4')-IIb sequence was flanked by directly repeated copies of ISCR6 in all but one of the strains studied, consistent with ISCR6-mediated gene acquisition. Tn6061 was chromosomally located in six strains and plasmid-borne in the remaining isolate, suggesting horizontal acquisition. Duplication-insertion of IS6100, that ended Tn6061, was responsible for large chromosomal inversions. Acquisition of Tn6061 and chromosomal inversions are further examples of intricate mechanisms that contribute to the genome plasticity of P. aeruginosa.
KeywordMeSH Terms
Chromosome Inversion
Chromosomes, Bacterial
DNA Transposable Elements
313. Jeong  JH, Shin  KS, Lee  JW, Park  EJ, Son  SY,     ( 2009 )

Analysis of a novel class 1 integron containing metallo-beta-lactamase gene VIM-2 in Pseudomonas aeruginosa.

Journal of microbiology (Seoul, Korea) 47 (6)
PMID : 20127470  :   DOI  :   10.1007/s12275-008-0272-2    
Abstract >>
Carbapenems such as imipenem are stable to most beta-lactamases. Recently, increased numbers of carbapenemase producing Gram-negative bacterial strains have been isolated because of the increased use of cabapenems. In this respect, control of these infectious carbapenemase producing Gram-negative bacteria and understanding their resistance mechanism are becoming more important. These carbapenem-hydrolyzing beta-lactamase genes have been reported to exist mostly as gene cassettes in an integron. This implies that antibiotic resistance genes may be transferred to other bacteria via the integron. In the present study, we identified and analyzed an integron containing VIM-2 type metallo-beta-lactamase gene in a carbapenemase producing Pseudomonas aeruginosa. In addition, the possibility of resistance spread by integron located in a plasmid was tested. Among glucose non-fermenting Gram-negative bacilli with reduced imipenem susceptibility (MIC > or = 8 microg/ml) isolated from Korean patients, P. aeruginosa 1082 showed resistance to most beta-lactams, cephalosporin, and aminoglycoside. We found that P. aeruginosa 1082 was inhibited by EDTA in EDTA double disk synergy test which means that this strain produces metallo-beta-lactamase. Class 1 integron containing bla (VIM-2) (carbapenem resistance gene), qacF (quaternary ammonium compound resistance gene), aacA4 (aminoglycoside resistance gene), catB3 (chloramphenicol resistance gene), bla (oxa-30) (extended-spectrum beta-lactam resistance gene), and aadAl (aminoglycoside resistance gene) gene cassettes was detected in P. aeruginosa 1082. The size of the integron was 5,246 bp and the structure and arrangement of the integron was a novel one in comparison with other integrons found in other P. aeruginosa. The integron could be transferred to Escherichia coli JM109 from P. aeruginosa 1082 possibly via self-transferable plasmid DNA. The integron and a bla (VIM-2) gene were detected in the plasmid DNA of the transconjugants whose imipenem resistance was slightly increased as a result of accepting the integron from the donor strain.
KeywordMeSH Terms
beta-Lactam Resistance
Integrons
314. Rahman  RN, Geok  LP, Wong  CF, Basri  M, Salleh  AB,     ( 2010 )

Molecular investigation of a gene encoding organic solvent-tolerant alkaline protease from Pseudomonas aeruginosa strain K.

Journal of basic microbiology 50 (2)
PMID : 20082370  :   DOI  :   10.1002/jobm.200900133    
Abstract >>
A gene encoding an organic solvent-stable protease was amplified from Pseudomonas aeruginosa strain K by polymerase chain reaction using consensus primers based on multiple sequence alignment of alkaline and metalloprotease genes from Pseudomonas species. The gene, which consisted of 1440 bp nucleotides and deduced 479 amino acid residues, was successfully expressed in pGEX-4T-1 expression system in the presence of 1.0 mM IPTG, after an incubation of 6 h at 37 degrees C. Under these conditions, the recombinant strain K protease was, subsequently, released into the periplasm of E. coli BL21 (DE3) with an optimum proteolytic activity detected at 1.0112 U/ml. To date, this is the first reported expression of alkaline protease (aprA) with such remarkable property in Escherichia coli.
KeywordMeSH Terms
315. Petrovski  S, Stanisich  VA,     ( 2010 )

Tn502 and Tn512 are res site hunters that provide evidence of resolvase-independent transposition to random sites.

Journal of bacteriology 192 (7)
PMID : 20118251  :   DOI  :   10.1128/JB.01322-09     PMC  :   PMC2838034    
Abstract >>
In this study, we report on the transposition behavior of the mercury(II) resistance transposons Tn502 and Tn512, which are members of the Tn5053 family. These transposons exhibit targeted and oriented insertion in the par region of plasmid RP1, since par-encoded components, namely, the ParA resolvase and its cognate res region, are essential for such transposition. Tn502 and, under some circumstances, Tn512 can transpose when par is absent, providing evidence for an alternative, par-independent pathway of transposition. We show that the alternative pathway proceeds by a two-step replicative process involving random target selection and orientation of insertion, leading to the formation of cointegrates as the predominant product of the first stage of transposition. Cointegrates remain unresolved because the transposon-encoded (TniR) recombination system is relatively inefficient, as is the host-encoded (RecA) system. In the presence of the res-ParA recombination system, TniR-mediated (and RecA-mediated) cointegrate resolution is highly efficient, enabling resolution both of cointegrates involving functional transposons (Tn502 and Tn512) and of defective elements (In0 and In2). These findings implicate the target-encoded accessory functions in the second stage of transposition as well as in the first. We also show that the par-independent pathway enables the formation of deletions in the target molecule.
KeywordMeSH Terms
DNA Transposable Elements
Plasmids
Recombination, Genetic
316. Fournier  D, Hocquet  D, Dehecq  B, Cholley  P, Plésiat  P,     ( 2010 )

Detection of a new extended-spectrum oxacillinase in Pseudomonas aeruginosa.

The Journal of antimicrobial chemotherapy 65 (2)
PMID : 20008045  :   DOI  :   10.1093/jac/dkp438    
Abstract >>
N/A
KeywordMeSH Terms
beta-Lactam Resistance
317. Madan  B, Mishra  P,     ( 2010 )

Co-expression of the lipase and foldase of Pseudomonas aeruginosa to a functional lipase in Escherichia coli.

Applied microbiology and biotechnology 85 (3)
PMID : 19629472  :   DOI  :   10.1007/s00253-009-2131-4    
Abstract >>
The lipA gene, a structural gene encoding for protein of molecular mass 48 kDa, and lipB gene, encoding for a lipase-specific chaperone with molecular mass of 35 kDa, of Pseudomonas aeruginosa B2264 were co-expressed in heterologous host Escherichia coli BL21 (DE3) to obtain in vivo expression of functional lipase. The recombinant lipase was expressed with histidine tag at its N terminus and was purified to homogeneity using nickel affinity chromatography. The amino acid sequence of LipA and LipB of P. aeruginosa B2264 was 99-100% identical with the corresponding sequence of LipA and LipB of P. aeruginosa LST-03 and P. aeruginosa PA01, but it has less identity with Pseudomonas cepacia (Burkholderia cepacia) as it showed only 37.6% and 23.3% identity with the B. cepacia LipA and LipB sequence, respectively. The molecular mass of the recombinant lipase was found to be 48 kDa. The recombinant lipase exhibited optimal activity at pH 8.0 and 37 degrees C, though it was active between pH 5.0 and pH 9.0 and up to 45 degrees C. K (m) and V (max) values for recombinant P. aeruginosa lipase were found to be 151.5 +/- 29 microM and 217 +/- 22.5 micromol min(-1) mg(-1) protein, respectively.
KeywordMeSH Terms
318. Yang  TH, Kwon  MA, Song  JK, Pan  JG, Rhee  JS,     ( 2010 )

Functional display of Pseudomonas and Burkholderia lipases using a translocator domain of EstA autotransporter on the cell surface of Escherichia coli.

Journal of biotechnology 146 (3)
PMID : 20138931  :   DOI  :   10.1016/j.jbiotec.2010.01.022    
Abstract >>
Functional expression of the industrially important Pseudomonas and Burkholderia lipases, such as those from P. aeruginosa, B. cepacia and P. fluorescens, was achieved on the cell surface of Escherichia coli using the C-terminal translocator moiety (EstATu) of autotransporter protein (EstA) from P. putida. In particular, lipases which required a lipase-specific foldase (Lif) for their proper folding were for the first time displayed in the active form by coexpression of the Lif protein.
KeywordMeSH Terms
319. Kotsakis  SD, Papagiannitsis  CC, Tzelepi  E, Legakis  NJ, Miriagou  V, Tzouvelekis  LS,     ( 2010 )

GES-13, a beta-lactamase variant possessing Lys-104 and Asn-170 in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 54 (3)
PMID : 20065056  :   DOI  :   10.1128/AAC.01561-09     PMC  :   PMC2825989    
Abstract >>
GES-13 beta-lactamase, a novel GES variant possessing Lys-104 and Asn-170, was identified in Pseudomonas aeruginosa. bla(GES-13) was the single gene cassette of a class 1 integron probably located in the chromosome. GES-13 efficiently hydrolyzed broad-spectrum cephalosporins and aztreonam. Imipenem was a potent inhibitor of GES-13 but was not hydrolyzed at measurable rates.
KeywordMeSH Terms
beta-Lactamases
Genetic Variation
320. Tato  M, Coque  TM, Baquero  F, Cantón  R,     ( 2010 )

Dispersal of carbapenemase blaVIM-1 gene associated with different Tn402 variants, mercury transposons, and conjugative plasmids in Enterobacteriaceae and Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 54 (1)
PMID : 19901094  :   DOI  :   10.1128/AAC.00783-09     PMC  :   PMC2798558    
Abstract >>
The emergence of bla(VIM-1) within four different genetic platforms from distinct Enterobacteriaceae and Pseudomonas aeruginosa isolates in an area with a low prevalence of metallo-beta-lactamase producers is reported. Forty-three VIM-1-producing isolates (including 19 Enterobacter cloacae, 2 Escherichia coli, and 2 P. aeruginosa isolates, 18 Klebsiella pneumoniae isolate, and 2 Klebsiella oxytoca isolate) recovered from 2005 to 2007 and corresponding to 15 pulsed-field gel electrophoresis types were studied. The Enterobacteriaceae isolates corresponded to a hospital outbreak, and the P. aeruginosa isolates were sporadically recovered. The genetic context of the integrons carrying bla(VIM-1) (arbitrarily designated types A, B, C, and D) was characterized by PCR mapping based on known Tn402 and mercury transposons and further sequencing. Among Enterobacteriaceae isolates, bla(VIM-1) was part of integrons located either in an In2-Tn402 element linked to Tn21 (type A; In110-bla(VIM-1)-aacA4-aadA1) or in a Tn402 transposon lacking the whole tni module [type B; In113-bla(VIM-1)-aacA4-dhfrII (also called dfrB1)-aadA1-catB2] and the transposon was associated with an IncHI2 or IncI1 plasmid, respectively. Among P. aeruginosa isolates, bla(VIM-1) was part of a new gene cassette array located in a defective Tn402 transposon carrying either tniBDelta3 and tniA (type C; bla(VIM-1)-aadA1) or tniC and DeltatniQ (type D; bla(VIM-1)-aadB), and both Tn402 variants were associated with conjugative plasmids of 30 kb. The dissemination of bla(VIM-1) was associated with different genetic structures and bacterial hosts, depicting a complex emergence and evolutionary network scenario in our facility, Ram?n y Cajal University Hospital, Madrid, Spain. Knowledge of the complex epidemiology of bla(VIM-1) is necessary to control this emerging threat.
KeywordMeSH Terms
321. Samuelsen  O, Toleman  MA, Sundsfjord  A, Rydberg  J, Leegaard  TM, Walder  M, Lia  A, Ranheim  TE, Rajendra  Y, Hermansen  NO, Walsh  TR, Giske  CG,     ( 2010 )

Molecular epidemiology of metallo-beta-lactamase-producing Pseudomonas aeruginosa isolates from Norway and Sweden shows import of international clones and local clonal expansion.

Antimicrobial agents and chemotherapy 54 (1)
PMID : 19884381  :   DOI  :   10.1128/AAC.00824-09     PMC  :   PMC2798561    
Abstract >>
Scandinavia is considered a region with a low prevalence of antimicrobial resistance. However, the number of multidrug-resistant (MDR) Gram-negative bacteria is increasing, including metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa. In this study MBL-producing P. aeruginosa isolates identified in Norway (n = 4) and Sweden (n = 9) from 1999 to 2007 were characterized. Two international clonal complexes (CC), CC111 (n = 8) and CC235 (n = 2), previously associated with MBL-producing isolates, were dominant. CC111 isolates (ST111/229; serotype O12; bla(VIM-2)) included clonally related isolates identified in Sk?ne County, Sweden (n = 6), and two isolates associated with importation from Greece and Denmark. In all CC111 isolates, bla(VIM-2) was located in integron In59.2 or In59 variants. The two CC235 isolates (ST235/ST230; serotype O11; bla(VIM-4)) were imported from Greece and Cyprus, were possibly clonally related, and carried bla(VIM-4) in two different integron structures. Three isolates imported from Ghana (ST233; serotype O6; bla(VIM-2)), Tunisia (ST654; serotype O11; bla(VIM-2)), and Thailand (ST260; serotype O6; bla(IMP-14)) were clonally unrelated. ST233 was part of a new CC (CC233) that included other MBL-producing isolates, while ST654 could also be part of a new CC associated with MBL producers. In the isolates imported from Ghana and Tunisia, bla(VIM-2) was part of unusual integron structures lacking the 3' conserved segment and associated with transposons. The bla(VIM) gene was found to be located on the chromosome in all isolates. Known risk factors for acquisition of MBL were reported for all patients except one. The findings suggest that both import of successful international clones and local clonal expansion contribute to the emergence of MBL-producing P. aeruginosa in Scandinavia.
KeywordMeSH Terms
322. Mavrodi  DV, Peever  TL, Mavrodi  OV, Parejko  JA, Raaijmakers  JM, Lemanceau  P, Mazurier  S, Heide  L, Blankenfeldt  W, Weller  DM, Thomashow  LS,     ( 2010 )

Diversity and evolution of the phenazine biosynthesis pathway.

Applied and environmental microbiology 76 (3)
PMID : 20008172  :   DOI  :   10.1128/AEM.02009-09     PMC  :   PMC2813009    
Abstract >>
Phenazines are versatile secondary metabolites of bacterial origin that function in biological control of plant pathogens and contribute to the ecological fitness and pathogenicity of the producing strains. In this study, we employed a collection of 94 strains having various geographic, environmental, and clinical origins to study the distribution and evolution of phenazine genes in members of the genera Pseudomonas, Burkholderia, Pectobacterium, Brevibacterium, and Streptomyces. Our results confirmed the diversity of phenazine producers and revealed that most of them appear to be soil-dwelling and/or plant-associated species. Genome analyses and comparisons of phylogenies inferred from sequences of the key phenazine biosynthesis (phzF) and housekeeping (rrs, recA, rpoB, atpD, and gyrB) genes revealed that the evolution and dispersal of phenazine genes are driven by mechanisms ranging from conservation in Pseudomonas spp. to horizontal gene transfer in Burkholderia spp. and Pectobacterium spp. DNA extracted from cereal crop rhizospheres and screened for the presence of phzF contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington state, which provided the first evidence of United States soils enriched in indigenous phenazine-producing bacteria.
KeywordMeSH Terms
Genes, Bacterial
323. Poirel  L, Docquier  JD, De Luca  F, Verlinde  A, Ide  L, Rossolini  GM, Nordmann  P,     ( 2010 )

BEL-2, an extended-spectrum beta-lactamase with increased activity toward expanded-spectrum cephalosporins in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 54 (1)
PMID : 19884378  :   DOI  :   10.1128/AAC.00859-09     PMC  :   PMC2798549    
Abstract >>
A Pseudomonas aeruginosa isolate recovered in Belgium produced a novel extended-spectrum ss-lactamase, BEL-2, differing from BEL-1 by a single Leu162Phe substitution. That modification significantly altered the kinetic properties of the enzyme, increasing its affinity for expanded-spectrum cephalosporins. The bla(BEL-2) gene was identified from a P. aeruginosa isolate clonally related to another bla(BEL-1)-positive isolate.
KeywordMeSH Terms
324. Yong  YC, Zhong  JJ,     ( 2009 )

A genetically engineered whole-cell pigment-based bacterial biosensing system for quantification of N-butyryl homoserine lactone quorum sensing signal.

Biosensors & bioelectronics 25 (1)
PMID : 19574033  :   DOI  :   10.1016/j.bios.2009.06.010    
Abstract >>
N-acyl homoserine lactone (AHL) is a widely conserved quorum sensing (QS) signal of gram-negative bacteria and has received attention in fighting against human diseases and environmental pollution. However, a method for quantifying AHL is lacking although it is urgently required for diagnosis and bioprocess manipulation. This work screened out an aromatics degrader Pseudomonas aeruginosa for biosensing system development, which produced a blue-green pigment regulated by the RhlI-RhlR QS system. By taking advantage of the recognition of N-butyryl homoserine lactone (BHL, the signal molecule of RhlI-RhlR QS system and an AHL) by the product of rhlR, a new whole-cell biosensor P. aeruginosa Delta rhlIR/pYC-rhlR (rhlI(-)rhlR(++)) was developed. It was constructed through abolishing its BHL production by in-frame deletion of rhlIR and over-expressing rhlR by introducing a multi-copy plasmid pYC-rhlR into Delta rhlIR. By using the pigment production which responded to exogenous BHL as biosensor output, BHL quantification in samples was simply done spectrophotometrically. Under optimum conditions, the calibration curve had the limit of detection (LOD), the 50% activation/effect concentration, the limit of quantification (LOQ), and the quantitative detection range of 1.3 nM, 2.77+/-0.45 microM, 5.7 nM and 0.11-49.7 microM, respectively. The biosensor output was stable, culture samples could be stored 10 days under -20 degrees C, and this sensing system was resistant to interferences by toxic aromatic pollutants. It was successfully applied to environmental samples even without extraction. The new whole-cell biosensing system provided a simple, stable, toxic pollutants-tolerant, and cost-effective tool for quantitative investigation of the QS signals' role in environmental processes.
KeywordMeSH Terms
Genetic Engineering
325. Nguyen  Y, Jackson  SG, Aidoo  F, Junop  M, Burrows  LL,     ( 2010 )

Structural characterization of novel Pseudomonas aeruginosa type IV pilins.

Journal of molecular biology 395 (3)
PMID : 19895819  :   DOI  :   10.1016/j.jmb.2009.10.070    
Abstract >>
Pseudomonas aeruginosa type IV pili, composed of PilA subunits, are used for attachment and twitching motility on surfaces. P. aeruginosa strains express one of five phylogenetically distinct PilA proteins, four of which are associated with accessory proteins that are involved either in pilin posttranslational modification or in modulation of pilus retraction dynamics. Full understanding of pilin diversity is crucial for the development of a broadly protective pilus-based vaccine. Here, we report the 1.6-A X-ray crystal structure of an N-terminally truncated form of the novel PilA from strain Pa110594 (group V), which represents the first non-group II pilin structure solved. Although it maintains the typical T4a pilin fold, with a long N-terminal alpha-helix and four-stranded antiparallel beta-sheet connected to the C-terminus by a disulfide-bonded loop, the presence of an extra helix in the alphabeta-loop and a disulfide-bonded loop with helical character gives the structure T4b pilin characteristics. Despite the presence of T4b features, the structure of PilA from strain Pa110594 is most similar to the Neisseria gonorrhoeae pilin and is also predicted to assemble into a fiber similar to the GC pilus, based on our comparative pilus modeling. Interactions between surface-exposed areas of the pilin are suggested to contribute to pilus fiber stability. The non-synonymous sequence changes between group III and V pilins are clustered in the same surface-exposed areas, possibly having an effect on accessory protein interactions. However, based on our high-confidence model of group III PilA(PA14), compensatory changes allow for maintenance of a similar shape.
KeywordMeSH Terms
326. Juan  C, Zamorano  L, Pérez  JL, Ge  Y, Oliver  A, N/A  N/A, N/A  N/A,     ( 2010 )

Activity of a new antipseudomonal cephalosporin, CXA-101 (FR264205), against carbapenem-resistant and multidrug-resistant Pseudomonas aeruginosa clinical strains.

Antimicrobial agents and chemotherapy 54 (2)
PMID : 19933793  :   DOI  :   10.1128/AAC.00834-09     PMC  :   PMC2812131    
Abstract >>
The activity of the new cephalosporin CXA-101 (CXA), previously designated FR264205, was evaluated against a collection of 236 carbapenem-resistant P. aeruginosa isolates, including 165 different clonal types, from a Spanish multicenter (127-hospital) study. The MICs of CXA were compared to the susceptibility results for antipseudomonal penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones. The MIC of CXA in combination with tazobactam (4 and 8 microg/ml) was determined for strains with high CXA MICs. The presence of acquired beta-lactamases was investigated by isoelectric focusing and PCR amplification followed by sequencing. Additional beta-lactamase genes were identified by cloning and sequencing. The CXA MIC50/MIC90 for the complete collection of carbapenem-resistant P. aeruginosa isolates was 1/4 microg/ml, with 95.3% of the isolates showing an MIC of 8 microg/ml produced a horizontally acquired beta-lactamase, including the metallo-beta-lactamase (MBL) VIM-2 (one strain), the extended-spectrum beta-lactamase (ESBL) PER-1 (one strain), several extended-spectrum OXA enzymes (OXA-101 [one strain], OXA-17 [two strains], and a newly described OXA-2 derivative [W159R] designated OXA-144 [four strains]), and a new BEL variant (BEL-3) ESBL (one strain), as identified by cloning and sequencing. Synergy with tazobactam in these 11 strains was limited, although 8 microg/ml reduced the mean CXA MIC by 2-fold. CXA is highly active against carbapenem-resistant P. aeruginosa isolates, including MDR strains. Resistance was restricted to still-uncommon strains producing an acquired MBL or ESBL.
KeywordMeSH Terms
327. Roy Chowdhury  P, Merlino  J, Labbate  M, Cheong  EY, Gottlieb  T, Stokes  HW,     ( 2009 )

Tn6060, a transposon from a genomic island in a Pseudomonas aeruginosa clinical isolate that includes two class 1 integrons.

Antimicrobial agents and chemotherapy 53 (12)
PMID : 19752283  :   DOI  :   10.1128/AAC.00687-09     PMC  :   PMC2786367    
Abstract >>
A 25,441-bp transposon was recovered from a Pseudomonas aeruginosa clinical isolate. While the transposition module was >99% identical to sequence of Tn1403, the element had been subject to rearrangements, with two In70.2-like class 1 integrons inserted into it in an unusual "tail-to-tail" configuration. One cassette array was the same as that in In70.2; however, the second was different, generating a transposon that collectively includes six resistance cassettes.
KeywordMeSH Terms
328. Barrow  K, Kwon  DH,     ( 2009 )

Alterations in two-component regulatory systems of phoPQ and pmrAB are associated with polymyxin B resistance in clinical isolates of Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 53 (12)
PMID : 19752280  :   DOI  :   10.1128/AAC.00893-09     PMC  :   PMC2786363    
Abstract >>
Polymyxins are often the only option to treat acquired multidrug-resistant Pseudomonas aeruginosa. Polymyxin susceptibility in P. aeruginosa PAO1 is associated with the lipopolysaccharide structure that is determined by arnBCADTEF and modulated by phoPQ and pmrAB. We examined five clonally unrelated clinical isolates of polymyxin B-resistant P. aeruginosa to investigate the molecular basis of polymyxin resistance. All isolates grew with 4 microg/ml polymyxin B (MIC, 8 microg/ml), whereas P. aeruginosa PAO1 grew with 0.25 mug/ml polymyxin B (MIC, 0.5 microg/ml). The resistant isolates were converted to susceptible ones (the MICs fell from 8 to 0.5 microg/ml) following the introduction of phoPQ (four isolates) and pmrAB (one isolate), which had been cloned from strain PAO1. DNA sequence analysis revealed that a single-nucleotide substitution in three isolates replaced a single amino acid of PhoQ, the deletion of 17 nucleotides in one isolate truncated the protein of PhoQ, and two nucleotide substitutions in one isolate replaced two amino acids of PmrB. The involvement of these amino acid substitutions or the truncated protein of PhoQ and PmrB in polymyxin B resistance was confirmed using strain PAO1 lacking phoPQ or pmrAB that was transformed by phoPQ or pmrAB containing the amino acid substitutions or the truncated protein. The resistant clinical isolates were sensitized by the inactivation of arnBCADTEF (the MICs fell from 8 to 0.5 microg/ml). These results suggest that polymyxin B resistance among clinical isolates of P. aeruginosa is associated with alterations in two-component regulatory systems of phoPQ or pmrAB.
KeywordMeSH Terms
329. Sampaleanu  LM, Bonanno  JB, Ayers  M, Koo  J, Tammam  S, Burley  SK, Almo  SC, Burrows  LL, Howell  PL,     ( 2009 )

Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex.

Journal of molecular biology 394 (1)
PMID : 19857646  :   DOI  :   10.1016/j.jmb.2009.09.037    
Abstract >>
Type IV pili (T4P) are bacterial virulence factors responsible for attachment to surfaces and for twitching motility, a motion that involves a succession of pilus extension and retraction cycles. In the opportunistic pathogen Pseudomonas aeruginosa, the PilM/N/O/P proteins are essential for T4P biogenesis, and genetic and biochemical analyses strongly suggest that they form an inner-membrane complex. Here, we show through co-expression and biochemical analysis that the periplasmic domains of PilN and PilO interact to form a heterodimer. The structure of residues 69-201 of the periplasmic domain of PilO was determined to 2.2 A resolution and reveals the presence of a homodimer in the asymmetric unit. Each monomer consists of two N-terminal coiled coils and a C-terminal ferredoxin-like domain. This structure was used to generate homology models of PilN and the PilN/O heterodimer. Our structural analysis suggests that in vivo PilN/O heterodimerization would require changes in the orientation of the first N-terminal coiled coil, which leads to two alternative models for the role of the transmembrane domains in the PilN/O interaction. Analysis of PilN/O orthologues in the type II secretion system EpsL/M revealed significant similarities in their secondary structures and the tertiary structures of PilO and EpsM, although the way these proteins interact to form inner-membrane complexes appears to be different in T4P and type II secretion. Our analysis suggests that PilN interacts directly, via its N-terminal tail, with the cytoplasmic protein PilM. This work shows a direct interaction between the periplasmic domains of PilN and PilO, with PilO playing a key role in the proper folding of PilN. Our results suggest that PilN/O heterodimers form the foundation of the inner-membrane PilM/N/O/P complex, which is critical for the assembly of a functional T4P complex.
KeywordMeSH Terms
Protein Multimerization
330. Juan  C, Mulet  X, Zamorano  L, Albertí  S, Pérez  JL, Oliver  A,     ( 2009 )

Detection of the novel extended-spectrum beta-lactamase OXA-161 from a plasmid-located integron in Pseudomonas aeruginosa clinical isolates from Spain.

Antimicrobial agents and chemotherapy 53 (12)
PMID : 19770278  :   DOI  :   10.1128/AAC.00822-09     PMC  :   PMC2786339    
Abstract >>
Two clonally related Pseudomonas aeruginosa isolates, recovered from two patients admitted to a pediatric intensive care unit, were found to harbor a new OXA-2 variant (Asn148Asp), designated OXA-161. The plasmid location of bla(OXA-161) was demonstrated through electroporation to PAO1, and its codification in a class I integron (together with aacA4) was demonstrated through PCR and sequencing. bla(OXA-2) and bla(OXA-161) were cloned in parallel to demonstrate the extended-spectrum beta-lactamase properties of OXA-161, conferring resistance to ceftazidime and reduced susceptibility to cefepime and aztreonam.
KeywordMeSH Terms
331. Giardina  G, Rinaldo  S, Castiglione  N, Caruso  M, Cutruzzol?  F,     ( 2009 )

A dramatic conformational rearrangement is necessary for the activation of DNR from Pseudomonas aeruginosa. Crystal structure of wild-type DNR.

Proteins 77 (1)
PMID : 19415759  :   DOI  :   10.1002/prot.22428    
Abstract >>
The opportunistic pathogen Pseudomonas aeruginosa can grow in low oxygen, because it is capable of anaerobic respiration using nitrate as a terminal electron acceptor (denitrification). An intermediate of the denitrification pathway is nitric oxide, a compound that may become cytotoxic at high concentration. The intracellular levels of nitric oxide are tightly controlled by regulating the expression of the enzymes responsible for its synthesis and degradation (nitrite and nitric oxide reductases). In this article, we present the crystallographic structure of the wild-type dissimilative nitrate respiration regulator (DNR), a master regulator controlling expression of the denitrification machinery and a putative target for new therapeutic strategies. Comparison with other structures among the CRP-FNR class of regulators reveals that DNR has crystallized in a conformation that has never been observed before. In particular, the sensing domain of DNR has undergone a rotation of more than 50 degrees with respect to the other structures. This suggests that DNR may undergo an unexpected and very large conformational rearrangement on activation.
KeywordMeSH Terms
332. Xu  H, Su  Z, Wang  S, Dai  X, Chen  J, Kong  F, Li  Y, Peng  S, Shao  Q, Lu  L, Ezaki  T,     ( 2009 )

Four novel resistance integron gene-cassette occurrences in bacterial isolates from zhenjiang, china.

Current microbiology 59 (2)
PMID : 19365688  :   DOI  :   10.1007/s00284-009-9405-z    
Abstract >>
Integrons, which are widely distributed among bacteria and are strongly associated with resistance, are specialized genetic elements that are capable of capturing, integrating, and mobilizing gene cassette. In this work, we investigated classes 1, 2, and 3 integrons associated integrases genes in 365 bacteria isolates, amplified and analyzed the structure of class 1 integron, detected 8 resistant gene cassettes [dfr17, aadA5, aadA1, aadA2, dhfrI, aadB, aac(6')-II, and pse-I], and found four novel gene-cassette arrays. We also found that commensal bacteria in the common microenvironment had the same integron gene cassette, which provided direct evidence that integron was an important horizontal transmission element.
KeywordMeSH Terms
Drug Resistance, Bacterial
Integrons
333. Viedma  E, Juan  C, Acosta  J, Zamorano  L, Otero  JR, Sanz  F, Chaves  F, Oliver  A,     ( 2009 )

Nosocomial spread of colistin-only-sensitive sequence type 235 Pseudomonas aeruginosa isolates producing the extended-spectrum beta-lactamases GES-1 and GES-5 in Spain.

Antimicrobial agents and chemotherapy 53 (11)
PMID : 19738007  :   DOI  :   10.1128/AAC.00900-09     PMC  :   PMC2772357    
Abstract >>
The mechanisms responsible for the increasing prevalence of colistin-only-sensitive (COS) Pseudomonas aeruginosa isolates in a Spanish hospital were investigated. Pulsed-field gel electrophoresis revealed that 24 (50%) of the studied isolates belonged to the same clone, identified as the internationally spread sequence type 235 (ST235) through multilocus sequence typing. In addition to several mutational resistance mechanisms, an integron containing seven resistance determinants was detected. Remarkably, the extended-spectrum beta-lactamase GES-1 and its Gly170Ser carbapenem-hydrolyzing derivative GES-5 were first documented to be encoded in a single integron. This work is the first to describe GES enzymes in Spain and adds them to the growing list of beta-lactamases of concern (PER, VIM, and OXA) detected in ST235 clone isolates.
KeywordMeSH Terms
334. Kitao  T, Miyoshi-Akiyama  T, Kirikae  T,     ( 2009 )

AAC(6')-Iaf, a novel aminoglycoside 6'-N-acetyltransferase from multidrug-resistant Pseudomonas aeruginosa clinical isolates.

Antimicrobial agents and chemotherapy 53 (6)
PMID : 19349516  :   DOI  :   10.1128/AAC.01360-08     PMC  :   PMC2687227    
Abstract >>
We report here the characterization of a novel aminoglycoside resistance gene, aac(6')-Iaf, present in two multidrug-resistant (MDR) Pseudomonas aeruginosa clinical isolates. These isolates, IMCJ798 and IMCJ799, were independently obtained from two patients, one with a urinary tract infection and the other with a decubitus ulcer, in a hospital located in the western part of Japan. Although the antibiotic resistance profiles of IMCJ798 and IMCJ799 were similar to that of MDR P. aeruginosa IMCJ2.S1, which caused outbreaks in the eastern part of Japan, the pulsed-field gel electrophoresis patterns for these isolates were different from that for IMCJ2.S1. Both IMCJ798 and IMCJ799 were found to contain a novel chromosomal class 1 integron, In123, which included aac(6')-Iaf as the first cassette gene. The encoded protein, AAC(6')-Iaf, was found to consist of 183 amino acids, with 91 and 87% identity to AAC(6')-Iq and AAC(6')-Im, respectively. IMCJ798, IMCJ799, and Escherichia coli transformants carrying a plasmid containing the aac(6')-Iaf gene and its upstream region were highly resistant to amikacin, dibekacin, and kanamycin but not to gentamicin. The production of AAC(6')-Iaf in these strains was confirmed by Western blot analysis. Thin-layer chromatography indicated that AAC(6')-Iaf is a functional acetyltransferase that specifically modifies the amino groups at the 6' positions of aminoglycosides. Collectively, these findings indicate that AAC(6')-Iaf contributes to aminoglycoside resistance.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
335. Cheng  M, Takenaka  S, Aoki  S, Murakami  S, Aoki  K,     ( 2009 )

Purification and characterization of an eggshell membrane decomposing protease from Pseudomonas aeruginosa strain ME-4.

Journal of bioscience and bioengineering 107 (4)
PMID : 19332295  :   DOI  :   10.1016/j.jbiosc.2008.12.010    
Abstract >>
A bacterial strain, ME-4, isolated from farm soil and identified as Pseudomonas aeruginosa, grew well on a medium containing eggshell membrane (ESM). P. aeruginosa strain ME-4 decomposed the ESM by producing an extracellular protease able to solubilize it. The protease was purified to homogeneity from culture supernatant by fractionation with (NH(4))(2)SO(4), as well as CM52 cellulose and DE52 cellulose column chromatography, with a final yield of 47%. The molecular mass of the enzyme was 33 kDa. The isolated enzyme was a metalloprotease and was strongly inhibited by EDTA, o-phenanthroline, and phosphoramidon. The enzyme inhibited by these reagents was reactivated in the presence of several metal ions. The enzyme acted on various proteins and showed higher activity with collagen than collagenase from Clostridium histolyticum. Results of assays with the FRETS combinatorial libraries revealed that the enzyme preferred Ser at the P1 position and Lys at the P2 position. It also preferred hydrophobic amino acid residues at the P1' and P2' positions. The enzyme showed a much higher solubilization activity with the ESM substrate than commercially obtained enzymes. The enzyme decomposed ESM to produce water-soluble peptides, Val-Leu-Pro-Pro and (X)-Val-Pro-Pro, and a free amino acid, tryptophan.
KeywordMeSH Terms
336. Rodríguez-Martínez  JM, Poirel  L, Nordmann  P,     ( 2009 )

Extended-spectrum cephalosporinases in Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 53 (5)
PMID : 19258272  :   DOI  :   10.1128/AAC.01410-08     PMC  :   PMC2681535    
Abstract >>
The characterization of AmpC-type beta-lactamases was performed in a collection of 32 clinical Pseudomonas aeruginosa isolates with intermediate susceptibility or resistance to imipenem and ceftazidime. Twenty-one out of those 32 isolates overexpressed AmpC beta-lactamase, and the MICs of ceftazidime and imipenem were reduced after cloxacillin addition. Cloning and sequencing identified 10 AmpC beta-lactamase variants. Reduced susceptibility to imipenem, ceftazidime, and cefepime was observed only with recombinant P. aeruginosa strains expressing an AmpC beta-lactamase that had an alanine residue at position 105. The catalytic efficiencies (k(cat)/K(m)) of the AmpC variants possessing this residue were increased against oxyiminocephalosporins and imipenem. In addition, we show here that those AmpC variants constitute a favorable background for the in vitro selection of imipenem-resistant strains. This report identified a novel resistance mechanism that may contribute to imipenem resistance in P. aeruginosa.
KeywordMeSH Terms
337. Wu  Y, Li  H, Li  J, Huang  ZH,     ( 2008 )

Detection of Pseudomonas aeruginosa carried a new array of gene cassettes within class 1 integron isolated from a teaching hospital in Nanjing, China.

Journal of microbiology (Seoul, Korea) 46 (6)
PMID : 19107398  :   DOI  :   10.1007/s12275-008-0021-6    
Abstract >>
We report here novel array of gene cassettes found in single variable region of class 1 integron disseminated in Pseudomonas aeruginosa isolated from a teaching hospital in Nanjing, Jiangsu Province, China. 29 of 47 (61%) P. aeruginosa strains were confirmed haboured class 1 integron, and all the positive strains have the same variable region confirmed by PCR and RFLP methods. The variable region contained an unreported order of four gene cassettes aac(6')-II-aadA13-cmlA8-oxa-10. Of those, cmlA8 gene was a variant of cmlA5 encoding non-enzymatic protein which putatively confer resistance to chloramphenicol. Susceptibility testing revealed multidrug-resistant mechanisms were involved in the class 1 integron positive clinical isolates. And the class 1 integron located on an about 15 kb transferable plasmid was certified by conjugation experiment and plasmid DNA analysis. The macro restriction profile indicated those clinical strains were clonally related.
KeywordMeSH Terms
Hospitals, Teaching
338. Xu  G, Ryan  C, Kiefel  MJ, Wilson  JC, Taylor  GL,     ( 2009 )

Structural studies on the Pseudomonas aeruginosa sialidase-like enzyme PA2794 suggest substrate and mechanistic variations.

Journal of molecular biology 386 (3)
PMID : 19166860  :   DOI  :   10.1016/j.jmb.2008.12.084    
Abstract >>
Pseudomonas aeruginosa encodes an enzyme (PA2794) that is annotated as a sialidase (or neuraminidase), as it possesses three bacterial neuraminidase repeats that are a signature of nonviral sialidases. A recent report showed that when the gene encoding this sialidase is knocked out, this led to a reduction in biofilm production in the lungs of mice, and it was suggested that the enzyme recognizes pseudaminic acid, a sialic acid analogue that decorates the flagella of Pseudomonas, Helicobacter, and Campylobacter species. Here, we present the crystal structure of the P. aeruginosa enzyme and show that it adopts a trimeric structure, partly held together by an immunoglobulin-like trimerization domain that is C-terminal to a classical beta-propeller sialidase domain. The recombinant enzyme does not show any sialidase activity with the standard fluorogenic sialic-acid-based substrate. The proposed active site contains certain conserved features of a sialidase: a nucleophilic tyrosine with its associated glutamic acid, and two of the usual three arginines that interact with the carboxylic acid group of the substrate, but is missing the first arginine and the aspartic acid that acts as an acid/base in all sialidases studied to date. We show, by in silico docking, that the active site may accommodate pseudaminic acid but not sialic acid and that this is due, in part, to a phenylalanine in the hydrophobic pocket that selects for the alternative stereochemistry of pseudaminic acid at C5 compared to sialic acid. Mutation of this phenylalanine to an alanine converts the enzyme into a sialidase, albeit a poor one, which we confirm by kinetics and NMR, and this allowed us to probe the function of other amino acids. We propose that a histidine plays the role of the acid/base, whose state is altered through a charge-relay system involving a novel His-Tyr-Glu triad. The location of this relay system precludes the presence of one of the three arginines usually found in a sialidase active site.
KeywordMeSH Terms
339. Hsiao  YS, Parker  D, Ratner  AJ, Prince  A, Tong  L,     ( 2009 )

Crystal structures of respiratory pathogen neuraminidases.

Biochemical and biophysical research communications 380 (3)
PMID : 19284989  :   DOI  :   10.1016/j.bbrc.2009.01.108     PMC  :   PMC3836282    
Abstract >>
Currently there is pressing need to develop novel therapeutic agents for the treatment of infections by the human respiratory pathogens Pseudomonas aeruginosa and Streptococcus pneumoniae. The neuraminidases of these pathogens are important for host colonization in animal models of infection and are attractive targets for drug discovery. To aid in the development of inhibitors against these neuraminidases, we have determined the crystal structures of the P. aeruginosa enzyme NanPs and S. pneumoniae enzyme NanA at 1.6 and 1.7A resolution, respectively. In situ proteolysis with trypsin was essential for the crystallization of our recombinant NanA. The active site regions of the two enzymes are strikingly different. NanA contains a deep pocket that is similar to that in canonical neuraminidases, while the NanPs active site is much more open. The comparative studies suggest that NanPs may not be a classical neuraminidase, and may have distinct natural substrates and physiological functions. This work represents an important step in the development of drugs to prevent respiratory tract colonization by these two pathogens.
KeywordMeSH Terms
340. Damron  FH, Qiu  D, Yu  HD,     ( 2009 )

The Pseudomonas aeruginosa sensor kinase KinB negatively controls alginate production through AlgW-dependent MucA proteolysis.

Journal of bacteriology 191 (7)
PMID : 19168621  :   DOI  :   10.1128/JB.01490-08     PMC  :   PMC2655532    
Abstract >>
Mucoidy, or overproduction of the exopolysaccharide known as alginate, in Pseudomonas aeruginosa is a poor prognosticator for lung infections in cystic fibrosis. Mutation of the anti-sigma factor MucA is a well-accepted mechanism for mucoid conversion. However, certain clinical mucoid strains of P. aeruginosa have a wild-type (wt) mucA. Here, we describe a loss-of-function mutation in kinB that causes overproduction of alginate in the wt mucA strain PAO1. KinB is the cognate histidine kinase for the transcriptional activator AlgB. Increased alginate production due to inactivation of kinB was correlated with high expression at the alginate-related promoters P(algU) and P(algD). Deletion of alternative sigma factor RpoN (sigma(54)) or the response regulator AlgB in kinB mutants decreased alginate production to wt nonmucoid levels. Mucoidy was restored in the kinB algB double mutant by expression of wt AlgB or phosphorylation-defective AlgB.D59N, indicating that phosphorylation of AlgB was not required for alginate overproduction when kinB was inactivated. The inactivation of the DegS-like protease AlgW in the kinB mutant caused loss of alginate production and an accumulation of the hemagglutinin (HA)-tagged MucA. Furthermore, we observed that the kinB mutation increased the rate of HA-MucA degradation. Our results also indicate that AlgW-mediated MucA degradation required algB and rpoN in the kinB mutant. Collectively, these studies indicate that KinB is a negative regulator of alginate production in wt mucA strain PAO1.
KeywordMeSH Terms
Down-Regulation
341. Li  H, Walsh  TR, Toleman  MA,     ( 2009 )

Molecular analysis of the sequences surrounding blaOXA-45 reveals acquisition of this gene by Pseudomonas aeruginosa via a novel ISCR element, ISCR5.

Antimicrobial agents and chemotherapy 53 (3)
PMID : 19064894  :   DOI  :   10.1128/AAC.00480-08     PMC  :   PMC2650518    
Abstract >>
The bla(OXA-45) gene of Pseudomonas aeruginosa 07-406 is driven by a promoter found within a truncated ISPme1 insertion sequence. The gene is located between nonidentical repeats of a new ISCR element, ISCR5, which is likely responsible for its acquisition. Sequence analysis indicates that ISCR5 is a chimera of ISCR3 and ISCR16.
KeywordMeSH Terms
Genes, Bacterial
342. Alontaga  AY, Rodriguez  JC, Schönbrunn  E, Becker  A, Funke  T, Yukl  ET, Hayashi  T, Stobaugh  J, Moënne-Loccoz  P, Rivera  M,     ( 2009 )

Structural characterization of the hemophore HasAp from Pseudomonas aeruginosa: NMR spectroscopy reveals protein-protein interactions between Holo-HasAp and hemoglobin.

Biochemistry 48 (1)
PMID : 19072037  :   DOI  :   10.1021/bi801860g     PMC  :   PMC2666852    
Abstract >>
Pseudomonas aeruginosa secretes a 205 residue long hemophore (full-length HasAp) that is subsequently cleaved at the C'-terminal domain to produce mainly a 184 residue long truncated HasAp that scavenges heme [Letoff?, S., Redeker, V., and Wandersman, C. (1998) Mol. Microbiol. 28, 1223-1234]. HasAp has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The X-ray crystal structure of truncated HasAp revealed a polypeptide alphabeta fold and a ferriheme coordinated axially by His32 and Tyr75, with the side chain of His83 poised to accept a hydrogen bond from the Tyr75 phenolic acid group. NMR investigations conducted with full-length HasAp showed that the carboxyl-terminal tail (21 residues) is disordered and conformationally flexible. NMR spectroscopic investigations aimed at studying a complex between apo-HasAp and human methemoglobin were stymied by the rapid heme capture by the hemophore. In an effort to circumvent this problem NMR spectroscopy was used to monitor the titration of 15N-labeled holo-HasAp with hemoglobin. These studies allowed identification of a specific area on the surface of truncated HasAp, encompassing the axial ligand His32 loop that serves as a transient site of interaction with hemoglobin. These findings are discussed in the context of a putative encounter complex between apo-HasAp and hemoglobin that leads to efficient hemoglobin-heme capture by the hemophore. Similar experiments conducted with full-length 15N-labeled HasAp and hemoglobin revealed a transient interaction site in full-length HasAp similar to that observed in the truncated hemophore. The spectral perturbations observed while investigating these interactions, however, are weaker than those observed for the interactions between hemoglobin and truncated HasAp, suggesting that the disordered tail in the full-length HasAp must be proteolyzed in the extracellular milieu to make HasAp a more efficient hemophore.
KeywordMeSH Terms
343. Chao  Z, Xiao-Feng  W, Dan-Hong  S, Jin-Ping  Y, Nan-Shan  Z,     ( 2008 )

Outbreak of Pseudomonas aeruginosa producing IMP-9-type metallo-beta-lactamase in Guangzhou, China.

International journal of antimicrobial agents 32 (4)
PMID : 18619819  :   DOI  :   10.1016/j.ijantimicag.2008.04.008    
Abstract >>
N/A
KeywordMeSH Terms
Disease Outbreaks
Drug Resistance, Multiple, Bacterial
344. Libisch  B, Poirel  L, Lepsanovic  Z, Mirovic  V, Balogh  B, Pászti  J, Hunyadi  Z, Dobák  A, Füzi  M, Nordmann  P,     ( 2008 )

Identification of PER-1 extended-spectrum beta-lactamase producing Pseudomonas aeruginosa clinical isolates of the international clonal complex CC11 from Hungary and Serbia.

FEMS immunology and medical microbiology 54 (3)
PMID : 19049645  :   DOI  :   10.1111/j.1574-695X.2008.00483.x    
Abstract >>
PER-1 extended-spectrum beta-lactamase-producing Pseudomonas aeruginosa clinical isolates from Budapest, Hungary, and Belgrade, Serbia, were characterized by molecular methods. Two PER-1-positive isolates were recovered from sporadic cases in Budapest and a small cluster of PER-1-positive infections involving four patients were identified at a Belgrade hospital. A class 1 integron harbouring a bla(OXA-2)beta-lactamase gene and four other gene cassettes was detected in both the Budapest and the Belgrade isolates. The two P. aeruginosa isolates from Budapest also carried another class 1 integron containing bla(OXA-74), aac(6')-Ib-cr and cmlA7 genes in its variable region. The aac(6')-Ib-cr fluoroquinolone-acetylating aminoglycoside acetyltransferase gene is described here for the first time in P. aeruginosa. Multilocus sequence typing (MLST) revealed that the PER-1 positive P. aeruginosa isolates identified in this study display ST235, a sequence type that belongs to clonal complex CC11. Two bla(PER-1)-positive P. aeruginosa reference isolates from France and Belgium could also be assigned to complex CC11 by MLST. Our results underscore the role of complex CC11 in the dissemination of bla(PER-1) among P. aeruginosa clinical isolates.
KeywordMeSH Terms
345. Patzer  JA, Walsh  TR, Weeks  J, Dzierzanowska  D, Toleman  MA,     ( 2009 )

Emergence and persistence of integron structures harbouring VIM genes in the Children's Memorial Health Institute, Warsaw, Poland, 1998-2006.

The Journal of antimicrobial chemotherapy 63 (2)
PMID : 19095681  :   DOI  :   10.1093/jac/dkn512    
Abstract >>
The aim was to perform a genetically detailed study of the emergence of metallo-beta-lactamase (MBL) genes in Pseudomonas spp. in the Children's Memorial Health Institute over a 9 year period. Carbapenem-resistant Pseudomonas spp. isolates were collected from 1998 to 2006 and screened for MBL production. MBL-positive isolates were further investigated by a combination of genetic techniques including PCR, genomic location experiments using pulsed-field gel electrophoresis (PFGE) of I-Ceu1, S1 and SpeI digests, and sequencing. Of the 20 MBL-containing Pseudomonas isolates collected from 1998 to 2006, 16 Pseudomonas aeruginosa isolates contained an identical class 1 integron structure. Two P. aeruginosa isolates contained the bla(VIM-2) gene, and two Pseudomonas putida isolates harboured the bla(VIM-4) gene cassette in different integron structures. PFGE analysis indicated that all bla(VIM-4)-containing P. aeruginosa isolates were closely related, whereas the P. putida isolates were not. All MBL genes in this study were chromosomally encoded, and all isolates harboured only one class 1 integron. The bla(VIM-2) isolates were clonal, and the genetic structure supporting the bla(VIM-2) gene was found in an identical chromosomal position. MBL gene emergence in this hospital has paralleled a 6-fold increase in carbapenem usage. We have found an increase in MBL gene diversity, MBL host organisms and MBL genetic support structures in the hospital over the 9 year study period. There is also compelling evidence of the persistence of individual strains in the hospital throughout the study period. This suggests that once MBL genes have emerged in a hospital environment, they are difficult to remove.
KeywordMeSH Terms
Integrons
346. Siarkou  VI, Vitti  D, Protonotariou  E, Ikonomidis  A, Sofianou  D,     ( 2009 )

Molecular epidemiology of outbreak-related pseudomonas aeruginosa strains carrying the novel variant blaVIM-17 metallo-beta-lactamase gene.

Antimicrobial agents and chemotherapy 53 (4)
PMID : 19164147  :   DOI  :   10.1128/AAC.01230-08     PMC  :   PMC2663106    
Abstract >>
A study was designed to investigate the molecular epidemiological characteristics of multidrug-resistant outbreak-related Pseudomonas aeruginosa isolates collected in a university hospital in northern Greece. Of 29 nonreplicate P. aeruginosa isolates resistant to carbapenems and ceftazidime, 14 were positive for metallo-beta-lactamase production. PCR analyses with primers specific for bla(VIM) and bla(IMP) revealed that 13 isolates carried a novel bla(VIM-2) gene variant, designated bla(VIM-17), and only 1 isolate carried bla(VIM-2), a gene predominant among P. aeruginosa strains in Greek hospitals. Pulsed-field gel electrophoresis of XbaI-digested genomic DNAs showed a close genetic relationship for 12 of 13 bla(VIM-17)-carrying outbreak-related isolates, which were of the O11 serotype; the clonally unrelated isolate carrying bla(VIM-17) was of the O12 serotype. PCR mapping strategies for the detection of class 1 integrons and sequencing approaches revealed the presence of integrons containing one bla(VIM) cassette flanked by two aacA29 cassettes. These integrons were similar but not identical to In59 (GenBank accession number AF263519) initially described in France. All isolates carrying bla(VIM-17), regardless of their genetic profile, had an identical integron, named In59.3, indicating that although the hospital outbreak was mainly due to clonal dissemination, the horizontal transmission of the bla(VIM-17)-containing integron among P. aeruginosa isolates should also have occurred. An outbreak-related isolate and a control strain, both of which carried the bla(VIM-2) gene but which were clonally distinct, had an identical integron, named In59.2, which differed only at the level of the bla(VIM) gene from In59.3 integrons, suggesting a common ancestry. The spread of the bla(VIM-17)-containing integron in clonally unrelated P. aeruginosa isolates without any evidence of plasmid carriage is probably associated with a transposon.
KeywordMeSH Terms
Disease Outbreaks
347. Juan  C, Beceiro  A, Gutiérrez  O, Albertí  S, Garau  M, Pérez  JL, Bou  G, Oliver  A,     ( 2008 )

Characterization of the new metallo-beta-lactamase VIM-13 and its integron-borne gene from a Pseudomonas aeruginosa clinical isolate in Spain.

Antimicrobial agents and chemotherapy 52 (10)
PMID : 18644957  :   DOI  :   10.1128/AAC.00465-08     PMC  :   PMC2565874    
Abstract >>
During a survey conducted to evaluate the incidence of class B carbapenemase (metallo-beta-lactamase [MBL])-producing Pseudomonas aeruginosa strains from hospitals in Majorca, Spain, five clinical isolates showed a positive Etest MBL screening test result. In one of them, strain PA-SL2, the presence of a new bla(VIM) derivative (bla(VIM-13)) was detected by PCR amplification with bla(VIM-1)-specific primers followed by sequencing. The bla(VIM-13)-producing isolate showed resistance to all beta-lactams (except aztreonam), gentamicin, tobramycin, and ciprofloxacin. VIM-13 exhibited 93% and 88% amino acid sequence identities with VIM-1 and VIM-2, respectively. bla(VIM-13) was cloned in parallel with bla(VIM-1), and the resistance profile conferred was analyzed both in Escherichia coli and in P. aeruginosa backgrounds. Compared to VIM-1, VIM-13 conferred slightly higher levels of resistance to piperacillin and lower levels of resistance to ceftazidime and cefepime. VIM-13 and VIM-1 were purified in parallel as well, and their kinetic parameters were compared. The k(cat)/K(m) ratios for the antibiotics mentioned above were in good agreement with the MIC data. Furthermore, EDTA inhibited the activity of VIM-13 approximately 25 times less than it inhibited the activity of VIM-1. VIM-13 was harbored in a class 1 integron, along with a new variant (Ala108Thr) of the aminoglycoside-modifying enzyme encoding gene aacA4, which confers resistance to gentamicin and tobramycin. Finally, the VIM-13 integron was apparently located in the chromosome, since transformation and conjugation experiments consistently yielded negative results and the bla(VIM-13) probe hybridized only with the genomic DNA.
KeywordMeSH Terms
Genes, Bacterial
348. Gambello  MJ, Iglewski  BH,     ( 1991 )

Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression.

Journal of bacteriology 173 (9)
PMID : 1902216  :   DOI  :   10.1128/jb.173.9.3000-3009.1991     PMC  :   PMC207884    
Abstract >>
We report the discovery of the lasR gene, which positively regulates elastase expression in Pseudomonas aeruginosa PAO1. The lasR gene was cloned by its ability to restore a positive elastase phenotype in strain PA103, a strain which possesses the elastase structural gene (lasB) but fails to synthesize the enzyme. Nucleotide sequence analysis revealed an open reading frame of 716 nucleotides encoding a protein of approximately 27 kDa. A labeled LasR protein of 27 kDa was detected in Escherichia coli by using a T7 RNA polymerase expression system. A chromosomal deletion mutant of the lasR gene was constructed in PAO1 by gene replacement. This mutant (PAO-R1) is devoid of elastolytic activity and elastase antigen. The deduced amino acid sequence of LasR is 27% homologous to the positive activator LuxR of Vibrio fischeri and the suspected activator 28K-UvrC of E. coli. Northern (RNA) analysis of total cellular RNA from PAO1, PAO-R1, and PAO-R1 containing the lasR gene on a multicopy plasmid (pMG1.7) revealed that a functional lasR gene is required for transcription of the elastase structural gene (lasB).
KeywordMeSH Terms
349. Battle  SE, Rello  J, Hauser  AR,     ( 2009 )

Genomic islands of Pseudomonas aeruginosa.

FEMS microbiology letters 290 (1)
PMID : 19025565  :   DOI  :   10.1111/j.1574-6968.2008.01406.x     PMC  :   PMC2648531    
Abstract >>
Key to Pseudomonas aeruginosa's ability to thrive in a diversity of niches is the presence of numerous genomic islands that confer adaptive traits upon individual strains. We reasoned that P. aeruginosa strains capable of surviving in the harsh environments of multiple hosts would therefore represent rich sources of genomic islands. To this end, we identified a strain, PSE9, that was virulent in both animals and plants. Subtractive hybridization was used to compare the genome of PSE9 with the less virulent strain PAO1. Nine genomic islands were identified in PSE9 that were absent in PAO1; seven of these had not been described previously. One of these seven islands, designated P. aeruginosa genomic island (PAGI)-5, has already been shown to carry numerous interesting ORFs, including several required for virulence in mammals. Here we describe the remaining six genomic islands, PAGI-6, -7, -8, -9, -10, and -11, which include a prophage element and two Rhs elements.
KeywordMeSH Terms
350. Naas  T, Namdari  F, Bogaerts  P, Huang  TD, Glupczynski  Y, Nordmann  P,     ( 2008 )

Genetic structure associated with blaOXA-18, encoding a clavulanic acid-inhibited extended-spectrum oxacillinase.

Antimicrobial agents and chemotherapy 52 (11)
PMID : 18663027  :   DOI  :   10.1128/AAC.00403-08     PMC  :   PMC2573139    
Abstract >>
The genetic environment of the bla(OXA-18) gene encoding a peculiar clavulanic acid-inhibitable Ambler class D extended-spectrum beta-lactamase was determined from the prototype OXA-18-producing Pseudomonas aeruginosa MUS clinical isolate. An 8.2-kb genomic DNA fragment containing bla(OXA-18) was cloned from P. aeruginosa MUS. Although most oxacillinases are located in integrons, bla(OXA-18) lacked gene cassette-specific features. It was bracketed by two duplicated sequences containing ISCR19, a novel insertion sequence of the ISCR family of mobile elements; DeltaintI1, a truncated integrase gene; and a truncated Deltaaac6'-Ib gene cassette. It is likely that ISCR19 was at the origin of the bla(OXA-18) gene mobilization by a rolling-circle transposition event followed by homologous recombination. Furthermore, analysis of the cloned genomic DNA fragment revealed the presence of the integron-containing bla(OXA-20) gene. Concomitantly, three P. aeruginosa clinical isolates, displaying a synergy image as determined by double-disk diffusion tests on cloxacillin-containing plates, were isolated from three patients hospitalized in different wards over a 9-month period at the Saint-Luc University hospital (Brussels, Belgium). These isolates were positive by PCR for bla(OXA-18) and bla(OXA-20) genes, genetically related to P. aeruginosa MUS as determined by pulsed-field gel electrophoresis, and carried the same bla(OXA-18)/bla(OXA-20)-associated genetic structures. This report characterized the genetic elements likely at the origin of bla(OXA-18) gene mobilization in P. aeruginosa and suggests the spread of oxacillin-type extended-spectrum beta-lactamases in P. aeruginosa at the Saint-Luc University hospital of Brussels, Belgium.
KeywordMeSH Terms
Genes, Bacterial
351. Xu  Z, Li  L, Shirtliff  ME, Alam  MJ, Yamasaki  S, Shi  L,     ( 2009 )

Occurrence and characteristics of class 1 and 2 integrons in Pseudomonas aeruginosa isolates from patients in southern China.

Journal of clinical microbiology 47 (1)
PMID : 19020065  :   DOI  :   10.1128/JCM.02027-08     PMC  :   PMC2620863    
Abstract >>
Class 1 and 2 integrons were detected in 45.8% (54/118) and 19.5% (23/118) of our tested Pseudomonas aeruginosa isolates, respectively. Three strains were positive for both the integrons. This is the first report of class 2 integrons in P. aeruginosa and also of isolates carrying class 1 and 2 integrons simultaneously.
KeywordMeSH Terms
Integrons
352. Qiu  D, Eisinger  VM, Head  NE, Pier  GB, Yu  HD,     ( 2008 )

ClpXP proteases positively regulate alginate overexpression and mucoid conversion in Pseudomonas aeruginosa.

Microbiology (Reading, England) 154 (Pt 7)
PMID : 18599839  :   DOI  :   10.1099/mic.0.2008/017368-0     PMC  :   PMC2995304    
Abstract >>
Overproduction of the exopolysaccharide alginate and conversion to a mucoid phenotype in Pseudomonas aeruginosa are markers for the onset of chronic lung infection in cystic fibrosis (CF). Alginate production is regulated by the extracytoplasmic function (ECF) sigma factor AlgU/T and the cognate anti-sigma factor MucA. Many clinical mucoid isolates carry loss-of-function mutations in mucA. These mutations, including the most common mucA22 allele, cause C-terminal truncations in MucA, indicating that an inability to regulate AlgU activity by MucA is associated with conversion to the mucoid phenotype. Here we report that a mutation in a stable mucoid strain derived from the parental strain PAO1, designated PAO581, that does not contain the mucA22 allele, was due to a single-base deletion in mucA (DeltaT180), generating another type of C-terminal truncation. A global mariner transposon screen in PAO581 for non-mucoid isolates led to the identification of three regulators of alginate production, clpP (PA1801), clpX (PA1802), and a clpP paralogue (PA3326, designated clpP2). The PAO581 null mutants of clpP, clpX and clpP2 showed decreased AlgU transcriptional activity and an accumulation of haemagglutinin (HA)-tagged N-terminal MucA protein with an apparent molecular mass of 15 kDa. The clpP and clpX mutants of a CF mucoid isolate revert to the non-mucoid phenotype. The ClpXP and ClpP2 proteins appear to be part of a proteolytic network that degrades the cytoplasmic portion of truncated MucA proteins to release the sequestered AlgU, which drives alginate biosynthesis.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
353. Ryoo  NH, Lee  K, Lim  JB, Lee  YH, Bae  IK, Jeong  SH,     ( 2009 )

Outbreak by meropenem-resistant Pseudomonas aeruginosa producing IMP-6 metallo-beta-lactamase in a Korean hospital.

Diagnostic microbiology and infectious disease 63 (1)
PMID : 18993011  :   DOI  :   10.1016/j.diagmicrobio.2008.08.019    
Abstract >>
Pseudomonas aeruginosa isolates containing the bla(IMP-6) gene were recovered from 5 patients hospitalized at a tertiary-care hospital in Korea. The bla(IMP-6) gene was in a class 1 integron containing 5 different insert gene cassettes. All of the isolates showed identical pattern in SpeI macrorestriction analysis.
KeywordMeSH Terms
354. Li  H, Toleman  MA, Bennett  PM, Jones  RN, Walsh  TR,     ( 2008 )

Complete Sequence of p07-406, a 24,179-base-pair plasmid harboring the blaVIM-7 metallo-beta-lactamase gene in a Pseudomonas aeruginosa isolate from the United States.

Antimicrobial agents and chemotherapy 52 (9)
PMID : 18591274  :   DOI  :   10.1128/AAC.01093-07     PMC  :   PMC2533458    
Abstract >>
An outbreak involving a Pseudomonas aeruginosa strain that was resistant to all tested antimicrobials except polymyxin B occurred in a hospital in Houston, TX. Previous studies on this strain showed that it possesses a novel mobile metallo-beta-lactamase (MBL) gene, designated bla(VIM-7), located on a plasmid (p07-406). Here, we report the complete sequence, annotation, and functional characterization of this plasmid. p07-406 is 24,179 bp in length, and 29 open reading frames were identified related to known or putatively recognized proteins. Analysis of this plasmid showed it to be comprised of four distinct regions: (i) a region of 5,200 bp having a Tn501-like mercuric resistance (mer) transposon upstream of the replication region; (ii) a Tn3-like transposon carrying a truncated integron with a bla(VIM-7) gene and an insertion sequence inserted at the other end of this transposon; (iii) a region of four genes, upstream of the Tn3-like transposon, possessing very high similarity to plasmid pXcB from Xanthomonas campestris pv. citri commonly associated with plants; (iv) a backbone sequence similar to the backbone structure of the IncP group plasmid Rms149, pB10, and R751. This is the first plasmid to be sequenced carrying an MBL gene and highlights the amelioration of DNA segments from disparate origins, most noticeably from plant pathogens.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Sequence Analysis, DNA
355. Samuelsen  O, Buarø  L, Toleman  MA, Giske  CG, Hermansen  NO, Walsh  TR, Sundsfjord  A,     ( 2009 )

The first metallo-beta-lactamase identified in norway is associated with a TniC-like transposon in a Pseudomonas aeruginosa isolate of sequence type 233 imported from Ghana.

Antimicrobial agents and chemotherapy 53 (1)
PMID : 19015364  :   DOI  :   10.1128/AAC.00785-08     PMC  :   PMC2612188    
Abstract >>
N/A
KeywordMeSH Terms
356. Wolter  DJ, Kurpiel  PM, Woodford  N, Palepou  MF, Goering  RV, Hanson  ND,     ( 2009 )

Phenotypic and enzymatic comparative analysis of the novel KPC variant KPC-5 and its evolutionary variants, KPC-2 and KPC-4.

Antimicrobial agents and chemotherapy 53 (2)
PMID : 19015357  :   DOI  :   10.1128/AAC.00734-08     PMC  :   PMC2630594    
Abstract >>
A novel Klebsiella pneumoniae carbapenemase (KPC) variant, designated bla(KPC-5), was discovered in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate from Puerto Rico. Characterization of the upstream region of bla(KPC-5) showed significant differences from the flanking regions of other bla(KPC) variants. Comparison of amino acid sequences with those of other KPC enzymes revealed that KPC-5 was an intermediate between KPC-2 and KPC-4, differing from KPC-2 by a single amino acid substitution (Pro(103)-->Arg), while KPC-4 contained Pro(103)-->Arg plus an additional amino acid change (Val(239)-->Gly). Transformation studies with an Escherichia coli recipient strain showed differences in the properties of the KPC variants. KPC-4 and KPC-5 both had pIs of 7.65, in contrast with the pI of 6.7 for KPC-2. KPC-2 transformants were less susceptible to the carbapenems than KPC-4 and KPC-5 transformants. These data correlated with higher rates of imipenem hydrolysis for KPC-2 than for KPC-4 and KPC-5. However, KPC-4 and KPC-5 transformants had higher ceftazidime MICs, and the enzymes from these transformants had slightly better hydrolysis of this drug than KPC-2. KPC-4 and KPC-5 were more sensitive than KPC-2 to inhibition by clavulanic acid in both susceptibility testing and hydrolysis assays. Thus, KPC enzymes may be evolving through stepwise mutations to alter their spectra of activity.
KeywordMeSH Terms
357. Lin  X, Xu  W, Huang  K, Mei  X, Liang  Z, Li  Z, Guo  J, Luo  Y,     ( 2009 )

Cloning, expression and characterization of recombinant elastase from Pseudomonas aeruginosa in Picha pastoris.

Protein expression and purification 63 (2)
PMID : 18952459  :   DOI  :   10.1016/j.pep.2007.12.011    
Abstract >>
The gene lasB from Pseudomonas aeruginosa, which encoded elastase, was cloned and firstly successfully expressed in Pichia pastoris stain KM71 under the control of AOX promoter. The effects on the recombinant elastase activities of different pH, different temperatures and different metal ions were assayed. The full-length gene (1497 bp) encodes a preproenzyme including an N-terminal signal peptide (23 aa), a propeptide (197 aa) and mature elastase (301 aa). The recombinant elastase was secreted into culture supernatants using signal sequence from lasB and showed a single band at about 34 kDa by SDS-PAGE. The recombinant elastase expression hit the highest level of approximately 450 mg/L and the specific elastolytic activity of the recombinant elastase was 130 U/ml, which was approximately 26-fold higher than that of elastase obtained from P. aeruginosa. The optimal temperature and pH of the recombinant elastase was 28 degrees C and 7.4, respectively. The enzyme possessed high resistance to heat, and can be activated by Ca(2+). These enzyme properties suggested that it could be produced in an industrial scale and has the potential to be a commercial enzyme.
KeywordMeSH Terms
358. Papagiannitsis  CC, Tzouvelekis  LS, Miriagou  V,     ( 2009 )

Relative strengths of the class 1 integron promoter hybrid 2 and the combinations of strong and hybrid 1 with an active p2 promoter.

Antimicrobial agents and chemotherapy 53 (1)
PMID : 19001114  :   DOI  :   10.1128/AAC.00912-08     PMC  :   PMC2612174    
Abstract >>
The relative strengths of the uncommon promoters hybrid 2, hybrid 1 with an active P2 promoter (hybrid 1+P2), and strong+P2, which drive transcription of resistance genes in class 1 integrons, were evaluated using bla(GES-1) as a reporter gene cassette. Hybrid 2 was stronger than hybrid 1. Coupling P2 with the strong promoter and with hybrid 1 caused a measurable increase in GES-1 expression.
KeywordMeSH Terms
359. Karanam  VR, Reddy  HP, Subba Raju  BV, Rao  JC, Kavikishore  PB, Vijayalakshmi  M,     ( 2008 )

Detection of indicator pathogens from pharmaceutical finished products and raw materials using multiplex PCR and comparison with conventional microbiological methods.

Journal of industrial microbiology & biotechnology 35 (9)
PMID : 18521641  :   DOI  :   10.1007/s10295-008-0376-z    
Abstract >>
A multiplex PCR assay was devised and compared with standard conventional methods for quality evaluation of pharmaceutical raw materials and finished products with low levels of microbial contamination. Samples which were artificially contaminated with <10 colony forming units of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella species and possibly contaminated samples were incubated for 16 h with different enrichment media. Primers that deduce 559 bp fragment of the 16S rRNA gene was employed in amplifying E. coli species, similarly invasion protein gene with 275 bp fragment size was used as target for detecting Salmonella spp., in case of S. aureus a 461 bp amplicon from m-RNA nuclease gene, and an 709 bp fragment from oprL gene was used for amplifying P. aeruginosa. The detection limits for artificially contaminants by multiplex PCR was 1 CFU/g, where as in case of conventional method the detection limit was >2 CFU/g. Similarly, when tested with possibly contaminated samples, 35% were detected for E. coli, Salmonella spp., S. aureus and P. aeruginosa species with multiplex PCR, while only 21% were detected with standard conventional microbial methods. Multiplex PCR assay provides sensitive and reliable results and allows for the cost-effective detection of all four bacterial pathogens in single reaction tube.
KeywordMeSH Terms
360. Schneider  I, Keuleyan  E, Rasshofer  R, Markovska  R, Queenan  AM, Bauernfeind  A,     ( 2008 )

VIM-15 and VIM-16, two new VIM-2-like metallo-beta-lactamases in Pseudomonas aeruginosa isolates from Bulgaria and Germany.

Antimicrobial agents and chemotherapy 52 (8)
PMID : 18519714  :   DOI  :   10.1128/AAC.00175-08     PMC  :   PMC2493107    
Abstract >>
Two Pseudomonas aeruginosa urine isolates from Bulgaria and Germany produced two new VIM-2 variants. VIM-15 had one amino acid substitution (Tyr218Phe) which caused a significant increase in hydrolytic efficiency. The substitution Ser54Leu, characterizing VIM-16, showed no influence on enzyme activity. Both genes were part of class I integrons located in the chromosome.
KeywordMeSH Terms
361. Arai  H, Igarashi  Y, Kodama  T,     ( 1991 )

Anaerobically induced expression of the nitrite reductase cytochrome c-551 operon from Pseudomonas aeruginosa.

FEBS letters 280 (2)
PMID : 1849489  :   DOI  :   10.1016/0014-5793(91)80329-2    
Abstract >>
The nitrite reductase gene (denA) and the cytochrome c-551 gene (denB) are located only 50 bp apart from each other in the Pseudomonas aeruginosa chromosome. We report evidence that these two genes are co-transcribed as an operon only under anaerobic (denitrifying) conditions. The nucleotide sequence of the promoter (regulatory) region of the operon is highly AT-rich and contains a sequence closely resembling the consensus FNR binding site in E. coli.
KeywordMeSH Terms
Bacterial Proteins
Operon
Promoter Regions, Genetic
362. Vézina  G, Levesque  RC,     ( 1991 )

Molecular characterization of the class II multiresistance transposable element Tn1403 from Pseudomonas aeruginosa.

Antimicrobial agents and chemotherapy 35 (2)
PMID : 1850969  :   DOI  :   10.1128/aac.35.2.313     PMC  :   PMC244998    
Abstract >>
Transposon Tn1403 is a 19.9-kb multiresistance class II transposable element originally found on the RPL11 plasmid from a clinical isolate of Pseudomonas aeruginosa. It encodes resistance to ampicillin (PSE-1 beta-lactamase), streptomycin and spectinomycin (aadA and aphC), and chloramphenicol (cat). It has structural homology with the tnpM and tnpI sequences of Tn21 and inverted repeats and res and tnpR sequences of Tn501, but it has no structural homology nor functional complementation with the resolvase gene of Tn21 or Tn3. Sequence analysis revealed long inverted repeats at each extremity of Tn1403 containing 38-bp inverted repeats that were 97.4% similar to those of Tn1721 and 5-bp direct repeats. Transposition assays showed a low frequency of transposition (3.5 x 10(-6)) compared with that of Tn3 (3.3 x 10(-3)) and no resolution of cointegrates.
KeywordMeSH Terms
DNA Transposable Elements
Transposon Resolvases
363. Sevastsyanovich  YR, Krasowiak  R, Bingle  LE, Haines  AS, Sokolov  SL, Kosheleva  IA, Leuchuk  AA, Titok  MA, Smalla  K, Thomas  CM,     ( 2008 )

Diversity of IncP-9 plasmids of Pseudomonas.

Microbiology (Reading, England) 154 (Pt 10)
PMID : 18832300  :   DOI  :   10.1099/mic.0.2008/017939-0     PMC  :   PMC2885752    
Abstract >>
IncP-9 plasmids are important vehicles for degradation and resistance genes that contribute to the adaptability of Pseudomonas species in a variety of natural habitats. The three completely sequenced IncP-9 plasmids, pWW0, pDTG1 and NAH7, show extensive homology in replication, partitioning and transfer loci (an approximately 25 kb region) and to a lesser extent in the remaining backbone segments. We used PCR, DNA sequencing, hybridization and phylogenetic analyses to investigate the genetic diversity of 30 IncP-9 plasmids as well as the possibility of recombination between plasmids belonging to this family. Phylogenetic analysis of rep and oriV sequences revealed nine plasmid subgroups with 7-35 % divergence between them. Only one phenotypic character was normally associated with each subgroup, except for the IncP-9beta cluster, which included naphthalene- and toluene-degradation plasmids. The PCR and hybridization analysis using pWW0- and pDTG1-specific primers and probes targeting selected backbone loci showed that members of different IncP-9 subgroups have considerable similarity in their overall organization, supporting the existence of a conserved ancestral IncP-9 sequence. The results suggested that some IncP-9 plasmids are the product of recombination between plasmids of different IncP-9 subgroups but demonstrated clearly that insertion of degradative transposons has occurred on multiple occasions, indicating that association of this phenotype with these plasmids is not simply the result of divergent evolution from a single successful ancestral degradative plasmid.
KeywordMeSH Terms
Genetic Variation
364. Libisch  B, Balogh  B, Füzi  M,     ( 2009 )

Identification of two multidrug-resistant Pseudomonas aeruginosa clonal lineages with a countrywide distribution in Hungary.

Current microbiology 58 (2)
PMID : 18946702  :   DOI  :   10.1007/s00284-008-9285-7    
Abstract >>
The aim of this study was to identify class 1 integrons from extended-spectrum and metallo-beta-lactamase-negative, multidrug-resistant Pseudomonas aeruginosa clinical isolates from Hungary and to characterize the isolates by phenotypic and molecular methods. Fourteen selected P. aeruginosa isolates resistant to ceftazidime, gentamicin, and ciprofloxacin were subjected to serotyping, random amplification of polymorphic DNA (RAPD), integron content analysis, and a phenotypic test to detect high-level production of AmpC. Four representative isolates were further analyzed by multilocus sequence typing. Two P. aeruginosa multidrug-resistant clonal lineages were identified with a countrywide distribution. The first lineage is characterized by serotype O4, RAPD genotype A, sequence type ST175, and the presence of a class 1 integron harbouring aadB and aadA13 gene cassettes in its variable region. The second lineage is characterized by serotype O6, RAPD genotype B, sequence type ST395, and a class 1 integron carrying a single aadB cassette. The corresponding isolates were recovered from altogether 11 towns in Hungary. ST175 and ST395 are the presently calculated founders of two distinct P. aeruginosa clonal complexes that appear to have a wide geographical distribution also outside Hungary. The multidrug-resistant phenotype associated with these two clonal lineages might have contributed to an increase in their frequency and to their subsequent diversification. Both P. aeruginosa lineages displayed > or =8-fold synergy with boronic acid/ceftazidime combinations, suggesting an AmpC-mediated resistance to ceftazidime. Our observations underscore the role of class 1 integrons in the spread of aminoglycoside resistance by clonal dissemination among P. aeruginosa clinical isolates in Hungary.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
365. Jellouli  K, Bayoudh  A, Manni  L, Agrebi  R, Nasri  M,     ( 2008 )

Purification, biochemical and molecular characterization of a metalloprotease from Pseudomonas aeruginosa MN7 grown on shrimp wastes.

Applied microbiology and biotechnology 79 (6)
PMID : 18512057  :   DOI  :   10.1007/s00253-008-1517-z    
Abstract >>
A protease-producing bacterium was isolated and identified as Pseudomonas aeruginosa MN7. The strain was found to produce proteases when it was grown in media containing only shrimp waste powder (SWP), indicating that it can obtain its carbon, nitrogen, and salts requirements directly from shrimp waste. The use of 60 g/l SWP resulted in a high protease production. Elastase, the major protease produced by P. aeruginosa MN7, was purified from the culture supernatant to homogeneity using acetone precipitation, Sephadex G-75 gel filtration, and ultrafiltration using a 10-kDa cut-off membrane, with a 5.2-fold increase in specific activity and 38.4% recovery. The molecular weight of the purified elastase was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The optimum temperature and pH for protease activity were 60 degrees C and 8.0, respectively. The activity of the enzyme was totally lost in the presence of ethylene glycol tetraacetic acid, suggesting that the purified enzyme is a metalloprotease. The purified enzyme was highly stable in the presence of organic solvents, retaining 100% of its initial activity after 60 days of incubation at 30 degrees C in the presence of dimethyl sulfoxide and methanol. The lasB gene, encoding the MN7 elastase, was isolated and its DNA sequence was determined.
KeywordMeSH Terms
366. Lee  MF, Peng  CF, Hsu  HJ, Chen  YH,     ( 2008 )

Molecular characterisation of the metallo-beta-lactamase genes in imipenem-resistant Gram-negative bacteria from a university hospital in southern Taiwan.

International journal of antimicrobial agents 32 (6)
PMID : 18804966  :   DOI  :   10.1016/j.ijantimicag.2008.07.009    
Abstract >>
In this study, 260 non-replicate imipenem-resistant Gram-negative bacteria isolated between January 2002 and December 2006 were subjected to a screening test for detection of metallo-beta-lactamase (MBL) using the Etest containing imipenem and ethylene diamine tetra-acetic acid (EDTA). Among the 260 strains, 123 (47.3%) appeared to produce MBL. Of these 123 strains, 113 (91.9%) were found by polymerase chain reaction (PCR) to carry MBL genes of types blaVIM-2, blaVIM-3, blaVIM-11 (blaVIM-11a), blaIMP-8 and novel blaIMP-24. One strain of Serratia marcescens harboured two MBL genes (blaVIM-11 and blaIMP-8) simultaneously. Of the 123 strains, 116 strains (94.3%) carrying the intI1 gene and 21 strains carrying integron-associated blaVIM-3, blaVIM-11 and blaIMP-8 genes were identified among Acinetobacter baumannii, Pseudomonas aeruginosa, Acinetobacter haemolyticus and S. marcescens. Using pulsed-field gel electrophoresis (PFGE) and Southern hybridisation with the blaVIM gene probe for I-CeuI-digested genomic DNA, P. aeruginosa 9527 strain harboured two class 1 integron-associated MBL genes in the chromosome, including blaVIM-3-orf2-aacA4 and novel bla(VIM-3)-orf2-aacA4-aadB-aacA4. This is the first description of the blaVIM-11 gene spreading among P. aeruginosa and A. baumannii strains in southern Taiwan. This finding suggests that clinical spread of this blaVIM-11 gene is a matter of great concern for carbapenem resistance in southern Taiwan.
KeywordMeSH Terms
367. Jeannot  K, Elsen  S, Köhler  T, Attree  I, van Delden  C, Plésiat  P,     ( 2008 )

Resistance and virulence of Pseudomonas aeruginosa clinical strains overproducing the MexCD-OprJ efflux pump.

Antimicrobial agents and chemotherapy 52 (7)
PMID : 18474583  :   DOI  :   10.1128/AAC.01107-07     PMC  :   PMC2443911    
Abstract >>
Since their initial description 2 decades ago, MexCD-OprJ-overproducing efflux mutants of Pseudomonas aeruginosa (also called nfxB mutants) have rarely been described in the clinical setting. Screening of 110 nonreplicate clinical isolates showing moderate resistance to ciprofloxacin (MIC from 0.5 microg/ml to 4 microg/ml) yielded only four mutants (3.6%) of that type harboring various alterations in the repressor gene nfxB. MexCD-OprJ upregulation correlated with an increased resistance to ciprofloxacin, cefepime, and chloramphenicol in most of the clinical strains, concomitant with a higher susceptibility to ticarcillin, aztreonam, imipenem, and aminoglycosides. Evidence was obtained that this increased susceptibility to aminoglycosides results from the impaired activity of efflux pump MexXY-OprM. Furthermore, MexCD-OprJ upregulation was found to impair bacterial growth and to have a strain-specific, variable impact on rhamnolipid, elastase, phospholipase C, and pyocyanin production. Review of patient files indicated that the four nfxB mutants were responsible for confirmed cases of infection and emerged during long-term therapy with ciprofloxacin. Taken together, these data show that, while rather infrequent among P. aeruginosa strains with low-level resistance to ciprofloxacin, MexCD-OprJ-overproducing mutants may be isolated after single therapy with fluoroquinolones and may be pathogenic.
KeywordMeSH Terms
368. Garza-Ramos  U, Morfin-Otero  R, Sader  HS, Jones  RN, Hernández  E, Rodriguez-Noriega  E, Sanchez  A, Carrillo  B, Esparza-Ahumada  S, Silva-Sanchez  J,     ( 2008 )

Metallo-beta-lactamase gene bla(IMP-15) in a class 1 integron, In95, from Pseudomonas aeruginosa clinical isolates from a hospital in Mexico.

Antimicrobial agents and chemotherapy 52 (8)
PMID : 18490501  :   DOI  :   10.1128/AAC.00679-07     PMC  :   PMC2493115    
Abstract >>
During 2003, 40 carbapenem-resistant Pseudomonas aeruginosa clinical isolates collected in a Mexican tertiary-care hospital were screened for metallo-beta-lactamase production. Thirteen isolates produced IMP-15, and 12 had a single pulsed-field gel electrophoresis pattern. The bla(IMP-15) gene cassette was inserted in a plasmid-borne integron with a unique array of gene cassettes and was named In95.
KeywordMeSH Terms
369. Labuschagne  Cde J, Weldhagen  GF, Ehlers  MM, Dove  MG,     ( 2008 )

Emergence of class 1 integron-associated GES-5 and GES-5-like extended-spectrum beta-lactamases in clinical isolates of Pseudomonas aeruginosa in South Africa.

International journal of antimicrobial agents 31 (6)
PMID : 18436436  :   DOI  :   10.1016/j.ijantimicag.2008.01.020    
Abstract >>
Several different Guiana extended-spectrum (GES) enzymes have been described occurring in Enterobacteriaceae and Pseudomonas aeruginosa worldwide. Polymerase chain reaction and gene sequencing analysis confirmed bla(GES) genes identified in three P. aeruginosa clinical isolates from South Africa as bla(GES-5) and bla(GES-5)-like, respectively. Compared with GES-1, the GES-5-like protein exhibited an A21E amino acid change, a novel mutation not previously described in this family. Integron structures identified upstream from the bla(GES-5) and bla(GES-5)-like genes were found to be identical to bla(GES-2)-carrying integrons described previously from the same geographical region. These findings confirm the establishment and persistence of integron-associated GES-type extended-spectrum beta-lactamases (ESBLs) in the South African nosocomial environment. This study describes the first isolation of class 1 integron-associated bla(GES-5) and the emergence of a novel GES-5-like ESBL in South Africa.
KeywordMeSH Terms
370. Battle  SE, Meyer  F, Rello  J, Kung  VL, Hauser  AR,     ( 2008 )

Hybrid pathogenicity island PAGI-5 contributes to the highly virulent phenotype of a Pseudomonas aeruginosa isolate in mammals.

Journal of bacteriology 190 (21)
PMID : 18757543  :   DOI  :   10.1128/JB.00785-08     PMC  :   PMC2580712    
Abstract >>
Most known virulence determinants of Pseudomonas aeruginosa are remarkably conserved in this bacterium's core genome, yet individual strains differ significantly in virulence. One explanation for this discrepancy is that pathogenicity islands, regions of DNA found in some strains but not in others, contribute to the overall virulence of P. aeruginosa. Here we employed a strategy in which the virulence of a panel of P. aeruginosa isolates was tested in mouse and plant models of disease, and a highly virulent isolate, PSE9, was chosen for comparison by subtractive hybridization to a less virulent strain, PAO1. The resulting subtractive hybridization sequences were used as tags to identify genomic islands found in PSE9 but absent in PAO1. One 99-kb island, designated P. aeruginosa genomic island 5 (PAGI-5), was a hybrid of the known P. aeruginosa island PAPI-1 and novel sequences. Whereas the PAPI-1-like sequences were found in most tested isolates, the novel sequences were found only in the most virulent isolates. Deletional analysis confirmed that some of these novel sequences contributed to the highly virulent phenotype of PSE9. These results indicate that targeting highly virulent strains of P. aeruginosa may be a useful strategy for identifying pathogenicity islands and novel virulence determinants.
KeywordMeSH Terms
371. Giardina  G, Rinaldo  S, Johnson  KA, Di Matteo  A, Brunori  M, Cutruzzol?  F,     ( 2008 )

NO sensing in Pseudomonas aeruginosa: structure of the transcriptional regulator DNR.

Journal of molecular biology 378 (5)
PMID : 18420222  :   DOI  :   10.1016/j.jmb.2008.03.013    
Abstract >>
All denitrifying bacteria can keep the steady-state concentrations of nitrite and nitric oxide (NO) below cytotoxic levels, controlling the expression of the denitrification gene clusters by redox signaling, mainly through transcriptional regulators belonging either to the DNR (dissimilative nitrate respiration regulator) or to the NnrR (nitrite and nitric oxide reductase regulator) subgroups of the FNR (fumarate and nitrate reductase regulatory protein)-CRP (cAMP receptor protein) superfamily. The NO dependence of the transcriptional activity of promoters regulated by these transcription factors has suggested that they may act as NO sensors in vivo. Despite great interest in the regulation of denitrification, which in Pseudomonas aeruginosa is strictly related to virulence, functional and structural characterization of these NO sensors is still lacking. Here we present the three-dimensional structure of the sensor domain of the DNR from P. aeruginosa at 2.1 A resolution. This is the first structure of a putative NO-sensing bacterial transcriptional regulator and reveals the presence of a large hydrophobic cavity that may be the cofactor binding site. Parallel spectroscopic evidence indicates that apo-DNR binds heme in vitro and that the heme-bound form reacts with carbon monoxide and NO, thus supporting the hypothesis that NO sensing involves gas binding to the ferrous heme. Preliminary experiments indicate that heterologous expression of the heme-containing DNR yields a protein able to bind DNA in vitro.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Protein Structure, Tertiary
Pseudomonas aeruginosa
Transcription, Genetic
372. Williams  SR, Gebhart  D, Martin  DW, Scholl  D,     ( 2008 )

Retargeting R-type pyocins to generate novel bactericidal protein complexes.

Applied and environmental microbiology 74 (12)
PMID : 18441117  :   DOI  :   10.1128/AEM.00141-08     PMC  :   PMC2446544    
Abstract >>
R-type pyocins are high-molecular-weight bacteriocins that resemble bacteriophage tail structures and are produced by some Pseudomonas aeruginosa strains. R-type pyocins kill by dissipating the bacterial membrane potential after binding. The high-potency, single-hit bactericidal kinetics of R-type pyocins suggest that they could be effective antimicrobials. However, the limited antibacterial spectra of natural R-type pyocins would ultimately compromise their clinical utility. The spectra of these protein complexes are determined in large part by their tail fibers. By replacing the pyocin tail fibers with tail fibers of Pseudomonas phage PS17, we changed the bactericidal specificity of R2 pyocin particles to a different subset of P. aeruginosa strains, including some resistant to PS17 phage. We further extended this idea by fusing parts of R2 tail fibers with parts of tail fibers from phages that infect other bacteria, including Escherichia coli and Yersinia pestis, changing the killing spectrum of pyocins from P. aeruginosa to the bacterial genus, species, or strain that serves as a host for the donor phage. The assembly of active R-type pyocins requires chaperones specific for the C-terminal portion of the tail fiber. Natural and retargeted R-type pyocins exhibit narrow bactericidal spectra and thus can be expected to cause little collateral damage to the healthy microbiotae and not to promote the horizontal spread of multidrug resistance among bacteria. Engineered R-type pyocins may offer a novel alternative to traditional antibiotics in some infections.
KeywordMeSH Terms
373. Hayden  HS, Gillett  W, Saenphimmachak  C, Lim  R, Zhou  Y, Jacobs  MA, Chang  J, Rohmer  L, D'Argenio  DA, Palmieri  A, Levy  R, Haugen  E, Wong  GK, Brittnacher  MJ, Burns  JL, Miller  SI, Olson  MV, Kaul  R,     ( 2008 )

Large-insert genome analysis technology detects structural variation in Pseudomonas aeruginosa clinical strains from cystic fibrosis patients.

Genomics 91 (6)
PMID : 18445516  :   DOI  :   10.1016/j.ygeno.2008.02.005     PMC  :   PMC2587363    
Abstract >>
Large-insert genome analysis (LIGAN) is a broadly applicable, high-throughput technology designed to characterize genome-scale structural variation. Fosmid paired-end sequences and DNA fingerprints from a query genome are compared to a reference sequence using the Genomic Variation Analysis (GenVal) suite of software tools to pinpoint locations of insertions, deletions, and rearrangements. Fosmids spanning regions that contain new structural variants can then be sequenced. Clonal pairs of Pseudomonas aeruginosa isolates from four cystic fibrosis patients were used to validate the LIGAN technology. Approximately 1.5 Mb of inserted sequences were identified, including 743 kb containing 615 ORFs that are absent from published P. aeruginosa genomes. Six rearrangement breakpoints and 220 kb of deleted sequences were also identified. Our study expands the "genome universe" of P. aeruginosa and validates a technology that complements emerging, short-read sequencing methods that are better suited to characterizing single-nucleotide polymorphisms than structural variation.
KeywordMeSH Terms
Genome, Bacterial
374. Khan  NH, Ahsan  M, Yoshizawa  S, Hosoya  S,