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1. McKay  GA, Woods  DE, MacDonald  KL, Poole  K,     ( 2003 )

Role of phosphoglucomutase of Stenotrophomonas maltophilia in lipopolysaccharide biosynthesis, virulence, and antibiotic resistance.

Infection and immunity 71 (6)
PMID : 12761084  :   DOI  :   10.1128/iai.71.6.3068-3075.2003     PMC  :   PMC155759    
Abstract >>
A homologue of the algC gene, responsible for the production of a phosphoglucomutase (PGM) associated with LPS and alginate biosynthesis in Pseudomonas aeruginosa, spgM, was cloned from Stenotrophomonas maltophilia. The spgM gene was shown to encode a bifunctional enzyme with both PGM and phosphomannomutase activities. Mutants lacking spgM produced less LPS than the SpgM(+) parent strain and had a tendency for shorter O polysaccharide chains. No changes in LPS chemistry were obvious as a result of the loss of spgM. Significantly, however, spgM mutants displayed a modest increase in susceptibility to several antimicrobial agents and were completely avirulent in an animal model of infection. The latter finding may relate to the resultant serum sensitivity of spgM mutants which, unlike the wild-type parent strain, were rapidly killed by human serum. These data highlight the contribution made by LPS to the antimicrobial resistance and virulence of S. maltophilia.
KeywordMeSH Terms
2. Sánchez  P, Alonso  A, Martinez  JL,     ( 2002 )

Cloning and characterization of SmeT, a repressor of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF.

Antimicrobial agents and chemotherapy 46 (11)
PMID : 12384340  :   DOI  :   10.1128/aac.46.11.3386-3393.2002     PMC  :   PMC128709    
Abstract >>
We report on the cloning of the gene smeT, which encodes the transcriptional regulator of the Stenotrophomonas maltophilia efflux pump SmeDEF. SmeT belongs to the TetR and AcrR family of transcriptional regulators. The smeT gene is located upstream from the structural operon of the pump genes smeDEF and is divergently transcribed from those genes. Experiments with S. maltophilia and the heterologous host Escherichia coli have demonstrated that SmeT is a transcriptional repressor. S1 nuclease mapping has demonstrated that expression of smeT is driven by a single promoter lying close to the 5' end of the gene and that expression of smeDEF is driven by an unique promoter that overlaps with promoter PSMET: The level of expression of smeT is higher in smeDEF-overproducing S. maltophilia strain D457R, which suggests that SmeT represses its own expression. Band-shifting assays have shown that wild-type strain S. maltophilia D457 contains a cellular factor(s) capable of binding to the intergenic smeT-smeD region. That cellular factor(s) was absent from smeDEF-overproducing S. maltophilia strain D457R. The sequence of smeT from D457R showed a point mutation that led to a Leu166Gln change within the SmeT protein. This change allowed overexpression of both smeDEF and smeT in D457R. It was noteworthy that expression of wild-type SmeT did not fully complement the smeT mutation in D457R. This suggests that the wild-type protein is not dominant over the mutant SmeT.
KeywordMeSH Terms
3. Labahn  J, Neumann  S, Büldt  G, Kula  MR, Granzin  J,     ( 2002 )

An alternative mechanism for amidase signature enzymes.

Journal of molecular biology 322 (5)
PMID : 12367528  :   DOI  :   10.1016/s0022-2836(02)00886-0    
Abstract >>
The peptide amidase from Stenotrophomonas maltophilia catalyses predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Peptide bonds or amide functions in amino acid side-chains are not hydrolysed. This specificity makes peptide amidase (Pam) interesting for different biotechnological applications. Pam belongs to the amidase signature (AS) family. It is the first protein within this family whose tertiary structure has been solved. The structure of the native Pam has been determined with a resolution of 1.4A and in complex with the competitive inhibitor chymostatin at a resolution of 1.8A. Chymostatin, which forms acyl adducts with many serine proteases, binds non-covalently to this enzyme.Pam folds as a very compact single-domain protein. The AS sequence represents a core domain that is covered by alpha-helices. This AS domain contains the catalytic residues. It is topologically homologous to the phosphoinositol phosphatase domain. The structural data do not support the recently proposed Ser-Lys catalytic dyad mechanism for AS enzymes. Our results are in agreement with the role of Ser226 as the primary nucleophile but differ concerning the roles of Ser202 and Lys123: Ser202, with direct contact both to the substrate molecule and to Ser226, presumably serves as an acid/bases catalyst. Lys123, with direct contact to Ser202 but no contact to Ser226 or the substrate molecule, most likely acts as an acid catalyst.
KeywordMeSH Terms
4. Windhorst  S, Frank  E, Georgieva  DN, Genov  N, Buck  F, Borowski  P, Weber  W,     ( 2002 )

The major extracellular protease of the nosocomial pathogen Stenotrophomonas maltophilia: characterization of the protein and molecular cloning of the gene.

The Journal of biological chemistry 277 (13)
PMID : 11796713  :   DOI  :   10.1074/jbc.M109525200    
Abstract >>
Stenotrophomonas maltophilia is increasingly emerging as a multiresistant pathogen in the hospital environment. In immunosuppressed patients, these bacteria may cause severe infections associated with tissue lesions such as pulmonary hemorrhage. This suggests proteolysis as a possible pathogenic mechanism in these infections. This study describes a protease with broad specificity secreted by S. maltophilia. The gene, termed StmPr1, codes for a 63-kDa precursor that is processed to the mature protein of 47 kDa. The enzyme is an alkaline serine protease that, by sequence homology and enzymic properties, can be further classified as a new member of the family of subtilases. It differs from the classic subtilisins in molecular size, in substrate specificity, and probably in the architecture of the active site. The StmPr1 protease is able to degrade several human proteins from serum and connective tissue. Furthermore, pan-protease inhibitors such as alpha(1)-antitrypsin and alpha(2)-macroglobulin were unable to abolish the activity of the bacterial protease. The data support the interpretation that the extracellular protease of S. maltophilia functions as a pathogenic factor and thus could serve as a target for the development of therapeutic agents.
KeywordMeSH Terms
5. Li  XZ, Zhang  L, Poole  K,     ( 2002 )

SmeC, an outer membrane multidrug efflux protein of Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 46 (2)
PMID : 11796339  :   DOI  :   10.1128/aac.46.2.333-343.2002     PMC  :   PMC127032    
Abstract >>
A homologue of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa, smeABC, was cloned from Stenotrophomonas maltophilia by using, as a probe, a PCR product amplified from this organism with primers based on the mexB sequence. The smeABC genes were hyperexpressed in a mutant strain displaying resistance to several antimicrobials, including aminoglycosides, beta-lactams, and fluoroquinolones. Deletions in smeC but not smeB compromised this resistance, suggesting that SmeC contributed to the multidrug resistance of the mutant as part of another, as-yet-unidentified multidrug efflux system. Consistent with SmeC functioning independently of SmeAB, a promoter activity was identified upstream of smeC. Upstream of the smeABC genes, a putative two-gene operon, smeSR, encoding homologues of bacterial two-component regulatory systems was identified. The cloned smeR gene activated expression of a smeA-lacZ fusion, indicating that SmeR positively regulates expression of the smeABC genes. Consistent with this, the multidrug resistance of the SmeABC-hyperexpressing mutant was compromised by deletion of smeR. Intriguingly, SmeC expression in S. maltophilia paralleled a beta-lactamase activity provided by a C-terminally truncated L2 enzyme, which was apparently responsible for the beta-lactam resistance of the SmeABC-hyperexpressing mutant. This represents the first report of coregulation of an efflux resistance determinant and a beta-lactamase.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Operon
6. Neumann  S, Kula  MR,     ( 2002 )

Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia.

Applied microbiology and biotechnology 58 (6)
PMID : 12021798  :   DOI  :   10.1007/s00253-002-0943-6    
Abstract >>
The peptide amidase (Pam) from the gram-negative bacterium Stenotrophomonas maltophilia catalyzes predominantly the hydrolysis of the C-terminal amide bond in peptide amides. Its gene (pam) was isolated by Southern hybridization using a DNA probe derived from the known N-terminal amino acid sequence. Pam is a member of the amidase signature family and was identified as a periplasmic protein by an N-terminal signal peptide found in the gene. The processed protein consists of 503 amino acids with a molecular mass of 53.5 kDa. The recombinant enzyme with a C-terminal His(6) tag has a monomeric structure and its isoelectric point is 6.3. The dipeptide amide L-Ala- L-Phe-NH(2) is hydrolyzed in the absence of cofactors to L-Ala- L-Phe-OH and ammonia with V(max)=194 U/mg and K(m) <0.5 mM. The natural function of Pam remains unclear. Chymostatin (K(i)<0.3 microM) and Pefabloc SC (K(i) not determined) were identified as inhibitors. When the gene was expressed in Escherichia coli on a 12-l scale, the specific activity in the crude extract was 60 U/mg, compared to 0.24 U/mg in S. maltophilia. In the expression system, Pam made up about 31% of the total soluble cell protein. From 75 g wet cells, 2.1 g of >95% pure enzyme was obtained, which corresponds to a total activity of 416,000 units.
KeywordMeSH Terms
7. Zhang  L, Li  XZ, Poole  K,     ( 2001 )

SmeDEF multidrug efflux pump contributes to intrinsic multidrug resistance in Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 45 (12)
PMID : 11709330  :   DOI  :   10.1128/AAC.45.12.3497-3503.2001     PMC  :   PMC90859    
Abstract >>
Stenotrophomonas maltophilia is an emerging nosocomial pathogen that displays high-level intrinsic resistance to a variety of structurally unrelated antimicrobial agents. Efflux mechanisms are known to contribute to acquired multidrug resistance in this organism, and indeed, one such multidrug efflux system, SmeDEF, was recently identified. Still, the importance of SmeDEF to intrinsic antibiotic resistance in S. maltophilia had not yet been determined. Reverse transcription-PCR confirmed expression of the smeDEF genes in wild-type S. maltophilia, and deletion of smeE or smeF in wild-type strains rendered the mutants hypersusceptible to several antimicrobials, suggesting that SmeDEF contributes to intrinsic antimicrobial resistance in this organism. Expression of smeDEF was also enhanced in an in vitro-selected multidrug-resistant mutant, although deletion of smeF but not of smeE in these mutants compromised antimicrobial resistance. Apparently, hyperexpressed SmeF is capable of functioning with additional multidrug efflux components to promote multidrug resistance in S. maltophilia.
KeywordMeSH Terms
8. Kobayashi  DY, Reedy  RM, Bick  J, Oudemans  PV,     ( 2002 )

Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control.

Applied and environmental microbiology 68 (3)
PMID : 11872449  :   DOI  :   10.1128/aem.68.3.1047-1054.2002     PMC  :   PMC123742    
Abstract >>
A chitinase gene was cloned on a 2.8-kb DNA fragment from Stenotrophomonas maltophilia strain 34S1 by heterologous expression in Burkholderia cepacia. Sequence analysis of this fragment identified an open reading frame encoding a deduced protein of 700 amino acids. Removal of the signal peptide sequence resulted in a predicted protein that was 68 kDa in size. Analysis of the sequence indicated that the chitinase contained a catalytic domain belonging to family 18 of glycosyl hydrolases. Three putative binding domains, a chitin binding domain, a novel polycystic kidney disease (PKD) domain, and a fibronectin type III domain, were also identified within the sequence. Pairwise comparisons of each domain to the most closely related sequences found in database searches clearly demonstrated variation in gene sources and the species from which related sequences originated. A 51-kDa protein with chitinolytic activity was purified from culture filtrates of S. maltophilia strain 34S1 by hydrophobic interaction chromatography. Although the protein was significantly smaller than the size predicted from the sequence, the N-terminal sequence verified that the first 15 amino acids were identical to the deduced sequence of the mature protein encoded by chiA. Marker exchange mutagenesis of chiA resulted in mutant strain C5, which was devoid of chitinolytic activity and lacked the 51-kDa protein in culture filtrates. Strain C5 was also reduced in the ability to suppress summer patch disease on Kentucky bluegrass, supporting a role for the enzyme in the biocontrol activity of S. maltophilia.
KeywordMeSH Terms
Magnaporthe
Pest Control, Biological
9. Avison  MB, Higgins  CS, von Heldreich  CJ, Bennett  PM, Walsh  TR,     ( 2001 )

Plasmid location and molecular heterogeneity of the L1 and L2 beta-lactamase genes of Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 45 (2)
PMID : 11158734  :   DOI  :   10.1128/AAC.45.2.413-419.2001     PMC  :   PMC90306    
Abstract >>
An approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia. This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 beta-lactamase genes. The location of L1 and L2 on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems. Sequence analysis of the 10 L1 genes revealed three novel genes, L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1 (L1a), respectively. The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lower k(cat)/K(m) ratios respectively). L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a. Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences. Three novel L2 genes (L2b, L2c, and L2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275 L2 (L2a) by 4, 9, and 25%, respectively. Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found. It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid.
KeywordMeSH Terms
10. Avison  MB, von Heldreich  CJ, Higgins  CS, Bennett  PM, Walsh  TR,     ( 2000 )

A TEM-2beta-lactamase encoded on an active Tn1-like transposon in the genome of a clinical isolate of Stenotrophomonas maltophilia.

The Journal of antimicrobial chemotherapy 46 (6)
PMID : 11102404  :   DOI  :   10.1093/jac/46.6.879    
Abstract >>
A constitutively expressed beta-lactamase gene from a clinical isolate of Stenotrophomonas maltophilia, J675Ia, has been cloned. Its DNA sequence is almost identical to that of bla(TEM2) (one nucleotide change) and the expressed enzyme is a Bush type 2a penicillinase with an amino acid sequence identical to that of TEM-2. The bla(TEM) gene was present within a novel Tn1/Tn3-type transposon in the genome of isolate J675Ia and the transposon was able to mobilize bla(TEM) on to the broad host-range conjugative plasmid, R388. When transferred to an Escherichia coli recipient, R388::Tn conferred high-level ampicillin resistance. This represents the first identification of a TEM beta-lactamase in S. maltophilia and the first evidence that this important clinical pathogen is able to act as a reservoir for mobile beta-lactamase genes in the hospital environment.
KeywordMeSH Terms
DNA Transposable Elements
Genome, Bacterial
11. 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
12. Sanchez  P, Alonso  A,     ( 2000 )

Stenotrophomonas maltophilia D457R contains a cluster of genes from gram-positive bacteria involved in antibiotic and heavy metal resistance.

Antimicrobial agents and chemotherapy 44 (7)
PMID : 10858330  :   DOI  :   10.1128/aac.44.7.1778-1782.2000     PMC  :   PMC89961    
Abstract >>
A cluster of genes involved in antibiotic and heavy metal resistance has been characterized from a clinical isolate of the gram-negative bacterium Stenotrophomonas maltophilia. These genes include a macrolide phosphotransferase (mphBM) and a cadmium efflux determinant (cadA), together with the gene cadC coding for its transcriptional regulator. The cadC cadA region is flanked by a truncated IS257 sequence and a region coding for a bin3 invertase. Despite their presence in a gram-negative bacterium, these genetic elements share a common gram-positive origin. The possible origin of these determinants as a remnant composite transposon as well as the role of gene transfer between gram-positive and gram-negative bacteria for the acquisition of antibiotic resistance determinants in chronic, mixed infections is discussed.
KeywordMeSH Terms
Drosophila Proteins
Multigene Family
Transcription Factors
13. Alonso  A, Martínez  JL,     ( 2000 )

Cloning and characterization of SmeDEF, a novel multidrug efflux pump from Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 44 (11)
PMID : 11036026  :   DOI  :   10.1128/aac.44.11.3079-3086.2000     PMC  :   PMC101606    
Abstract >>
Stenotrophomonas maltophilia is a nosocomial bacterial pathogen intrinsically resistant to several antibiotics. The mechanisms involved in this intrinsic multiresistance phenotype are poorly understood. A library of chromosomal DNA from a spontaneous multidrug-resistant S. maltophilia D457R mutant (A. Alonso and J. L. Martinez, Antimicrob. Agents Chemother. 41:1140-1142, 1997) was screened for complementation of erythromycin susceptibility on an antibiotic-hypersusceptible Escherichia coli DeltaacrAB strain. Cloning and further analysis revealed that a 6-kbp region constituting a transcriptional unit was capable of complementing the antibiotic-susceptible phenotype of an E. coli DeltaacrAB strain. We identified three open reading frames, smeD, smeE and smeF, which code for members of the membrane fusion protein, resistance nodulation division, and outer membrane factor families, respectively. Drug susceptibility assays indicated that the SmeDEF system cloned in E. coli mediates resistance to a wide range of antibiotics. Ethidium bromide and norfloxacin accumulation experiments in the presence and in the absence of carbonyl cyanide m-chlorophenylhydrazone showed that this system constitutes a drug efflux pump dependent on the membrane proton motive force. The presence of high levels of smeDEF mRNA in the multiresistant D457R mutant was consistent with the high levels of SmeF (formerly Omp54) observed in the same strain. In contrast, transcription levels of smeDEF in the D457 strain were tiny, which correlates with the low levels of SmeF observed for this strain. Also, for both the D457 and D457R strains, we observed growth phase-dependent regulation in which the highest level of transcription corresponded to early exponential phase, with transcription decreasing throughout the growth curve to undetectable levels at 24 h.
KeywordMeSH Terms
Bacterial Proteins
Membrane Transport Proteins
14. Aazaz  A, Wang  G,     ( 2000 )

Cloning and overexpression of a tyrosinase gene mel from Pseudomonas maltophila.

FEMS microbiology letters 185 (1)
PMID : 10731602  :   DOI  :   10.1111/j.1574-6968.2000.tb09035.x    
Abstract >>
The tyrosinase gene (mel), which is responsible for melanin formation, was isolated by shotgun cloning of SalI fragments of Pseudomonas maltophila DNA. A 0.7-kb SalI fragment in the recombinant plasmid pWSY8 imparted the ability to synthesize melanin to an Escherichia coli host HB101. The nucleotide sequence of this DNA fragment revealed an open reading frame of 504 bp, encoding a protein of 169 amino acids. The fragment containing the mel gene was then cloned into an expression plasmid pPAS1 under the control of a promoter isolated from the host, P. maltophilia AT18. This strain increased the melanin production by 70.6% compared with the strain HB101/pWSY8, in which the cloned mel gene was under the control of the lac promoter from the vector pUC18.
KeywordMeSH Terms
15. Li  XZ, Zhang  L,     ( 2000 )

Multiple antibiotic resistance in Stenotrophomonas maltophilia: involvement of a multidrug efflux system.

Antimicrobial agents and chemotherapy 44 (2)
PMID : 10639352  :   DOI  :   10.1128/aac.44.2.287-293.2000     PMC  :   PMC89673    
Abstract >>
Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics, although the mechanisms of resistance are generally poorly understood. Multidrug resistant (MDR) strains were readily selected by plating a sensitive reference strain of the organism individually onto a variety of antibiotics, including tetracycline, chloramphenicol, ciprofloxacin, and norfloxacin. Tetracycline-selected MDR strains typically showed cross-resistance to erythromycin and fluoroquinolones and, in some instances, aminoglycosides. MDR mutants selected with the other agents generally displayed resistance to chloramphenicol and fluoroquinolones only, although two MDR strains (e.g., K1385) were also resistant to erythromycin and hypersusceptible to aminoglycosides. Many of the MDR strains expressed either moderate or high levels of a novel outer membrane protein (OMP) of ca. 50 kDa molecular mass, a phenotype typical of MDR strains of Pseudomonas aeruginosa hyperexpressing drug efflux systems. Indeed, the 50-kDa OMP of these S. maltophilia MDR strains reacted with antibody to OprM, the outer membrane component of the MexAB-OprM MDR efflux system of P. aeruginosa. Similarly, a ca. 110-kDa cytoplasmic membrane protein of these MDR strains also reacted with antibody to the MexB component of the P. aeruginosa pump. The outer and cytoplasmic membranes of several clinical S. maltophilia strains also reacted with the anti-OprM and anti-MexB antibodies. N-terminal amino acid sequencing of a cyanogen bromide-generated peptide of the 50-kDa OMP of MDR strain K1385, dubbed SmeM (Stenotrophomonas multidrug efflux), revealed it to be very similar to a number of outer membrane multidrug efflux components of P. aeruginosa and Pseudomonas putida. Deletion of the L1 and L2 beta-lactamase genes confirmed that these enzymes were responsible for the bulk of the beta-lactam resistance of K1385 and its parent. Still, overexpression of the MDR efflux mechanism in an L1- and L2-deficient derivative of K1385 did yield a modest increase in resistance to a few beta-lactams. These data are consistent with the MDR efflux mechanism(s) playing a role in the multidrug resistance of S. maltophilia.
KeywordMeSH Terms
Drug Resistance, Microbial
Drug Resistance, Multiple
16. Ploy  MC, Lambert  T,     ( 1999 )

Characterization of the chromosomal aac(6')-Iz gene of Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 43 (10)
PMID : 10508008  :   PMC  :   PMC89484    
Abstract >>
The aac(6')-Iz gene of Stenotrophomonas maltophilia BM2690 encoding an aminoglycoside 6'-N-acetyltransferase was characterized. The gene was identified as a coding sequence of 462 bp corresponding to a protein with a calculated mass of 16,506 Da, a value in good agreement with that of ca. 16,000 found by in vitro coupled transcription-translation. Analysis of the deduced amino acid sequence indicated that the protein was a member of the major subfamily of aminoglycoside 6'-N-acetyltransferases. The enzyme conferred resistance to amikacin but not to gentamicin, indicating that it was an AAC(6') of type I. The open reading frame upstream from the aac(6')-Iz gene was homologous to the fprA gene of Myxococcus xanthus (61% identity), which encodes a putative pyridoxine (pyridoxamine) 5'-phosphate oxidase. Pulsed-field gel electrophoresis of total DNA from BM2690 and S. maltophilia ATTC 13637 digested with XbaI, DraI, and SpeI followed by hybridization with rRNA and aac(6')-Iz-specific probes indicated that the gene was located in the chromosome. The aac(6')-Iz gene was detected by DNA-DNA hybridization in all 80 strains of S. maltophilia tested. The MICs of gentamicin against these strains of S. maltophilia were lower than those of amikacin, netilmicin, and tobramycin, indicating that production of AAC(6')-Iz contributes to aminoglycoside resistance in S. maltophilia.
KeywordMeSH Terms
17. Chen  ZW, Hassan-Abdulah  A, Zhao  G, Jorns  MS, Mathews  FS,     ( 2006 )

Heterotetrameric sarcosine oxidase: structure of a diflavin metalloenzyme at 1.85 A resolution.

Journal of molecular biology 360 (5)
PMID : 16820168  :   DOI  :   10.1016/j.jmb.2006.05.067    
Abstract >>
The crystal structure of heterotetrameric sarcosine oxidase (TSOX) from Pseudomonas maltophilia has been determined at 1.85 A resolution. TSOX contains three coenzymes (FAD, FMN and NAD+), four different subunits (alpha, 103 kDa; beta, 44 kDa; gamma, 21 kDa; delta, 11 kDa) and catalyzes the oxidation of sarcosine (N-methylglycine) to yield hydrogen peroxide, glycine and formaldehyde. In the presence of tetrahydrofolate, the oxidation of sarcosine is coupled to the formation of 5,10-methylenetetrahydrofolate. The NAD+ and putative folate binding sites are located in the alpha-subunit. The FAD binding site is in the beta-subunit. FMN is bound at the interface of the alpha and beta-subunits. The FAD and FMN rings are separated by a short segment of the beta-subunit with the closest atoms located 7.4 A apart. Sulfite, an inhibitor of oxygen reduction, is bound at the FMN site. 2-Furoate, a competitive inhibitor with respect to sarcosine, is bound at the FAD site. The sarcosine dehydrogenase and 5,10-methylenetetrahydrofolate synthase sites are 35 A apart but connected by a large internal cavity (approximately 10,000 A3). An unexpected zinc ion, coordinated by three cysteine and one histidine side-chains, is bound to the delta-subunit. The N-terminal half of the alpha subunit of TSOX (alphaA) is closely similar to the FAD-binding domain of glutathione reductase but with NAD+ replacing FAD. The C-terminal half of the alpha subunit of TSOX (alphaB) is similar to the C-terminal half of dimethylglycine oxidase and the T-protein of the glycine cleavage system, proteins that bind tetrahydrofolate. The beta-subunit of TSOX is very similar to monomeric sarcosine oxidase. The gamma-subunit is similar to the C-terminal sub-domain of alpha-TSOX. The delta-subunit shows little similarity with any PDB entry. The alphaA domain/beta-subunit sub-structure of TSOX closely resembles the alphabeta dimer of L-proline dehydrogenase, a heteroctameric protein (alphabeta)4 that shows highest overall similarity to TSOX.
KeywordMeSH Terms
Models, Molecular
18. Hu  X, Fukutani  A, Liu  X, Kimbara  K, Kawai  F,     ( 2007 )

Isolation of bacteria able to grow on both polyethylene glycol (PEG) and polypropylene glycol (PPG) and their PEG/PPG dehydrogenases.

Applied microbiology and biotechnology 73 (6)
PMID : 17043822  :   DOI  :   10.1007/s00253-006-0616-y    
Abstract >>
Two bacterial consortia growing on a random copolymer of ethylene glycol and propylene glycol units were obtained by enrichment cultures from various microbial samples. Six major strains included in both consortia were purified and identified as Sphingomonads, Pseudomonas sp. and Stenotrophomonas maltophilia. Three of them (Sphingobium sp. strain EK-1, Sphingopyxis macrogoltabida strain EY-1, and Pseudomonas sp. strain PE-2) utilized both PEG and polypropylene glycol (PPG) as a sole carbon source. Four PEG-utilizing bacteria had PEG dehydrogenase (PEG-DH) activity, which was induced by PEG. PCR products from DNA of these bacteria generated with primers designed from a PEG-DH gene (AB196775 for S. macrogoltabida strain 103) indicated the presence of a sequence that is the homologous to the PEG-DH gene (99% identity). On the other hand, five PPG-utilizing bacteria had PPG dehydrogenase (PPG-DH) activity, but the activity was constitutive. PCR of a PPG-DH gene was performed using primers designed from a polyvinyl alcohol dehydrogenase (PVA-DH) gene (AB190288 for Sphingomonas sp. strain 113P3) because a PPG-DH gene has not been cloned yet, but both PPG-DH and PVA-DH were active toward PPG and PVA (Mamoto et al. 2006). PCR products of the five strains did not have similarity to each other or to oxidoreductases including PVA-DH.
KeywordMeSH Terms
19. Huang  TP, Somers  EB, Wong  AC,     ( 2006 )

Differential biofilm formation and motility associated with lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes in Stenotrophomonas maltophilia.

Journal of bacteriology 188 (8)
PMID : 16585771  :   DOI  :   10.1128/JB.188.8.3116-3120.2006     PMC  :   PMC1446987    
Abstract >>
Stenotrophomonas maltophilia WR-C is capable of forming biofilm on polystyrene and glass. The lipopolysaccharide/exopolysaccharide-coupled biosynthetic genes rmlA, rmlC, and xanB are necessary for biofilm formation and twitching motility. Mutants with mutations in rmlAC and xanB display contrasting biofilm phenotypes on polystyrene and glass and differ in swimming motility.
KeywordMeSH Terms
20. Wang  X, Li  B, Herman  PL, Weeks  DP,     ( 1997 )

A Three-Component Enzyme System Catalyzes the O Demethylation of the Herbicide Dicamba in Pseudomonas maltophilia DI-6.

Applied and environmental microbiology 63 (4)
PMID : 16535584  :   PMC  :   PMC1389562    
Abstract >>
An enzyme activity which converts dicamba (2-methoxy-3,6-dichlorobenzoic acid) to 3,6-dichlorosalicylic acid in vitro has been detected in cell lysates of Pseudomonas maltophilia DI-6. Phenyl-Sepharose column chromatography of a partially purified lysate resulted in the separation of this enzyme into three separate protein components tentatively identified as an oxygenase, a ferredoxin, and a reductase. The activity of dicamba O-demethylase was dependent on oxygen and required NADH and Mg(sup2+).
KeywordMeSH Terms
21. Hassan-Abdallah  A, Zhao  G, Eschenbrenner  M, Chen  ZW, Mathews  FS, Jorns  MS,     ( 2005 )

Cloning, expression and crystallization of heterotetrameric sarcosine oxidase from Pseudomonas maltophilia.

Protein expression and purification 43 (1)
PMID : 15922624  :   DOI  :   10.1016/j.pep.2005.03.023     PMC  :   PMC1993822    
Abstract >>
Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional enzyme that catalyzes the oxidation of the methyl group in sarcosine (N-methylglycine) and transfer of the oxidized methyl group into the one-carbon metabolic pool. In addition to four different subunits, TSOX contains three coenzymes (FAD, FMN, and NAD) and a binding site for tetrahydrofolate, the coenzyme acceptor of the oxidized methyl group from sarcosine. Based on preliminary success in crystallization of the natural enzyme, the genes encoding the subunits for TSOX from Pseudomonas maltophilia (pTSOX) were cloned by functional screening of a genomic library. Recombinant enzyme exhibiting the same specific activity as natural pTSOX could not be isolated using a similar or identical purification procedure. This difficulty was overcome by affinity purification of recombinant pTSOX containing a C-terminal (His)(6) tag on the subunit (gamma) encoded by soxG, the gene located at the 3' end of the pTSOX operon. Affinity-purified pTSOX could not be crystallized, a problem traced to microheterogeneity in the recombinant enzyme where about half of the FMN is present in a modified form that is not found in the natural enzyme and may be a biosynthetic intermediate. The modified flavin was eliminated by expression of the recombinant enzyme in the presence of sarcosine, the same reagent used to induce expression of the natural enzyme. Homogenous recombinant pTSOX was isolated from cells grown in the presence of sarcosine by chromatography on affinity and hydrophobic interaction matrices. High quality crystals that diffract to 1.85 A resolution have been obtained.
KeywordMeSH Terms
22. Herman  PL, Behrens  M, Chakraborty  S, Chrastil  BM, Barycki  J, Weeks  DP,     ( 2005 )

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression.

The Journal of biological chemistry 280 (26)
PMID : 15855162  :   DOI  :   10.1074/jbc.M500597200    
Abstract >>
Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA (3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme (i.e. reductase(DIC), ferredoxin(DIC), and oxygenase(DIC)) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron oxygenases that function as monooxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenase(DIC) has strong similarities with the core alphasubunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.
KeywordMeSH Terms
23. Chakraborty  S, Behrens  M, Herman  PL, Arendsen  AF, Hagen  WR, Carlson  DL, Wang  XZ, Weeks  DP,     ( 2005 )

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: purification and characterization.

Archives of biochemistry and biophysics 437 (1)
PMID : 15820213  :   DOI  :   10.1016/j.abb.2005.02.024    
Abstract >>
Dicamba O-demethylase is a multicomponent enzyme that catalyzes the conversion of the herbicide 2-methoxy-3,6-dichlorobenzoic acid (dicamba) to 3,6-dichlorosalicylic acid (DCSA). The three components of the enzyme were purified and characterized. Oxygenase(DIC) is a homotrimer (alpha)3 with a subunit molecular mass of approximately 40 kDa. FerredoxinDIC and reductaseDIC are monomers with molecular weights of approximately 14 and 45 kDa, respectively. EPR spectroscopic analysis suggested the presence of a single [2Fe-2S](2+/1+) cluster in ferredoxinDIC and a single Rieske [2Fe-2S](2+; 1+) cluster within oxygenaseDIC. Consistent with the presence of a Rieske iron-sulfur cluster, oxygenaseDIC displayed a high reduction potential of E(m,7.0) = -21 mV whereas ferredoxinDIC exhibited a reduction potential of approximately E(m,7.0) = -171 mV. Optimal oxygenaseDIC activity in vitro depended on the addition of Fe2+. The identification of formaldehyde and DCSA as reaction products demonstrated that dicamba O-demethylase acts as a monooxygenase. Taken together, these data suggest that oxygenaseDIC is an important new member of the Rieske non-heme iron family of oxygenases.
KeywordMeSH Terms
24. Furushita  M, Okamoto  A, Maeda  T, Ohta  M, Shiba  T,     ( 2005 )

Isolation of multidrug-resistant Stenotrophomonas maltophilia from cultured yellowtail (Seriola quinqueradiata) from a marine fish farm.

Applied and environmental microbiology 71 (9)
PMID : 16151156  :   DOI  :   10.1128/AEM.71.9.5598-5600.2005     PMC  :   PMC1214673    
Abstract >>
Six strains of multidrug-resistant Stenotrophomonas maltophilia were isolated from cultured yellowtail. The strains were divided into two clusters based on the 16S rRNA genes, and all of them contained L1 metallo-beta-lactamase and L2 beta-lactamase genes. Differences in the intercluster divergence between the lactamase genes suggest that horizontal transfer of the genes occurred.
KeywordMeSH Terms
Aquaculture
Drug Resistance, Multiple, Bacterial
25. Grover  S, Odell  WD,     ( 1992 )

Partial characterization of the 30 kD Ig-binding protein from Pseudomonas maltophilia.

Biochemical and biophysical research communications 182 (3)
PMID : 1540156  :   DOI  :   10.1016/0006-291x(92)91841-d    
Abstract >>
We have previously demonstrated that Pseudomonas maltophilia (ATCC 13637) possess a 30 kDa cell wall protein which binds various subclasses of IgG's and IgA by their Fc region. The protein was solubilized by papain and purified by affinity chromatography on cyanogen bromide activated sepharose beads conjugated with human IgG. The eluent was electrophoresed on a 12% polyacrylamide gel under denaturing conditions, and the immunoactive bands identified by Western blot analysis, a second gel was stained with Coomassie blue. The affinity purified eluent was electrophoresed on a one-dimensional 15% polyacrylamide gel and stained with Coomassie blue. The protein band of interest was cut. The protein band was then digested in situ with Staphylococcus aureus V-8 protease. The peptide bands were separated by electrophoresis on a second one dimensional 15% polyacrylamide gel and then electroblotted into a polyvinylidine difluoride membrane. The bands were visualized by staining with Coomassie blue, cut out, and sequenced using an automated gas phase sequencer. Minimal amino acid composition was determined in a similar fashion. We have thus obtained partial N-terminal amino acid sequence data from the above method.
KeywordMeSH Terms
Prostatic Secretory Proteins
26. Gould  VC, Okazaki  A, Howe  RA, Avison  MB,     ( 2004 )

Analysis of sequence variation among smeDEF multi drug efflux pump genes and flanking DNA from defined 16S rRNA subgroups of clinical Stenotrophomonas maltophilia isolates.

The Journal of antimicrobial chemotherapy 54 (2)
PMID : 15254029  :   DOI  :   10.1093/jac/dkh367    
Abstract >>
To determine the level of variation in the smeDEF efflux pump and smeT transcriptional regulator genes among three defined 16S rRNA sequence subgroups of clinical Stenotrophomonas maltophilia isolates. smeDEF sequencing used a PCR genome walking approach. Determination of the sequence surrounding smeDEF used a flanking primer PCR method and specific primers anchored in smeD or smeF together with random primers. smeDEF is chromosomal and located in the same position in the chromosome in all three subgroups of isolates. Flanking smeD is a gene, smeT, encoding a putative transcriptional repressor for smeDEF. Variation at these loci among the isolates is considerably lower (up to 10%) than at intrinsic beta-lactamase loci (up to 30%) in the same isolates, implying greater functional constraint. The smeD-smeT intergenic region contains a highly conserved section, which maps with previously predicted promoter/operator regions, and a hypervariable untranslated region, which can be used to subgroup clinical isolates. These data provide further evidence that it is possible to group clinical isolates of the inherently variable species, S. maltophilia, based on genotypic properties. Isolate D457, in which most work concerning smeDEF expression has been performed, does not fall into S. maltophilia subgroup A, which is the most typical.
KeywordMeSH Terms
27. Schneider  KL, Marrero  G, Alvarez  AM, Presting  GG,     ( 2011 )

Classification of plant associated bacteria using RIF, a computationally derived DNA marker.

PloS one 6 (4)
PMID : 21533033  :   DOI  :   10.1371/journal.pone.0018496     PMC  :   PMC3080875    
Abstract >>
A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.
KeywordMeSH Terms
Genetic Markers
28. Lin  CW, Lin  HC, Huang  YW, Chung  TC, Yang  TC,     ( 2011 )

Inactivation of mrcA gene derepresses the basal-level expression of L1 and L2 �]-lactamases in Stenotrophomonas maltophilia.

The Journal of antimicrobial chemotherapy 66 (9)
PMID : 21719470  :   DOI  :   10.1093/jac/dkr276    
Abstract >>
To characterize the relationship between inactivation of the mrcA gene and �]-lactamase expression and �]-lactams resistance in Stenotrophomonas maltophilia KJ and to investigate the involvement of ampR, ampN-ampG, ampD(I) and creBC in this. The mrcA deletion mutant KJ�GmrcA was constructed to investigate the role of this putative penicillin-binding protein 1a (PBP1a) in �]-lactamase expression and �]-lactam resistance. The �GampR, �GampNG, �GampDI and �GcreBC alleles were introduced into KJ�GmrcA, and KJ�GDI�GBC and KJ�GDI�GmrcA�GBC were also constructed for comparison. All the mutants and their corresponding parent strains were assayed for �]-lactamase activities and MICs of �]-lactams. Inactivation of mrcA caused basal L1/L2 �]-lactamase production to increase by ?100-fold, but made little difference to cefuroxime-induced �]-lactamase activity and the MICs of �]-lactams. The �GmrcA-derived basal �]-lactamase hyperproduction was ampR and ampN-ampG dependent. Simultaneous inactivation of ampD(I) and mrcA did not augment �]-lactamase production over and above that seen in an ampD(I) mutant alone. Furthermore, we could find no evidence for a role of the creBC two-component regulatory system in �]-lactamase hyperproduction in a �GampD(I) or �GmrcA background. Inactivation of mrcA, predicted to encode PBP1a, causes basal L1/L2 �]-lactamase hyperproduction in S. maltophilia.
KeywordMeSH Terms
29. Hernández  A, Ruiz  FM, Romero  A, Martínez  JL,     ( 2011 )

The binding of triclosan to SmeT, the repressor of the multidrug efflux pump SmeDEF, induces antibiotic resistance in Stenotrophomonas maltophilia.

PLoS pathogens 7 (6)
PMID : 21738470  :   DOI  :   10.1371/journal.ppat.1002103     PMC  :   PMC3128119    
Abstract >>
The wide utilization of biocides poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that biocides can select, at least in laboratory experiments, antibiotic resistant bacteria. This situation has raised concerns, not just on scientists and clinicians, but also on regulatory agencies, which are demanding studies on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases and on human health. In the present article, we explored the possibility that the widely used biocide triclosan might induce antibiotic resistance using as a model the opportunistic pathogen Stenotrophomonas maltophilia. Biochemical, functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic- and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. To our knowledge this is the first crystal structure obtained for a transcriptional regulator bound to triclosan. This work provides the molecular basis for understanding the mechanisms allowing the induction of phenotypic resistance to antibiotics by triclosan.
KeywordMeSH Terms
Drug Resistance, Bacterial
30. Vasileuskaya-Schulz  Z, Kaiser  S, Maier  T, Kostrzewa  M, Jonas  D,     ( 2011 )

Delineation of Stenotrophomonas spp. by multi-locus sequence analysis and MALDI-TOF mass spectrometry.

Systematic and applied microbiology 34 (1)
PMID : 21247714  :   DOI  :   10.1016/j.syapm.2010.11.011    
Abstract >>
The genus Stenotrophomonas is genetically and phenotypically heterogeneous. Of the nine species now accepted, only S. maltophilia is of clinical importance. Based on DNA-sequences of seven house keeping genes, it encompasses genogroups of DNA-similarity below 97% that predominantly comprise strains of environmental origin. Therefore, in order to unravel the uneven distribution of environmental isolates within genogroups and reveal genetic relationships within the genus, there is need for an easy and reliable approach for the identification and delineation of Stenotrophomonas spp. In this first study, a multi-locus sequence analysis (MLSA) with seven housekeeping genes (atpD, gapA, guaA, mutM, nuoD, ppsA and recA) was applied for analysis of 21 S. maltophilia of environmental origin, Stenotrophomonas spp. and related genera. The genotypic findings were compared with the results of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Our MLSA provided reliable inter- and intra-species discrimination of all tested isolates that correlated with the MALDI-TOF mass spectrometry data. One distantly related genogroup of environmental S. maltophilia strains needs to be reclassified as S. rhizophila. However, there are still remaining delineated S. maltophilia genogroups of predominantly environmental origin. Our data provide further evidence that 'Pseudomonas'beteli is a heterotypic synonym of S. maltophilia. Based on MLSA and MALDI-TOF data, Stenotrophomonas sp. (DSM 2408) belongs to S. koreensis.
KeywordMeSH Terms
31. Hu  LF, Chang  X, Ye  Y, Wang  ZX, Shao  YB, Shi  W, Li  X, Li  JB,     ( 2011 )

Stenotrophomonas maltophilia resistance to trimethoprim/sulfamethoxazole mediated by acquisition of sul and dfrA genes in a plasmid-mediated class 1 integron.

International journal of antimicrobial agents 37 (3)
PMID : 21296557  :   DOI  :   10.1016/j.ijantimicag.2010.10.025    
Abstract >>
Stenotrophomonas maltophilia is becoming a more and more common cause of infections. In this study, the minimal inhibitory concentrations of trimethoprim/sulfamethoxazole (SXT), ceftazidime, minocycline, levofloxacin, chloramphenicol and ticarcillin/clavulanic acid were determined and the distribution of integrons and sul1, sul2 and dfrA genes was investigated in 102 S. maltophilia isolates collected from patients treated in 31 hospitals in Anhui, China, in the month of September in 2006-2008. The rate of resistance to SXT was up to 30.4%, and 64.7% of isolates were class 1 integron-positive. Sequencing data revealed the following novel gene cassettes embedded in class 1 integrons: dfrA17-aadA5; dfrA12-aadA2; aacA4-catB8-aadA1; aadB-aac(6')-II-bla(CARB-8); and arr-3-aacA4. This is the first report of the gene cassettes dfrA17-aadA5 and dfrA12-aadA2 and of sul2 genes in SXT-resistant S. maltophilia isolates in China. None of the SXT-susceptible S. maltophilia isolates were positive for sul2 or dfrA gene products by polymerase chain reaction (PCR), but PCR products for sul1 were detected in 27 SXT-susceptible and 25 SXT-resistant isolates. The findings from this study indicate that the sul1 gene, in combination with dfrA17 and dfrA12 gene cassettes and sul2 genes located within a 7.3kb plasmid, lead to a high rate of SXT resistance and also confirm the need for ongoing resistance surveillance.
KeywordMeSH Terms
Genes, Bacterial
Integrons
Plasmids
32. Mehnaz  S, Baig  DN, Lazarovits  G,     ( 2010 )

Genetic and phenotypic diversity of plant growth promoting rhizobacteria isolated from sugarcane plants growing in pakistan.

Journal of microbiology and biotechnology 20 (12)
PMID : 21193815  :  
Abstract >>
Bacteria were isolated from roots of sugarcane varieties grown in the fields of Punjab. They were identified by using API20E/NE bacterial identification kits and from sequences of 16S rRNA and amplicons of the cpn60 gene. The majority of bacteria were found to belong to the genera of Enterobacter, Pseudomonas, and Klebsiella, but members of genera Azospirillum, Rhizobium, Rahnella, Delftia, Caulobacter, Pannonibacter, Xanthomonas, and Stenotrophomonas were also found. The community, however, was dominated by members of the Pseudomonadaceae and Enterobacteriaceae, as representatives of these genera were found in samples from every variety and location examined. All isolates were tested for the presence of five enzymes and seven factors known to be associated with plant growth promotion. Ten isolates showed lipase activity and eight were positive for protease activity. Cellulase, chitinase, and pectinase were not detected in any strain. Nine strains showed nitrogen fixing ability (acetylene reduction assay) and 26 were capable of solubilizing phosphate. In the presence of 100 mg/l tryptophan, all strains except one produced indole acetic acid in the growth medium. All isolates were positive for ACC deaminase activity. Six strains produced homoserine lactones and three produced HCN and hexamate type siderophores. One isolate was capable of inhibiting the growth of 24 pathogenic fungal strains of Colletotrichum, Fusarium, Pythium, and Rhizoctonia spp. In tests of their abilities to grow under a range of temperature, pH, and NaCl concentrations, all isolates grew well on plates with 3% NaCl and most of them grew well at 4 to 41degrees C and at pH 11.
KeywordMeSH Terms
Genetic Variation
Rhizosphere
33. Nicoletti  M, Iacobino  A, Prosseda  G, Fiscarelli  E, Zarrilli  R, De Carolis  E, Petrucca  A, Nencioni  L, Colonna  B, Casalino  M,     ( 2011 )

Stenotrophomonas maltophilia strains from cystic fibrosis patients: genomic variability and molecular characterization of some virulence determinants.

International journal of medical microbiology : IJMM 301 (1)
PMID : 20952251  :   DOI  :   10.1016/j.ijmm.2010.07.003    
Abstract >>
The genetic relatedness of 52 Stenotrophomonas maltophilia strains, collected from various environmental and clinical sources, including cystic fibrosis (CF) patients, as well as the presence and the expression of some virulence-associated genes were studied. Pulsed-field gel electrophoresis (PFGE) analysis identified 47 profiles and three clusters of isolates with an identical PFGE pattern considered to be indistinguishable strains. Restriction fragment length polymorphism of the gyrB gene grouped the 52 strains into nine different profiles. Most CF clinical isolates (29 out of 41) showed profile 1, while the analysis of the hypervariable regions of the 16S rRNA gene revealed five distinct allelic variations, with the majority of CF isolates (23 out of 41) belonging to sequence group 1. Furthermore, the strains were characterized for motility and expression of virulence-associated genes, including genes encoding type-1 fimbriae, proteases (StmPr1 and StmPr2) and esterase. All S. maltophilia strains exhibited a very broad range of swimming and twitching motility, while none showed swarming motility. A complete smf-1 gene was PCR-amplified only from clinically derived S. maltophilia strains. Finally, the virulence of representative S. maltophilia strains impaired in the expression of proteases and esterase activities was evaluated by infecting larvae of the wax moth Galleria mellonella. The results obtained strongly indicate that the major extracellular protease StmPr1 may be a relevant virulence factor of S. maltophilia.
KeywordMeSH Terms
34. Li  D, Yu  T, Zhang  Y, Yang  M, Li  Z, Liu  M, Qi  R,     ( 2010 )

Antibiotic resistance characteristics of environmental bacteria from an oxytetracycline production wastewater treatment plant and the receiving river.

Applied and environmental microbiology 76 (11)
PMID : 20400569  :   DOI  :   10.1128/AEM.02964-09     PMC  :   PMC2876458    
Abstract >>
We characterized the bacterial populations in surface water receiving effluent from an oxytetracycline (OTC) production plant. Additional sampling sites included the receiving river water 5 km upstream and 20 km downstream from the discharge point. High levels of OTC were found in the wastewater (WW), and the antibiotic was still detectable in river water downstream (RWD), with undetectable levels in river water upstream (RWU). A total of 341 bacterial strains were isolated using nonselective media, with the majority being identified as Gammaproteobacteria. The MICs were determined for 10 antibiotics representing seven different classes of antibiotics, and the corresponding values were significantly higher for the WW and RWD isolates than for the RWU isolates. Almost all bacteria (97%) from the WW and RWD samples demonstrated multidrug-resistant (MDR) phenotypes, while in RWU samples, these were less frequent (28%). The WW and RWD isolates were analyzed for the presence of 23 tetracycline (tet) resistance genes. The majority of isolates (94.2% and 95.4% in WW and RWD, respectively) harbored the corresponding genes, with tet(A) being the most common (67.0%), followed by tet(W), tet(C), tet(J), tet(L), tet(D), tet(Y), and tet(K) (in the range between 21.0% and 40.6%). Class I integrons were detected in the majority of WW and RWD isolates (97.4% and 86.2%, respectively) but were not associated with the tet genes. We hypothesize that the strong selective pressure imposed by a high concentration of OTC contributes to the wide dissemination of tetracycline resistance genes and other antibiotic resistance genes, possibly through mobile genetic elements.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
Water Microbiology
Water Purification
35. Huang  YW, Lin  CW, Hu  RM, Lin  YT, Chung  TC, Yang  TC,     ( 2010 )

AmpN-AmpG operon is essential for expression of L1 and L2 beta-lactamases in Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 54 (6)
PMID : 20385866  :   DOI  :   10.1128/AAC.01283-09     PMC  :   PMC2876420    
Abstract >>
AmpG is an inner membrane permease which transports products of murein sacculus degradation from the periplasm into the cytosol in Gram-negative bacteria. This process is linked to induction of the chromosomal ampC beta-lactamase gene in some members of the Enterobacteriaceae and in Pseudomonas aeruginosa. In this study, the ampG homologue of Stenotrophomonas maltophilia KJ was analyzed. The ampG homologue and its upstream ampN gene form an operon and are cotranscribed under the control of the promoter P(ampN). Expression from P(ampN) was found to be independent of beta-lactam exposure and ampN and ampG products. A DeltaampN allele exerted a polar effect on the expression of ampG and resulted in a phenotype of null beta-lactamase inducibility. Complementation assays elucidated that an intact ampN-ampG operon is essential for beta-lactamase induction. Consistent with ampG of Escherichia coli, the ampN-ampG operon of S. maltophilia did not exhibit a gene dosage effect on beta-lactamase expression. The AmpG permease of E. coli could complement the beta-lactamase inducibility of ampN or ampG mutants of S. maltophilia, indicating that both species have the same precursor of activator ligand(s) for beta-lactamase induction.
KeywordMeSH Terms
Operon
36. Chang  YC, Huang  YW, Chiang  KH, Yang  TC, Chung  TC,     ( 2010 )

Introduction of an AmpR-L2 intergenic segment attenuates the induced beta-lactamase activity of Stenotrophomonas maltophilia.

European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology 29 (7)
PMID : 20397035  :   DOI  :   10.1007/s10096-010-0924-0    
Abstract >>
Expression of the L1 and L2 beta-lactamase genes is generally regulated by a LysR type regulator of the AmpR in Stenotrophomonas maltophilia. The ampR gene is located immediately upstream of L2 and is transcribed divergently, forming an ampR-L2 module. The ampR-L2 modules of 16 S. maltophilia isolates were analyzed, revealing that the ampR-L2 intergenic (IG) regions show a significant genetic diversity, whereas AmpR proteins are highly conserved. The induction potential of the different AmpR toward the different ampR-L2 IG regions was evaluated by introducing the various IG-xylE transcriptional fusion constructs into a wild S. maltophilia strain. The induction levels achieved in the various AmpR-IG pairs display quantitative differences; meanwhile, the host beta-lactamase activity, in some cases, is attenuated by the introduced IG segment. Similar beta-lactamase attenuation phenomenon was observed in Enterobacter cloacae with an ampR-L2 IG segment of S. maltophilia. A concept of oligonucleotides attenuator for the development of an antimicrobial agent is proposed.
KeywordMeSH Terms
Mutagenesis, Insertional
37. Gordon  NC, Wareham  DW,     ( 2010 )

Novel variants of the Smqnr family of quinolone resistance genes in clinical isolates of Stenotrophomonas maltophilia.

The Journal of antimicrobial chemotherapy 65 (3)
PMID : 20071366  :   DOI  :   10.1093/jac/dkp476    
Abstract >>
Recent analysis of Stenotrophomonas maltophilia has identified a novel family of resistance genes (Smqnr) encoding pentapeptide repeat proteins, which confer low-level resistance to quinolones. This study describes further novel variants present in clinical isolates of S. maltophilia and investigates their effect on resistance to a number of quinolones in an Escherichia coli host. PCR for Smqnr alleles was carried out on a selection of S. maltophilia from clinical specimens, and amplicons were cloned and transformed in E. coli TOP10 cells. Transformed colonies carrying the plasmid were tested for susceptibility to a range of quinolones by MIC determination. DNA sequences were determined and translated peptide sequences compared with known SmQnr sequences. Thirteen isolates were found to contain Smqnr alleles, of which six corresponded to previously identified Smqnr sequences, while seven were novel variants. Increases in quinolone MICs compared with wild-type E. coli TOP10 were seen for all strains transformed with Smqnr alleles. There is considerable diversity within Smqnr alleles. S. maltophilia may be a significant reservoir for the dissemination of quinolone resistance elements to Enterobacteriaceae.
KeywordMeSH Terms
Drug Resistance, Bacterial
Genes, Bacterial
38. D'Ordine  RL, Rydel  TJ, Storek  MJ, Sturman  EJ, Moshiri  F, Bartlett  RK, Brown  GR, Eilers  RJ, Dart  C, Qi  Y, Flasinski  S, Franklin  SJ,     ( 2009 )

Dicamba monooxygenase: structural insights into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation.

Journal of molecular biology 392 (2)
PMID : 19616009  :   DOI  :   10.1016/j.jmb.2009.07.022    
Abstract >>
Dicamba (2-methoxy-3,6-dichlorobenzoic acid) O-demethylase (DMO) is the terminal Rieske oxygenase of a three-component system that includes a ferredoxin and a reductase. It catalyzes the NADH-dependent oxidative demethylation of the broad leaf herbicide dicamba. DMO represents the first crystal structure of a Rieske non-heme iron oxygenase that performs an exocyclic monooxygenation, incorporating O(2) into a side-chain moiety and not a ring system. The structure reveals a 3-fold symmetric trimer (alpha(3)) in the crystallographic asymmetric unit with similar arrangement of neighboring inter-subunit Rieske domain and non-heme iron site enabling electron transport consistent with other structurally characterized Rieske oxygenases. While the Rieske domain is similar, differences are observed in the catalytic domain, which is smaller in sequence length than those described previously, yet possessing an active-site cavity of larger volume when compared to oxygenases with larger substrates. Consistent with the amphipathic substrate, the active site is designed to interact with both the carboxylate and aromatic ring with both key polar and hydrophobic interactions observed. DMO structures were solved with and without substrate (dicamba), product (3,6-dichlorosalicylic acid), and either cobalt or iron in the non-heme iron site. The substitution of cobalt for iron revealed an uncommon mode of non-heme iron binding trapped by the non-catalytic Co(2+), which, we postulate, may be transiently present in the native enzyme during the catalytic cycle. Thus, we present four DMO structures with resolutions ranging from 1.95 to 2.2 A, which, in sum, provide a snapshot of a dynamic enzyme where metal binding and substrate binding are coupled to observed structural changes in the non-heme iron and catalytic sites.
KeywordMeSH Terms
39. Dumitru  R, Jiang  WZ, Weeks  DP, Wilson  MA,     ( 2009 )

Crystal structure of dicamba monooxygenase: a Rieske nonheme oxygenase that catalyzes oxidative demethylation.

Journal of molecular biology 392 (2)
PMID : 19616011  :   DOI  :   10.1016/j.jmb.2009.07.021     PMC  :   PMC3109874    
Abstract >>
Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 A resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.
KeywordMeSH Terms
40. Yang  TC, Huang  YW, Hu  RM, Huang  SC, Lin  YT,     ( 2009 )

AmpDI is involved in expression of the chromosomal L1 and L2 beta-lactamases of Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 53 (7)
PMID : 19414581  :   DOI  :   10.1128/AAC.01513-08     PMC  :   PMC2704650    
Abstract >>
Two ampD homologues, ampD(I) and ampD(II), of Stenotrophomonas maltophilia have been cloned and analyzed. Comparative genomic analysis revealed that the genomic context of the ampD(II) genes is quite different, whereas that of the ampD(I) genes is more conserved in S. maltophilia strains. The ampD system of S. maltophilia is distinct from that of the Enterobacteriaceae and Pseudomonas aeruginosa in three respects. (i) AmpD(I) of S. maltophilia is not encoded in an ampDE operon, in contrast to what happens in the Enterobacteriaceae and P. aeruginosa. (ii) The AmpD systems of the Enterobacteriaceae and P. aeruginosa are generally involved in the regulation of ampR-linked ampC gene expression, while AmpD(I) of S. maltophilia is responsible for the regulation of two intrinsic beta-lactamase genes, of which the L2 gene, but not the L1 gene, is linked to ampR. (iii) S. maltophilia exhibits a one-step L1 and L2 gene derepression model involving ampD(I), distinct from the two- or three-step derepression of the Enterobacteriaceae and P. aeruginosa. Moreover, the ampD(I) and ampD(II) genes are constitutively expressed and not regulated by the inducer and AmpR protein, and the expression of ampD(II) is weaker than that of ampD(I). Finally, AmpD(II) is not associated with the derepression of beta-lactamases, and its role in S. maltophilia remains unclear.
KeywordMeSH Terms
41. Hernández  A, Maté  MJ, Sánchez-Díaz  PC, Romero  A, Rojo  F, Martínez  JL,     ( 2009 )

Structural and functional analysis of SmeT, the repressor of the Stenotrophomonas maltophilia multidrug efflux pump SmeDEF.

The Journal of biological chemistry 284 (21)
PMID : 19324881  :   DOI  :   10.1074/jbc.M809221200     PMC  :   PMC2682891    
Abstract >>
Stenotrophomonas maltophilia is an opportunistic pathogen characterized for its intrinsic low susceptibility to several antibiotics. Part of this low susceptibility relies on the expression of chromosomally encoded multidrug efflux pumps, with SmeDEF being the most relevant antibiotic resistance efflux pump so far studied in this bacterial species. Expression of smeDEF is down-regulated by the SmeT repressor, encoded upstream smeDEF, in its complementary DNA strand. In the present article we present the crystal structure of SmeT and analyze its interactions with its cognate operator. Like other members of the TetR family of transcriptional repressors, SmeT behaves as a dimer and presents some common structural features with other TetR proteins like TtgR, QacR, and TetR. Differing from other TetR proteins for which the structure is available, SmeT turned out to have two extensions at the N and C termini that might be relevant for its function. Besides, SmeT presents the smallest binding pocket so far described in the TetR family of transcriptional repressors, which may correlate with a specific type and range of effectors. In vitro studies revealed that SmeT binds to a 28-bp pseudopalindromic region, forming two complexes. This operator region was found to overlap the promoters of smeT and smeDEF. This finding is consistent with a role for SmeT simultaneously down-regulating smeT and smeDEF transcription, likely by steric hindrance on RNA polymerase binding to DNA.
KeywordMeSH Terms
Drug Resistance, Multiple, Bacterial
42. Kaiser  S, Biehler  K, Jonas  D,     ( 2009 )

A Stenotrophomonas maltophilia multilocus sequence typing scheme for inferring population structure.

Journal of bacteriology 191 (9)
PMID : 19251858  :   DOI  :   10.1128/JB.00892-08     PMC  :   PMC2681804    
Abstract >>
Stenotrophomonas maltophilia is an opportunistic, highly resistant, and ubiquitous pathogen. Strains have been assigned to genogroups using amplified fragment length polymorphism. Hence, isolates of environmental and clinical origin predominate in different groups. A multilocus sequence typing (MLST) scheme was developed using a highly diverse selection of 70 strains of various ecological origins from seven countries on all continents including strains of the 10 previously defined genogroups. Sequence data were assigned to 54 sequence types (ST) based on seven loci. Indices of association for all isolates and clinical isolates of 2.498 and 2.562 indicated a significant linkage disequilibrium, as well as high congruence of tree topologies from different loci. Potential recombination events were detected in one-sixth of all ST. Calculation of the mean divergence between and within predicted clusters confirmed previously defined groups and revealed five additional groups. Consideration of the different ecological origins showed that 18 out of 31 respiratory tract isolates, including 12 out of 19 isolates from cystic fibrosis (CF) patients, belonged to genogroup 6. In contrast, 16 invasive strains isolated from blood cultures were distributed among nine different genogroups. Three genogroups contained isolates of strictly environmental origin that also featured high sequence distances to other genogroups, including the S. maltophilia type strain. On the basis of this MLST scheme, isolates can be assigned to the genogroups of this species in order to further scrutinize the population structure of this species and to unravel the uneven distribution of environmental and clinical isolates obtained from infected, colonized, or CF patients.
KeywordMeSH Terms
Sequence Analysis, DNA
43. Adelowo  OO, Fagade  OE,     ( 2009 )

The tetracycline resistance gene tet39 is present in both Gram-negative and Gram-positive bacteria from a polluted river, Southwestern Nigeria.

Letters in applied microbiology 48 (2)
PMID : 19196439  :   DOI  :   10.1111/j.1472-765X.2008.02523.x    
Abstract >>
Previous analysis of tet39 suggests it may be present in other bacterial species. Hence, we investigated the host range of tet39 among bacterial from a poultry waste polluted river in Southwestern Nigeria. Thirteen resistant bacterial isolated from the water and sediment of the polluted river was investigated for the presence of tetracycline resistance genes tetA, tetB, tetC, tet39 and the transposon integrase gene of the Tn916/1545 family by PCR. While tetA, tetB, tetC and integrase genes cannot be detected in any of the organisms, tet39 was detected in eight of the tested organisms including three Gram-positive species. Sequence analysis showed the genes have high sequence identities (> or =99%) with tet39 of Acinetobacter sp. LUH5605, the first and only bacterial genus from which the gene has been reported to date. This is a novel observation. This study shows that apart from Acinetobacter, tet39 is present in other bacterial species tested in this study. This study adds to available information on the occurrence and distribution of tet39 among environmental bacteria and suggests that the gene has a broader host range than previously reported.
KeywordMeSH Terms
Tetracycline Resistance
Water Pollution
44. Shimizu  K, Kikuchi  K, Sasaki  T, Takahashi  N, Ohtsuka  M, Ono  Y, Hiramatsu  K,     ( 2008 )

Smqnr, a new chromosome-carried quinolone resistance gene in Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 52 (10)
PMID : 18644963  :   DOI  :   10.1128/AAC.00026-08     PMC  :   PMC2565915    
Abstract >>
A new chromosome-carried quinolone resistance gene from Stenotrophomonas maltophilia, Smqnr, was characterized. The gene was present in type strain CCUG 5866 and was also detected in 24 clinical isolates and showed some allelic diversity. The expression of Smqnr in Escherichia coli decreased the susceptibilities of the E. coli isolates to several fluoroquinolones.
KeywordMeSH Terms
Genes, Bacterial
45. Wang  C, Zhan  Q, Mi  Z, Huang  Z, Chen  G,     ( 2008 )

Distribution of the antiseptic-resistance gene qacEDelta1 in 283 clinical isolates of Gram-negative bacteria in China.

The Journal of hospital infection 69 (4)
PMID : 18511148  :   DOI  :   10.1016/j.jhin.2008.04.001    
Abstract >>
N/A
KeywordMeSH Terms
Drug Resistance, Bacterial
Genes, Bacterial
46. Young  JM, Park  DC, Shearman  HM, Fargier  E,     ( 2008 )

A multilocus sequence analysis of the genus Xanthomonas.

Systematic and applied microbiology 31 (5)
PMID : 18783906  :   DOI  :   10.1016/j.syapm.2008.06.004    
Abstract >>
A multilocus sequence analysis (MLSA) of strains representing all validly published Xanthomonas spp. (119 strains) was conducted using four genes; dnaK, fyuA, gyrB and rpoD, a total of 440 sequences. Xanthomonas spp. were divided into two groups similar to those indicated in earlier 16S rDNA comparative analyses, and they possibly represent distinct genera. The analysis clearly differentiated most species that have been established by DNA-DNA reassociation. A similarity matrix of the data indicated clear numerical differences that could form the basis for species differentiation in the future, as an alternative to DNA-DNA reassociation. Some species, X. cynarae, X. gardneri and X. hortorum, formed a single heterogeneous group that is in need of further investigation. X. gardneri appeared to be a synonym of X. cynarae. Recently proposed new species, X. alfalfae, X. citri, X. euvesicatoria, X. fuscans and X. perforans, were not clearly differentiated as species from X. axonopodis, and X. euvesicatoria and X. perforans are very probably synonyms. MLSA offers a powerful tool for further investigation of the classification of Xanthomonas. Based on the dataset produced, the method also offers a relatively simple way of identifying strains as members of known species, or of indicating their status as members of new species.
KeywordMeSH Terms
47. Nakajima  Y, Ito  K, Toshima  T, Egawa  T, Zheng  H, Oyama  H, Wu  YF, Takahashi  E, Kyono  K, Yoshimoto  T,     ( 2008 )

Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue.

Journal of bacteriology 190 (23)
PMID : 18820015  :   DOI  :   10.1128/JB.02010-07     PMC  :   PMC2583625    
Abstract >>
The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.
KeywordMeSH Terms
48. Sánchez  MB, Hernández  A, Rodríguez-Martínez  JM, Martínez-Martínez  L, Martínez  JL,     ( 2008 )

Predictive analysis of transmissible quinolone resistance indicates Stenotrophomonas maltophilia as a potential source of a novel family of Qnr determinants.

BMC microbiology 8 (N/A)
PMID : 18793450  :   DOI  :   10.1186/1471-2180-8-148     PMC  :   PMC2556341    
Abstract >>
Predicting antibiotic resistance before it emerges at clinical settings constitutes a novel approach for preventing and fighting resistance of bacterial pathogens. To analyse the possibility that novel plasmid-encoded quinolone resistance determinants (Qnr) can emerge and disseminate among bacterial pathogens, we searched the presence of those elements in nearly 1000 bacterial genomes and metagenomes. We have found a number of novel potential qnr genes in the chromosomes of aquatic bacteria and in metagenomes from marine organisms. Functional studies of the Stenotrophomonas maltophilia Smqnr gene show that plasmid-encoded SmQnr confers quinolone resistance upon its expression in a heterologous host. Altogether, the data presented in our work support the notion that predictive studies on antibiotic resistance are feasible, using currently available information on bacterial genomes and with the aid of bioinformatic and functional tools. Our results confirm that aquatic bacteria can be the origin of plasmid-encoded Qnr, and highlight the potential role of S. maltophilia as a source of novel Qnr determinants.
KeywordMeSH Terms
49. Hu  RM, Huang  KJ, Wu  LT, Hsiao  YJ, Yang  TC,     ( 2008 )

Induction of L1 and L2 beta-lactamases of Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 52 (3)
PMID : 18086856  :   DOI  :   10.1128/AAC.00682-07     PMC  :   PMC2258547    
Abstract >>
Isogenic L1 and L2 gene knockout mutants of Stenotrophomonas maltophilia KJ (KJDeltaL1 and KJDeltaL2, respectively) were constructed by xylE gene replacement. Induction kinetics of the L1 and L2 genes were evaluated by testing catechol 2,3-dioxygenase activity in the mutants. The results suggested that the induction of the L1 and L2 genes was differentially regulated.
KeywordMeSH Terms
Enzyme Induction
Gene Expression Regulation, Bacterial
50. Nauton  L, Kahn  R, Garau  G, Hernandez  JF, Dideberg  O,     ( 2008 )

Structural insights into the design of inhibitors for the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia.

Journal of molecular biology 375 (1)
PMID : 17999929  :   DOI  :   10.1016/j.jmb.2007.10.036    
Abstract >>
One mechanism by which bacteria can escape the action of beta-lactam antibiotics is the production of metallo-beta-lactamases. Inhibition of these enzymes should restore the action of these widely used antibiotics. The tetrameric enzyme L1 from Stenotrophomonas maltophilia was used as a model system to determine a series of high-resolution crystal structures of apo, mono and bi-metal substituted proteins as well as protein-inhibitor complexes. Unexpectedly, although the apo structure revealed only few significant structural differences from the holo structure, some inhibitors were shown to induce amino acid side-chain rotations in the tightly packed active site. Moreover, one inhibitor employs a new binding mode in order to interact with the di-zinc center. This structural information could prove essential in the process of elucidation of the mode of interaction between a putative lead compound and metallo-beta-lactamases, one of the main steps in structure-based drug design.
KeywordMeSH Terms
beta-Lactamase Inhibitors
51. Toleman  MA, Bennett  PM, Bennett  DM, Jones  RN, Walsh  TR,     ( 2007 )

Global emergence of trimethoprim/sulfamethoxazole resistance in Stenotrophomonas maltophilia mediated by acquisition of sul genes.

Emerging infectious diseases 13 (4)
PMID : 17553270  :   DOI  :   10.3201/eid1304.061378     PMC  :   PMC2725981    
Abstract >>
Trimethoprim/sulfamethoxazole (TMP/SMX) resistance remains a serious threat in the treatment of Stenotrophomonas maltophilia infections. We analyzed an international collection of 55 S. maltophilia TMP/SMX-sensitive (S) (n=30) and -resistant (R) (n=25) strains for integrons; sul1, sul2 and dhfr genes; and insertion element common region (ISCR) elements. sul1, as part of a class 1 integron, was detected in 17 of 25 TMP/SMX-R. Nine TMP/SMX-R strains carried sul2; 7 were on large plasmids. Five TMP/SMX-R isolates were positive for ISCR2, and 4 were linked to sul2; 2 others possessed ISCR3. Two ISCR2s were adjacent to floR. Six TMP/SMX-S isolates harbored novel ISCR elements, ISCR9 and ISCR10. Linkage of ISCR3, ISCR9, and ISCR10 to sul2 and dhfr genes was not demonstrated. The data from this study indicate that class 1 integrons and ISCR elements linked to sul2 genes can mediate TMP/SMX resistance in S. maltophilia and are geographically widespread, findings that reinforce the need for ongoing resistance surveillance.
KeywordMeSH Terms
52. Huang  TP, Wong  AC,     ( 2007 )

A cyclic AMP receptor protein-regulated cell-cell communication system mediates expression of a FecA homologue in Stenotrophomonas maltophilia.

Applied and environmental microbiology 73 (15)
PMID : 17574998  :   DOI  :   10.1128/AEM.00366-07     PMC  :   PMC1951048    
Abstract >>
Stenotrophomonas maltophilia WR-C possesses an rpf/diffusible signal factor (DSF) cell-cell communication system. It produces cis-Delta2-11-methyl-dodecenoic acid, a DSF, and seven structural derivatives, which require rpfF and rpfB for synthesis. Acquisition of iron from the environment is important for bacterial growth as well as the expression of virulence genes. We identified a gene homologous to fecA, which encodes a ferric citrate receptor that transports exogenous siderophore ferric citrate from the environment into the bacterial periplasm. Western blot analysis with anti-FecA-His(6) antibody showed that the FecA homologue was induced in the iron-depleted medium supplemented with a low concentration of ferric citrate. Deletion of rpfF or rpfB resulted in reduced FecA expression compared to the wild type. Synthetic DSF restored FecA expression by the DeltarpfF mutant to the wild-type level. Reverse transcription-PCR showed that the fecA transcript was decreased in the DeltarpfF mutant compared to the wild type. These data suggest that DSF affected the level of fecA mRNA. Transposon inactivation of crp, which encodes cyclic AMP (cAMP) receptor protein (CRP) resulted in reduced FecA expression and rpfF transcript level. Putative CRP binding sites were located upstream of the rpfF promoter, indicating that the effect of CRP on FecA is through the rpf/DSF pathway and by directly controlling rpfF. We propose that CRP may serve as a checkpoint for iron uptake, protease activity, and hemolysis in response to environmental changes such as changes in concentrations of glucose, cAMP, iron, or DSF.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Signal Transduction
53. Calvopiña  K, Hinchliffe  P, Brem  J, Heesom  KJ, Johnson  S, Cain  R, Lohans  CT, Fishwick  CWG, Schofield  CJ, Spencer  J, Avison  MB,     ( 2017 )

Structural/mechanistic insights into the efficacy of nonclassical �]-lactamase inhibitors against extensively drug resistant Stenotrophomonas maltophilia clinical isolates.

Molecular microbiology 106 (3)
PMID : 28876489  :   DOI  :   10.1111/mmi.13831    
Abstract >>
Clavulanic acid and avibactam are clinically deployed serine �]-lactamase inhibitors, important as a defence against antibacterial resistance. Bicyclic boronates are recently discovered inhibitors of serine and some metallo �]-lactamases. Here, we show that avibactam and a bicyclic boronate inhibit L2 (serine �]-lactamase) but not L1 (metallo �]-lactamase) from the extensively drug resistant human pathogen Stenotrophomonas maltophilia. X-ray crystallography revealed that both inhibitors bind L2 by covalent attachment to the nucleophilic serine. Both inhibitors reverse ceftazidime resistance in S. maltophilia because, unlike clavulanic acid, they do not induce L1 production. Ceftazidime/inhibitor resistant mutants hyperproduce L1, but retain aztreonam/inhibitor susceptibility because aztreonam is not an L1 substrate. Importantly, avibactam, but not the bicyclic boronate is deactivated by L1 at a low rate; the utility of avibactam might be compromised by mutations that increase this deactivation rate. These data rationalize the observed clinical efficacy of ceftazidime/avibactam plus aztreonam as combination therapy for S. maltophilia infections and confirm that aztreonam-like �]-lactams plus nonclassical �]-lactamase inhibitors, particularly avibactam-like and bicyclic boronate compounds, have potential for treating infections caused by this most intractable of drug resistant pathogens.
KeywordMeSH Terms
54. Hatti  K, Gulati  A, Srinivasan  N, Murthy  MR,     ( 2016 )

Determination of crystal structures of proteins of unknown identity using a marathon molecular replacement procedure: structure of Stenotrophomonas maltophilia phosphate-binding protein.

Acta crystallographica. Section D, Structural biology 72 (Pt 10)
PMID : 27710929  :   DOI  :   10.1107/S2059798316012419    
Abstract >>
During the past decade, the authors have collected a few X-ray diffraction data sets from protein crystals that appeared to be easy cases of molecular replacement but failed to yield structures even after extensive trials. Here, the use of a large-scale molecular replacement method that explores all structurally characterized domains as phasing models to determine the structure corresponding to two data sets collected at 1.9 and 2.3 ? resolution is reported. These two structures were of the same protein independently crystallized in 2007 and 2011. The structures derived are virtually identical and were found to consist of two compact globular domains connected by a hinge. The high resolution of one of these data sets enabled inference of the amino-acid sequence from the electron-density map. The deduced sequence is nearly identical to that of a protein from the multidrug-resistant bacterium Stenotrophomonas maltophilia. Although the structure of this protein has not been determined previously, it is homologous to the well studied DING proteins which mediate the cellular uptake of phosphate ions. The final electron-density maps from both of the data sets revealed a large density at the interface of the two globular domains that is likely to represent a phosphate ion. Thus, the structure is likely to be that of a phosphate-binding protein encoded by the S. maltophilia genome (SmPBP; PDB entry 5j1d). The nature of the phosphate-binding site of SmPBP closely resembles that of Pseudomonas fluorescens DING (PfluDING), which displays remarkable discrimination between the closely similar phosphate and arsenate ions. The results presented here illustrate that routine crystallization trials may occasionally lead to the serendipitous crystallization of a protein of unknown identity and brute-force molecular replacement through `fold space' might allow the identification of the unknown protein.
KeywordMeSH Terms
Stenotrophomonas maltophilia
molecular replacement
phosphate-binding proteins
serendipity
55. Madhaiyan  M, Alex  TH, Ngoh  ST, Prithiviraj  B, Ji  L,     ( 2015 )

Leaf-residing Methylobacterium species fix nitrogen and promote biomass and seed production in Jatropha curcas.

Biotechnology for biofuels 8 (N/A)
PMID : 26697111  :   DOI  :   10.1186/s13068-015-0404-y     PMC  :   PMC4687150    
Abstract >>
Jatropha curcas L. (Jatropha) is a potential biodiesel crop that can be cultivated on marginal land because of its strong tolerance to drought and low soil nutrient content. However, seed yield remains low. To enhance the commercial viability and green index of Jatropha biofuel, a systemic and coordinated approach must be adopted to improve seed oil and biomass productivity. Here, we present our investigations on the Jatropha-associated nitrogen-fixing bacteria with an aim to understand and exploit the unique biology of this plant from the perspective of plant-microbe interactions. An analysis of 1017 endophytic bacterial isolates derived from different parts of Jatropha revealed that diazotrophs were abundant and diversely distributed into five classes belonging to �\, �], �^-Proteobacteria, Actinobacteria and Firmicutes. Methylobacterium species accounted for 69.1 % of endophytic bacterial isolates in leaves and surprisingly, 30.2 % which were able to fix nitrogen that inhabit in leaves. Among the Methylobacterium isolates, strain L2-4 was characterized in detail. Phylogenetically, strain L2-4 is closely related to M. radiotolerans and showed strong molybdenum-iron dependent acetylene reduction (AR) activity in vitro and in planta. Foliar spray of L2-4 led to successful colonization on both leaf surface and in internal tissues of systemic leaves and significantly improved plant height, leaf number, chlorophyll content and stem volume. Importantly, seed production was improved by 222.2 and 96.3 % in plants potted in sterilized and non-sterilized soil, respectively. Seed yield increase was associated with an increase in female-male flower ratio. The ability of Methylobacterium to fix nitrogen and colonize leaf tissues serves as an important trait for Jatropha. This bacteria-plant interaction may significantly contribute to Jatropha's tolerance to low soil nutrient content. Strain L2-4 opens a new possibility to improve plant's nitrogen supply from the leaves and may be exploited to significantly improve the productivity and Green Index of Jatropha biofuel.
KeywordMeSH Terms
Biofuel
Culturable endophyte
Jatropha curcas L.
Methylobacterium
Nitrogen fixation
Biofuel
Culturable endophyte
Jatropha curcas L.
Methylobacterium
Nitrogen fixation
Biofuel
Culturable endophyte
Jatropha curcas L.
Methylobacterium
Nitrogen fixation
Biofuel
Culturable endophyte
Jatropha curcas L.
Methylobacterium
Nitrogen fixation
Biofuel
Culturable endophyte
Jatropha curcas L.
Methylobacterium
Nitrogen fixation
Biofuel
Culturable endophyte
Jatropha curcas L.
Methylobacterium
Nitrogen fixation
Biofuel
Culturable endophyte
Jatropha curcas L.
Methylobacterium
Nitrogen fixation
Biofuel
Culturable endophyte
Jatropha curcas L.
Methylobacterium
Nitrogen fixation
56. Peeters  C, Depoorter  E, Praet  J, Vandamme  P,     ( 2016 )

Extensive cultivation of soil and water samples yields various pathogens in patients with cystic fibrosis but not Burkholderia multivorans.

Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 15 (6)
PMID : 26996269  :   DOI  :   10.1016/j.jcf.2016.02.014    
Abstract >>
While the epidemiology of Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) patients suggests that Burkholderia multivorans is acquired from environmental sources, this species has rarely been isolated from soil and water samples. Multiple isolation strategies were applied to water and soil samples that were previously shown to be B. multivorans PCR positive. These included direct plating and liquid enrichment procedures and the use of selective media, acclimatizing recovery and co-cultivation with CF sputum. MALDI-TOF mass spectrometry and sequence analysis of 16S rRNA and housekeeping genes were used to identify all isolates. None of the approaches yielded B. multivorans isolates. Other Burkholderia species, several Gram-negative non-fermenting bacteria (including Cupriavidus, Inquilinus, Pandoraea, Pseudomonas and Stenotrophomonas) and rapidly growing mycobacteria (including Mycobacterium chelonae) were all isolated from water and soil samples. The use of Bcc isolation media yielded a surprisingly wide array of rare but often clinically relevant CF pathogens, confirming that soil and water are reservoirs of these infectious agents.
KeywordMeSH Terms
Burkholderia multivorans
Gram-negative non-fermenters
Isolation
Rapidly growing mycobacteria
Soil
Water
Burkholderia multivorans
Gram-negative non-fermenters
Isolation
Rapidly growing mycobacteria
Soil
Water
Burkholderia multivorans
Gram-negative non-fermenters
Isolation
Rapidly growing mycobacteria
Soil
Water
Burkholderia multivorans
Gram-negative non-fermenters
Isolation
Rapidly growing mycobacteria
Soil
Water
Burkholderia Infections
Cystic Fibrosis
Respiratory Tract Infections
Soil Microbiology
Water Microbiology
57. Liu  MC, Tsai  YL, Huang  YW, Chen  HY, Hsueh  PR, Lai  SY, Chen  LC, Chou  YH, Lin  WY, Liaw  SJ,     ( 2016 )

Stenotrophomonas maltophilia PhoP, a Two-Component Response Regulator, Involved in Antimicrobial Susceptibilities.

PloS one 11 (5)
PMID : 27159404  :   DOI  :   10.1371/journal.pone.0153753     PMC  :   PMC4861329    
Abstract >>
Stenotrophomonas maltophilia, a gram-negative bacterium, has increasingly emerged as an important nosocomial pathogen. It is well-known for resistance to a variety of antimicrobial agents including cationic antimicrobial polypeptides (CAPs). Resistance to polymyxin B, a kind of CAPs, is known to be controlled by the two-component system PhoPQ. To unravel the role of PhoPQ in polymyxin B resistance of S. maltophilia, a phoP mutant was constructed. We found MICs of polymyxin B, chloramphenicol, ampicillin, gentamicin, kanamycin, streptomycin and spectinomycin decreased 2-64 fold in the phoP mutant. Complementation of the phoP mutant by the wild-type phoP gene restored all of the MICs to the wild type levels. Expression of PhoP was shown to be autoregulated and responsive to Mg2+ levels. The polymyxin B and gentamicin killing tests indicated that pretreatment of low Mg2+ can protect the wild-type S. maltophilia from killing but not phoP mutant. Interestingly, we found phoP mutant had a decrease in expression of SmeZ, an efflux transporter protein for aminoglycosides in S. maltophilia. Moreover, phoP mutant showed increased permeability in the cell membrane relative to the wild-type. In summary, we demonstrated the two-component regulator PhoP of S. maltophilia is involved in antimicrobial susceptibilities and low Mg2+ serves as a signal for triggering the pathway. Both the alteration in membrane permeability and downregulation of SmeZ efflux transporter in the phoP mutant contributed to the increased drug susceptibilities of S. maltophilia, in particular for aminoglycosides. This is the first report to describe the role of the Mg2+-sensing PhoP signaling pathway of S. maltophilia in regulation of the SmeZ efflux transporter and in antimicrobial susceptibilities. This study suggests PhoPQ TCS may serve as a target for development of antimicrobial agents against multidrug-resistant S. maltophilia.
KeywordMeSH Terms
58. Tacão  M, Correia  A, Henriques  IS,     ( 2015 )

Low Prevalence of Carbapenem-Resistant Bacteria in River Water: Resistance Is Mostly Related to Intrinsic Mechanisms.

Microbial drug resistance (Larchmont, N.Y.) 21 (5)
PMID : 26430939  :   DOI  :   10.1089/mdr.2015.0072    
Abstract >>
Carbapenems are last-resort antibiotics to handle serious infections caused by multiresistant bacteria. The incidence of resistance to these antibiotics has been increasing and new resistance mechanisms have emerged. The dissemination of carbapenem resistance in the environment has been overlooked. The main goal of this research was to assess the prevalence and diversity of carbapenem-resistant bacteria in riverine ecosystems. The presence of frequently reported carbapenemase-encoding genes was inspected. The proportion of imipenem-resistant bacteria was on average 2.24 CFU/ml. Imipenem-resistant strains (n=110) were identified as Pseudomonas spp., Stenotrophomonas maltophilia, Aeromonas spp., Chromobacterium haemolyticum, Shewanella xiamenensis, and members of Enterobacteriaceae. Carbapenem-resistant bacteria were highly resistant to other beta-lactams such as quinolones, aminoglycosides, chloramphenicol, tetracyclines, and sulfamethoxazole/trimethoprim. Carbapenem resistance was mostly associated with intrinsically resistant bacteria. As intrinsic resistance mechanisms, we have identified the blaCphA gene in 77.3% of Aeromonas spp., blaL1 in all S. maltophilia, and blaOXA-48-like in all S. xiamenensis. As acquired resistance mechanisms, we have detected the blaVIM-2 gene in six Pseudomonas spp. (5.45%). Integrons with gene cassettes encoding resistance to aminoglycosides (aacA and aacC genes), trimethoprim (dfrB1b), and carbapenems (blaVIM-2) were found in Pseudomonas spp. Results suggest that carbapenem resistance dissemination in riverine ecosystems is still at an early stage. Nevertheless, monitoring these aquatic compartments for the presence of resistance genes and its host organisms is essential to outline strategies to minimize resistance dissemination.
KeywordMeSH Terms
Water Microbiology
59. Kim  EM, Seo  JH, Baek  K, Kim  BG,     ( 2015 )

Characterization of two-step deglycosylation via oxidation by glycoside oxidoreductase and defining their subfamily.

Scientific reports 5 (N/A)
PMID : 26057169  :   DOI  :   10.1038/srep10877     PMC  :   PMC4650693    
Abstract >>
Herein, we report a two-step deglycosylation mediated by the oxidation of glycoside which is different from traditional glycoside hydrolase (GH) mechanism. Previously, we reported a novel flavin adenine dinucleotide (FAD)-dependent glycoside oxidoreductase (FAD-GO) having deglycosylation activity. Various features of the reaction of FAD-GO such as including mechanism and catalytic residue and substrate specificity were studied. In addition, classification of novel FAD-GO subfamily was attempted. Deglycosylation of glycoside was performed spontaneously via oxidation of 3-OH of glycone moiety by FAD-GO mediated oxidation reaction. His493 residue was identified as a catalytic residue for the oxidation step. Interestingly, this enzyme has broad glycone and aglycon specificities. For the classification of FAD-GO enzyme subfamily, putative FAD-GOs were screened based on the FAD-GO from Rhizobium sp. GIN611 (gi 365822256) using BLAST search. The homologs of R. sp. GIN611 included the putative FAD-GOs from Stenotrophomonas strains, Sphingobacterium strains, Agrobacterium tumefaciens str. C58, and etc. All the cloned FAD-GOs from the three strains catalyzed the deglycosylation via enzymatic oxidation. Based on their substrate specificities, deglycosylation and oxidation activities to various ginsenosides, the FAD-GO subfamily members can be utilized as novel biocatalysts for the production of various aglycones.
KeywordMeSH Terms
60. He  T, Shen  J, Schwarz  S, Wu  C, Wang  Y,     ( 2015 )

Characterization of a genomic island in Stenotrophomonas maltophilia that carries a novel floR gene variant.

The Journal of antimicrobial chemotherapy 70 (4)
PMID : 25477328  :   DOI  :   10.1093/jac/dku491    
Abstract >>
To characterize the chromosomally encoded novel floR gene variant floRv from Stenotrophomonas maltophilia of porcine origin and elucidate the gene order and content of the floRv-flanking regions in an MDR genomic island (GI). Whole genome sequencing was used to identify the unknown florfenicol resistance gene in S. maltophilia strain GZP-Sm1. The candidate gene was cloned into pMD19-T and Escherichia coli transformants carrying this vector were tested for phenicol MICs. Flanking sequences of the florfenicol resistance gene were identified by a de novo assembly and a primer walking strategy. GZP-Sm1 carried a floR gene variant, designated floRv. E. coli clones carrying this gene were resistant to chloramphenicol and florfenicol. The deduced 404 amino acid FloRv protein showed 84.1%-91.8% amino acid identity to various FloR proteins. The gene floRv was located in an MDR region within a 40 226 bp GI region. Six resistance genes, including floRv (phenicol resistance), tetR-tetA(A) (tetracycline resistance), strA/strB (streptomycin resistance), sul1 (sulphonamide resistance) and aadA2 (streptomycin/spectinomycin resistance), were located in this MDR region. PCR analysis revealed that the GI was not stable and could be excised from the chromosome as a circular intermediate. The floRv gene was identified in a porcine S. maltophilia isolate. Six resistance genes including floRv were located in a novel GI. As an opportunistic pathogen in animals and humans, S. maltophilia might act as a resistance gene reservoir in farm environments. Its contribution to the spread of resistance genes to other pathogens should be monitored.
KeywordMeSH Terms
MDR
food animals
integration
phenicol exporter genes
Drug Resistance, Bacterial
Genomic Islands
61. Huedo  P, Yero  D, Martínez-Servat  S, Estibariz  I, Planell  R, Martínez  P, Ruyra  A, Roher  N, Roca  I, Vila  J, Daura  X, Gibert  I,     ( 2014 )

Two different rpf clusters distributed among a population of Stenotrophomonas maltophilia clinical strains display differential diffusible signal factor production and virulence regulation.

Journal of bacteriology 196 (13)
PMID : 24769700  :   DOI  :   10.1128/JB.01540-14     PMC  :   PMC4054175    
Abstract >>
The quorum-sensing (QS) system present in the emerging nosocomial pathogen Stenotrophomonas maltophilia is based on the signaling molecule diffusible signal factor (DSF). Production and detection of DSF are governed by the rpf cluster, which encodes the synthase RpfF and the sensor RpfC, among other components. Despite a well-studied system, little is known about its implication in virulence regulation in S. maltophilia. Here, we have analyzed the rpfF gene from 82 S. maltophilia clinical isolates. Although rpfF was found to be present in all of the strains, it showed substantial variation, with two populations (rpfF-1 and rpfF-2) clearly distinguishable by the N-terminal region of the protein. Analysis of rpfC in seven complete genome sequences revealed a corresponding variability in the N-terminal transmembrane domain of its product, suggesting that each RpfF variant has an associated RpfC variant. We show that only RpfC-RpfF-1 variant strains display detectable DSF production. Heterologous rpfF complementation of �GrpfF mutants of a representative strain of each variant suggests that RpfF-2 is, however, functional and that the observed DSF-deficient phenotype of RpfC-RpfF-2 variant strains is due to permanent repression of RpfF-2 by RpfC-2. This is corroborated by the �GrpfC mutant of the RpfC-RpfF-2 representative strain. In line with this observations, deletion of rpfF from the RpfC-RpfF-1 strain leads to an increase in biofilm formation, a decrease in swarming motility, and relative attenuation in the Caenorhabditis elegans and zebrafish infection models, whereas deletion of the same gene from the representative RpfC-RpfF-2 strain has no significant effect on these virulence-related phenotypes.
KeywordMeSH Terms
Multigene Family
62. Huang  YW, Hu  RM, Chu  FY, Lin  HR, Yang  TC,     ( 2013 )

Characterization of a major facilitator superfamily (MFS) tripartite efflux pump EmrCABsm from Stenotrophomonas maltophilia.

The Journal of antimicrobial chemotherapy 68 (11)
PMID : 23794602  :   DOI  :   10.1093/jac/dkt250    
Abstract >>
To characterize the emrRCABsm operon of Stenotrophomonas maltophilia. The presence of the emrRCABsm operon was verified by RT-PCR. The regulatory role of EmrRsm was investigated by �GemrRsm mutant construction and promoter transcriptional fusion assay. A susceptibility test was employed to assess the substrate spectrum of the EmrCABsm efflux pump. The requirement for each component of the EmrCABsm pump was assessed by individual mutant construction and susceptibility testing. The expression of the emrRCABsm operon was evaluated by an induction assay, using different compounds as inducers. emrRsm, emrCsm, emrAsm and emrBsm formed a four-member operon that was negatively regulated by the MarR-type transcriptional regulator EmrRsm. The emrRCABsm operon was intrinsically poorly expressed and the EmrCAB pump favoured extrusion of the uncoupling agents carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and tetrachlorosalicylanilide (TCS), and the hydrophobic antibiotics nalidixic acid and erythromycin. However, the emrRCABsm operon could not be derepressed by CCCP, nalidixic acid, TCS, 2-chlorophenylhydrazine hydrochloride or salicylate, which are known to be possible inducers for MarR-type regulons. Each component of the EmrCABsm pump was apparently essential for pump function. The EmrRsm-regulated EmrCABsm efflux pump is involved in the extrusion of hydrophobic compounds.
KeywordMeSH Terms
antibiotic resistance
bacteria
efflux pump
63. Liu  J, Chen  P, Zheng  C, Huang  YP,     ( 2013 )

Characterization of maltocin P28, a novel phage tail-like bacteriocin from Stenotrophomonas maltophilia.

Applied and environmental microbiology 79 (18)
PMID : 23835182  :   DOI  :   10.1128/AEM.01648-13     PMC  :   PMC3754179    
Abstract >>
Stenotrophomonas maltophilia is an important global opportunistic pathogen for which limited therapeutics are available because of the emergence of multidrug-resistant strains. A novel bacteriocin, maltocin P28, which is produced by S. maltophilia strain P28, may be the first identified phage tail-like bacteriocin from S. maltophilia. Maltocin P28 resembles a contractile but nonflexible phage tail structure based on electron microscopy, and it is sensitive to trypsin, proteinase K, and heat. SDS-PAGE analysis of maltocin P28 revealed two major protein bands of approximately 43 and 20 kDa. The N-terminal amino acid residues of these two major subunits were sequenced, and the maltocin P28 gene cluster was located on the S. maltophilia P28 chromosome. Our sequence analysis results indicate that this maltocin gene cluster consists of 23 open reading frames (ORFs), and that its gene organization is similar to that of the P2 phage genome and R2 pyocin gene cluster. ORF17 and ORF18 encode the two major structural proteins, which correspond to gpFI (tail sheath) and gpFII (tail tube) of P2 phage, respectively. We found that maltocin P28 had bactericidal activity against 38 of 81 tested S. maltophilia strains. Therefore, maltocin P28 is a promising therapeutic substitute for antibiotics for S. maltophilia infections.
KeywordMeSH Terms
64. Lee  SI, Choi  SH, Lee  EY,     ( 2012 )

Molecular cloning, purification, and characterization of a novel polyMG-specific alginate lyase responsible for alginate MG block degradation in Stenotrophomas maltophilia KJ-2.

Applied microbiology and biotechnology 95 (6)
PMID : 22805784  :   DOI  :   10.1007/s00253-012-4266-y    
Abstract >>
A gene for a polyMG-specific alginate lyase possessing a novel structure was identified and cloned from Stenotrophomas maltophilia KJ-2 by using PCR with homologous nucleotide sequences-based primers. The recombinant alginate lyase consisting of 475 amino acids was purified on Ni-Sepharose column and exhibited the highest activity at pH 8 and 40 �XC. Interestingly, the recombinant alginate lyase was expected to have a similar catalytic active site of chondroitin B lyase but did not show chondroitin lyase activity. In the test of substrate specificity, the recombinant alginate lyase preferentially degraded the glycosidic bond of polyMG-block than polyM-block and polyG-block. The chemical structures of the degraded alginate oligosaccharides were elucidated to have mannuronate (M) at the reducing end on the basis of NMR analysis, supporting that KJ-2 polyMG-specific alginate lyase preferably degraded the glycosidic bond in M-G linkage than that in G-M linkage. The KJ-2 polyMG-specific alginate lyase can be used in combination with other alginate lyases for a synergistic saccharification of alginate.
KeywordMeSH Terms
Cloning, Molecular
65. Nunvar  J, Drevinek  P, Licha  I,     ( 2012 )

DNA profiling of Stenotrophomonas maltophilia by PCR targeted to its species-specific repetitive palindromic sequences.

Letters in applied microbiology 54 (1)
PMID : 22044359  :   DOI  :   10.1111/j.1472-765X.2011.03172.x    
Abstract >>
The aim of this study was to develop a simple protocol for a PCR-based fingerprinting of Stenotrophomonas maltophilia (SmrepPCR) that utilizes primers complementary to repetitive extragenic palindromic elements (REPs) of this micro-organism. The relatedness of 34 isolates of environmental and clinical origin was investigated by two SmrepPCRs with two different primers, gyrB sequencing and XbaI macrorestriction followed by pulsed-field gel electrophoresis. While SmrepPCR (with primer DIR) results matched data obtained from the analysis of gyrB nucleotide sequences and identified several clonal complexes, XbaI macrorestriction showed high level of heterogeneity between isolates. The macrorestriction-based clustering of isolates did not correspond to both gyrB and DIR-SmrepPCR grouping. Our results show that SmrepPCR-inferred relationship of isolates is in a good agreement with sequence-based methods. The combined information from all methods used suggests that rapid evolution of S. maltophilia genomes might be predominantly due to high rate of rearrangements caused by mobile genetic elements. The presented method is an inexpensive and easy to perform alternative to genotype S. maltophilia isolates and to study their population genetics. SmrepPCR demonstrates the usefulness of species-specific repetitive elements in genomic analyses.
KeywordMeSH Terms
DNA Fingerprinting
Inverted Repeat Sequences
66. Chen  CH, Huang  CC, Chung  TC, Hu  RM, Huang  YW, Yang  TC,     ( 2011 )

Contribution of resistance-nodulation-division efflux pump operon smeU1-V-W-U2-X to multidrug resistance of Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 55 (12)
PMID : 21930878  :   DOI  :   10.1128/AAC.00317-11     PMC  :   PMC3232770    
Abstract >>
KJ09C, a multidrug-resistant mutant of Stenotrophomonas maltophilia KJ, was generated by in vitro selection with chloramphenicol. The multidrug-resistant phenotype of KJ09C was attributed to overexpression of a resistance nodulation division (RND)-type efflux system encoded by an operon consisting of five genes: smeU1, smeV, smeW, smeU2, and smeX. Proteins encoded by smeV, smeW, and smeX were similar to the membrane fusion protein, RND transporter, and outer membrane protein, respectively, of known RND-type systems. The proteins encoded by smeU1 and smeU2 were found to belong to the family of short-chain dehydrogenases/reductases. Mutant KJ09C exhibited increased resistance to chloramphenicol, quinolones, and tetracyclines and susceptibility to aminoglycosides; susceptibility to �]-lactams and erythromycin was not affected. The expression of the smeU1-V-W-U2-X operon was regulated by the divergently transcribed LysR-type regulator gene smeRv. Overexpression of the SmeVWX pump contributed to the acquired resistance to chloramphenicol, quinolones, and tetracyclines. Inactivation of smeV and smeW completely abolished the activity of the SmeVWX pump, whereas inactivation of smeX alone decreased the activity of the SmeVWX pump. The enhanced aminoglycoside susceptibility observed in KJ09C resulted from SmeX overexpression.
KeywordMeSH Terms
67. Ribitsch  D, Heumann  S, Karl  W, Gerlach  J, Leber  R, Birner-Gruenberger  R, Gruber  K, Eiteljoerg  I, Remler  P, Siegert  P, Lange  J, Maurer  KH, Berg  G, Guebitz  GM, Schwab  H,     ( 2012 )

Extracellular serine proteases from Stenotrophomonas maltophilia: Screening, isolation and heterologous expression in E. coli.

Journal of biotechnology 157 (1)
PMID : 21983234  :   DOI  :   10.1016/j.jbiotec.2011.09.025    
Abstract >>
A large strain collection comprising antagonistic bacteria was screened for novel detergent proteases. Several strains displayed protease activity on agar plates containing skim milk but were inactive in liquid media. Encapsulation of cells in alginate beads induced protease production. Stenotrophomonas maltophilia emerged as best performer under washing conditions. For identification of wash-active proteases, four extracellular serine proteases called StmPr1, StmPr2, StmPr3 and StmPr4 were cloned. StmPr2 and StmPr4 were sufficiently overexpressed in E. coli. Expression of StmPr1 and StmPr3 resulted in unprocessed, insoluble protein. Truncation of most of the C-terminal domain which has been identified by enzyme modeling succeeded in expression of soluble, active StmPr1 but failed in case of StmPr3. From laundry application tests StmPr2 turned out to be a highly wash-active protease at 45�XC. Specific activity of StmPr2 determined with suc-L-Ala-L-Ala-L-Pro-l-Phe-p-nitroanilide as the substrate was 17��2U/mg. In addition we determined the kinetic parameters and cleavage preferences of protease StmPr2.
KeywordMeSH Terms
68. Svensson-Stadler  LA, Mihaylova  SA, Moore  ER,     ( 2012 )

Stenotrophomonas interspecies differentiation and identification by gyrB sequence analysis.

FEMS microbiology letters 327 (1)
PMID : 22092789  :   DOI  :   10.1111/j.1574-6968.2011.02452.x    
Abstract >>
Stenotrophomonas species are found commonly in environmental and clinical samples; Stenotrophomonas maltophilia is an important opportunistic pathogen of humans. Traditional phenotyping protocols, as well as genotyping by 16S rRNA gene sequence analysis, do not reliably distinguish the species of Stenotrophomonas. Sequence analyses of two targeted PCR-amplified regions of the gyrB gene, which encodes the �]-subunit of DNA gyrase, enabled resolution and identification of these species. Most type strains of the different species of Stenotrophomonas exhibited more than 7% dissimilarity in the gyrB gene sequences. Among these, strains identified as the same species exhibited sequence dissimilarities up to 4.6% and 5.9% for the two regions, respectively. Strains identified as S. maltophilia, with 16S rRNA gene sequence similarities > 99.0%, were grouped within a 'S. maltophilia complex'; these organisms exhibited gyrB similarities as low as 93%. Many of these strains possessed genomic DNA similarities with the type strain of S. maltophilia CCUG 5866(T) below 70%. These data, including gyrB sequence comparisons, indicate that strains identified as S. maltophilia may comprise distinct, new species.
KeywordMeSH Terms
Environmental Microbiology
69. Zhang  R, Sun  Q, Hu  YJ, Yu  H, Li  Y, Shen  Q, Li  GX, Cao  JM, Yang  W, Wang  Q, Zhou  HW, Hu  YY, Chen  GX,     ( 2012 )

Detection of the Smqnr quinolone protection gene and its prevalence in clinical isolates of Stenotrophomonas maltophilia in China.

Journal of medical microbiology 61 (Pt 4)
PMID : 22096133  :   DOI  :   10.1099/jmm.0.037309-0    
Abstract >>
The aim of this study was to detect novel variants of the Stenotrophomonas maltophilia Smqnr gene family and analyse the prevalence of Smqnr genes in clinical isolates of S. maltophilia in China. In total, 442 clinical isolates of S. maltophilia were collected from nine hospitals in four provinces in China. Antimicrobial susceptibility testing against six commonly used antibiotics was performed on these isolates. The sequences of the Smqnr genes amplified by PCR were aligned with those of known Smqnr genes in GenBank and an Smqnr database. The resistance rate against co-trimoxazole was highest at 48.6 %, followed by resistance rates against ceftazidime, chloramphenicol, ticarcillin/clavulanate and tigecycline at 28.7, 21.3, 19.0 and 16.1 %, respectively. The highest susceptibility was shown to levofloxacin, with a resistance rate of just 6.1 %. Smqnr genes were detected in 114 isolates, and comprised 11 previously identified genes and 20 new variants, bringing the total number of known Smqnr genes to 47. The 20 novel Smqnr genes were designated Smqnr28-47 and the encoded proteins showed only 1-12 amino acid differences among each other. The most common Smqnr genes in China were Smqnr8 and its variant Smqnr35 with prevalences of 17.5 % (20/114) and 13.2 % (15/114), respectively. Both the known and the novel Smqnr genes were discovered in both quinolone non-sensitive and sensitive isolates with similar frequency, suggesting that the Smqnr gene makes little contribution to quinolone resistance in this organism.
KeywordMeSH Terms
70. Huang  YW, Hu  RM, Lin  CW, Chung  TC, Yang  TC,     ( 2012 )

NagZ-dependent and NagZ-independent mechanisms for �]-lactamase expression in Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 56 (4)
PMID : 22252801  :   DOI  :   10.1128/AAC.05645-11     PMC  :   PMC3318340    
Abstract >>
�]-N-Acetylglucosaminidase (NagZ), encoded by the nagZ gene, is a critical enzyme for basal-level ampC derepression (ampC expression in the absence of �]-lactam challenge) in ampD and dacB mutants of Pseudomonas aeruginosa. Three mutants with a phenotype of basal-level L1 and L2 �]-lactamase derepression in Stenotrophomonas maltophilia have been reported, including KJ�GDI (ampD(I) mutant), KJ�GmrcA (mrcA mutant), and KJ�GDI�GmrcA (ampD(I) and mrcA double mutant). In this study, nagZ of S. maltophilia was characterized, and its roles in basal-level �]-lactamase derepression, induced �]-lactamase activities, and �]-lactam resistance of KJ�GDI, KJ�GmrcA, and KJ�GDI�GmrcA were evaluated. Expression of the nagZ gene was constitutive and not regulated by AmpR, AmpD(I), AmpN, AmpG, PBP1a, and NagZ. Introduction of �GnagZ into KJ�GDI nearly abolished basal-level derepressed �]-lactamase activity; conversely, introduction of �GnagZ into KJ�GmrcA did not affect it. At least two activator ligands (ALs) are thus considered responsible for �]-lactamase expression in the S. maltophilia system, specifically, the NagZ-dependent (AL1) and NagZ-independent (AL2) ligands responsible for the basal-level derepressed �]-lactamase activities of KJ�GDI and KJ�GmrcA, respectively. The contributions of AL1 and AL2 to the induced �]-lactamase activities may vary with the types of �]-lactams. nagZ inactivation did not affect aztreonam-, cefoxitin-, and carbenicillin-induced �]-lactamase activities, but it attenuated cefuroxime- and piperacillin-induced �]-lactamase activities. Introduction of �GnagZ into KJ, KJ�GDI, KJ�GmrcA, and KJ�GDI�GmrcA did not significantly change the MICs of the �]-lactams tested except that the MICs of cefuroxime and piperacillin moderately decreased in strains KJ�GZ and KJ�GDI�GZ (nagZ mutants).
KeywordMeSH Terms
71.     ( 1997 )

Phylogeny of mercury resistance (mer) operons of gram-negative bacteria isolated from the fecal flora of primates.

Applied and environmental microbiology 63 (3)
PMID : 9055422  :   PMC  :   PMC168397    
Abstract >>
Nine polymorphic mer loci carried by 185 gram-negative fecal bacterial strains from humans and nonhuman primates are described. The loci were characterized with specific intragenic and intergenic PCR primers to amplify distinct regions covering approximately 80% of the typical gram-negative mer locus. These loci were grouped phylogenetically with respect to each other and with respect to seven previously sequenced mer operons from gram-negative bacteria (the latter designated loci 1, 2, 3, 6, 7, 8, and delta 8 by us here for the purpose of this analysis). Six of the mer loci recovered from primates are similar either to these previously sequenced mer loci or to another locus recently observed in environmental isolates (locus 4), and three are novel (loci 5, 9, and 10). We have observed merC, or a merC-like gene, or merF on the 5' side of merA in all of the loci except that of Tn501 (here designated mer locus 6). The merB gene was observed occasionally, always on the 3' side of merA. Unlike the initial example of a merB-containing mer locus carried by plasmid pDU1358 (locus 8), all the natural primate loci carrying merB also had large deletions of the central region of the operon (and were therefore designated locus delta 8). Four of the loci we describe (loci 2, 5, 9, and 10) have no region of homology to merB from pDU1358 and yet strains carrying them were phenylmercury resistant. Two of these loci (loci 5 and 10) also lacked merD, the putative secondary regulator of operon expression. Phylogenetic comparison of character states derived from PCR product data grouped those loci which have merC into one clade; these are locus 1 (including Tn21), locus 3, and locus 4. The mer loci which lack merC grouped into a second clade: locus 6 (including Tn501) and locus 2. Outlying groups lacked merD or possessed merB. While these mer operons are characterized by considerable polymorphism, our ability to discern coherent clades suggests that recombination is not entirely random and indeed may be focused on the immediate 5' and 3' proximal regions of merA. Our observations confirm and extend the idea that the mer operon is a genetic mosaic and has a predominance of insertions and/or deletions of functional genes immediately before and after the merA gene. chi sites are found in several of the sequenced operons and may be involved in the abundant reassortments we observe for mer genes.
KeywordMeSH Terms
Operon
72.     ( 1997 )

Structural studies of malate dehydrogenases (MDHs): MDHs in Brevundimonas species are the first reported MDHs in Proteobacteria which resemble lactate dehydrogenases in primary structure.

Journal of bacteriology 179 (12)
PMID : 9190829  :   DOI  :   10.1128/jb.179.12.4066-4070.1997     PMC  :   PMC179222    
Abstract >>
The N-terminal sequences of malate dehydrogenases from 10 bacterial strains, representing seven genera of Proteobacteria, were determined. Of these, the enzyme sequences of species classified in the genus Brevundimonas clearly resembled those malate dehydrogenases with greatest similarity to lactate dehydrogenases. Additional evidence from subunit molecular weights, peptide mapping, and enzyme mobilities suggested that malate dehydrogenases from species of the genus Brevundimonas were structurally distinct from others in the study.
KeywordMeSH Terms
73.     ( 1997 )

Sequence analysis and enzyme kinetics of the L2 serine beta-lactamase from Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 41 (7)
PMID : 9210666  :   PMC  :   PMC163940    
Abstract >>
The L2 serine active-site beta-lactamase from Stenotrophomonas maltophilia has been classified as a clavulanic acid-sensitive cephalosporinase. The gene encoding this enzyme from S. maltophilia 1275 IID has been cloned on a 3.3-kb fragment into pK18 under the control of a Ptac promoter to generate recombinant plasmid pUB5840; when expressed in Escherichia coli, this gene confers resistance to cephalosporins and penicillins. Sequence analysis has revealed an open reading frame (ORF) of 909 bp with a GC content of 71.6%, comparable to that of the L1 metallo-beta-lactamase gene (68.4%) from the same bacterium. The ORF encodes an unmodified protein of 303 amino acids with a predicted molecular mass of 31.5 kDa, accommodating a putative leader peptide of 27 amino acids. Comparison of the amino acid sequence with those of other beta-lactamases showed it to be most closely related (54% identity) to the BLA-A beta-lactamase from Yersinia enterocolitica. Sequence identity is most obvious near the STXK active-site motif and the SDN loop motif common to all serine active-site penicillinases. Sequences outside the conserved regions display low homology with comparable regions of other class A penicillinases. Kinetics of the enzyme from the cloned gene demonstrated an increase in activity with cefotaxime but markedly less activity with imipenem than previously reported. Hence, the S. maltophilia L2 beta-lactamase is an inducible Ambler class A beta-lactamase which would account for the sensitivity to clavulanic acid.
KeywordMeSH Terms
74.     ( 1997 )

Association of mercury resistance with antibiotic resistance in the gram-negative fecal bacteria of primates.

Applied and environmental microbiology 63 (11)
PMID : 9361435  :   PMC  :   PMC168768    
Abstract >>
Gram-negative fecal bacterial from three longitudinal Hg exposure experiments and from two independent survey collections were examined for their carriage of the mercury resistance (mer) locus. The occurrence of antibiotic resistance was also assessed in both mercury-resistant (Hgr) and mercury-susceptible (Hgs) isolates from the same collections. The longitudinal studies involved exposure of the intestinal flora to Hg released from amalgam "silver" dental restorations in six monkeys. Hgr strains were recovered before the installation of amalgams, and frequently these became the dominant strains while amalgams were installed. Such persistent Hgr strains always carried the same mer locus throughout the experiments. In both the longitudinal and survey collections, certain mer loci were preferentially associated with one genus, whereas other mer loci were recovered from many genera. In general, strains with any mer locus were more likely to be multiresistant than were strains without mer loci; this clustering tendency was also seen for antibiotic resistance genes. However, the association of antibiotic multiresistance with mer loci was not random; regardless of source, certain mer loci occurred in highly multiresistant strains (with as many as seven antibiotic resistances), whereas other mer loci were found in strains without any antibiotic resistance. The majority of highly multiresistant Hgr strains also carried genes characteristic of an integron, a novel genetic element which enables the formation of tandem arrays of antibiotic resistance genes. Hgr strains lacking antibiotic resistance showed no evidence of integron components.
KeywordMeSH Terms
75.     ( 1993 )

Partial nucleotide sequence of the Xanthomonas maltophilia chorionic gonadotropin-like receptor.

Biochemical and biophysical research communications 190 (2)
PMID : 8427582  :   DOI  :   10.1006/bbrc.1993.1057    
Abstract >>
Xanthomonas maltophilia possesses a unique high-affinity binding site which binds human chorionic gonadotropin (hCG), but not human luteinizing hormone (hLH) or other glycoprotein hormones. We designed primers from the known nucleotide sequence of the human LH/CG receptor, spanning an area extending from transmembrane region 2 to transmembrane region 6. Genomic DNA extracted from Xanthomonas maltophilia was used to obtain a PCR amplified product using the above primers. The primary amplification product was cloned in a pCR11 TA cloning vector, and the partial nucleotide sequence of the gene determined. This determined sequence showed 73% identity with the human, as well as the rat LH/CG receptor. Comparison of the translated protein sequence with the human, rat and porcine receptor protein sequences showed a 52% similarity.
KeywordMeSH Terms
76.     ( 1996 )

Dipeptidyl peptidase IV from Xanthomonas maltophilia: sequencing and expression of the enzyme gene and characterization of the expressed enzyme.

Journal of biochemistry 120 (6)
PMID : 9010758  :   DOI  :   10.1093/oxfordjournals.jbchem.a021529    
Abstract >>
The dipeptidyl peptidase IV [EC 3.4.14.5] gene of Xanthomonas maltophilia, expressed in Escherichia coli, was cloned by the shotgun method. Nucleotide sequence analysis revealed an open reading frame of 2,223 bp, coding for a protein of 741 amino acids with a predicted molecular weight of 82,080. The expressed enzyme was extracted with SDS, and the solubilized enzyme was purified about 1,030-fold on columns of Toyopearl HW65C, DEAE-Toyopearl twice, and hydroxyapatite, with an activity recovery of 50%. The enzyme hydrolyzed a proline-containing peptide at the penultimate position, and was inhibited by diisopropyl phosphofluoridate. The enzyme was most active at pH 8.5, and was stable between at pH 7.0 and 9.0. The molecular weight of the purified enzyme was estimated to be 83,000 and 165,000 by SDS-PAGE and gel filtration, respectively.
KeywordMeSH Terms
77.     ( 1993 )

A bacterial protein has homology with human chorionic gonadotropin (hCG).

Biochemical and biophysical research communications 193 (3)
PMID : 8323559  :   DOI  :   10.1006/bbrc.1993.1702    
Abstract >>
Studies from our laboratory have demonstrated the presence of a 48.5 kD cell wall protein in the bacterium, Xanthomonas maltophilia, which immunologically resembles the beta subunit of human chorionic gonadotropin. Primers were designed from the amino acid sequences of enzymatically cleaved peptide fragments of this protein. These primers were used to obtain PCR amplified products, which were subsequently cloned in a PCR11TA cloning vector, and a 492 base pair nucleotide sequence was obtained with a 164 amino acid open reading frame. When this nucleotide sequence was aligned with exon 2 of genes 5 and 6 of the beta hCG gene, a 53% homology was observed. The translated protein sequence had a 35% homology with hCG and a 25% homology with human luteinizing hormone.
KeywordMeSH Terms
78.     ( 1994 )

Sequence analysis of the L1 metallo-beta-lactamase from Xanthomonas maltophilia.

Biochimica et biophysica acta 1218 (2)
PMID : 8018721  :   DOI  :   10.1016/0167-4781(94)90011-6    
Abstract >>
The amino acid sequence deduced from the L1 beta-lactamase gene of Xanthomonas maltophilia shows a significant variation from that of the CphA and Blm metallo-beta-lactamases of Aeromonas hydrophila and Bacillus cereus, respectively. Whilst the N-terminus of the L1 protein shows some similarity, particularly at the histidine residues previously suggested as a zinc-binding motif, the C-terminus of the protein demonstrates very little similarity. Such differences amongst this group of enzymes would argue for at least three subclasses within the Group 3 beta-lactamases. However, in order to predict their phylogenetic ancestry more sequence data are required from other possible metallo-beta-lactamases.
KeywordMeSH Terms
79.     ( 1996 )

Physical structure and expression of alkBA encoding alkane hydroxylase and rubredoxin reductase from Pseudomonas maltophilia.

Biochemical and biophysical research communications 218 (1)
PMID : 8573125  :   DOI  :   10.1006/bbrc.1996.0004    
Abstract >>
The structural genes of the Pseudomonas maltophilia alk system, which are localized on the OCT plasmid were cloned as a 4.2-kilobase pair Hind III fragment. This fragment contains sequences for alkane hydroxylase gene (alkB) and rubredoxin reductase gene (alkA), respectively. The alkB gene encodes a 373-amino acid polypeptide (47.4 kD) that can be expressed at high levels in Pseudomonas and Escherichia coli. The alkBA genes were complemented with alkane hydroxylation in both bacteria. This result shows that alkBA gene is essential for alkane hydroxylation since chromosomal loci have been encoded for other enzymes involved in fatty acid oxidation.
KeywordMeSH Terms
Genes, Bacterial
80. Grover  S, Woodward  SR, Odell  WD,     ( 1995 )

Complete sequence of the gene encoding a chorionic gonadotropin-like protein from Xanthomonas maltophilia.

Gene 156 (1)
PMID : 7537705  :   DOI  :   10.1016/0378-1119(95)00056-c    
Abstract >>
Our laboratory has previously reported that: (i) Xanthomonas maltophilia (Xm) produces a protein which has immunological resemblance to the beta-subunit of human chorionic gonadotropin (hCG) and (ii) possesses a high-affinity receptor which binds holo hCG, and the endogenous ligand, Xm chorionic gonadotropin (xCG), but does not bind human luteinizing hormone (hLH). We have also previously published a 492-bp partial nucleotide sequence of the gene (xcg) coding for xCG. We report herein the entire xcg sequence of 1362 bp, which codes for a 48-kDa protein. This sequence confirmed the 492-bp sequence, as well as two partial amino acid (aa) sequences which we have previously reported. The sequence has a region which is homologous to aa 56-139 of the beta-subunit of hCG, and a second region homologous to the C-terminal tail of hCG. This is the first report of a prokaryotic gene homologous to the hCG beta-subunit-encoding gene.
KeywordMeSH Terms
Bacterial Proteins
Sequence Homology, Amino Acid
81. Bicknell  R, Emanuel  EL, Gagnon  J, Waley  SG,     ( 1985 )

The production and molecular properties of the zinc beta-lactamase of Pseudomonas maltophilia IID 1275.

The Biochemical journal 229 (3)
PMID : 3931629  :   DOI  :   10.1042/bj2290791     PMC  :   PMC1145126    
Abstract >>
The production and purification of a tetrameric zinc beta-lactamase from Pseudomonas maltophilia IID 1275 were greatly improved. Three charge variants were isolated by chromatofocusing. The subunits each contain two atomic proportions of zinc and (in two of the variants) one residue of cysteine. The thiol group is not required for activity, nor does it appear to bind to the metal. Replacement of zinc by cobalt, cadmium or nickel takes place at a measurable rate, and gives enzymes that are less active than the zinc enzyme. The properties of this enzyme differ from those of the other known zinc beta-lactamase, beta-lactamase II from Bacillus cereus. The amino acid sequence of the N-terminal 32 residues was determined; there is no similarity to the N-terminal sequences of other beta-lactamases.
KeywordMeSH Terms
82. Reina  JC, Torres  M, Llamas  I,     ( 2019 )

Stenotrophomonas maltophilia AHL-Degrading Strains Isolated from Marine Invertebrate Microbiota Attenuate the Virulence of Pectobacterium carotovorum and Vibrio coralliilyticus.

Marine biotechnology (New York, N.Y.) 21 (2)
PMID : 30762152  :   DOI  :   10.1007/s10126-019-09879-w    
Abstract >>
Many Gram-negative aquacultural and agricultural pathogens control virulence factor expression through a quorum-sensing (QS) mechanism involving the production of N-acylhomoserine (AHL) signalling molecules. Thus, the interruption of QS systems by the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), has been proposed as a novel strategy to combat these infections. Given that the symbiotic bacteria of marine invertebrates are considered to be an important source of new bioactive molecules, this study explores the presence of AHL-degrading bacteria among 827 strains previously isolated from the microbiota of anemones and holothurians. Four of these strains (M3-1, M1-14, M3-13 and M9-54-2), belonging to the species Stenotrophomonas maltophilia, were selected on the basis of their ability to degrade a broad range of AHLs, and the enzymes involved in their activity were identified. Strain M9-54-2, which showed the strongest AHL-degrading activity, was selected for further study. High-performance liquid chromatography-mass-spectrometry confirmed that the QQ enzyme is not a lactonase. Strain M9-54-2 degraded AHL accumulation and reduced the production of enzymatic activity in Pectobacterium carotovorum CECT 225T and Vibrio coralliilyticus VibC-Oc-193 in in vitro co-cultivation experiments. The effect of AHL inactivation was confirmed by a reduction in potato tuber maceration and brine shrimp (Artemia salina) mortality caused by P. carotovorum and Vibrio coralliilyticus, respectively. This study strengthens the evidence of marine organisms as an underexplored and promising source of QQ enzymes, useful to prevent infections in aquaculture and agriculture. To our knowledge, this is the first time that anemones and holothurians have been studied for this purpose.
KeywordMeSH Terms
Acylase
N-Acylhomoserine lactones
Quorum quenching
Stenotrophomonas
Virulence
83. Lira  F, Berg  G, Martínez  JL,     ( 2017 )

Double-Face Meets the Bacterial World: The Opportunistic Pathogen Stenotrophomonas maltophilia.

Frontiers in microbiology 8 (N/A)
PMID : 29170656  :   DOI  :   10.3389/fmicb.2017.02190     PMC  :   PMC5684188    
Abstract >>
Most studies on bacterial virulence focus on the pathogen itself. However, it is important to recall that the in-host behavior and the virulence of bacterial pathogens constitute a complex situation that depends on both the microorganisms and the infected host. While healthy people (the community) is infected by classical pathogenic microorganisms, able to cope with the anti-infection defenses of the host, in the case of people with basal diseases, debilitated or immunodepressed, the range of pathogens able to cause infection is wider and includes the so-named opportunistic pathogens, which lack the inherent ability to cause disease in healthy hosts and rarely produce infections in the community. Some of the most relevant opportunistic pathogens, as Stenotrophomonas maltophilia, have an environmental origin and, in occasions, present interesting biotechnological properties. Consequently, it is important knowing whether S. maltophilia isolates recovered from infections constitute a specific phylogenetic branch that has evolved toward acquiring a virulent phenotype as it happens in the case of classical pathogens or rather, any member of this bacterial species is capable of producing infection and its pathogenic behavior is mainly a consequence of the host situation. To address this question, we analyzed a set of environmental and clinical S. maltophilia strains. Our results indicate that this opportunistic pathogen presents a large core genome and that the distribution of genes in general, and of known virulence determinants in particular, is similar among environmental and clinical isolates. The majority of genes not belonging to the S. maltophilia core genome are present in just one or two of the analyzed strains. This indicates that, more than speciation into different lineages (virulent and environmental), the evolution of S. maltophilia is based in the strain-specific acquisition of genes, likely involved in the adaptation of this bacterial species to different microniches. In addition, both environmental and clinical isolates present low susceptibility to several antimicrobials. Altogether our results support that S. maltophilia does not present a specific evolutionary branch toward virulence and most likely infection is mainly the consequence of the impaired anti-infective response of the infected patients.
KeywordMeSH Terms
Stenotrophomonas maltophilia
antibiotic resistance
comparative genomics
core genome
opportunistic pathogens
pangenome
84. Harmon  DE, Miranda  OA, McCarley  A, Eshaghian  M, Carlson  N, Ruiz  C,     ( 2019 )

Prevalence and characterization of carbapenem-resistant bacteria in water bodies in the Los Angeles-Southern California area.

MicrobiologyOpen 8 (4)
PMID : 29987921  :   DOI  :   10.1002/mbo3.692     PMC  :   PMC6460273    
Abstract >>
Carbapenems are �]-lactam antibiotics used in healthcare settings as last resort drugs to treat infections caused by antibiotic-resistant bacteria. Carbapenem-resistant bacteria are increasingly being isolated from healthcare facilities; however, little is known about their distribution or prevalence in the environment, especially in the United States, where their distribution in water environments from the West Coast has not been studied before. The aim of this study was to determine the prevalence of carbapenem-resistant bacteria and carbapenemase genes in water bodies from the Los Angeles area (California, USA). All samples that were analyzed contained carbapenem-resistant bacteria with a frequency of between 0.1 and 324 carbapenem-resistant cfu per 100 mls of water. We identified 76 carbapenem-resistant or -intermediate isolates, most of which were also resistant to noncarbapenem antibiotics, as different strains of Enterobacter asburiae, Aeromonas veronii, Cupriavidus gilardii, Pseudomonas, and Stenotrophomonas species. Of them, 52 isolates were carbapenemase-producers. Furthermore, PCR and sequence analysis to identify the carbapenemase gene of these carbapenemase-producing isolates revealed that all Enterobacter asburiae isolates had a blaIMI -2 gene 100% identical to the reference sequence, and all Stenotrophomonas maltophlia isolates had a blaL1 gene 83%-99% identical to the reference blaL1 . Our findings indicate that water environments in Southern California are an important reservoir of bacteria-resistant to carbapenems and other antibiotics, including bacteria carrying intrinsic and acquired carbapenemase genes.
KeywordMeSH Terms
Aeromonas
Cupriavidus
Enterobacter
Pseudomonas
Stenotrophomonas
carbapenem
carbapenem-resistant
carbapenemase
Aeromonas
Cupriavidus
Enterobacter
Pseudomonas
Stenotrophomonas
carbapenem
carbapenem-resistant
carbapenemase
Aeromonas
Cupriavidus
Enterobacter
Pseudomonas
Stenotrophomonas
carbapenem
carbapenem-resistant
carbapenemase
Aeromonas
Cupriavidus
Enterobacter
Pseudomonas
Stenotrophomonas
carbapenem
carbapenem-resistant
carbapenemase
Aeromonas
Cupriavidus
Enterobacter
Pseudomonas
Stenotrophomonas
carbapenem
carbapenem-resistant
carbapenemase
Drug Resistance, Bacterial
85. Kim  YJ, Park  JH, Seo  KH,     ( 2018 )

Presence of Stenotrophomonas maltophilia exhibiting high genetic similarity to clinical isolates in final effluents of pig farm wastewater treatment plants.

International journal of hygiene and environmental health 221 (2)
PMID : 29254843  :   DOI  :   10.1016/j.ijheh.2017.12.002    
Abstract >>
Although the prevalence of community-acquired Stenotrophomonas maltophilia infections is sharply increasing, the sources and likely transmission routes of this bacterium are poorly understood. We studied the significance of the presence of S. maltophilia in final effluents and receiving rivers of pig farm wastewater treatment plants (WWTPs). The loads and antibiotic resistance profiles of S. maltophilia in final effluents were assessed. Antibiotic resistance determinants and biofilm formation genes were detected by PCR, and genetic similarity to clinical isolates was investigated using multilocus sequence typing (MLST). S. maltophilia was recovered from final effluents at two of three farms and one corresponding receiving river. Tests of resistance to antibiotics recommended for S. maltophilia infection revealed that for each agent, at least one isolate was classified as resistant or intermediate, with the exception of minocycline. Furthermore, multidrug resistant S. maltophilia susceptible to antibiotics of only two categories was isolated and found to carry the sul2 gene, conferring trimethoprim/sulfamethoxazole resistance. All isolates carried spgM, encoding a major factor in biofilm formation. MLST revealed that isolates of the same sequence type (ST; ST189) were present in both effluent and receiving river samples, and phylogenetic analysis showed that all of the STs identified in this study clustered with clinical isolates. Moreover, one isolate (ST192) recovered in this investigation demonstrated 99.61% sequence identity with a clinical isolate (ST98) associated with a fatal infection in South Korea. Thus, the pathogenicity of the isolates reported here is likely similar to that of those from clinical environments, and WWTPs may play a role as a source of S. maltophilia from which this bacterium spreads to human communities. To the best of our knowledge, this represents the first report of S. maltophilia in pig farm WWTPs. Our results indicate that nationwide epidemiological investigations are needed to examine the possible link between WWTP-derived S. maltophilia and hospital- and community-acquired infections.
KeywordMeSH Terms
Pig farm
Stenotrophomonas maltophilia
Waste water treatment plant
Swine
86.     ( 2013 )

High activity catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 as a useful tool in cis,cis-muconic acid production.

Antonie van Leeuwenhoek 103 (6)
PMID : 23536173  :   DOI  :   10.1007/s10482-013-9910-8     PMC  :   PMC3656225    
Abstract >>
This is the first report of a catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 with high activity against catechol and its methyl derivatives. This enzyme was maximally active at pH 8.0 and 40 �XC and the half-life of the enzyme at this temperature was 3 h. Kinetic studies showed that the value of K m and V max was 12.8 �gM and 1,218.8 U/mg of protein, respectively. During our studies on kinetic properties of the catechol 1,2-dioxygenase we observed substrate inhibition at >80 �gM. The nucleotide sequence of the gene encoding the S. maltophilia strain KB2 catechol 1,2-dioxygenase has high identity with other catA genes from members of the genus Pseudomonas. The deduced 314-residue sequence of the enzyme corresponds to a protein of molecular mass 34.5 kDa. This enzyme was inhibited by competitive inhibitors (phenol derivatives) only by ca. 30 %. High tolerance against condition changes is desirable in industrial processes. Our data suggest that this enzyme could be of use as a tool in production of cis,cis-muconic acid and its derivatives.
KeywordMeSH Terms
87. Bastard  K, Perret  A, Mariage  A, Bessonnet  T, Pinet-Turpault  A, Petit  JL, Darii  E, Bazire  P, Vergne-Vaxelaire  C, Brewee  C, Debard  A, Pellouin  V, Besnard-Gonnet  M, Artiguenave  F, Médigue  C, Vallenet  D, Danchin  A, Zaparucha  A, Weissenbach  J, Salanoubat  M, de Berardinis  V,     ( 2017 )

Parallel evolution of non-homologous isofunctional enzymes in methionine biosynthesis.

Nature chemical biology 13 (8)
PMID : 28581482  :   DOI  :   10.1038/nchembio.2397    
Abstract >>
Experimental validation of enzyme function is crucial for genome interpretation, but it remains challenging because it cannot be scaled up to accommodate the constant accumulation of genome sequences. We tackled this issue for the MetA and MetX enzyme families, phylogenetically unrelated families of acyl-L-homoserine transferases involved in L-methionine biosynthesis. Members of these families are prone to incorrect annotation because MetX and MetA enzymes are assumed to always use acetyl-CoA and succinyl-CoA, respectively. We determined the enzymatic activities of 100 enzymes from diverse species, and interpreted the results by structural classification of active sites based on protein structure modeling. We predict that >60% of the 10,000 sequences from these families currently present in databases are incorrectly annotated, and suggest that acetyl-CoA was originally the sole substrate of these isofunctional enzymes, which evolved to use exclusively succinyl-CoA in the most recent bacteria. We also uncovered a divergent subgroup of MetX enzymes in fungi that participate only in L-cysteine biosynthesis as O-succinyl-L-serine transferases.
KeywordMeSH Terms
Evolution, Molecular
88.     ( 2013 )

Role of the pcm-tolCsm operon in the multidrug resistance of Stenotrophomonas maltophilia.

The Journal of antimicrobial chemotherapy 68 (9)
PMID : 23629016  :   DOI  :   10.1093/jac/dkt148    
Abstract >>
To elucidate the role of the pcm-tolCsm operon in the multidrug resistance of Stenotrophomonas maltophilia. The presence of the pcm-tolCsm operon was verified by RT-PCR. The phylogenetic relationship between the outer membrane proteins known to be involved in functional tripartite efflux in Escherichia coli, Pseudomonas aeruginosa and S. maltophilia was analysed. The contribution of TolCsm to resistance to a variety of compounds was investigated by susceptibility testing of the �GtolCsm mutant. The role of pcm in the expression and function of tolCsm was assessed by quantitative real-time PCR and complementation assay. The pcm and tolCsm genes formed an operon. TolCsm of S. maltophilia, OpmH of P. aeruginosa and TolC of E. coli formed a distinguishing phylogenetic TolC-like clade. TolCsm deletion increased the susceptibility of S. maltophilia KJ2 to several antimicrobial agents (aminoglycoside, macrolide, �]-lactam, chloramphenicol, nalidixic acid, doxycycline and trimethoprim/sulfamethoxazole) and chemical compounds (acriflavine, carbonyl cyanide 3-chlorophenylhydrazone, crystal violet, fusaric acid, menadione, Paraquat, plumbagin, SDS and tetrachlorosalicylanilide). The in-frame deletion of pcm caused a polar effect on the expression of tolCsm, which compromised the resistance to amikacin and gentamicin. Nevertheless, the presence of the PCM protein made an insignificant contribution to the function of TolCsm in the resistance to amikacin and gentamicin. The pcm-tolCsm operon makes a significant contribution to the multidrug resistance of S. maltophilia.
KeywordMeSH Terms
RND-type efflux pumps
outer membrane proteins
tripartite efflux pumps
Drug Resistance, Multiple, Bacterial
Operon
89.     ( 2012 )

An inducible fusaric acid tripartite efflux pump contributes to the fusaric acid resistance in Stenotrophomonas maltophilia.

PloS one 7 (12)
PMID : 23236431  :   DOI  :   10.1371/journal.pone.0051053     PMC  :   PMC3517613    
Abstract >>
Fusaric acid (5-butylpicolinic acid), a mycotoxin, is noxious to some microorganisms. Stenotrophomonas maltophilia displays an intrinsic resistance to fusaric acid. This study aims to elucidate the mechanism responsible for the intrinsic fusaric acid resistance in S. maltophilia. A putative fusaric acid resistance-involved regulon fuaR-fuaABC was identified by the survey of the whole genome sequence of S. maltophilia K279a. The fuaABC operon was verified by reverse transcriptase-PCR. The contribution of the fuaABC operon to the antimicrobial resistance was evaluated by comparing the antimicrobials susceptibility between the wild-type strain and fuaABC knock-out mutant. The regulatory role of fuaR in the expression of the fuaABC operon was assessed by promoter transcription fusion assay. The fuaABC operon was inducibly expressed by fusaric acid and the inducibility was fuaR dependent. FuaR functioned as a repressor of the fuaABC operon in absence of a fusaric acid inducer and as an activator in its presence. Overexpression of the fuaABC operon contributed to the fusaric acid resistance. A novel tripartite fusaric acid efflux pump, FuaABC, was identified in this study. Distinct from the formally classification, the FuaABC may constitute a new type of subfamily of the tripartite efflux pump.
KeywordMeSH Terms
Drug Resistance, Bacterial
90.     ( 2012 )

Novel ISCR1-linked resistance genes found in multidrug-resistant Gram-negative bacteria in southern China.

International journal of antimicrobial agents 40 (5)
PMID : 22890194  :   DOI  :   10.1016/j.ijantimicag.2012.06.016    
Abstract >>
Non-duplicate multidrug-resistant (MDR) Gram-negative bacteria (n=1329) isolated from southern China between January 2008 and December 2009 were investigated for the presence of ISCR1 as well as characterisation of ISCR1-linked resistance genes. Of 433 ISCR1-positive strains, 151 appeared to carry ISCR1-linked resistance genes. Seven different ISCR1-linked resistance gene arrays were identified by restriction fragment length polymorphism (RFLP) and DNA sequencing analysis. Many of these arrays are reported in some species for the first time. A total of 12 genes, including a novel ABC transporter (GenBank accession no. GU944725), qnrA1, qnrB2, qnrB6, bla(DHA-1), ampR, bla(CTX-M-9), bla(PER-1), insB, sapA-like peptide transport periplasmic protein, putative glutathione S-transferase and short-chain dehydrogenase/reductase, were detected. This study was the first to employ PCR-RFLP using HinfI and RsaI to analyse ISCR1-linked genes. ISCR1 was widely disseminated among MDR Gram-negative bacteria and was in close association with quinolone resistance and �]-lactamase genes (class A and class C) in southern China.
KeywordMeSH Terms
DNA Transposable Elements
Drug Resistance, Multiple, Bacterial
Genes, Bacterial
91.     ( 1998 )

The crystal structure of the L1 metallo-beta-lactamase from Stenotrophomonas maltophilia at 1.7 A resolution.

Journal of molecular biology 284 (1)
PMID : 9811546  :   DOI  :   10.1006/jmbi.1998.2148    
Abstract >>
The structure of the L1 metallo-beta-lactamase from the opportunistic pathogen Stenotrophomonas maltophilia has been determined at 1.7 A resolution by the multiwavelength anomalous dispersion (MAD) approach exploiting both the intrinsic binuclear zinc centre and incorporated selenomethionine residues. L1 is unique amongst all known beta-lactamases in that it exists as a tetramer. The protein exhibits the alphabeta/betaalpha fold found only in the metallo-beta-lactamases and displays several unique features not previously observed in these enzymes. These include a disulphide bridge and two substantially elongated loops connected to the active site of the enzyme. Two closely spaced zinc ions are bound at the active site with tetrahedral (Zn1) and trigonal bipyramidal (Zn2) co-ordination, respectively; these are bridged by a water molecule which we propose acts as the nucleophile in the hydrolytic reaction. Ligation of the second zinc ion involves both residues and geometry which have not been previously observed in the metallo-beta-lactamases. Simulated binding of the substrates ampicillin, ceftazidime and imipenem suggests that the substrate is able to bind to the enzyme in a variety of different conformations whose common features are direct interactions of the beta-lactam carbonyl oxygen and nitrogen with the zinc ions and of the beta-lactam carboxylate with Ser187. We describe a catalytic mechanism whose principal features are a nucleophilic attack of the bridging water on the beta-lactam carbonyl carbon, electrostatic stabilisation of a negatively charged tetrahedral transition state and protonation of the beta-lactam nitrogen by a second water molecule co-ordinated by Zn2. Further, we propose that direct metal:substrate interactions provide a substantial contribution to substrate binding and that this may explain the lack of specificity which is a feature of this class of enzyme.
KeywordMeSH Terms
92.     ( 1998 )

Molecular heterogeneity of the L-1 metallo-beta-lactamase family from Stenotrophomonas maltophilia.

Antimicrobial agents and chemotherapy 42 (5)
PMID : 9593158  :   PMC  :   PMC105789    
Abstract >>
We have determined the nucleotide sequence of the blaS gene encoding the carbapenem-hydrolyzing L-1 beta-lactamase from Stenotrophomonas maltophilia GN12873. Analysis of the DNA and deduced amino acid sequences identified a product of 290 amino acids. Comparisons of the L-1 amino acid sequence with those of other zinc beta-lactamases showed 88.6% identity with the L-1 enzyme from S. maltophilia IID1275 and less than 20% identity with other class B metalloenzymes.
KeywordMeSH Terms

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