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1. Chang  MC, Chang  SY, Chen  SL, Chuang  SM,     ( 1992 )

Cloning and expression in Escherichia coli of the gene encoding an extracellular deoxyribonuclease (DNase) from Aeromonas hydrophila.

Gene 122 (1)
PMID : 1452026  :   DOI  :   10.1016/0378-1119(92)90046-r    
Abstract >>
The gene encoding an extracellular DNase from Aeromonas hydrophila CHC-1 has been cloned and sequenced. Following expression of the dns in Escherichia coli, it was revealed that some of the cloned enzyme was present in the cell-free extracellular supernatant fluid, and there was no cell lysis and concurrent release of cytoplasmic or periplasmic proteins. Therefore, results suggest that E. coli cells were capable of secreting the DNase extracellularly, albeit very inefficiently. The dns is transcribed from its own promoter in E. coli, and expressed as a 25-kDa product, as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of the culture supernatant preparations followed by a DNA-hydrolysis assay. Nucleotide sequence analysis predicted a single open reading frame of 690 bp encoding a 230-amino acid (aa) polypeptide, with a potential 20-aa signal peptide located at the N terminus of the predicted protein. The deduced aa sequence of the entire protein is highly homologous with that of the DNase of Vibrio cholerae.
KeywordMeSH Terms
2. van der Goot  FG, Lakey  J, Pattus  F, Kay  CM, Sorokine  O, Van Dorsselaer  A, Buckley  JT,     ( 1992 )

Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.

Biochemistry 31 (36)
PMID : 1382579  :   DOI  :   10.1021/bi00151a026    
Abstract >>
The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.
KeywordMeSH Terms
3. Kokka  RP, Vedros  NA, Janda  JM,     ( 1992 )

Immunochemical analysis and possible biological role of an Aeromonas hydrophila surface array protein in septicaemia.

Journal of general microbiology 138 (6)
PMID : 1382113  :   DOI  :   10.1099/00221287-138-6-1229    
Abstract >>
The biochemical, immunological, and biological properties of an S layer purified from an Aeromonas hydrophila strain (AH-342) involved in a case of bacteraemia were investigated. The S layer selectively removed from the cell surface was composed of a single acidic (pI 4.56) protein subunit (surface array protein, SAP) with a molecular mass of approximately 52 kDa. Amino acid analysis of this 52 kDa protein indicated a molecule composed of 498 amino acids with 46% hydrophobic residues. No cysteine residues were detected. The first 35 residues of the N-terminus were sequenced by Edman degradation; only 4-24% homology was noted between this sequence and those previously published for SAPs of Aeromonas salmonicida (A450) and a strain of A. hydrophila (TF7) originally isolated from a moribund fish. Polyclonal antibodies raised against AH-342 SAP were genospecific, reacting only against S layers produced by A. hydrophila strains and not those from Aeromonas veronii. Acute serum from the bacteraemic patient from whom AH-342 was isolated reacted strongly with the SAP of AH-342 in immunoblot studies. Purified SAP, when intraperitoneally co-inoculated with SAP- strains of A. hydrophila into Swiss-Webster mice, could reduce the 50% lethal dose by approximately 30-70 fold. The results suggest that the SAP of A. hydrophila strains may play an important role in systemic dissemination after invasion through the gastrointestinal mucosa.
KeywordMeSH Terms
Bacterial Proteins
4. Marks  P, McGeehan  J, Wilson  G, Errington  N, Kneale  G,     ( 2003 )

Purification and characterisation of a novel DNA methyltransferase, M.AhdI.

Nucleic acids research 31 (11)
PMID : 12771207  :   DOI  :   10.1093/nar/gkg399     PMC  :   PMC156732    
Abstract >>
We have cloned the M and S genes of the restriction-modification (R-M) system AhdI and have purified the resulting methyltransferase to homogeneity. M.AhdI is found to form a 170 kDa tetrameric enzyme having a subunit stoichiometry M2S2 (where the M and S subunits are responsible for methylation and DNA sequence specificity, respectively). Sedimentation equilibrium experiments show that the tetrameric enzyme dissociates to form a heterodimer at low concentration, with K(d) approximately 2 microM. The intact (tetrameric) enzyme binds specifically to a 30 bp DNA duplex containing the AhdI recognition sequence GACN5GTC with high affinity (K(d) approximately 50 nM), but at low enzyme concentration the DNA binding activity is governed by the dissociation of the tetramer into dimers, leading to a sigmoidal DNA binding curve. In contrast, only non-specific binding is observed if the duplex lacks the recognition sequence. Methylation activity of the purified enzyme was assessed by its ability to prevent restriction by the cognate endonuclease. The subunit structure of the M.AhdI methyltransferase resembles that of type I MTases, in contrast to the R.AhdI endonuclease which is typical of type II systems. AhdI appears to be a novel R-M system with properties intermediate between simple type II systems and more complex type I systems, and may represent an intermediate in the evolution of R-M systems.
KeywordMeSH Terms
5. Ho  AS, Sohel  I, Schoolnik  GK,     ( 1992 )

Cloning and characterization of fxp, the flexible pilin gene of Aeromonas hydrophila.

Molecular microbiology 6 (18)
PMID : 1360140  :   DOI  :   10.1111/j.1365-2958.1992.tb01449.x    
Abstract >>
The flexible pilus of Aeromonas hydrophila is a morphologically and biochemically unique organelle which binds eukaryotic cell surfaces and whose expression is induced by specific physiochemical conditions. fxp, the structural gene coding for the flexible pilus subunit, was localized on a 7.6kb plasmid of A. hydrophila strain AH26. A putative Shine-Dalgarno sequence and -10 and -35 regions were identified, a signal peptide sequence delineated, and the coding sequence compared with other bacterial sequences and found to be unique. Plasmid and chromosomal DNA was prepared from 66 other Aeromonas strains and 12 strains from other bacterial genera and examined by Southern blot hybridization using a labelled fxp oligonucleotide and the 7.6kb plasmid as probes. No hybridizing sequences were identified except in the original strain, AH26. It is proposed that fxp codes for a highly evolved organelle, possibly widely distributed in nature, but that it is carried on a genetic element that is rapidly lost from most strains upon in vitro cultivation and storage.
KeywordMeSH Terms
Genes, Bacterial
6. Niumsup  P, Simm  AM, Nurmahomed  K, Walsh  TR, Bennett  PM, Avison  MB,     ( 2003 )

Genetic linkage of the penicillinase gene, amp, and blrAB, encoding the regulator of beta-lactamase expression in Aeromonas spp.

The Journal of antimicrobial chemotherapy 51 (6)
PMID : 12746371  :   DOI  :   10.1093/jac/dkg247    
Abstract >>
Aeromonas hydrophila T429125, a human clinical isolate, possesses three coordinately inducible beta-lactamases encoded by ampH (class D beta-lactamase), cepH (class C beta-lactamase) and imiH (class B beta-lactamase). We report that upstream of ampH there are two genes, blrA and blrB, encoding a putative two-component regulatory system. PCR studies revealed the same blrAB-amp gene arrangement in all Aeromonas spp. isolates tested; namely, Aeromonas veronii bv. sobria, Aeromonas jandaei, Aeromonas mediae, Aeromonas salmonicida and Aeromonas trota. A dominant mutation in the predicted BlrB kinase domain results in beta-lactamase overexpression in A. hydrophila T429125, but in other beta-lactamase-overexpressing mutants blrAB remains intact. Relative to the parent strain, A. hydrophila T429125, beta-lactamase- overexpressing mutants show a clear hierarchy of increased beta-lactamase expression: ImiH > CepH > AmpH. The same hierarchy is seen following beta-lactam challenge of A. hydrophila T429125, and correlates with the number of blr-tag sequences (TTCAC) found upstream of each beta-lactamase gene: ampH (one), cepH (two) and imiH (three).
KeywordMeSH Terms
7. Hirono  I, Aoki  T, Asao  T, Kozaki  S,     ( 1992 )

Nucleotide sequences and characterization of haemolysin genes from Aeromonas hydrophila and Aeromonas sobria.

Microbial pathogenesis 13 (6)
PMID : 1302284  :  
Abstract >>
Extracellular haemolysin is thought to be one of the important virulence factors in Aeromonas infection. Two extracellular haemolysin genes (AHH3 and AHH4) from Aeromonas hydrophila strain 28SA, one (AHH5) from A. hydrophila strain AH-1 and one (ASA1) from Aeromonas sobria strain 33 were cloned into cosmid and plasmid vector DNA in Escherichia coli. The nucleotide sequences of the open reading frames of AHH3 and AHH4 are both 1476 basepairs (bp), whereas AHH5 and ASA1 are 1455 and 1467 bp in length, respectively. The deduced amino acid sequences of AHH3, AHH4, AHH5 and the previously reported aerolysin from A. hydrophila showed a significant degree of sequence homology of over 90% each. The amino acid identity of the ASA1 haemolysin and those from A. hydrophila and Aeromonas trota aerolysins ranged from 58-68%. From DNA hybridization analysis using our cloned haemolysin genes as probes, we found that the AHH5 and ASA1 DNA probes hybridized with about 31 and 75% strains of motile Aeromonas species, respectively. The activity of haemolysins of cloned genes were different in medium agar containing various erythrocytes.
KeywordMeSH Terms
8. Altarriba  M, Merino  S, Gavín  R, Canals  R, Rabaan  A, Shaw  JG, Tomás  JM,     ( 2003 )

A polar flagella operon (flg) of Aeromonas hydrophila contains genes required for lateral flagella expression.

Microbial pathogenesis 34 (5)
PMID : 12732473  :  
Abstract >>
Aeromonas spp. are pathogens of both humans and poikilothermic animals, causing a variety of diseases. Certain strains are able to produce two distinct types of flagella; polar flagella for swimming in liquid and lateral flagella for swarming over surfaces. Although, both types of flagella have been associated as colonisation factors, little is known about their organisation and expression. Here we characterised a complete flagellar locus of Aeromonas hydrophila (flg) containing 16 genes, this was analogous to region 1 of the Vibrio parahaemolyticus polar flagellum, with the difference that no flagellin genes were found on A. hydrophila while V. parahaemolyticus showed three flagellin genes. The flg region was present in all Aeromonas strain tested. Defined insertion mutants in flgL, were unable to swim, had a drastic reduction in swarming, lateral flagella, HEp-2 cell adhesion and biofilm formation. Mutations in flgN caused a drastic reduction in lateral flagella, inability to swarm, but these strains were still able to swim. Whereas the cheV mutants still produced both types of flagella and were able to swim and swarm. These results suggest that FlgN is required for lateral flagella formation and swarming motility, but not for polar flagellum-mediated swimming.
KeywordMeSH Terms
9. Zhang  YL, Lau  YL, Arakawa  E, Leung  KY,     ( 2003 )

Detection and genetic analysis of group II capsules in Aeromonas hydrophila.

Microbiology (Reading, England) 149 (Pt 4)
PMID : 12686647  :   DOI  :   10.1099/mic.0.26144-0    
Abstract >>
The genetic organization and sequences of the group II capsule gene cluster of Aeromonas hydrophila PPD134/91 have been determined previously. The purified capsular polysaccharides can increase the ability of avirulent strain PPD35/85 to survive in naive tilapia serum but have no inhibitory effect on the adhesion of PPD134/91 to carp epithelial cells. In this study, the presence of group II capsules among 33 randomly chosen A. hydrophila strains was examined by electron microscopy and genetic analysis. Ten strains were found to produce group II capsules. A PCR detection system was developed to identify two types of group II capsules (IIA and IIB) based on their genetic organization in the region II gene clusters. Group IIA capsules in the authors' collection of A. hydrophila strains are mainly found in the O : 18 and O : 34 serogroups, while group IIB capsules are found in the O : 21 and O : 27 serogroups. The presence of group II capsules in A. hydrophila strongly correlates with the serum and phagocyte survival abilities (seven out of ten strains). The results indicate that the authors' PCR detection system can constitute a reliable assay for the classification of group II capsules in A. hydrophila.
KeywordMeSH Terms
10. Sha  J, Galindo  CL, Pancholi  V, Popov  VL, Zhao  Y, Houston  CW, Chopra  AK,     ( 2003 )

Differential expression of the enolase gene under in vivo versus in vitro growth conditions of Aeromonas hydrophila.

Microbial pathogenesis 34 (4)
PMID : 12668143  :  
Abstract >>
Aeromonas hydrophila is an emerging human pathogen that leads to gastroenteritis and other invasive diseases. By using a murine peritoneal culture (MPC) model, we identified via restriction fragment differential display PCR (RFDDPCR) five genes of A. hydrophila that were differentially expressed under in vivo versus in vitro growth conditions. The gene encoding enolase was among those five genes that were differentially up regulated. Enolase is a glycolytic enzyme and its surface expression was recently shown to be important in the pathogenesis of a gram-positive bacterium Streptococcus pyogenes. By Western blot analysis and Immunogold staining, we demonstrated secretion and surface expression of enolase in A. hydrophila. We also showed that the whole cells of A. hydrophila had strong enolase activity. Using an enzyme-linked immunosorbant assay and sandwich Western blot analysis, we demonstrated binding of enolase to human plasminogen, which is involved in the fibrinolytic system of the host. We cloned the A. hydrophila enolase gene, which exhibited 62% homology at the DNA level and 57% homology at the amino acid level when compared to S. pyogenes enolase. This is a first report describing the increased expression of enolase gene in vivo that could potentially contribute to the pathogenesis of A. hydrophila infections.
KeywordMeSH Terms
Genes, Bacterial
11. Pidiyar  VJ, Jangid  K, Dayananda  KM, Kaznowski  A, Gonzalez  JM, Patole  MS, Shouche  YS,     ( 2003 )

Phylogenetic affiliation of Aeromonas culicicola MTCC 3249(T) based on gyrB gene sequence and PCR-amplicon sequence analysis of cytolytic enterotoxin gene.

Systematic and applied microbiology 26 (2)
PMID : 12866846  :   DOI  :   10.1078/072320203322346047    
Abstract >>
We determined the gyrB gene sequences of all 17 hybridizations groups of Aeromonas. Phylogenetic trees showing the evolutionary relatedness of gyrB and 16S rRNA genes in the type strains of Aeromonas were compared. Using this approach, we determined the phylogenetic position of Aeromonas culicicola MTCC 3249(T), isolated from midgut of Culex quinquefasciatus. In the gyrB based-analysis A. culicicola MTCC 3249(T) grouped with A. veronii whereas, it grouped with A. jandaei in the 16S rRNA based tree. The number of nucleotide differences in 16S rRNA sequences was less than found with the gyrB sequence data. Most of the observed nucleotide differences in the gyrB gene were synonymous. The Cophenetic Correlation Coefficient (CCC) for gyrB sequences was 0.87 indicating this gene to be a better molecular chronometer compared to 16S rRNA for delineation of Aeromonas species. This strain was found to be positive for the cytolytic enterotoxin gene. PCR-Amplicon Sequence Analysis (PCR-ASA) of this gene showed that the isolate is affiliated to type I and is potentially pathogenic. These PCR-ASA results agreed in part with the gyrB sequence results.
KeywordMeSH Terms
Genes, Bacterial
12. Wang  G, Clark  CG, Liu  C, Pucknell  C, Munro  CK, Kruk  TM, Caldeira  R, Woodward  DL, Rodgers  FG,     ( 2003 )

Detection and characterization of the hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR.

Journal of clinical microbiology 41 (3)
PMID : 12624028  :   DOI  :   10.1128/jcm.41.3.1048-1054.2003     PMC  :   PMC150266    
Abstract >>
A multiplex PCR assay was designed to amplify the Aeromonas hydrophila and A. veronii bv. sobria hemolysin and aerolysin genes. The assay was evaluated by using 121 clinical isolates and 7 reference strains of Aeromonas spp., and these were divided into five genotypes on the basis of the results of the multiplex PCR. The five genotypes were characterized as type 1 for those carrying the ahh1 gene only (36% of isolates), type 2 for those carrying the asa1 gene only (8.5% of isolates), type 3 for those carrying both the ahh1 and the asa1 genes (4% of isolates), type 4 for those carrying the ahh1 gene and the A. hydrophila aerA (aerolysin) gene (37.5% of isolates), and type 5 for those in which no hemolysin genes were detected (14% of isolates). The most common single hemolysin gene carried among all the Aeromonas isolates examined was ahh1, with 99 of 128 (77%) of isolates testing positive for this gene either alone or in combination with other hemolysin genes. Phenotypic expression of toxins was evaluated in a Vero cell culture cytotoxicity assay. These results indicated that there is a statistically significant correlation between the cytotoxin titers and the hemolysin genotype. Isolates belonging to genotype 4 (carrying both the ahh1 gene and the aerolysin and hemolysin aerA genes) expressed higher cytotoxin titers than isolates of the other genotypes (P < 0.001). These isolates were more cytotoxic in cell culture and may have greater clinical significance.
KeywordMeSH Terms
13. Jeanteur  D, Gletsu  N, Pattus  F, Buckley  JT,     ( 1992 )

Purification of Aeromonas hydrophila major outer-membrane proteins: N-terminal sequence analysis and channel-forming properties.

Molecular microbiology 6 (22)
PMID : 1283000  :   DOI  :   10.1111/j.1365-2958.1992.tb02203.x    
Abstract >>
Four outer-membrane proteins of Aeromonas hydrophila were purified and their N-terminal sequences and channel-forming properties were determined. Three could be matched with proteins from other species. One was a maltoporin, as its level increased when cells were grown in maltose-containing media, and the channel it formed was blocked by maltose. Another was like OmpF and OmpC of Escherichia coli, except that its channel fluctuated much more rapidly. The third protein, which was produced in low-phosphate medium, exhibited several properties of the general anion porin PhoE. The fourth showed no similarity to any known proteins. It had a unique N-terminus and it formed small sharply-defined cation-selective channels. Two other proteins which corresponded to OmpW of Vibrio cholerae and E. coli OmpA were partly characterized.
KeywordMeSH Terms
Ion Channels
14. Yáñez  MA, Catalán  V, Apráiz  D, Figueras  MJ, Martínez-Murcia  AJ,     ( 2003 )

Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences.

International journal of systematic and evolutionary microbiology 53 (Pt 3)
PMID : 12807216  :   DOI  :   10.1099/ijs.0.02443-0    
Abstract >>
The phylogenetic relationships of all known species of the genus Aeromonas were investigated by using the sequence of gyrB, a gene that encodes the B-subunit of DNA gyrase. Nucleotide sequences of gyrB were determined from 53 Aeromonas strains, including some new isolates, which were also characterized by analysis of the 16S rDNA variable regions. The results support the recognition of the family Aeromonadaceae, as distinct from Plesiomonas shigelloides and other enteric bacteria. This phylogenetic marker revealed strain groupings that are consistent with the taxonomic organization of all Aeromonas species described to date. In particular, gyrB results agreed with 16S rDNA analysis; moreover, the former showed a higher capacity to differentiate between species. The present analysis was useful for the elucidation of reported discrepancies between different DNA-DNA hybridization sets. Additionally, due to the sequence diversity found at the intraspecies level, gyrB is proposed as a useful target for simultaneous identification of species and strains. In conclusion, the gyrB gene has proved to be an excellent molecular chronometer for phylogenetic studies of the genus Aeromonas.
KeywordMeSH Terms
Phylogeny
Sequence Analysis, DNA
15. Merino  S, Gavín  R, Vilches  S, Shaw  JG, Tomás  JM,     ( 2003 )

A colonization factor (production of lateral flagella) of mesophilic Aeromonas spp. is inactive in Aeromonas salmonicida strains.

Applied and environmental microbiology 69 (1)
PMID : 12514057  :   DOI  :   10.1128/aem.69.1.663-667.2003     PMC  :   PMC152462    
Abstract >>
The nine laf (lateral flagellum) genes of mesophilic aeromonads are in the Aeromonas salmonicida genome. The laf genes are functional, except for lafA (flagellin gene), which was inactivated by transposase 8 (IS3 family). A pathogenic characteristic of mesophilic aeromonads (lateral flagella) is abolished in this specialized pathogen with a narrow host range.
KeywordMeSH Terms
16. Tsitrin  Y, Morton  CJ, el-Bez  C, Paumard  P, Velluz  MC, Adrian  M, Dubochet  J, Parker  MW, Lanzavecchia  S, van der Goot  FG,     ( 2002 )

Conversion of a transmembrane to a water-soluble protein complex by a single point mutation.

Nature structural biology 9 (10)
PMID : 12219082  :   DOI  :   10.1038/nsb839    
Abstract >>
Proteins exist in one of two generally incompatible states: either membrane associated or soluble. Pore-forming proteins are exceptional because they are synthesized as a water-soluble molecule but end up being located in the membrane -- that is, they are nonconstitutive membrane proteins. Here we report the pronounced effect of the single point mutation Y221G of the pore-forming toxin aerolysin. This mutation blocks the hemolytic activity of the toxin but does not affect its initial structure, its ability to bind to cell-surface receptors or its capacity to form heptamers, which constitute the channel-forming unit. The overall structure of the Y221G protein as analyzed by cryo-negative staining EM and three-dimensional reconstruction is remarkably similar to that of the wild type heptamer. The mutant protein forms a mushroom-shaped complex whose stem domain is thought to be within the membrane in the wild type toxin. In contrast to the wild type heptamer, which is a hydrophobic complex, the Y221G heptamer is fully hydrophilic. This point mutation has, therefore, converted a normally membrane-embedded toxin into a soluble complex.
KeywordMeSH Terms
Point Mutation
17. Goñi-Urriza  M, Arpin  C, Capdepuy  M, Dubois  V, Caumette  P, Quentin  C,     ( 2002 )

Type II topoisomerase quinolone resistance-determining regions of Aeromonas caviae, A. hydrophila, and A. sobria complexes and mutations associated with quinolone resistance.

Antimicrobial agents and chemotherapy 46 (2)
PMID : 11796341  :   DOI  :   10.1128/aac.46.2.350-359.2002     PMC  :   PMC127024    
Abstract >>
Most Aeromonas strains isolated from two European rivers were previously found to be resistant to nalidixic acid. In order to elucidate the mechanism of this resistance, 20 strains of Aeromonas caviae (n = 10), A. hydrophila (n = 5), and A. sobria (n = 5) complexes, including 3 reference strains and 17 environmental isolates, were investigated. Fragments of the gyrA, gyrB, parC, and parE genes encompassing the quinolone resistance-determining regions (QRDRs) were amplified by PCR and sequenced. Results obtained for the six sensitive strains showed that the GyrA, GyrB, ParC, and ParE QRDR fragments of Aeromonas spp. were highly conserved (> or =96.1% identity), despite some genetic polymorphism; they were most closely related to those of Vibrio spp., Pseudomonas spp., and members of the family Enterobacteriaceae (72.4 to 97.1% homology). All 14 environmental resistant strains carried a point mutation in the GyrA QRDR at codon 83, leading to the substitution Ser-83-->Ile (10 strains) or Ser-83-->Arg. In addition, seven strains harbored a mutation in the ParC QRDR either at position 80 (five strains), generating a Ser-80-->Ile (three strains) or Ser-80-->Arg change, or at position 84, yielding a Glu-84-->Lys modification. No amino acid alterations were discovered in the GyrB and ParE QRDRs. Double gyrA-parC missense mutations were associated with higher levels of quinolone resistance compared with the levels associated with single gyrA mutations. The most resistant strains probably had an additional mechanism(s) of resistance, such as decreased accumulation of the drugs. Our data suggest that, in mesophilic Aeromonas spp., as in other gram-negative bacteria, gyrase and topoisomerase IV are the primary and secondary targets for quinolones, respectively.
KeywordMeSH Terms
18. Zhang  YL, Arakawa  E, Leung  KY,     ( 2002 )

Novel Aeromonas hydrophila PPD134/91 genes involved in O-antigen and capsule biosynthesis.

Infection and immunity 70 (5)
PMID : 11953367  :   DOI  :   10.1128/iai.70.5.2326-2335.2002     PMC  :   PMC127894    
Abstract >>
The sequences of the O-antigen and capsule gene clusters of the virulent Aeromonas hydrophila strain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11 A. hydrophila strains of different serotypes. A. hydrophila PPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster of A. hydrophila PPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster of Escherichia coli and other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulent A. hydrophila strain PPD35/85 to survive in na?ve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion of A. hydrophila PPD134/91 to carp epithelial cells.
KeywordMeSH Terms
Multigene Family
19. Leclère  V, Chotteau-Lelièvre  A, Gancel  F, Imbert  M, Blondeau  R,     ( 2001 )

Occurrence of two superoxide dismutases in Aeromonas hydrophila: molecular cloning and differential expression of the sodA and sodB genes.

Microbiology (Reading, England) 147 (Pt 11)
PMID : 11700360  :   DOI  :   10.1099/00221287-147-11-3105    
Abstract >>
Aeromonas spp., considered as emerging opportunistic pathogens, belong to the family Vibrionaceae. Among the criteria currently used for their classification is the presence of a single FeSOD (iron-containing superoxide dismutase), which distinguishes them from Enterobacteriacea. In this paper the cloning of the sodA and sodB genes encoding two different SODs in Aeromonas hydrophila ATCC 7966 is reported. The sodB gene encoded an FeSOD (196 amino acids, 21.5 kDa), was constitutively expressed and showed 75% homology with the E. coli FeSOD. The sodA gene encoded a protein of 206 amino acids (22.5 kDa) with MnSOD (manganese-containing SOD) activity and showed 55% homology with the Escherichia coli MnSOD. The MnSOD of A. hydrophila was detected only during the stationary phase of growth under high aeration or when induced by lack of iron. Nevertheless, paraquat had no detectable effect on its production. The amino-terminal part of the Mn-containing protein contained a putative signal sequence which could permit a periplasmic localization.
KeywordMeSH Terms
20. Sha  J, Kozlova  EV, Chopra  AK,     ( 2002 )

Role of various enterotoxins in Aeromonas hydrophila-induced gastroenteritis: generation of enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity.

Infection and immunity 70 (4)
PMID : 11895956  :   DOI  :   10.1128/iai.70.4.1924-1935.2002     PMC  :   PMC127858    
Abstract >>
Three enterotoxins from the Aeromonas hydrophila diarrheal isolate SSU have been molecularly characterized in our laboratory. One of these enterotoxins is cytotoxic in nature, whereas the other two are cytotonic enterotoxins, one of them heat labile and the other heat stable. Earlier, by developing an isogenic mutant, we demonstrated the role of a cytotoxic enterotoxin in causing systemic infection in mice. In the present study, we evaluated the role of these three enterotoxins in evoking diarrhea in a murine model by developing various combinations of enterotoxin gene-deficient mutants by marker-exchange mutagenesis. A total of six isogenic mutants were prepared in a cytotoxic enterotoxin gene (act)-positive or -negative background strain of A. hydrophila. We developed two single knockouts with truncation in either the heat-labile (alt) or the heat-stable (ast) cytotonic enterotoxin gene; three double knockouts with truncations of genes encoding (i) alt and ast, (ii) act and alt, and (iii) act and ast genes; and a triple-knockout mutant with truncation in all three genes, act, alt, and ast. The identity of these isogenic mutants developed by double-crossover homologous recombination was confirmed by Southern blot analysis. Northern and Western blot analyses revealed that the expression of different enterotoxin genes in the mutants was correspondingly abrogated. We tested the biological activity of these mutants in a diet-restricted and antibiotic-treated mouse model with a ligated ileal loop assay. Our data indicated that all of these mutants had significantly reduced capacity to evoke fluid secretion compared to that of wild-type A. hydrophila; the triple-knockout mutant failed to induce any detectable level of fluid secretion. The biological activity of selected A. hydrophila mutants was restored after complementation. Taken together, we have established a role for three enterotoxins in A. hydrophila-induced gastroenteritis in a mouse model with the greatest contribution from the cytotoxic enterotoxin Act, followed by the Alt and Ast cytotonic enterotoxins.
KeywordMeSH Terms
Escherichia coli Proteins
21. Wu  ML, Chuang  YC, Chen  JP, Chen  CS, Chang  MC,     ( 2001 )

Identification and characterization of the three chitin-binding domains within the multidomain chitinase Chi92 from Aeromonas hydrophila JP101.

Applied and environmental microbiology 67 (11)
PMID : 11679332  :   DOI  :   10.1128/AEM.67.11.5100-5106.2001     PMC  :   PMC93277    
Abstract >>
The gene (chi92) encoding the extracellular chitinase of Aeromonas hydrophila JP101 has been cloned and expressed in Escherichia coli. The mature form of Chi92 is an 842-amino-acid (89.830-kDa) modular enzyme comprised of a family 18 catalytic domain, an unknown-function region (the A region), and three chitin-binding domains (ChBDs; Chi92-N, ChBD(CI), and ChBD(CII)). The C-terminally repeated ChBDs, ChBD(CI) and ChBD(CII), were grouped into family V of cellulose-binding domains on the basis of sequence homology. Chitin binding and enzyme activity studies with C-terminally truncated Chi92 derivatives lacking ChBDs demonstrated that the ChBDs are responsible for its adhesion to unprocessed and colloidal chitins. Further adsorption experiments with glutathione S-transferase (GST) fusion proteins (GST-CI and GST-CICII) demonstrated that a single ChBD (ChBD(CI)) could promote efficient chitin and cellulose binding. In contrast to the two C-terminal ChBDs, the Chi92-N domain is similar to ChiN of Serratia marcescens ChiA, which has been proposed to participate in chitin binding. A truncated derivative of Chi92 that contained only a catalytic domain and Chi92-N still exhibited insoluble-chitin-binding and hydrolytic activities. Thus, it appears that Chi92 contains Chi92-N as the third ChBD in addition to two ChBDs (ChBD(CI) and ChBD(CII)).
KeywordMeSH Terms
22. Kidd  SP, Pemberton  JM,     ( 2002 )

The cloning and characterization of a second alpha-amylase of A. hydrophila JMP636.

Journal of applied microbiology 92 (2)
PMID : 11849357  :  
Abstract >>
The aim of this study was to identify, clone and characterize the second amylase of Aeromonas hydrophila JMP636, AmyB, and to compare it to AmyA. The amylase activity of A. hydrophila JMP636 is encoded by multiple genes. A second genetically distinct amylase gene, amyB, has been cloned and expressed from its own promoter in Escherichia coli. AmyB is a large alpha-amylase of 668 amino acids. Outside the conserved domains of alpha-amylases there is limited sequence relationship between the two alpha-amylases of A. hydrophila JMP636 AmyA and AmyB. Significant (80%) similarity exists between amyB and an alpha-amylase of A. hydrophila strain MCC-1. Differences in either the functional properties or activity under different environmental conditions as possible explanations for multiple copies of amylases in JMP636 is less likely after an examination of several physical properties, with each of the properties being very similar for both enzymes (optimal pH and temperature, heat instability). However the reaction end products and substrate specificity did vary enough to give a possible reason for the two enzymes being present. Both enzymes were confirmed to be alpha-type amylases. AmyB has been isolated, characterized and then compared to AmyA. The amylase phenotype is rarely encoded by more than one enzyme within one strain, this study therefore allows the better understanding of the unusual amylase production by A. hydrophila.
KeywordMeSH Terms
Bacterial Proteins
23. Sha  J, Lu  M, Chopra  AK,     ( 2001 )

Regulation of the cytotoxic enterotoxin gene in Aeromonas hydrophila: characterization of an iron uptake regulator.

Infection and immunity 69 (10)
PMID : 11553581  :   DOI  :   10.1128/IAI.69.10.6370-6381.2001     PMC  :   PMC98772    
Abstract >>
The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of Aeromonas hydrophila is aerolysin related and crucial to the pathogenesis of Aeromonas infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (act), a portion of the act gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (phoA) of Escherichia coli. The act::phoA reporter gene was then introduced into the chromosome of A. hydrophila by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the act promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The act promoter exhibited optimal activity at pH 7.0 and at 37 degrees C, and maximal PhoA activity was noted when the culture was aerated. Using a Vibrio cholerae iron uptake regulator gene (fur) as a probe, a 2.6-kb SalI/HindIII DNA fragment from an A. hydrophila chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the fur gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of V. cholerae. Complementation experiments demonstrated that the A. hydrophila fur gene could restore iron regulation in an E. coli fur-minus mutant. Using the suicide vector pDMS197, we generated a fur isogenic mutant of wild-type A. hydrophila SSU. Northern blot analysis data indicated that the repression in the transcription of the act gene by iron was relieved in the fur isogenic mutant. Further, iron regulation in the fur isogenic mutant of A. hydrophila could be restored by complementation. These results are important in understanding the regulation of the act gene under in vivo conditions.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
24. Merino  S, Gavín  R, Altarriba  M, Izquierdo  L, Maguire  ME, Tomás  JM,     ( 2001 )

The MgtE Mg2+ transport protein is involved in Aeromonas hydrophila adherence.

FEMS microbiology letters 198 (2)
PMID : 11430413  :   DOI  :   10.1111/j.1574-6968.2001.tb10641.x    
Abstract >>
Aeromonas hydrophila AH-3 strains carrying mutations in mgtE, which encodes a Mg2+ and Co2+ transport system, showed a 50% reduction of in vitro adherence to HEp-2 cells, a reduction in swarming in semisolid swarming agar, and decrease in biofilm formation of over 60% in comparison to the wild-type strain. The cloned A. hydrophila mgtE expressed from a plasmid complements a Salmonella typhimurium strain deleted for all Mg2+ transporters both phenotypically and by measurement of 57Co2+ uptake. Likewise, plasmid-borne mgtE was able to complement the changes observed in A. hydrophila mgtE mutants. We suggest that MgtE and thus Mg2+ and possibly Co2+ have a role in A. hydrophila related to their swarming ability and related consequences such as adherence and biofilm formation.
KeywordMeSH Terms
25. Merino  S, Altarriba  M, Gavín  R, Izquierdo  L, Tomás  JM,     ( 2001 )

The cell division genes (ftsE and X) of Aeromonas hydrophila and their relationship with opsonophagocytosis.

FEMS microbiology letters 198 (2)
PMID : 11430412  :   DOI  :   10.1111/j.1574-6968.2001.tb10640.x    
Abstract >>
A transposon mutant from Aeromonas hydrophila AH-3 was obtained which was highly resistant to opsonophagocytosis. The mutation was identified in the ftsE gene and we characterised the operon ftsY, E and X from this bacterium. These genes, as in enteric bacteria, are neighbours to rpoH. The A. hydrophilia ftsE and X genes were fully able to complement Escherichia coli ftsE mutants, and also complement the opsonophagocytosis-resistant phenotype of the A. hydrophila mutant strain. This phenotype seems to be related to the filamentous phenotype at 37 degrees C exhibited by the A. hydrophila ftsE mutant.
KeywordMeSH Terms
ATP-Binding Cassette Transporters
Escherichia coli Proteins
Operon
26. Avison  MB, Niumsup  P, Walsh  TR, Bennett  PM,     ( 2000 )

Aeromonas hydrophila AmpH and CepH beta-lactamases: derepressed expression in mutants of Escherichia coli lacking creB.

The Journal of antimicrobial chemotherapy 46 (5)
PMID : 11062187  :   DOI  :   10.1093/jac/46.5.695    
Abstract >>
The class 1 cephalosporinase (CepH) and class 2d oxacillinase (AmpH) from an Aeromonas hydrophila clinical isolate, strain T429125, have been cloned and sequenced. Both enzymes are typical of their equivalents in other species of Aeromonas Both cloned beta-lactamase genes were expressed at a low level in a standard laboratory Escherichia coli strain, but when cloned into a cre deletion E. coli mutant, they were expressed at significantly higher levels. Specific disruption of the creB gene resulted in similar increased levels of beta-lactamase expression, so it was concluded that CreB represses the transcription of ampH and cepH in a cre(+) E. coli strain. The expression of cepH was four times that of ampH in the deltacre mutant because of an additional factor encoded on the cloned T429125 chromosomal fragment containing cepH. This factor was able to trans-activate expression of co-resident ampH in the deltacre mutant such that expression of the two genes was approximately equal. The entire cepH-containing fragment was sequenced, but it contained no genes that were obviously related to any known class of DNA-binding protein.
KeywordMeSH Terms
Escherichia coli Proteins
Penicillin-Binding Proteins
27. Ramaiah  N, Hill  RT, Chun  J, Ravel  J, Matte  MH, Straube  WL, Colwell  RR,     ( 2000 )

Use of a chiA probe for detection of chitinase genes in bacteria from the Chesapeake Bay(1).

FEMS microbiology ecology 34 (1)
PMID : 11053737  :   DOI  :   10.1111/j.1574-6941.2000.tb00755.x    
Abstract >>
PCR primers specific for the chiA gene were designed by alignment and selection of highly conserved regions of chiA sequences from Serratia marcescens, Alteromonas sp., Bacillus circulans and Aeromonas caviae. These primers were used to amplify a 225 bp fragment of the chiA gene from Vibrio harveyi to produce a chiA gene probe. The chiA PCR primers and probe were used to detect the presence of the chiA gene in an assemblage of 53 reference strains and gave consistent results. Selected chiA fragments amplified by PCR were cloned and sequenced from nine known strains and from Chesapeake Bay isolates 6d and 11d. This confirmed the specificity and utility of the primers for detection of chiA-positive environmental strains. Over 1000 bacterial isolates from Chesapeake Bay water samples were tested for the presence of the chiA gene which was found to be present in 5-41% (average 21%) of the culturable bacterial community. The approach developed in this study was valuable for isolation and enumeration of chiA-positive bacteria in environmental samples.
KeywordMeSH Terms
28. Ong  CT, Zhang  YL,     ( 2000 )

Molecular analysis of genetic differences between virulent and avirulent strains of Aeromonas hydrophila isolated from diseased fish.

Microbiology (Reading, England) 146 (Pt 4) (N/A)
PMID : 10784058  :   DOI  :   10.1099/00221287-146-4-999    
Abstract >>
Aeromonas hydrophila, a normal inhabitant of aquatic environments, is an opportunistic pathogen of a variety of aquatic and terrestrial animals, including humans. A. hydrophila PPD134/91 is defined as virulent whereas PPD35/85 is defined as avirulent on the basis of their different LD50 values in fish. Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains. Sixty-nine genomic regions of differences were absent in PPD35/85, and the DNA sequences of these regions were determined. Sixteen ORFs encoded by 23 fragments showed high homology to known proteins of other bacteria. ORFs encoded by the remaining 46 fragments were identified as new proteins of A. hydrophila, showing no significant homology to any known proteins. Among these PPD134/91-specific genes, 22 DNA fragments (21 ORFs) were present in most of the eight virulent strains studied but mostly absent in the seven avirulent strains, suggesting that they are universal virulence genes in A. hydrophila. The PPD134/91-specific genes included five known virulence factors of A. hydrophila: haemolysin (hlyA), protease (oligopeptidase A), outer-membrane protein (Omp), multidrug-resistance protein and histone-like protein (HU-2). Another 47 DNA fragments (44 ORFs) were mainly present in PPD134/91, indicating the heterogeneity among motile aeromonads. Some of these fragments encoded virulence determinants. These included genes for the synthesis of O-antigen and type II restriction/modification system. The results indicated that SSH is successful in identifying genetic differences and virulence genes among different strains of A. hydrophila.
KeywordMeSH Terms
Genome, Bacterial
29. Temprano  A, Sánchez  M, Hernanz  C, Yugueros  J, Cascón  A,     ( 2000 )

A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila.

Infection and immunity 68 (6)
PMID : 10816468  :   DOI  :   10.1128/iai.68.6.3233-3241.2000     PMC  :   PMC97569    
Abstract >>
Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in rainbow trout. A gene encoding an elastolytic activity, ahyB, was cloned from Aeromonas hydrophila AG2 into pUC18 and expressed in Escherichia coli and in the nonproteolytic species Aeromonas salmonicida subsp. masoucida. Nucleotide sequence analysis of the ahyB gene revealed an open reading frame of 1,764 nucleotides with coding capacity for a 588-amino-acid protein with a molecular weight of 62,728. The first 13 N-terminal amino acids of the purified protease completely match those deduced from DNA sequence starting at AAG (Lys-184). This finding indicated that AhyB is synthesized as a preproprotein with a 19-amino-acid signal peptide, a 164-amino-acid N-terminal propeptide, and a 405-amino-acid intermediate which is further processed into a mature protease and a C-terminal propeptide. The protease hydrolyzed casein and elastin and showed a high sequence similarity to other metalloproteases, especially with the mature form of the Pseudomonas aeruginosa elastase (52% identity), Helicobacter pylori zinc metalloprotease (61% identity), or proteases from several species of Vibrio (52 to 53% identity). The gene ahyB was insertionally inactivated, and the construct was used to create an isogenic ahyB mutant of A. hydrophila. These first reports of a defined mutation in an extracellular protease of A. hydrophila demonstrate an important role in pathogenesis.
KeywordMeSH Terms
Oncorhynchus mykiss
30. Merino  S, Aguilar  A, Nogueras  MM,     ( 2000 )

Cloning, sequencing, and role in serum susceptibility of porin II from mesophilic Aeromonas hydrophila.

Infection and immunity 68 (4)
PMID : 10722573  :   DOI  :   10.1128/iai.68.4.1849-1854.2000     PMC  :   PMC97357    
Abstract >>
We cloned and sequenced the structural gene for Aeromonas hydrophila porin II from strain AH-3 (serogroup O:34). The genetic position of this gene, like that of ompF in Escherichia coli, is adjacent to aspC and transcribed in the same direction. However, upstream of the porin II gene no similarities with E. coli were found. We obtained defined insertion mutants in porin II gene either in A. hydrophila (O:34) or A. veronii sobria (serogroup O:11) serum-resistant or -sensitive strains. Furthermore, we complemented these mutants with a plasmid harboring only the porin II gene, which allowed us to define the role of porin II as an important surface molecule involved in serum susceptibility and C1q binding in these strains.
KeywordMeSH Terms
31. Hirama  T,     ( 1999 )

Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance.

The Journal of biological chemistry 274 (32)
PMID : 10428840  :   DOI  :   10.1074/jbc.274.32.22604    
Abstract >>
Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosphatidylinositol (GPI) anchors on host cell-surface structures. The nature of the receptors and the location of the receptor-binding sites on the toxin molecule were investigated using surface plasmon resonance. Aerolysin bound to the GPI-anchored proteins Thy-1, variant surface glycoprotein, and contactin with similar rate constants and affinities. Enzymatic removal of N-linked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin. Aerolysin is a bilobal protein, and both lobes were shown to be required for optimal binding. The large lobe by itself bound Thy-1 with an affinity that was at least 10-fold weaker than that of the whole toxin, whereas the small lobe bound the GPI-anchored protein at least 1000-fold more weakly than the intact toxin. Mutation analyses provided further evidence that both lobes were involved in GPI anchor binding, with certain single amino acid substitutions in either domain leading to reductions in affinity of as much as 100-fold. A variant with single amino acid substitutions in both lobes of the protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weakly to immobilized proaerolysin, suggesting that interactions with cell-surface carbohydrate structures other than GPI anchors may partially mediate toxin binding to host cells.
KeywordMeSH Terms
32. Tomás  JM, Swift  S, Aguilar  A, Nogueras  MM, Regue  M,     ( 1999 )

Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34.

Infection and immunity 67 (8)
PMID : 10417167  :   PMC  :   PMC96688    
Abstract >>
Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5alpha. Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process.
KeywordMeSH Terms
33. Dodd  HN, Pemberton  JM,     ( 1999 )

The gene encoding a periplasmic deoxyribonuclease from Aeromonas hydrophila.

FEMS microbiology letters 173 (1)
PMID : 10220879  :   DOI  :   10.1111/j.1574-6968.1999.tb13482.x    
Abstract >>
A gene encoding a deoxyribonuclease, dnsH, was cloned from Aeromonas hydrophila JMP636. The predicted mature protein was very similar to the previously described extracellular Dns from this organism and an N-terminal region corresponding to a large putative signal sequence was predicted for the JMP636 protein. Inactivation of dnsII demonstrated that the DnsH protein was not present extracellularly in this strain. As DnsH degraded plasmid DNA and was believed to have a periplasmic location, a dnsH mutant was constructed to determine whether electroporation of A. hydrophila with plasmid DNA could be achieved. No transformants were detected. From SDS-PAGE studies, at least two additional DNases remain to be characterised from A. hydrophila JMP636.
KeywordMeSH Terms
Bacterial Proteins
34. Erova  TE, Sha  J, Horneman  AJ, Borchardt  MA, Khajanchi  BK, Fadl  AA, Chopra  AK,     ( 2007 )

Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila.

FEMS microbiology letters 275 (2)
PMID : 17725618  :   DOI  :   10.1111/j.1574-6968.2007.00895.x    
Abstract >>
A clinical strain SSU of Aeromonas hydrophila produces a potent cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. A new gene, which encoded a hemolysin of 439-amino acid residues with a molecular mass of 49 kDa, was identified. This hemolysin (HlyA) was detected based on the observation that the act gene minus mutant of A. hydrophila SSU still had residual hemolytic activity. The new hemolysin gene (hlyA) was cloned, sequenced, and overexpressed in Escherichia coli. The hlyA gene exhibited 96% identity with its homolog found in a recently annotated genome sequence of an environmental isolate, namely the type strain ATCC 7966 of A. hydrophila subspecies hydrophila. The hlyA gene did not exhibit any homology with other known hemolysins and aerolysin genes detected in Aeromonas isolates. However, this hemolysin exhibited significant homology with hemolysin of Vibrio vulnificus as well as with the cystathionine beta synthase domain protein of Shewanella oneidensis. The HlyA protein was activated only after treatment with trypsin and the resulting hemolytic activity was not neutralizable with antibodies to Act. The presence of the hlyA gene in clinical and water Aeromonas isolates was investigated and DNA fingerprint analysis was performed to demonstrate its possible role in Aeromonas virulence.
KeywordMeSH Terms
Hemolysin Proteins
35. Vilches  S, Wilhelms  M, Yu  HB, Leung  KY, Tomás  JM, Merino  S,     ( 2008 )

Aeromonas hydrophila AH-3 AexT is an ADP-ribosylating toxin secreted through the type III secretion system.

Microbial pathogenesis 44 (1)
PMID : 17689917  :   DOI  :   10.1016/j.micpath.2007.06.004    
Abstract >>
We cloned and sequenced an ADP-ribosylating toxin (AexT) from a mesophilic Aeromonas hydrophila strain AH-3 with a type III secretion system (T3SS). This toxin only showed homology, in genes and proteins, with the first half of A. salmonicida AexT. The A. hydrophila AexT showed ADP-ribosyltransferase activity, translocation through the T3SS system, and this A. hydrophila T3SS system is inducible under calcium-depleted conditions. The A. hydrophila aexT mutant showed a slight reduction in their virulence assayed by several methods when compared to the wild-type strain, while an A. hydrophila T3SS mutant is highly reduced in virulence on the same assays. The A. hydrophila AexT is the first described and the smallest T3SS effector toxin found in mesophilic Aeromonas with a functional T3SS.
KeywordMeSH Terms
36. Vilches  S, Canals  R, Wilhelms  M, Saló  MT, Knirel  YA, Vinogradov  E, Merino  S, Tomás  JM,     ( 2007 )

Mesophilic Aeromonas UDP-glucose pyrophosphorylase (GalU) mutants show two types of lipopolysaccharide structures and reduced virulence.

Microbiology (Reading, England) 153 (Pt 8)
PMID : 17660404  :   DOI  :   10.1099/mic.0.2007/006437-0    
Abstract >>
A mutation in galU that causes the lack of O34-antigen lipopolysaccharide (LPS) in Aeromonas hydrophila strain AH-3 was identified. It was proved that A. hydrophila GalU is a UDP-glucose pyrophosphorylase responsible for synthesis of UDP-glucose from glucose 1-phosphate and UTP. The galU mutant from this strain showed two types of LPS structures, represented by two bands on LPS gels. The first one (slow-migrating band in gels) corresponds to a rough strain having the complete core, with two significant differences: it lacks the terminal galactose residue from the LPS-core and 4-amino-4-deoxyarabinose residues from phosphate groups in lipid A. The second one (fast-migrating band in gels) corresponds to a deeply truncated structure with the LPS-core restricted to one 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) and three l-glycero-d-manno-heptose residues. galU mutants in several motile mesophilic Aeromonas strains from serotypes O1, O2, O11, O18, O21 and O44 were also devoid of the O-antigen LPS. The galU mutation reduced to less than 1 % the survival of these Aeromonas strains in serum, decreased the ability of these strains to adhere and reduced by 1.5 or 2 log units the virulence of Aeromonas serotype O34 strains in a septicaemia model in either fish or mice. All the changes observed in the galU mutants were rescued by the introduction of the corresponding single wild-type gene.
KeywordMeSH Terms
37. Horsfall  LE, Garau  G, Liénard  BM, Dideberg  O, Schofield  CJ, Frère  JM, Galleni  M,     ( 2007 )

Competitive inhibitors of the CphA metallo-beta-lactamase from Aeromonas hydrophila.

Antimicrobial agents and chemotherapy 51 (6)
PMID : 17307979  :   DOI  :   10.1128/AAC.00866-06     PMC  :   PMC1891371    
Abstract >>
Various inhibitors of metallo-beta-lactamases have been reported; however, none are effective for all subgroups. Those that have been found to inhibit the enzymes of subclass B2 (catalytically active with one zinc) either contain a thiol (and show less inhibition towards this subgroup than towards the dizinc members of B1 and B3) or are inactivators behaving as substrates for the dizinc family members. The present work reveals that certain pyridine carboxylates are competitive inhibitors of CphA, a subclass B2 enzyme. X-ray crystallographic analyses demonstrate that pyridine-2,4-dicarboxylic acid chelates the zinc ion in a bidentate manner within the active site. Salts of these compounds are already available and undergoing biomedical testing for various nonrelated purposes. Pyridine carboxylates appear to be useful templates for the development of more-complex, selective, nontoxic inhibitors of subclass B2 metallo-beta-lactamases.
KeywordMeSH Terms
beta-Lactamase Inhibitors
38. Hou  XL, Cao  QY, Pan  JC, Chen  Z,     ( 2006 )

[Classification and identification of vibrio cholerae and vibrio parahaemolyticus isolates based on gyrB gene phylogenetic analysis].

Wei sheng wu xue bao = Acta microbiologica Sinica 46 (6)
PMID : 17302148  :  
Abstract >>
In order to validate the usefulness of gyrB genotype for the classification and identification of Vibrio cholerae and Vibrio parahaemolyticus isolates, the phylogenetic analysis of 13 V. cholerae, 8 V. parahaemolyticus, 2 Aeromonas hydrophila and 1 Plesiomonas shigelloides strains was carried out using the partial coding sequence of gyrB, a gene that encodes the B subunit of DNA gyrase (topoisomerase type II) in bacteria. These strains were separately clustered at species level and typed by the DNA sequences of reference strains from GenBank. CtxA positive V. cholerae strains including 8 clincical isolates of 0139 and 2 clinical isolates of 01 formed one cluster. Four V. parahaemolyticus strains of 1 isolate from 2002 Zhejiang outbreak patient (tdh positive), 2 clinical isoltates from 2004 and 1 strain from Japan were grouped with an environmental isolate (trh positive) from 2001. GyrB genotype is applicable to species identification of V. cholerae, V. parahaemolyticus, A. hydrophila and P. shigelloides isolates. The ctxA positive 0139 and 01 group of V. cholerae are closely related, as reflected by gyrB sequence divergence. Furthermore, the toxigenic V. parahaemolyticus strain isolated from environments may be the potential pathogen to the local prevalent and sporadic cases.
KeywordMeSH Terms
39. Kosti?  T, Weilharter  A, Rubino  S, Delogu  G, Uzzau  S, Rudi  K, Sessitsch  A, Bodrossy  L,     ( 2007 )

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens.

Analytical biochemistry 360 (2)
PMID : 17123456  :   DOI  :   10.1016/j.ab.2006.09.026    
Abstract >>
A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.
KeywordMeSH Terms
40. Canals  R, Jiménez  N, Vilches  S, Regué  M, Merino  S, Tomás  JM,     ( 2007 )

Role of Gne and GalE in the virulence of Aeromonas hydrophila serotype O34.

Journal of bacteriology 189 (2)
PMID : 17098903  :   DOI  :   10.1128/JB.01260-06     PMC  :   PMC1797372    
Abstract >>
The mesophilic Aeromonas hydrophila AH-3 (serotype O34) strain shows two different UDP-hexose epimerases in its genome: GalE (EC 3.1.5.2) and Gne (EC 3.1.5.7). Similar homologues were detected in the different mesophilic Aeromonas strains tested. GalE shows only UDP-galactose 4-epimerase activity, while Gne is able to perform a dual activity (mainly UDP-N-acetyl galactosamine 4-epimerase and also UDP-galactose 4-epimerase). We studied the activities in vitro of both epimerases and also in vivo through the lipopolysaccharide (LPS) structure of A. hydrophila gne mutants, A. hydrophila galE mutants, A. hydrophila galE-gne double mutants, and independently complemented mutants with both genes. Furthermore, the enzymatic activity in vivo, which renders different LPS structures on the mentioned A. hydrophila mutant strains or the complemented mutants, allowed us to confirm a clear relationship between the virulence of these strains and the presence/absence of the O34 antigen LPS.
KeywordMeSH Terms
41. Saavedra  MJ, Figueras  MJ, Martínez-Murcia  AJ,     ( 2006 )

Updated phylogeny of the genus Aeromonas.

International journal of systematic and evolutionary microbiology 56 (Pt 10)
PMID : 17012583  :   DOI  :   10.1099/ijs.0.64351-0    
Abstract >>
Recent phylogenetic studies of the genus Aeromonas based on gyrB and rpoD gene sequences have improved the phylogeny based on 16S rRNA gene sequences first published in 1992, particularly in the ability to split closely related species. These studies did not include the recently described species Aeromonas simiae and Aeromonas molluscorum and only a single strain of Aeromonas culicicola was available for analysis at that time. In the present work, these Aeromonas species and newly isolated strains of A. culicicola were examined. Sequence analysis indicates that A. simiae and A. molluscorum belong to non-described phylogenetic lines of descent within this genus, which supports the original description of both species. The most closely related species are Aeromonas schubertii and Aeromonas encheleia, respectively, which is consistent with 16S rRNA gene sequencing results. However, while the five strains of A. molluscorum showed nucleotide differences in their gyrB and rpoD gene sequences, the only two known A. simiae strains exhibited identical gene sequences, suggesting that they are isolates of the same strain. On the basis of the rpoD gene sequence phylogeny, A. culicicola strains from the original description and new isolates from drinking water and ornamental fish clustered within the species Aeromonas veronii, suggesting inconsistencies with previous results. Other strains with previously controversial taxonomy and new isolates from other studies were included in this study in order to clarify their phylogenetic affiliation at the species level.
KeywordMeSH Terms
Phylogeny
42. Lan  X, Zhang  X, Hu  J, Shimosaka  M,     ( 2006 )

Cloning, expression, and characterization of a chitinase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9.

Bioscience, biotechnology, and biochemistry 70 (10)
PMID : 17031053  :   DOI  :   10.1271/bbb.60169    
Abstract >>
The chitinolytic bacterium Aeromonas hydrophila strain SUWA-9, which was isolated from freshwater in Lake Suwa (Nagano Prefecture, Japan), produced several kinds of chitin-degrading enzymes. A gene coding for an endo-type chitinase (chiA) was isolated from SUWA-9. The chiA ORF encodes a polypeptide of 865 amino acid residues with a molecular mass of 91.6 kDa. The deduced amino acid sequence showed high similarity to those of bacterial chitinases classified into family 18 of glycosyl hydrolases. chiA was expressed in Escherichia coli and the recombinant chitinase (ChiA) was purified and examined. The enzyme hydrolyzed N-acetylchitooligomers from trimer to pentamer and produced monomer and dimer as a final product. It also reacted toward colloidal chitin and chitosan with a low degree of deacetylation. When cells of SUWA-9 were grown in the presence of colloidal chitin, a 60 kDa-truncated form of ChiA that had lost the C-terminal chitin-binding domain was secreted.
KeywordMeSH Terms
43. Pillai  L, Sha  J, Erova  TE, Fadl  AA, Khajanchi  BK, Chopra  AK,     ( 2006 )

Molecular and functional characterization of a ToxR-regulated lipoprotein from a clinical isolate of Aeromonas hydrophila.

Infection and immunity 74 (7)
PMID : 16790746  :   DOI  :   10.1128/IAI.00402-06     PMC  :   PMC1489700    
Abstract >>
Human diseases caused by species of Aeromonas have been classified into two major groups: septicemia and gastroenteritis. In this study, we reported the molecular and functional characterization of a new virulence factor, ToxR-regulated lipoprotein, or TagA, from a diarrheal isolate, SSU, of Aeromonas hydrophila. The tagA gene of A. hydrophila exhibited 60% identity with that of a recently identified stcE gene from Escherichia coli O157:H7, which encoded a protein (StcE) that provided serum resistance to the bacterium and prevented erythrocyte lysis by controlling classical pathway of complement activation by cleaving the complement C1-esterase inhibitor (C1-INH). We purified A. hydrophila TagA as a histidine-tagged fusion protein (rTagA) from E. coli DE3 strain using a T7 promoter-based pET30 expression vector and nickel affinity column chromatography. rTagA cleaved C1-INH in a time-dependent manner. The tagA isogenic mutant of A. hydrophila, unlike its corresponding wild-type (WT) or the complemented strain, was unable to cleave C1-INH, which is required to potentiate the C1-INH-mediated lysis of host and bacterial cells. We indeed demonstrated colocalization of C1-INH and TagA on the bacterial surface by confocal fluorescence microscopy, which ultimately resulted in increased serum resistance of the WT bacterium. Likewise, we delineated the role of TagA in contributing to the enhanced ability of C1-INH to inhibit the classical complement-mediated lysis of erythrocytes. Importantly, we provided evidence that the tagA mutant was significantly less virulent in a mouse model of infection (60%) than the WT bacterium at two 50% lethal doses, which resulted in 100% mortality within 48 h. Taken together, our data provided new information on the role of TagA as a virulence factor in bacterial pathogenesis. This is the first report of TagA characterization from any species of Aeromonas.
KeywordMeSH Terms
44. Hu  F, You  S,     ( 2007 )

Inactivation of type I polyhydroxyalkanoate synthase in Aeromonas hydrophila resulted in discovery of another potential PHA synthase.

Journal of industrial microbiology & biotechnology 34 (3)
PMID : 17043804  :   DOI  :   10.1007/s10295-006-0180-6    
Abstract >>
Aeromonas hydrophila CGMCC 0911 possessing type I polyhydroxyalkanoate (PHA) synthase (PhaC) produced only PHBHHx from lauric acid but not from glucose. Medium-chain-length (mcl) PHA was produced from lauric acid or glucose only when PhaC of A. hydrophila was inactivated, indicating the existence of another PHA synthase in the wild type. Using PCR cloning strategy, the potential PHA synthase gene (phaC (mcl)) was obtained from genomic DNA of the wild type and exhibited strong homology to type II PHA synthase genes of Pseudomonas strains. The phaC (mcl) gene was PCR subcloned into plasmid pBBR1MCS2 and expressed in a PHA-negative mutant of Pseudomonas putida. Recombinant P. putida synthesized mcl PHA from gluconate or octanoate. This result proved that wild type A. hydrophila possessed another type II PHA synthase, which was responsible for the synthesis of mcl PHA, besides type I PHA synthase.
KeywordMeSH Terms
45. Iacovache  I, Paumard  P, Scheib  H, Lesieur  C, Sakai  N, Matile  S, Parker  MW, van der Goot  FG,     ( 2006 )

A rivet model for channel formation by aerolysin-like pore-forming toxins.

The EMBO journal 25 (3)
PMID : 16424900  :   DOI  :   10.1038/sj.emboj.7600959     PMC  :   PMC1383540    
Abstract >>
The bacterial toxin aerolysin kills cells by forming heptameric channels, of unknown structure, in the plasma membrane. Using disulfide trapping and cysteine scanning mutagenesis coupled to thiol-specific labeling on lipid bilayers, we identify a loop that lines the channel. This loop has an alternating pattern of charged and uncharged residues, suggesting that the transmembrane region has a beta-barrel configuration, as observed for Staphylococcal alpha-toxin. Surprisingly, we found that the turn of the beta-hairpin is composed of a stretch of five hydrophobic residues. We show that this hydrophobic turn drives membrane insertion of the developing channel and propose that, once the lipid bilayer has been crossed, it folds back parallel to the plane of the membrane in a rivet-like fashion. This rivet-like conformation was modeled and sequence alignments suggest that such channel riveting may operate for many other pore-forming toxins.
KeywordMeSH Terms
Models, Molecular
46. Erova  TE, Pillai  L, Fadl  AA, Sha  J, Wang  S, Galindo  CL, Chopra  AK,     ( 2006 )

DNA adenine methyltransferase influences the virulence of Aeromonas hydrophila.

Infection and immunity 74 (1)
PMID : 16368997  :   DOI  :   10.1128/IAI.74.1.410-424.2006     PMC  :   PMC1346675    
Abstract >>
Among the various virulence factors produced by Aeromonas hydrophila, a type II secretion system (T2SS)-secreted cytotoxic enterotoxin (Act) and the T3SS are crucial in the pathogenesis of Aeromonas-associated infections. Our laboratory molecularly characterized both Act and the T3SS from a diarrheal isolate, SSU of A. hydrophila, and defined the role of some regulatory genes in modulating the biological effects of Act. In this study, we cloned, sequenced, and expressed the DNA adenine methyltransferase gene of A. hydrophila SSU (dam(AhSSU)) in a T7 promoter-based vector system using Escherichia coli ER2566 as a host strain, which could alter the virulence potential of A. hydrophila. Recombinant Dam, designated as M.AhySSUDam, was produced as a histidine-tagged fusion protein and purified from an E. coli cell lysate using nickel affinity chromatography. The purified Dam had methyltransferase activity, based on its ability to transfer a methyl group from S-adenosyl-l-methionine to N(6)-methyladenine-free lambda DNA and to protect methylated lambda DNA from digestion with DpnII but not against the DpnI restriction enzyme. The dam gene was essential for the viability of the bacterium, and overproduction of Dam in A. hydrophila SSU, using an arabinose-inducible, P(BAD) promoter-based system, reduced the virulence of this pathogen. Specifically, overproduction of M.AhySSUDam decreased the motility of the bacterium by 58%. Likewise, the T3SS-associated cytotoxicity, as measured by the release of lactate dehydrogenase enzyme in murine macrophages infected with the Dam-overproducing strain, was diminished by 55% compared to that of a control A. hydrophila SSU strain harboring the pBAD vector alone. On the contrary, cytotoxic and hemolytic activities associated with Act as well as the protease activity in the culture supernatant of a Dam-overproducing strain were increased by 10-, 3-, and 2.4-fold, respectively, compared to those of the control A. hydrophila SSU strain. The Dam-overproducing strain was not lethal to mice (100% survival) when given by the intraperitoneal route at a dose twice that of the 50% lethal dose, which within 2 to 3 days killed 100% of the animals inoculated with the A. hydrophila control strain. Taken together, our data indicated alteration of A. hydrophila virulence by overproduction of Dam.
KeywordMeSH Terms
47. Canals  R, Jiménez  N, Vilches  S, Regué  M, Merino  S, Tomás  JM,     ( 2006 )

The UDP N-acetylgalactosamine 4-epimerase gene is essential for mesophilic Aeromonas hydrophila serotype O34 virulence.

Infection and immunity 74 (1)
PMID : 16369010  :   DOI  :   10.1128/IAI.74.1.537-548.2006     PMC  :   PMC1346635    
Abstract >>
Mesophilic Aeromonas hydrophila strains of serotype O34 typically express smooth lipopolysaccharide (LPS) on their surface. A single mutation in the gene that codes for UDP N-acetylgalactosamine 4-epimerase (gne) confers the O(-) phenotype (LPS without O-antigen molecules) on a strain in serotypes O18 and O34, but not in serotypes O1 and O2. The gne gene is present in all the mesophilic Aeromonas strains tested. No changes were observed for the LPS core in a gne mutant from A. hydrophila strain AH-3 (serotype O34). O34 antigen LPS contains N-acetylgalactosamine, while no such sugar residue forms part of the LPS core from A. hydrophila AH-3. Some of the pathogenic features of A. hydrophila AH-3 gne mutants are drastically reduced (serum resistance or adhesion to Hep-2 cells), and the gne mutants are less virulent for fish and mice compared to the wild-type strain. Strain AH-3, like other mesophilic Aeromonas strains, possess two kinds of flagella, and the absence of O34 antigen molecules by gne mutation in this strain reduced motility without any effect on the biogenesis of both polar and lateral flagella. The reintroduction of the single wild-type gne gene in the corresponding mutants completely restored the wild-type phenotype (presence of smooth LPS) independently of the O wild-type serotype, restored the virulence of the wild-type strain, and restored motility (either swimming or swarming).
KeywordMeSH Terms
48. Canals  R, Altarriba  M, Vilches  S, Horsburgh  G, Shaw  JG, Tomás  JM, Merino  S,     ( 2006 )

Analysis of the lateral flagellar gene system of Aeromonas hydrophila AH-3.

Journal of bacteriology 188 (3)
PMID : 16428388  :   DOI  :   10.1128/JB.188.3.852-862.2006     PMC  :   PMC1347325    
Abstract >>
Mesophilic Aeromonas strains express a polar flagellum in all culture conditions, and certain strains produce lateral flagella on semisolid media or on surfaces. Although Aeromonas lateral flagella have been described as a colonization factor, little is known about their organization and expression. Here we characterized the complete lateral flagellar gene cluster of Aeromonas hydrophila AH-3 containing 38 genes, 9 of which (lafA-U) have been reported previously. Among the flgLL and lafA structural genes we found a modification accessory factor gene (maf-5) that is involved in formation of lateral flagella; this is the first time that such a gene has been described for lateral flagellar gene systems. All Aeromonas lateral flagellar genes were located in a unique chromosomal region, in contrast to Vibrio parahaemolyticus, in which the analogous genes are distributed in two different chromosomal regions. In A. hydrophila mutations in flhAL, lafK, fliJL, flgNL, flgEL, and maf-5 resulted in a loss of lateral flagella and reductions in adherence and biofilm formation, but they did not affect polar flagellum synthesis. Furthermore, we also cloned and sequenced the A. hydrophila AH-3 alternative sigma factor sigma54 (rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella.
KeywordMeSH Terms
49. Sen  K,     ( 2005 )

Development of a rapid identification method for Aeromonas species by multiplex-PCR.

Canadian journal of microbiology 51 (11)
PMID : 16333335  :   DOI  :   10.1139/w05-089    
Abstract >>
Existing biochemical methods cannot distinguish among some species of Aeromonads, while genetic methods are labor intensive. In this study, primers were developed to three genes of Aeromonas: lipase, elastase, and DNA gyraseB. In addition, six previously described primer sets, five corresponding to species-specific signature regions of the 16S rRNA gene from A. veronii, A. popoffii, A. caviae, A. jandaei, and A. schubertii, respectively, and one corresponding to A. hydrophila specific lipase (hydrolipase), were chosen. The primer sets were combined in a series of multiplex-PCR (mPCR) assays against 38 previously characterized strains. Following PCR, each species was distinguished by the production of a unique combination of amplicons. When the assays were tested using 63 drinking water isolates, there was complete agreement in the species identification (ID) for 59 isolates, with ID established by biochemical assays. Sequencing the gyrB and the 16S rRNA gene from the remaining four strains established that the ID obtained by mPCR was correct for three strains. For only one strain, no consensus ID could be obtained. A rapid and reliable method for identification of different Aeromonas species is proposed that does not require restriction enzyme digestions, thus simplifying and speeding up the process.
KeywordMeSH Terms
50. Jiang  B, Howard  SP,     ( 1992 )

The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway.

Molecular microbiology 6 (10)
PMID : 1640836  :   DOI  :   10.1111/j.1365-2958.1992.tb00856.x    
Abstract >>
The Aeromonas hydrophila Tn5-751 insertion mutant L1.97 is unable to secrete extracellular proteins, and is fragile because of defective assembly of its outer membrane. A KpnI 4.1 kb fragment, which complements this mutant when supplied with an exogenous promoter, was isolated and sequenced. It contains two complete genes, exeE and exeF, plus fragments of two others and may form part of an operon. The exeE and exeF open reading frames encode 501-residue M(r) 55,882 and 388-residue M(r) 43,431 proteins, respectively. These genes were expressed in vitro and their initiation codons verified by deletion analysis. Tn5-751 had inserted near the centre of the exeE gene in the L1.97 strain. Subclones of the KpnI 4.1 kb fragment which contained only the exeE gene fully complemented the mutation, indicating that its function is required both for extracellular secretion and outer membrane assembly. ExeE and ExeF are highly similar to other proteins which have been shown to be involved in extracellular secretion, suggesting that an additional export apparatus beyond that required for inner membrane translocation may be part of the physiology of many Gram-negative bacteria.
KeywordMeSH Terms
Bacterial Proteins
Genes, Bacterial
51. Canals  R, Ramirez  S, Vilches  S, Horsburgh  G, Shaw  JG, Tomás  JM, Merino  S,     ( 2006 )

Polar flagellum biogenesis in Aeromonas hydrophila.

Journal of bacteriology 188 (2)
PMID : 16385045  :   DOI  :   10.1128/JB.188.2.542-555.2006     PMC  :   PMC1347287    
Abstract >>
Mesophilic Aeromonas spp. constitutively express a single polar flagellum that helps the bacteria move to more favorable environments and is an important virulence and colonization factor. Certain strains can also produce multiple lateral flagella in semisolid media or over surfaces. We have previously reported 16 genes (flgN to flgL) that constitute region 1 of the Aeromonas hydrophila AH-3 polar flagellum biogenesis gene clusters. We identified 39 new polar flagellum genes distributed in four noncontiguous chromosome regions (regions 2 to 5). Region 2 contained six genes (flaA to maf-1), including a modification accessory factor gene (maf-1) that has not been previously reported and is thought to be involved in glycosylation of polar flagellum filament. Region 3 contained 29 genes (fliE to orf29), most of which are involved in flagellum basal body formation and chemotaxis. Region 4 contained a single gene involved in the motor stator formation (motX), and region 5 contained the three master regulatory genes for the A. hydrophila polar flagella (flrA to flrC). Mutations in the flaH, maf-1, fliM, flhA, fliA, and flrC genes, as well as the double mutant flaA flaB, all caused loss of polar flagella and reduction in adherence and biofilm formation. A defined mutation in the pomB stator gene did not affect polar flagellum motility, in contrast to the motX mutant, which was unable to swim even though it expressed a polar flagellum. Mutations in all of these genes did not affect lateral flagellum synthesis or swarming motility, showing that both A. hydrophila flagellum systems are entirely distinct.
KeywordMeSH Terms
52. Poole  TL, Callaway  TR, Bischoff  KM, Warnes  CE, Nisbet  DJ,     ( 2006 )

Macrolide inactivation gene cluster mphA-mrx-mphR adjacent to a class 1 integron in Aeromonas hydrophila isolated from a diarrhoeic pig in Oklahoma.

The Journal of antimicrobial chemotherapy 57 (1)
PMID : 16339607  :   DOI  :   10.1093/jac/dki421    
Abstract >>
To characterize a multidrug-resistant Aeromonas hydrophila isolate (CVM861) that possesses a high-level macrolide inactivation gene cluster (mphA-mrx-mphR), previously only reported in Escherichia coli. PCR fragment length mapping, gene sequencing and Southern blotting were used to map the mphA-mrx-mphR gene cluster and flanking elements in CVM861. Conjugation experiments were done to determine whether the multidrug resistance genetic element was mobile. The mphA-mrx-mphR gene cluster mapped downstream of a class 1 integron and upstream of an aph(3') gene, and was present on a Tn21-like element. The gene order determined by sequencing was intI1-dhfrXII-orfF-aadA2-qacDeltaE-sul1-orf5Delta178-tnpA-mphR-mrx-mphA. Horizontal transmission of high-level macrolide resistance from CVM861 to E. coli 47011 was inconsistent; however, a composite plasmid possessing the mphA gene cluster was transferred at a conjugation frequency of 2.02 x 10(-5) per recipient. An mphA-mrx-mphR gene cluster was present downstream of the In2 integron located on a Tn21-like transposon in an A. hydrophila isolate. Whether this recombination event resulted in the truncation of the orf5 sequence is unknown. The presence of other resistance genes downstream of the mphA-mrx-mphR gene cluster suggests that multiple recombination events have occurred on this genetic element. This is the first known report of the mphA-mrx-mphR gene cluster carried by A. hydrophila and the first known isolation of this cluster in the United States.
KeywordMeSH Terms
Genes, Bacterial
53. Sperisen  P, Schmid  CD, Bucher  P, Zilian  O,     ( 2005 )

Stealth proteins: in silico identification of a novel protein family rendering bacterial pathogens invisible to host immune defense.

PLoS computational biology 1 (1��6��)
PMID : 16299590  :   DOI  :   10.1371/journal.pcbi.0010063     PMC  :   PMC1285062    
Abstract >>
There are a variety of bacterial defense strategies to survive in a hostile environment. Generation of extracellular polysaccharides has proved to be a simple but effective strategy against the host's innate immune system. A comparative genomics approach led us to identify a new protein family termed Stealth, most likely involved in the synthesis of extracellular polysaccharides. This protein family is characterized by a series of domains conserved across phylogeny from bacteria to eukaryotes. In bacteria, Stealth (previously characterized as SacB, XcbA, or WefC) is encoded by subsets of strains mainly colonizing multicellular organisms, with evidence for a protective effect against the host innate immune defense. More specifically, integrating all the available information about Stealth proteins in bacteria, we propose that Stealth is a D-hexose-1-phosphoryl transferase involved in the synthesis of polysaccharides. In the animal kingdom, Stealth is strongly conserved across evolution from social amoebas to simple and complex multicellular organisms, such as Dictyostelium discoideum, hydra, and human. Based on the occurrence of Stealth in most Eukaryotes and a subset of Prokaryotes together with its potential role in extracellular polysaccharide synthesis, we propose that metazoan Stealth functions to regulate the innate immune system. Moreover, there is good reason to speculate that the acquisition and spread of Stealth could be responsible for future epidemic outbreaks of infectious diseases caused by a large variety of eubacterial pathogens. Our in silico identification of a homologous protein in the human host will help to elucidate the causes of Stealth-dependent virulence. At a more basic level, the characterization of the molecular and cellular function of Stealth proteins may shed light on fundamental mechanisms of innate immune defense against microbial invasion.
KeywordMeSH Terms
Computational Biology
54. Sha  J, Pillai  L, Fadl  AA, Galindo  CL, Erova  TE, Chopra  AK,     ( 2005 )

The type III secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila.

Infection and immunity 73 (10)
PMID : 16177316  :   DOI  :   10.1128/IAI.73.10.6446-6457.2005     PMC  :   PMC1230953    
Abstract >>
Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.
KeywordMeSH Terms
55. McGeehan  JE, Streeter  SD, Papapanagiotou  I, Fox  GC, Kneale  GG,     ( 2005 )

High-resolution crystal structure of the restriction-modification controller protein C.AhdI from Aeromonas hydrophila.

Journal of molecular biology 346 (3)
PMID : 15713456  :   DOI  :   10.1016/j.jmb.2004.12.025    
Abstract >>
Restriction-modification (R-M) systems serve to protect the host bacterium from invading bacteriophage. The multi-component system includes a methyltransferase, which recognizes and methylates a specific DNA sequence, and an endonuclease which recognises the same sequence and cleaves within or close to this site. The endonuclease will only cleave DNA that is unmethylated at the specific site, thus host DNA is protected while non-host DNA is cleaved. However, following DNA replication, expression of the endonuclease must be delayed until the host DNA is appropriately methylated. In many R-M systems, this regulation is achieved at the transcriptional level via the controller protein, or C-protein. We have solved the first X-ray structure of an R-M controller protein, C.AhdI, to 1.69 A resolution using selenomethionine MAD. C.AhdI is part of a Type IIH R-M system from the pathogen Aeromonas hydrophila. The structure reveals an all-alpha protein that contains a classical helix-turn-helix (HTH) domain and can be assigned to the Xre family of transcriptional regulators. Unlike its monomeric structural homologues, an extended helix generates an interface that results in dimerisation of the free protein. The dimer is electrostatically polarised and a positively charged surface corresponds to the position of the DNA recognition helices of the HTH domain. Comparison with the structure of the lambda cI ternary complex suggests that C.AhdI activates transcription through direct contact with the sigma70 subunit of RNA polymerase.
KeywordMeSH Terms
56. Yu  HB, Zhang  YL, Lau  YL, Yao  F, Vilches  S, Merino  S, Tomas  JM, Howard  SP, Leung  KY,     ( 2005 )

Identification and characterization of putative virulence genes and gene clusters in Aeromonas hydrophila PPD134/91.

Applied and environmental microbiology 71 (8)
PMID : 16085838  :   DOI  :   10.1128/AEM.71.8.4469-4477.2005     PMC  :   PMC1183340    
Abstract >>
Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Furthermore, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.
KeywordMeSH Terms
Multigene Family
57. Lu  X, Zhang  W, Jian  J, Wu  Q, Chen  GQ,     ( 2005 )

Molecular cloning and functional analysis of two polyhydroxyalkanoate synthases from two strains of Aeromonas hydrophila spp.

FEMS microbiology letters 243 (1)
PMID : 15668013  :   DOI  :   10.1016/j.femsle.2004.11.051    
Abstract >>
Polyhydroxyalkanoate (PHA) synthase genes (phaC) were cloned from two Aeromonas hydrophila strains named WQ and 4AK5, respectively. Both strains are able to produce PHBHHx copolyesters consisting of 3-hydroxybutyrate (3HB) and 3-hydroxyhexanoate (3HHx). Sequence analysis showed that there was only 2 bp difference between these two PHA synthase genes, corresponding to two-amino acid difference at positions of 437 and 458 of the two synthases. PHA productivity and its monomer content produced by A. hydrophila WQ and A. hydrophila 4AK5 were quite different. A. hydrophila WQ accumulated 33% PHBHHx of its cell dry weight (CDW) with 5 mol% 3HHx in the copolyester when cultured in lauric acid for 48 h. Yet A. hydrophila 4AK5 was able to produce 43% PHBHHx of the CDW with 14 mol% 3HHx under the same condition. Hetero-expression of PHA synthase genes of A. hydrophila WQ and A. hydrophila 4AK5, respectively, in Escherichia coli XL1-Blue led to PHBHHx accumulation of 24% and 39% of the CDW and the 3HHx content in PHBHHx were 6 and 15 mol%, respectively. This indicated that the function of these two PHA synthases were different due to these two different residues at positions of 437 and 458. Site specific mutation was carried out to change these two amino acid residues. Results showed that the changes on either of the two amino acids negatively affected the PHA productivity.
KeywordMeSH Terms
Cloning, Molecular
58. Garau  G, Bebrone  C, Anne  C, Galleni  M, Frère  JM, Dideberg  O,     ( 2005 )

A metallo-beta-lactamase enzyme in action: crystal structures of the monozinc carbapenemase CphA and its complex with biapenem.

Journal of molecular biology 345 (4)
PMID : 15588826  :   DOI  :   10.1016/j.jmb.2004.10.070    
Abstract >>
One strategy developed by bacteria to resist the action of beta-lactam antibiotics is the expression of metallo-beta-lactamases. CphA from Aeromonas hydrophila is a member of a clinically important subclass of metallo-beta-lactamases that have only one zinc ion in their active site and for which no structure is available. The crystal structures of wild-type CphA and its N220G mutant show the structural features of the active site of this enzyme, which is modeled specifically for carbapenem hydrolysis. The structure of CphA after reaction with a carbapenem substrate, biapenem, reveals that the enzyme traps a reaction intermediate in the active site. These three X-ray structures have allowed us to propose how the enzyme recognizes carbapenems and suggest a mechanistic pathway for hydrolysis of the beta-lactam. This will be relevant for the design of metallo-beta-lactamase inhibitors as well as of antibiotics that escape their hydrolytic activity.
KeywordMeSH Terms
59. Vilches  S, Urgell  C, Merino  S, Chacón  MR, Soler  L, Castro-Escarpulli  G, Figueras  MJ, Tomás  JM,     ( 2004 )

Complete type III secretion system of a mesophilic Aeromonas hydrophila strain.

Applied and environmental microbiology 70 (11)
PMID : 15528564  :   DOI  :   10.1128/AEM.70.11.6914-6919.2004     PMC  :   PMC525241    
Abstract >>
We have investigated the existence and genetic organization of a functional type III secretion system (TTSS) in a mesophilic Aeromonas strain by initially using the Aeromonas hydrophila strain AH-3. We report for the first time the complete TTSS DNA sequence of an Aeromonas strain that comprises 35 genes organized in a similar disposition as that in Pseudomonas aeruginosa. Using several gene probes, we also determined the presence of a TTSS in clinical or environmental strains of different Aeromonas species: A. hydrophila, A. veronii, and A. caviae. By using one of the TTSS genes (ascV), we were able to obtain a defined insertion mutant in strain AH-3 (AH-3AscV), which showed reduced toxicity and virulence in comparison with the wild-type strain. Complementation of the mutant strain with a plasmid vector carrying ascV was fully able to restore the wild-type toxicity and virulence.
KeywordMeSH Terms
Multigene Family
Sequence Analysis, DNA
60. Soler  L, Yáñez  MA, Chacon  MR, Aguilera-Arreola  MG, Catalán  V, Figueras  MJ, Martínez-Murcia  AJ,     ( 2004 )

Phylogenetic analysis of the genus Aeromonas based on two housekeeping genes.

International journal of systematic and evolutionary microbiology 54 (Pt 5)
PMID : 15388703  :   DOI  :   10.1099/ijs.0.03048-0    
Abstract >>
The phylogenetic relationships of all known species of the genus Aeromonas, and especially Aeromonas bestiarum and Aeromonas salmonicida, were investigated on 70 strains using the rpoD sequence, which encodes the sigma70 factor. This analysis was complemented with the sequence of gyrB, which has already proven useful for determining the phylogenetic relationships in the genus. Nucleotide sequences of rpoD and gyrB showed that both genes had similar substitution rates (< 2 %) and a similar number of variable positions (34 % for rpoD versus 32 % for gyrB). Strain groupings by analysis of rpoD, gyrB and a combination of both genes were consistent with the taxonomic organization of all Aeromonas species described to date. However, the simultaneous analysis of both clocks improved the reliability and the power to differentiate, in particular, closely related taxa. At the inter-species level, gyrB showed a better resolution for differentiating Aeromonas sp. HG11/Aeromonas encheleia and Aeromonas veronii/Aeromonas culicicola/Aeromonas allosaccharophila, while rpoD more clearly differentiated A. salmonicida from A. bestiarum. The analysis of rpoD provided initial evidence for clear phylogenetic divergence between the latter two species.
KeywordMeSH Terms
Phylogeny
61. Lan  X, Ozawa  N, Nishiwaki  N, Kodaira  R, Okazaki  M, Shimosaka  M,     ( 2004 )

Purification, cloning, and sequence analysis of beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9.

Bioscience, biotechnology, and biochemistry 68 (5)
PMID : 15170113  :   DOI  :   10.1271/bbb.68.1082    
Abstract >>
A chitinolytic bacterium was isolated from Lake Suwa and identified as Aeromonas hydrophila strain SUWA-9. The strain grew well on a synthetic medium containing colloidal chitin as sole carbon source. Chitin-degrading activity was induced by colloidal chitin or N-acetylglucosamine (GlcNAc). Most of the activity, however, was not detected in culture fluid but was associated with cells. A beta-N-acetylglucosaminidase was purified after it was solubilized from cells by sonication. The purified enzyme hydrolyzed N-acetylchitooligomers from dimer to pentamer and produced GlcNAc as a final product. The enzyme also hydrolyzed synthetic substrates such as p-nitrophenyl (pNP)-N-acetyl-beta-D-glucosaminide and pNP-N-acetyl-beta-D-galactosaminide. A gene coding for the purified beta-N-acetylglucosaminidase was isolated. The ORF identified is 2661 nucleotides long and encodes a precursor protein of 887 amino acids including a signal peptide of 22 amino acid residues. The amino acid sequence deduced showed a high similarity to those of bacterial beta-N-acetylhexosaminidases classified in family 20 of glycosyl hydrolases.
KeywordMeSH Terms
62. Fang  HM, Ge  R, Sin  YM,     ( 2004 )

Cloning, characterisation and expression of Aeromonas hydrophila major adhesin.

Fish & shellfish immunology 16 (5)
PMID : 15110338  :   DOI  :   10.1016/j.fsi.2003.10.003    
Abstract >>
Aeromonas hydrophila, an important pathogen in fish, is believed to cause diseases by adhesive and enterotoxic mechanisms. The adhesion is a prerequisite for successful invasion. In this study, the gene of a 43 kDa major adhesin (designated as AHA1) was cloned and expressed. Nucleotide sequence analysis of AHA1 revealed an open reading frame encoding a polypeptide of 373 amino acids with a 20-amino-acid putative signal peptide (molecular weight 40,737 Da). The amino acid sequences of Aha1p showed a very high homology with the other two outer membrane proteins of A. hydrophila. Using the T-5 expression system, this major adhesin Aha1p was expressed in Escherichia coli. The purified recombinant adhesin could competitively inhibit A. hydrophila from invading fish epithelial cells in vitro. Western-blot analysis showed that this major adhesin is a very conserved antigen among various strains of Aeromonas. When used to immunise blue gourami, the recombinant adhesin could confer significant protection to fish against experimental A. hydrophila challenge.
KeywordMeSH Terms
Gene Expression
63. Epple  HJ, Mankertz  J, Ignatius  R, Liesenfeld  O, Fromm  M, Zeitz  M, Chakraborty  T, Schulzke  JD,     ( 2004 )

Aeromonas hydrophila beta-hemolysin induces active chloride secretion in colon epithelial cells (HT-29/B6).

Infection and immunity 72 (8)
PMID : 15271947  :   DOI  :   10.1128/IAI.72.8.4848-4858.2004     PMC  :   PMC470692    
Abstract >>
The diarrheal mechanisms in Aeromonas enteritis are not completely understood. In this study we investigated the effect of aeromonads and of their secretory products on ion secretion and barrier function of monolayers of human intestinal cells (HT-29/B6). Ion secretion was determined as a short-circuit current (I(SC)) of HT-29/B6 monolayers mounted in Ussing-type chambers. Transepithelial resistance (R(t)) served as a measure of permeability. A diarrheal strain of Aeromonas hydrophila (strain Sb) added to the mucosal side of HT-29/B6 monolayers induced a significant I(SC) (39 +/- 3 microA/cm(2)) and decreased the R(t) to approximately 10% of the initial value. A qualitatively identical response was obtained with sterile supernatant of strain Sb, and Aeromonas supernatant also induced a significant I(SC) in totally stripped human colon. Tracer flux and ion replacement studies revealed the I(SC) to be mainly accounted for by electrogenic Cl(-) secretion. Supernatant applied serosally completely abolished basal I(SC). The supernatant-induced I(SC) was inhibited by the protein kinase C inhibitor chelerythrine, whereas a protein kinase A inhibitor (H8) and a Ca(2+) chelator (BAPTA-AM) had no effect. Physicochemical properties indicated that the supernatant's active compound was an aerolysin-related Aeromonas beta-hemolysin. Accordingly, identical I(SC) and R(t) responses were obtained with Escherichia coli lysates harboring the cloned beta-hemolysin gene from strain SB or the aerA gene encoding for aerolysin. Sequence comparison revealed a 64% homology between aerolysin and the beta-hemolysin cloned from Aeromonas sp. strain Sb. In conclusion, beta-hemolysin secreted by pathogenic aeromonads induces active Cl(-) secretion in the intestinal epithelium, possibly by channel insertion into the apical membrane and by activation of protein kinase C.
KeywordMeSH Terms
64. Yu  HB, Rao  PS, Lee  HC, Vilches  S, Merino  S, Tomas  JM, Leung  KY,     ( 2004 )

A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis.

Infection and immunity 72 (3)
PMID : 14977925  :   DOI  :   10.1128/iai.72.3.1248-1256.2004     PMC  :   PMC356039    
Abstract >>
Aeromonas hydrophila is a gram-negative opportunistic pathogen in fish and humans. Many bacterial pathogens of animals and plants have been shown to inject anti-host virulence determinants into the hosts via a type III secretion system (TTSS). Degenerate primers based on lcrD family genes that are present in every known TTSS allowed us to locate the TTSS gene cluster in A. hydrophila AH-1. A series of genome walking steps helped in the identification of 25 open reading frames that encode proteins homologous to those in TTSSs in other bacteria. PCR-based analysis showed the presence of lcrD homologs (ascV) in all of the 33 strains of A. hydrophila isolated from various sources. Insertional inactivation of two of the TTSS genes (aopB and aopD) led to decreased cytotoxicity in carp epithelial cells, increased phagocytosis, and reduced virulence in blue gourami. These results show that a TTSS is required for A. hydrophila pathogenesis. This is the first report of sequencing and characterization of TTSS gene clusters from A. hydrophila. The TTSS identified here may help in developing suitable vaccines as well as in further understanding of the pathogenesis of A. hydrophila.
KeywordMeSH Terms
65. Sha  J, Kozlova  EV, Fadl  AA, Olano  JP, Houston  CW, Peterson  JW, Chopra  AK,     ( 2004 )

Molecular characterization of a glucose-inhibited division gene, gidA, that regulates cytotoxic enterotoxin of Aeromonas hydrophila.

Infection and immunity 72 (2)
PMID : 14742556  :   DOI  :   10.1128/iai.72.2.1084-1095.2004     PMC  :   PMC321642    
Abstract >>
By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
66. Gueneau de Novoa  P, Williams  KP,     ( 2004 )

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

Nucleic acids research 32 (Database issue)
PMID : 14681369  :   DOI  :   10.1093/nar/gkh102     PMC  :   PMC308836    
Abstract >>
tmRNA combines tRNA- and mRNA-like properties and ameliorates problems arising from stalled ribosomes. Research on the mechanism, structure and biology of tmRNA is served by the tmRNA website (http://www.indiana.edu/~ tmrna), a collection of sequences, alignments, secondary structures and other information. Because many of these sequences are not in GenBank, a BLAST server has been added; another new feature is an abbreviated alignment for the tRNA-like domain only. Many tmRNA sequences from plastids have been added, five found in public sequence data and another 10 generated by direct sequencing; detection in early-branching members of the green plastid lineage brings coverage to all three primary plastid lineages. The new sequences include the shortest known tmRNA sequence. While bacterial tmRNAs usually have a lone pseudoknot upstream of the mRNA segment and a string of three or four pseudoknots downstream, plastid tmRNAs collectively show loss of pseudoknots at both postions. The pseudoknot-string region is also too short to contain the usual pseudoknot number in another new entry, the tmRNA sequence from a bacterial endosymbiont of insect cells, Tremblaya princeps. Pseudoknots may optimize tmRNA function in free-living bacteria, yet become dispensible when the endosymbiotic lifestyle relaxes selective pressure for fast growth.
KeywordMeSH Terms
Databases, Nucleic Acid
Evolution, Molecular
Internet
67. Chatterjee  C, Kumar  S, Chakraborty  S, Tan  YW, Leung  KY, Sivaraman  J, Mok  YK,     ( 2011 )

Crystal structure of the heteromolecular chaperone, AscE-AscG, from the type III secretion system in Aeromonas hydrophila.

PloS one 6 (4)
PMID : 21559439  :   DOI  :   10.1371/journal.pone.0019208     PMC  :   PMC3084799    
Abstract >>
The putative needle complex subunit AscF forms a ternary complex with the chaperones AscE and AscG in the type III secretion system of Aeromonas hydrophila so as to avoid premature assembly. Previously, we demonstrated that the C-terminal region of AscG (residues 62-116) in the hetero-molecular chaperone, AscE-AscG, is disordered and susceptible to limited protease digestion. Here, we report the crystal structure of the ordered AscG(1-61) region in complex with AscE at 2.4 ? resolution. Helices �\2 and �\3 of AscE in the AscE-AscG(1-61) complex assumes a helix-turn-helix conformation in an anti-parallel fashion similar to that in apo AscE. However, in the presence of AscG, an additional N-terminal helix �\1 in AscE (residues 4-12) is observed. PscG or YscG in the crystal structures of PscE-PscF-PscG or YscE-YscF-YscG, respectively, assumes a typical tetratricopeptide repeat (TPR) fold with three TPR repeats and one C-terminal capping helix. By comparison, AscG in AscE-AscG(1-61) comprises three anti-parallel helices that resembles the N-terminal TPR repeats in the corresponding region of PscG or YscG in PscE-PscF-PscG or YscE-YscF-YscG. Thermal denaturation of AscE-AscG and AscE-AscG(1-61) complexes demonstrates that the C-terminal disordered region does not contribute to the thermal stability of the overall complex. The N-terminal region of the AscG in the AscE-AscG complex is ordered and assumes a structure similar to those in the corresponding regions of PscE-PscG-PscF or YscE-YscF-YscG complexes. While the C-terminal region of AscG in the AscE-AscG complex is disordered and will assume its structure only in the presence of the substrate AscF. We hypothesize that AscE act as a chaperone of the chaperone to keep AscG in a stable but partially disordered state for interaction with AscF.
KeywordMeSH Terms
68. Iacovache  I, Degiacomi  MT, Pernot  L, Ho  S, Schiltz  M, Dal Peraro  M, van der Goot  FG,     ( 2011 )

Dual chaperone role of the C-terminal propeptide in folding and oligomerization of the pore-forming toxin aerolysin.

PLoS pathogens 7 (7)
PMID : 21779171  :   DOI  :   10.1371/journal.ppat.1002135     PMC  :   PMC3136475    
Abstract >>
Throughout evolution, one of the most ancient forms of aggression between cells or organisms has been the production of proteins or peptides affecting the permeability of the target cell membrane. This class of virulence factors includes the largest family of bacterial toxins, the pore-forming toxins (PFTs). PFTs are bistable structures that can exist in a soluble and a transmembrane state. It is unclear what drives biosynthetic folding towards the soluble state, a requirement that is essential to protect the PFT-producing cell. Here we have investigated the folding of aerolysin, produced by the human pathogen Aeromonas hydrophila, and more specifically the role of the C-terminal propeptide (CTP). By combining the predictive power of computational techniques with experimental validation using both structural and functional approaches, we show that the CTP prevents aggregation during biosynthetic folding. We identified specific residues that mediate binding of the CTP to the toxin. We show that the CTP is crucial for the control of the aerolysin activity, since it protects individual subunits from aggregation within the bacterium and later controls assembly of the quaternary pore-forming complex at the surface of the target host cell. The CTP is the first example of a C-terminal chain-linked chaperone with dual function.
KeywordMeSH Terms
Protein Folding
69. Fontes  MC, Saavedra  MJ, Martins  C, Martínez-Murcia  AJ,     ( 2011 )

Phylogenetic identification of Aeromonas from pigs slaughtered for consumption in slaughterhouses at the North of Portugal.

International journal of food microbiology 146 (2)
PMID : 21402427  :   DOI  :   10.1016/j.ijfoodmicro.2011.02.010    
Abstract >>
In the present study, 710 isolates of Aeromonas spp. have been collected from pig carcasses, diaphragm muscle, faeces, dehairing equipment and water in slaughterhouses at the North of Portugal. The isolates were obtained from a total of 154 samples. All presumptive Aeromonas isolates were subjected to ERIC-PCR analysis and those which presented a different pattern were taken and the species classified by gyrB gene sequencing. We have found the species A. hydrophila, A. salmonicida, A. bestiarum, A. caviae, A. media, A. veronii, A. allosaccharophila, A. simiae and A. aquariorum. To our knowledge, this extent of Aeromonas species diversity has not been previously described from meat or from the slaughter environment, perhaps due to the unreliability of available identification methods. A noticeable level of isolate redundancy (strains with identical gyrB sequence) from different samples collected in different dates was also obtained, indicating that only a few predominant strains of these species persist at the slaughter system. It is also important to emphasise the presence of Aeromonas species previously associated with illness in man.
KeywordMeSH Terms
70. Martinez-Murcia  AJ, Monera  A, Saavedra  MJ, Oncina  R, Lopez-Alvarez  M, Lara  E, Figueras  MJ,     ( 2011 )

Multilocus phylogenetic analysis of the genus Aeromonas.

Systematic and applied microbiology 34 (3)
PMID : 21353754  :   DOI  :   10.1016/j.syapm.2010.11.014    
Abstract >>
A broad multilocus phylogenetic analysis (MLPA) of the representative diversity of a genus offers the opportunity to incorporate concatenated inter-species phylogenies into bacterial systematics. Recent analyses based on single housekeeping genes have provided coherent phylogenies of Aeromonas. However, to date, a multi-gene phylogenetic analysis has never been tackled. In the present study, the intra- and inter-species phylogenetic relationships of 115 strains representing all Aeromonas species described to date were investigated by MLPA. The study included the independent analysis of seven single gene fragments (gyrB, rpoD, recA, dnaJ, gyrA, dnaX, and atpD), and the tree resulting from the concatenated 4705 bp sequence. The phylogenies obtained were consistent with each other, and clustering agreed with the Aeromonas taxonomy recognized to date. The highest clustering robustness was found for the concatenated tree (i.e. all Aeromonas species split into 100% bootstrap clusters). Both possible chronometric distortions and poor resolution encountered when using single-gene analysis were buffered in the concatenated MLPA tree. However, reliable phylogenetic species delineation required an MLPA including several "bona fide" strains representing all described species.
KeywordMeSH Terms
71. Molero  R, Wilhelms  M, Infanzón  B, Tomás  JM, Merino  S,     ( 2011 )

Aeromonas hydrophila motY is essential for polar flagellum function, and requires coordinate expression of motX and Pom proteins.

Microbiology (Reading, England) 157 (Pt 10)
PMID : 21737499  :   DOI  :   10.1099/mic.0.049544-0    
Abstract >>
By the analysis of the Aeromonas hydrophila ATCC7966(T) genome we identified A. hydrophila AH-3 MotY. A. hydrophila MotY, like MotX, is essential for the polar flagellum function energized by an electrochemical potential of Na(+) as coupling ion, but is not involved in lateral flagella function energized by the proton motive force. Thus, the A. hydrophila polar flagellum stator is a complex integrated by two essential proteins, MotX and MotY, which interact with one of two redundant pairs of proteins, PomAB and PomA(2)B(2). In an A. hydrophila motX mutant, polar flagellum motility is restored by motX complementation, but the ability of the A. hydrophila motY mutant to swim is not restored by introduction of the wild-type motY alone. However, its polar flagellum motility is restored when motX and -Y are expressed together from the same plasmid promoter. Finally, even though both the redundant A. hydrophila polar flagellum stators, PomAB and PomA(2)B(2), are energized by the Na(+) ion, they cannot be exchanged. Furthermore, Vibrio parahaemolyticus PomAB and Pseudomonas aeruginosa MotAB or MotCD are unable to restore swimming motility in A. hydrophila polar flagellum stator mutants.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
72. Regué  M, Tomás  JM, Merino  S, Jimenez  N, Molero  R, Bouamama  L,     ( 2011 )

A UDP-HexNAc:polyprenol-P GalNAc-1-P transferase (WecP) representing a new subgroup of the enzyme family.

Journal of bacteriology 193 (8)
PMID : 21335454  :   DOI  :   10.1128/JB.01441-10     PMC  :   PMC3133024    
Abstract >>
The Aeromonas hydrophila AH-3 WecP represents a new class of UDP-HexNAc:polyprenol-P HexNAc-1-P transferases. These enzymes use a membrane-associated polyprenol phosphate acceptor (undecaprenyl phosphate [Und-P]) and a cytoplasmic UDP-d-N-acetylhexosamine sugar nucleotide as the donor substrate. Until now, all the WecA enzymes tested were able to transfer UDP-GlcNAc to the Und-P. In this study, we present in vitro and in vivo proofs that A. hydrophila AH-3 WecP transfers GalNAc to Und-P and is unable to transfer GlcNAc to the same enzyme substrate. The molecular topology of WecP is more similar to that of WbaP (UDP-Gal polyprenol-P transferase) than to that of WecA (UDP-GlcNAc polyprenol-P transferase). WecP is the first UDP-HexNAc:polyprenol-P GalNAc-1-P transferase described.
KeywordMeSH Terms
73. Guan  R, Xiong  J, Huang  W, Guo  S,     ( 2011 )

Enhancement of protective immunity in European eel (Anguilla anguilla) against Aeromonas hydrophila and Aeromonas sobria by a recombinant Aeromonas outer membrane protein.

Acta biochimica et biophysica Sinica 43 (1)
PMID : 21148192  :   DOI  :   10.1093/abbs/gmq115    
Abstract >>
To develop a vaccine, which can simultaneously prevent the diseases caused by various pathogenic bacteria in fish, we try to find a conserved outer membrane protein (OMP) antigen from different bacterial pathogens. In this study, an OMP fragment of 747 bp (named as Omp-G), which was highly conserved in seven Aeromonas OMP sequences from the NCBI database, was amplified by PCR from one Aeromonas sobria strain (B10) and two Aeromonas hydrophila strains (B27 and B33) with the designed specific primers. The sequence was cloned into pGEX-2T (6 �� His-tag) vector, expressed in Escherichia coli system, and then the recombinant protein (named as rOmp-G) was purified with nickel chelating affinity chromatography. The purified rOmp-G showed a good immunogenicity in rabbits and well-conserved characteristics in these three pathogens by enzyme-linked immunosorbed assay. Furthermore, the rOmp-G also showed good immunogenicity in eels (Anguilla anguilla) for eliciting significantly increased specific antibodies (P < 0.01), and providing higher protection efficiencies (P < 0.05) after the pathogens challenge. The values of the relative percent survival in eels were 70% and 50% for two A. hydrophila strain challenge, and 75% for A. sobria strain challenge. This is the first report of a potential vaccination in eels that simultaneously provide protectiveness against different Aeromonas pathogens with a conserved partial OMP.
KeywordMeSH Terms
74. Lassaux  P, Hamel  M, Gulea  M, Delbrück  H, Mercuri  PS, Horsfall  L, Dehareng  D, Kupper  M, Frère  JM, Hoffmann  K, Galleni  M, Bebrone  C,     ( 2010 )

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

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

Aeromonas hydrophila PepO outer membrane endopeptidase activates human big endothelin-3 in vitro and induces skin ulcer in goldfish (Carassius auratus).

Veterinary microbiology 145 (1��2��)
PMID : 20456877  :   DOI  :   10.1016/j.vetmic.2010.03.009    
Abstract >>
In Aeromonas hydrophila, the gram-negative bacterial fish pathogen, PepO constitutes the thermoregulated outer membrane M13 family zinc endopeptidase, which is expressed maximally at 16 degrees C and is down-regulated above 30 degrees C. Cultivation of A. hydrophila at 16 degrees C enabled it to activate big endothelin (ET), the vasoconstrictor and ulcerogenic peptide naturally secreted from human vascular endothelial cell (HUVEC) culture. Furthermore, A. hydrophila PepO in vitro shows strong enzymatic preference for human big ET-3 rather than big ET-1 and big ET-2. At water temperature of 16+/-1 degrees C, intramuscular infection of goldfish, Carassius auratus, with wild-type A. hydrophila led to development of a pathognomonic big ulcer at the injection site while the PepO deficient mutant strain lost both its big ET endopeptidase activity in vitro as well as its ulcerogenic property in vivo. This is the first report of expression, subcellular localization and functional analysis of PepO metalloendopeptidase in A. hydrophila.
KeywordMeSH Terms
76. Carvalho-Castro  GA, Lopes  CO, Leal  CA, Cardoso  PG, Leite  RC, Figueiredo  HC,     ( 2010 )

Detection of type III secretion system genes in Aeromonas hydrophila and their relationship with virulence in Nile tilapia.

Veterinary microbiology 144 (3��4��)
PMID : 20185253  :   DOI  :   10.1016/j.vetmic.2010.01.021    
Abstract >>
The goals of this study were to develop a PCR technique to detect ascV and aopB genes from the type III secretion system (T3SS), to evaluate the frequency of these genes in Aeromonas hydrophila strains isolated from diseased fish and from aquaculture environments, and to determine the relationship between the presence of these genes and virulence of A. hydrophila in Nile tilapia. The PCR assay developed here successfully detected the target genes, showing three different profiles for the strains ascV+/aopB+, ascV+/aopB-, and ascV-/aopB-. A higher frequency of ascV+/aopB+ was verified in isolates from diseased fish compared to those from aquaculture environments (P<0.05). Among 64 isolates from diseased fish, ascV+/aopB+ (62.5%) was the most frequent profile (P<0.05) and caused more intensive mortality rates. Environmental strains containing the ascV+/aopB+ profile were less virulent than isolates from clinical cases. These results suggest that the presence of a functional T3SS probably increases the virulence of A. hydrophila. The PCR technique was shown to be a specific and efficient tool for detection of T3SS, and this technique can be used for virulence typing of A. hydrophila isolates.
KeywordMeSH Terms
Cichlids
77. Hilton  S, Buckley  JT,     ( 1991 )

Studies on the reaction mechanism of a microbial lipase/acyltransferase using chemical modification and site-directed mutagenesis.

The Journal of biological chemistry 266 (2)
PMID : 1985976  :  
Abstract >>
Aeromonas hydrophila releases a protein which is a member of the lipase superfamily, similar in reaction mechanism to the important mammalian plasma enzyme lecithin-cholesterol acyltransferase. We have used chemical modification and site-directed mutagenesis of the protein to identify amino acids which may be involved in catalysis. The enzyme was unaffected by phenylmethylsulfonyl fluoride, but it was almost completely inhibited by another serine-reactive compound, diethyl p-nitrophenyl phosphate. A serine selectively modified by this reagent was identified by sequencing the amino-terminal region of the protein. It was located at position 16, in the short consensus sequence shared by the enzyme with other lipases. When this serine was changed to asparagine the product was an enzymatically inert protein which nevertheless retained the surface activity of the wild-type enzyme, suggesting its ability to bind to substrate was not changed. Diethylpyrocarbonate treatment drastically reduced the rate of acyl transfer by the native enzyme, but this did not appear to be due to modification of an essential histidine, since inhibition was not reversed by addition of hydroxylamine. We have shown that only two of the histidines in the enzyme can be involved in catalysis (Hilton, S., McCubbin, W. D., Kay, C.M., and Buckley, J. T. (1990) Biochemistry, 29, 9072-9078). Replacing both of these with asparagines had little or no effect on enzyme activity. These results indicate that, in apparent contrast to other lipases, histidine does not participate in the reaction catalyzed by the microbial enzyme. Since catalysis was not inhibited by sulfhydryl reagents, we conclude that a free cysteine is also not required for activity. This may distinguish the microbial enzyme from the mammalian acyltransferase.
KeywordMeSH Terms
Mutagenesis, Site-Directed
78. Miñana-Galbis  D, Urbizu-Serrano  A, Farfán  M, Fusté  MC, Lorén  JG,     ( 2009 )

Phylogenetic analysis and identification of Aeromonas species based on sequencing of the cpn60 universal target.

International journal of systematic and evolutionary microbiology 59 (Pt 8)
PMID : 19567585  :   DOI  :   10.1099/ijs.0.005413-0    
Abstract >>
An analysis of the universal target (UT) sequence from the cpn60 gene was performed in order to evaluate its usefulness in phylogenetic and taxonomic studies and as an identification marker for the genus Aeromonas. Sequences of 555 bp, corresponding to the UT region, were obtained from a collection of 35 strains representing all of the species and subspecies of Aeromonas. From the analysis of these sequences, a range of divergence of 0-23.3% was obtained, with a mean of 11.2+/-0.9%. Comparative analyses between cpn60 and gyrB, rpoD and 16S rRNA gene sequences were carried out from the same Aeromonas strain collection. Sequences of the cpn60 UT region showed similar discriminatory power to gyrB and rpoD sequences. The phylogenetic relationships inferred from cpn60 sequence distances indicated an excellent correlation with the present affiliation of Aeromonas species with the exception of Aeromonas hydrophila subsp. dhakensis, which appeared in a separate phylogenetic line, and Aeromonas sharmana, which exhibited a very loose phylogenetic relationship to the genus Aeromonas. Sequencing of cpn60 from 33 additional Aeromonas strains also allowed us to establish intra- and interspecific threshold values. Intraspecific divergence rates were
KeywordMeSH Terms
79. Balsalobre  LC, Dropa  M, Matté  GR, Matté  MH,     ( 2009 )

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei.

Journal of water and health 7 (4)
PMID : 19590136  :   DOI  :   10.2166/wh.2009.082    
Abstract >>
Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hlyA/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hlyA/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.
KeywordMeSH Terms
Water Microbiology
80. Wang  X, Wang  Q, Xiao  J, Liu  Q, Wu  H, Xu  L, Zhang  Y,     ( 2009 )

Edwardsiella tarda T6SS component evpP is regulated by esrB and iron, and plays essential roles in the invasion of fish.

Fish & shellfish immunology 27 (3)
PMID : 19563898  :   DOI  :   10.1016/j.fsi.2009.06.013    
Abstract >>
Edwardsiella tarda is a gram-negative pathogen for hemorrhagic septicemia in a broad range of hosts. The type VI secretion system (T6SS) has recently been dissected in E. tarda to secrete EvpC, EvpI and a novel effector protein EvpP. In this study, sequencing and genetic alignments showed that evpP genes from different E. tarda isolates were highly similar and an evpP homolog was also found in Aeromonas hydrophila 0865 isolated from a diseased eel, suggesting the possible lateral gene transfer of evpP or the whole T6SS gene island. With reporter strains carrying gfp gene fused to the evpP promoter region, flow cytometric analysis revealed that transcription of evpP was positively regulated by either the two-component system EsrA-EsrB in E. tarda or the iron concentration in media. Compared with the parental strain, in-frame deletion of evpP in E. tarda EIB202 led to the significantly increased 50% lethal doses in zebrafish (Danio rerio) and Japanese flounder (Paralichthys olivaceus), decreased hemolytic activities, failure to adhere to mucus and reduced serum resistance, and complementation of an intact evpP gene restored these phenotypes in the evpP mutant. Investigation of infection kinetics indicated that the evpP deletion mutant was unable to proliferate in vivo, particularly in immune organs of fish. Moreover, the evpP deletion mutant exhibited incapacity to internalize in EPC cell model in vitro, demonstrating that EvpP in T6SS plays critical roles for invasion mechanism of E. tarda and merits as potential target for attenuated live vaccine construction.
KeywordMeSH Terms
81. Maiti  B, Raghunath  P, Karunasagar  I, Karunasagar  I,     ( 2009 )

Cloning and expression of an outer membrane protein OmpW of Aeromonas hydrophila and study of its distribution in Aeromonas spp.

Journal of applied microbiology 107 (4)
PMID : 19426281  :   DOI  :   10.1111/j.1365-2672.2009.04296.x    
Abstract >>
The main aims of this study were to clone and express an outer membrane protein (OMP), OmpW, of Aeromonas hydrophila and to study its distribution in Aeromonas spp. The gene encoding OmpW in A. hydrophila has been cloned and expressed in Escherichia coli. Primers were designed for amplification of full-length ompW gene and used for identification of this gene in different Aeromonas spp. Of the 42 Aeromonas strains tested, all the isolates were positive by polymerase chain reaction (PCR) except one strain of Aeromonas veronii biovar veronii (VTE338). None of the other gram-negative bacteria were positive by PCR with primers specific to ompW gene of A. hydrophila. Polyclonal antibodies were raised in rabbit against the purified recombinant protein and the reaction of these antibodies was confirmed by western blotting using the purified recombinant protein and 42 Aeromonas cultures grown at various salt concentrations. The ompW-based PCR method developed in this study was found to be 100% specific and 97% sensitive. Expression of OmpW protein of Aeromonas was found to be salt-dependant. Recombinant OmpW protein was found to be highly immunogenic in fish. To our knowledge, this is the first report on cloning and expression of OmpW protein of A. hydrophila. Full-length ompW gene amplification by PCR can be used for the detection of Aeromonas. Recombinant OmpW protein can be useful for vaccination of fish against Aeromonas spp.
KeywordMeSH Terms
82. Fricke  WF, Welch  TJ, McDermott  PF, Mammel  MK, LeClerc  JE, White  DG, Cebula  TA, Ravel  J,     ( 2009 )

Comparative genomics of the IncA/C multidrug resistance plasmid family.

Journal of bacteriology 191 (15)
PMID : 19482926  :   DOI  :   10.1128/JB.00189-09     PMC  :   PMC2715731    
Abstract >>
Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.
KeywordMeSH Terms
83. Ho  AS, Mietzner  TA, Smith  AJ, Schoolnik  GK,     ( 1990 )

The pili of Aeromonas hydrophila: identification of an environmentally regulated "mini pilin".

The Journal of experimental medicine 172 (3)
PMID : 1974915  :   DOI  :   10.1084/jem.172.3.795     PMC  :   PMC2188559    
Abstract >>
Ultrastructural studies of Aeromonas hydrophila strain AH26 revealed two distinctive pilus types: "straight" pili appear as brittle, rod-like filaments, whereas "flexible" pili are supple and curvilinear. Straight pili are produced constitutively under all tested conditions of growth. In contrast, the expression of flexible pili is regulated by physical and chemical variables, being produced at 22 vs. 37 degrees C, in a liquid vs. a solid medium, and when the availability of free-iron is reduced by the presence of deferoxamine mesylate. Both pilus proteins were purified and biochemically and functionally characterized. The major repeating subunit of the straight pilus is a 17,000-mol wt polypeptide with amino acid sequence homology with Escherichia coli type 1 and Pap pili. The flexible pilus filament is a homopolymer composed of a novel 46 amino acid polypeptide. Resistance of the flexible pilus filament to disaggregation using various chemical treatments was demonstrated; its stability as a polymer and its apparent mechanical strength seem to be conferred by a 20 amino acid hydrophobic, COOH-terminal domain. Purified straight pili lack hemagglutinating function. In contrast, purified flexible pili cause the agglutinin of human, guinea pig, ovine, bovine, and avian erythrocytes, although this property could only be demonstrated in the presence of divalent cations and was most evident at 4 vs. 22 degrees C. Taken together, these results suggest that the pathogenic and ecological roles of the flexible pilus are related to this species' existence as a free-living organism in aquatic environments and its ability to cause infections, both in cold-blooded vertebrates and the human intestine.
KeywordMeSH Terms
84. Khajanchi  BK, Sha  J, Kozlova  EV, Erova  TE, Suarez  G, Sierra  JC, Popov  VL, Horneman  AJ, Chopra  AK,     ( 2009 )

N-acylhomoserine lactones involved in quorum sensing control the type VI secretion system, biofilm formation, protease production, and in vivo virulence in a clinical isolate of Aeromonas hydrophila.

Microbiology (Reading, England) 155 (Pt 11)
PMID : 19729404  :   DOI  :   10.1099/mic.0.031575-0     PMC  :   PMC2888131    
Abstract >>
In this study, we delineated the role of N-acylhomoserine lactone(s) (AHLs)-mediated quorum sensing (QS) in the virulence of diarrhoeal isolate SSU of Aeromonas hydrophila by generating a double knockout Delta ahyRI mutant. Protease production was substantially reduced in the Delta ahyRI mutant when compared with that in the wild-type (WT) strain. Importantly, based on Western blot analysis, the Delta ahyRI mutant was unable to secrete type VI secretion system (T6SS)-associated effectors, namely haemolysin coregulated protein and the valine-glycine repeat family of proteins, while significant levels of these effectors were detected in the culture supernatant of the WT A. hydrophila. In contrast, the production and translocation of the type III secretion system (T3SS) effector AexU in human colonic epithelial cells were not affected when the ahyRI genes were deleted. Solid surface-associated biofilm formation was significantly reduced in the Delta ahyRI mutant when compared with that in the WT strain, as determined by a crystal violet staining assay. Scanning electron microscopic observations revealed that the Delta ahyRI mutant was also defective in the formation of structured biofilm, as it was less filamentous and produced a distinct exopolysaccharide on its surface when compared with the structured biofilm produced by the WT strain. These effects of AhyRI could be complemented either by expressing the ahyRI genes in trans or by the exogeneous addition of AHLs to the Delta ahyRI/ahyR(+) complemented strain. In a mouse lethality experiment, 50 % attenuation was observed when we deleted the ahyRI genes from the parental strain of A. hydrophila. Together, our data suggest that AHL-mediated QS modulates the virulence of A. hydrophila SSU by regulating the T6SS, metalloprotease production and biofilm formation.
KeywordMeSH Terms
Biofilms
Quorum Sensing
85. Bebrone  C, Delbrück  H, Kupper  MB, Schlömer  P, Willmann  C, Frère  JM, Fischer  R, Galleni  M, Hoffmann  KM,     ( 2009 )

The structure of the dizinc subclass B2 metallo-beta-lactamase CphA reveals that the second inhibitory zinc ion binds in the histidine site.

Antimicrobial agents and chemotherapy 53 (10)
PMID : 19651913  :   DOI  :   10.1128/AAC.00288-09     PMC  :   PMC2764157    
Abstract >>
Bacteria can defend themselves against beta-lactam antibiotics through the expression of class B beta-lactamases, which cleave the beta-lactam amide bond and render the molecule harmless. There are three subclasses of class B beta-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-beta-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.
KeywordMeSH Terms
86. Singh  V, Somvanshi  P, Rathore  G, Kapoor  D, Mishra  BN,     ( 2009 )

Gene cloning, expression and homology modeling of hemolysin gene from Aeromonas hydrophila.

Protein expression and purification 65 (1)
PMID : 19136063  :   DOI  :   10.1016/j.pep.2008.11.015    
Abstract >>
Hemolysin is a significant toxin secreted by Aeromonas hydrophila, which contributes pathogenicity of fish to humans. The complete ORF of hemolysin gene (1886 bp) was amplified using PCR. It was cloned in TA and sub-cloned in pET28a vector then transformed into Escherichia coli BL21(DE3) codon plus RP cells expressed by the induction with 1.0 mM of IPTG. The expected size of expressed protein was 68.0 kDa estimated by migration in 12% SDS-PAGE. Anti-His monoclonal antibodies were used to substantiate the recombinant protein by Western blotting. The percent similarity between hemolysin of A. hydrophila with other hemolytic toxins revealed that the hemolysin/aerolysin/cytotoxin sequence varied from 99.35 to 50.40%. Homology modeling was used to construct 3-D structure of hemolysin of A. hydrophila with the known crystal 3-D structure (PDB: 1XEZ). This protein can be used for immunoassays and it is suitable for vaccine candidate against A. hydrophila infection.
KeywordMeSH Terms
Cloning, Molecular
Gene Expression
Models, Molecular
87. Wilhelms  M, Vilches  S, Molero  R, Shaw  JG, Tomás  JM, Merino  S,     ( 2009 )

Two redundant sodium-driven stator motor proteins are involved in Aeromonas hydrophila polar flagellum rotation.

Journal of bacteriology 191 (7)
PMID : 19181813  :   DOI  :   10.1128/JB.01526-08     PMC  :   PMC2655530    
Abstract >>
Motility is an essential characteristic for mesophilic Aeromonas strains. We identified a new polar flagellum region (region 6) in the A. hydrophila AH-3 (serotype O34) chromosome that contained two additional polar stator genes, named pomA2 and pomB2. A. hydrophila PomA2 and PomB2 are highly homologous to other sodium-conducting polar flagellum stator motors as well as to the previously described A. hydrophila AH-3 PomA and PomB. pomAB and pomA2B2 were present in all the mesophilic Aeromonas strains tested and were independent of the strains' ability to produce lateral flagella. Unlike MotX, which is a stator protein that is essential for polar flagellum rotation, here we demonstrate that PomAB and PomA2B2 are redundant sets of proteins, as neither set on its own is essential for polar flagellum motility in either aqueous or high-viscosity environments. Both PomAB and PomA2B2 are sodium-coupled stator complexes, although PomA2B2 is more sensitive to low concentrations of sodium than PomAB. Furthermore, the level of transcription in aqueous and high-viscosity environments of pomA2B2 is reduced compared to that of pomAB. The A. hydrophila AH-3 polar flagellum is the first case described in which two redundant sodium-driven stator motor proteins (PomAB and PomA2B2) are found.
KeywordMeSH Terms
88. Tan  YW, Yu  HB, Leung  KY, Sivaraman  J, Mok  YK,     ( 2008 )

Structure of AscE and induced burial regions in AscE and AscG upon formation of the chaperone needle-subunit complex of type III secretion system in Aeromonas hydrophila.

Protein science : a publication of the Protein Society 17 (10)
PMID : 18662905  :   DOI  :   10.1110/ps.036798.108     PMC  :   PMC2548367    
Abstract >>
In the type III secretion system (T3SS) of Aeromonas hydrophila, the putative needle complex subunit AscF requires both putative chaperones AscE and AscG for formation of a ternary complex to avoid premature assembly. Here we report the crystal structure of AscE at 2.7 A resolution and the mapping of buried regions of AscE, AscG, and AscF in the AscEG and AscEFG complexes using limited protease digestion. The dimeric AscE is comprised of two helix-turn-helix monomers packed in an antiparallel fashion. The N-terminal 13 residues of AscE are buried only upon binding with AscG, but this region is found to be nonessential for the interaction. AscE functions as a monomer and can be coexpressed with AscG or with both AscG and AscF to form soluble complexes. The AscE binding region of AscG in the AscEG complex is identified to be within the N-terminal 61 residues of AscG. The exposed C-terminal substrate-binding region of AscG in the AscEG complex is induced to be buried only upon binding to AscF. However, the N-terminal 52 residues of AscF remain exposed even in the ternary AscEFG complex. On the other hand, the 35-residue C-terminal region of AscF in the complex is resistant to protease digestion in the AscEFG complex. Site-directed mutagenesis showed that two C-terminal hydrophobic residues, Ile83 and Leu84, of AscF are essential for chaperone binding.
KeywordMeSH Terms
89. Liénard  BM, Garau  G, Horsfall  L, Karsisiotis  AI, Damblon  C, Lassaux  P, Papamicael  C, Roberts  GC, Galleni  M, Dideberg  O, Frère  JM, Schofield  CJ,     ( 2008 )

Structural basis for the broad-spectrum inhibition of metallo-beta-lactamases by thiols.

Organic & biomolecular chemistry 6 (13)
PMID : 18563261  :   DOI  :   10.1039/b802311e    
Abstract >>
The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible.
KeywordMeSH Terms
beta-Lactamase Inhibitors
90. Kozlova  EV, Popov  VL, Sha  J, Foltz  SM, Erova  TE, Agar  SL, Horneman  AJ, Chopra  AK,     ( N/A )

Mutation in the S-ribosylhomocysteinase (luxS) gene involved in quorum sensing affects biofilm formation and virulence in a clinical isolate of Aeromonas hydrophila.

Microbial pathogenesis 45 (5��6��)
PMID : 18930130  :   DOI  :   10.1016/j.micpath.2008.08.007    
Abstract >>
A diarrheal isolate SSU of Aeromonas hydrophila produces a cytotoxic enterotoxin (Act) with cytotoxic, enterotoxic, and hemolytic activities. Our laboratory has characterized from the above Aeromonas strain, in addition to Act, the type 3- and T6-secretion systems and their effectors, as well as the genes shown to modulate the production of AI-1-like autoinducers, N-acylhomoserine lactones (AHLs) involved in quorum sensing (QS). In this study, we demonstrated the presence of an S-ribosylhomocysteinase (LuxS)-based autoinducer (AI)-2 QS system in A. hydrophila SSU and its contribution to bacterial virulence. The luxS isogenic mutant of A. hydrophila, which we prepared by marker exchange mutagenesis, showed an alteration in the dynamics and architecture of the biofilm formation, a decrease in the motility of the bacterium, and an enhanced virulence in the septicemic mouse model. Moreover, these effects of the mutation could be complemented. Enhanced production of the biofilm exopolysaccharide and filaments in the mutant strain were presumably the major causes of the observed phenotype. Our earlier studies indicated that the wild-type A. hydrophila with overproduction of DNA adenine methyltransferase (Dam) had significantly reduced motility, greater hemolytic activity associated with Act, and an enhanced ability to produce AI-1 lactones. Furthermore, such a Dam-overproducing strain was not lethal to mice. On the contrary, the luxS mutant with Dam overproduction showed an increased motility and had no effect on lactone production. In addition, the Dam-overproducing luxS mutant strain was not altered in its ability to induce lethality in a mouse model of infection when compared to the parental strain which overproduced Dam. We suggested that an altered gene expression in the luxS mutant of A. hydrophila SSU, as it related to biofilm formation and virulence, might be linked with the interruption of the bacterial metabolic pathway, specifically of methionine synthesis.
KeywordMeSH Terms
Biofilms
Mutation
Quorum Sensing
91. Massidda  O, Rossolini  GM, Satta  G,     ( 1991 )

The Aeromonas hydrophila cphA gene: molecular heterogeneity among class B metallo-beta-lactamases.

Journal of bacteriology 173 (15)
PMID : 1856163  :   DOI  :   10.1128/jb.173.15.4611-4617.1991     PMC  :   PMC208136    
Abstract >>
An Aeromonas hydrophila gene, named cphA, coding for a carbapenem-hydrolyzing metallo-beta-lactamase, was cloned in Escherichia coli by screening an Aeromonas genomic library for clones able to grow on imipenem-containing medium. From sequencing data, the cloned cphA gene appeared able to code for a polypeptide of 254 amino acids whose sequence includes a potential N-terminal leader sequence for targeting the protein to the periplasmic space. These data were in agreement with the molecular mass of the original Aeromonas enzyme and of the recombinant enzyme produced in E. coli, evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude beta-lactamase preparations followed by renaturation treatment for proteins separated in the gel and localization of protein bands showing carbapenem-hydrolyzing beta-lactamase activity by a modified iodometric technique. The deduced amino acid sequence of the CphA enzyme showed regions of partial homology with both the beta-lactamase II of Bacillus cereus and the CfiA beta-lactamase of Bacteroides fragilis. Sequence homologies were more pronounced in the regions encompassing the amino acid residues known in the enzyme of B. cereus to function as ligand-binding residues for the metal cofactor. The CphA enzyme, however, appeared to share a lower degree of similarity with the two other enzymes, which, in turn, seemed more closely related to each other. These results, therefore, suggest the existence of at least two molecular subclasses within molecular class B metallo-beta-lactamases.
KeywordMeSH Terms
Genes, Bacterial
92. Kadlec  K, Schwarz  S,     ( 2008 )

Analysis and distribution of class 1 and class 2 integrons and associated gene cassettes among Escherichia coli isolates from swine, horses, cats and dogs collected in the BfT-GermVet monitoring study.

The Journal of antimicrobial chemotherapy 62 (3)
PMID : 18550679  :   DOI  :   10.1093/jac/dkn233    
Abstract >>
In the BfT-GermVet monitoring study, 417 Escherichia coli isolates collected during 2004-06 in Germany from various disease conditions of pigs (n = 87), horses (n = 102) or cats/dogs (n = 228) were investigated for their susceptibility to 24 antimicrobial agents. This study dealt with the identification of integron-associated resistance genes among these isolates. Class 1 and class 2 integrons were detected by PCR. The variable parts of the integrons were cloned and sequenced. Transformation and conjugation experiments were conducted to confirm a plasmid location of the integrons. Class 1 and/or class 2 integrons, alone or in different combinations, were detected in 79 of the 417 E. coli isolates. Four trimethoprim resistance genes (dfrA1/12/14/17), five streptomycin/spectinomycin resistance genes (aadA1/2/4/5/6), two streptothricin resistance genes (estX, sat2), one gentamicin/tobramycin/kanamycin resistance gene (aadB) and one chloramphenicol resistance gene (catB3) were detected. Seven different cassette arrangements were identified within class 1 integrons: aadA1 (21 isolates), dfrA1 + aadA1 (18 isolates), dfrA17 + aadA5 (9 isolates), dfrA12 + orfF + aadA2 (8 isolates), aadB + aadA1 (1 isolate), dfrA14 + recombined aadA6 (1 isolate) and dfrA1 + catB3 + aadA4 (1 isolate). Three different cassette arrangements in class 2 integrons, dfrA1 + sat2 + aadA1 (24 isolates), estX + sat2 + aadA1 (6 isolates) and estX + sat2 + DeltaaadA1 (1 isolate), were identified. The plasmid location of class 1 and/or class 2 integrons was confirmed in 37 isolates. Class 1 and/or class 2 integrons carrying resistance gene cassettes were detected in 18.9% of the isolates tested. This molecular analysis complements the phenotypic susceptibility testing conducted in the BfT-GermVet monitoring study and helps to explain the persistence of resistance genes even without direct selective pressure.
KeywordMeSH Terms
Integrons
93. Kulinska  A, Czeredys  M, Hayes  F, Jagura-Burdzy  G,     ( 2008 )

Genomic and functional characterization of the modular broad-host-range RA3 plasmid, the archetype of the IncU group.

Applied and environmental microbiology 74 (13)
PMID : 18502921  :   DOI  :   10.1128/AEM.00229-08     PMC  :   PMC2446526    
Abstract >>
IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids.
KeywordMeSH Terms
Conjugation, Genetic
94. Reith  ME, Singh  RK, Curtis  B, Boyd  JM, Bouevitch  A, Kimball  J, Munholland  J, Murphy  C, Sarty  D, Williams  J, Nash  JH, Johnson  SC, Brown  LL,     ( 2008 )

The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen.

BMC genomics 9 (N/A)
PMID : 18801193  :   DOI  :   10.1186/1471-2164-9-427     PMC  :   PMC2556355    
Abstract >>
Aeromonas salmonicida subsp. salmonicida is a Gram-negative bacterium that is the causative agent of furunculosis, a bacterial septicaemia of salmonid fish. While other species of Aeromonas are opportunistic pathogens or are found in commensal or symbiotic relationships with animal hosts, A. salmonicida subsp. salmonicida causes disease in healthy fish. The genome sequence of A. salmonicida was determined to provide a better understanding of the virulence factors used by this pathogen to infect fish. The nucleotide sequences of the A. salmonicida subsp. salmonicida A449 chromosome and two large plasmids are characterized. The chromosome is 4,702,402 bp and encodes 4388 genes, while the two large plasmids are 166,749 and 155,098 bp with 178 and 164 genes, respectively. Notable features are a large inversion in the chromosome and, in one of the large plasmids, the presence of a Tn21 composite transposon containing mercury resistance genes and an In2 integron encoding genes for resistance to streptomycin/spectinomycin, quaternary ammonia compounds, sulphonamides and chloramphenicol. A large number of genes encoding potential virulence factors were identified; however, many appear to be pseudogenes since they contain insertion sequences, frameshifts or in-frame stop codons. A total of 170 pseudogenes and 88 insertion sequences (of ten different types) are found in the A. salmonicida genome. Comparison with the A. hydrophila ATCC 7966T genome reveals multiple large inversions in the chromosome as well as an approximately 9% difference in gene content indicating instances of single gene or operon loss or gain.A limited number of the pseudogenes found in A. salmonicida A449 were investigated in other Aeromonas strains and species. While nearly all the pseudogenes tested are present in A. salmonicida subsp. salmonicida strains, only about 25% were found in other A. salmonicida subspecies and none were detected in other Aeromonas species. Relative to the A. hydrophila ATCC 7966T genome, the A. salmonicida subsp. salmonicida genome has acquired multiple mobile genetic elements, undergone substantial rearrangement and developed a significant number of pseudogenes. These changes appear to be a consequence of adaptation to a specific host, salmonid fish, and provide insights into the mechanisms used by the bacterium for infection and avoidance of host defence systems.
KeywordMeSH Terms
Genome, Bacterial
95. Jimenez  N, Canals  R, Saló  MT, Vilches  S, Merino  S, Tomás  JM,     ( 2008 )

The Aeromonas hydrophila wb*O34 gene cluster: genetics and temperature regulation.

Journal of bacteriology 190 (12)
PMID : 18408022  :   DOI  :   10.1128/JB.00153-08     PMC  :   PMC2446748    
Abstract >>
The Aeromonas hydrophila wb*(O34) gene cluster of strain AH-3 (serotype O34) was cloned and sequenced. This cluster contains genes necessary for the production of O34-antigen lipopolysaccharide (LPS) in A. hydrophila. We determined, using either mutation or sequence homology, roles for the majority of genes in the cluster by using the chemical O34-antigen LPS structure obtained for strain AH-3. The O34-antigen LPS export system has been shown to be a Wzy-dependent pathway typical of heteropolysaccharide pathways. Furthermore, the production of A. hydrophila O34-antigen LPS in Escherichia coli K-12 strains is dependent on incorporation of the Gne enzyme (UDP-N-acetylgalactosamine 4-epimerase) necessary for the formation of UDP-galactosamine in these strains. By using rapid amplification of cDNA ends we were able to identify a transcription start site upstream of the terminal wzz gene, which showed differential transcription depending on the growth temperature of the strain. The Wzz protein is able to regulate the O34-antigen LPS chain length. The differential expression of this protein at different temperatures, which was substantially greater at 20 degrees C than at 37 degrees C, explains the previously observed differential production of O34-antigen LPS and its correlation with the virulence of A. hydrophila serotype O34 strains.
KeywordMeSH Terms
Multigene Family
96. Barghouthi  S, Payne  SM, Arceneaux  JE, Byers  BR,     ( 1991 )

Cloning, mutagenesis, and nucleotide sequence of a siderophore biosynthetic gene (amoA) from Aeromonas hydrophila.

Journal of bacteriology 173 (16)
PMID : 1830579  :   DOI  :   10.1128/jb.173.16.5121-5128.1991     PMC  :   PMC208203    
Abstract >>
Many isolates of the Aeromonas species produce amonabactin, a phenolate siderophore containing 2,3-dihydroxybenzoic acid (2,3-DHB). An amonabactin biosynthetic gene (amoA) was identified (in a Sau3A1 gene library of Aeromonas hydrophila 495A2 chromosomal DNA) by its complementation of the requirement of Escherichia coli SAB11 for exogenous 2,3-DHB to support siderophore (enterobactin) synthesis. The gene amoA was subcloned as a SalI-HindIII 3.4-kb DNA fragment into pSUP202, and the complete nucleotide sequence of amoA was determined. A putative iron-regulatory sequence resembling the Fur repressor protein-binding site overlapped a possible promoter region. A translational reading frame, beginning with valine and encoding 396 amino acids, was open for 1,188 bp. The C-terminal portion of the deduced amino acid sequence showed 58% identity and 79% similarity with the E. coli EntC protein (isochorismate synthetase), the first enzyme in the E. coli 2,3-DHB biosynthetic pathway, suggesting that amoA probably encodes a step in 2,3-DHB biosynthesis and is the A. hydrophila equivalent of the E. coli entC gene. An isogenic amonabactin-negative mutant, A. hydrophila SB22, was isolated after marker exchange mutagenesis with Tn5-inactivated amoA (amoA::Tn5). The mutant excreted neither 2,3-DHB nor amonabactin, was more sensitive than the wild-type to growth inhibition by iron restriction, and used amonabactin to overcome iron starvation.
KeywordMeSH Terms
Intramolecular Transferases
97. Sepe  A, Barbieri  P, Peduzzi  R, Demarta  A,     ( 2008 )

Evaluation of recA sequencing for the classification of Aeromonas strains at the genotype level.

Letters in applied microbiology 46 (4)
PMID : 18346137  :   DOI  :   10.1111/j.1472-765X.2008.02339.x    
Abstract >>
To evaluate the usefulness of partial recA sequences for the identification of Aeromonas strains at the genotype level. A partial recA sequence was obtained from 21 type or reference strains and 33 Aeromonas isolates, collected in the South of Switzerland from human, animal and aquatic environments. The 272 bp long recA fragments showed a mean interspecies divergence of 7.8% and allowed the classification of strains at genotype level. However, some discrepancies could be observed with other gene sequence based analyses in the classification of some strains. The 272 bp long recA fragment is a good molecular marker to infer taxonomy of members of the genus Aeromonas, even if the primers we chose for the amplification did not allow its direct sequencing. In the genus Aeromonas, nucleotide sequences of some protein-encoding genes have already been evaluated as molecular markers to be used in taxonomical and epidemiological researches. This study suggests the usefulness of a recA fragment as a further sequence to investigate for these purposes.
KeywordMeSH Terms
98. Libisch  B, Giske  CG, Kovács  B, Tóth  TG, Füzi  M,     ( 2008 )

Identification of the first VIM metallo-beta-lactamase-producing multiresistant Aeromonas hydrophila strain.

Journal of clinical microbiology 46 (5)
PMID : 18367570  :   DOI  :   10.1128/JCM.00047-08     PMC  :   PMC2395088    
Abstract >>
A VIM metallo-beta-lactamase-producing Aeromonas hydrophila strain carrying an integron-borne bla(VIM-4) gene was isolated from a cirrhotic patient's fecal sample in a Budapest hospital. The variable region of this integron is identical with that of a previously characterized integron from Pseudomonas aeruginosa clinical isolates in P?cs in southern Hungary.
KeywordMeSH Terms
99. Erova  TE, Kosykh  VG, Fadl  AA, Sha  J, Horneman  AJ, Chopra  AK,     ( 2008 )

Cold shock exoribonuclease R (VacB) is involved in Aeromonas hydrophila pathogenesis.

Journal of bacteriology 190 (10)
PMID : 18344363  :   DOI  :   10.1128/JB.00075-08     PMC  :   PMC2394994    
Abstract >>
In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966(T) of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4 degrees C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37 degrees C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD(50)). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD(50) dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.
KeywordMeSH Terms
Cold Temperature
100. Jimenez  N, Canals  R, Lacasta  A, Kondakova  AN, Lindner  B, Knirel  YA, Merino  S, Regué  M, Tomás  JM,     ( 2008 )

Molecular analysis of three Aeromonas hydrophila AH-3 (serotype O34) lipopolysaccharide core biosynthesis gene clusters.

Journal of bacteriology 190 (9)
PMID : 18310345  :   DOI  :   10.1128/JB.01874-07     PMC  :   PMC2347379    
Abstract >>
By the isolation of three different Aeromonas hydrophila strain AH-3 (serotype O34) mutants with an altered lipopolysaccharide (LPS) migration in gels, three genomic regions encompassing LPS core biosynthesis genes were identified and characterized. When possible, mutants were constructed using each gene from the three regions, containing seven, four, and two genes (regions 1 to 3, respectively). The mutant LPS core structures were elucidated by using mass spectrometry, methylation analysis, and comparison with the full core structure of an O-antigen-lacking AH-3 mutant previously established by us. Combining the gene sequence and complementation test data with the structural data and phenotypic characterization of the mutant LPSs enabled a presumptive assignment of all LPS core biosynthesis gene functions in A. hydrophila AH-3. The three regions and the genes contained are in complete agreement with the recently sequenced genome of A. hydrophila ATCC 7966. The functions of the A. hydrophila genes waaC in region 3 and waaF in region 2 were completely established, allowing the genome annotations of the two heptosyl transferase products not previously assigned. Having the functions of all genes involved with the LPS core biosynthesis and most corresponding single-gene mutants now allows experimental work on the role of the LPS core in the virulence of A. hydrophila.
KeywordMeSH Terms
Genes, Bacterial
101. Lan  X, Zhang  X, Kodaira  R, Zhou  Z, Shimosaka  M,     ( 2008 )

Gene cloning, expression, and characterization of a second beta-N-acetylglucosaminidase from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9.

Bioscience, biotechnology, and biochemistry 72 (2)
PMID : 18256480  :   DOI  :   10.1271/bbb.70573    
Abstract >>
A gene coding for a second beta-N-acetylglucosaminidase (nagB) was isolated from the chitinolytic bacterium Aeromonas hydrophila strain SUWA-9. The nagB open reading frame encoded a polypeptide of 618 amino acid residues with a molecular mass of 68.8 kDa. It did not contain a sequence characteristic of a signal peptide at the N-terminus. The deduced amino acid sequence showed high similarity to those of bacterial beta-N-acetylhexosaminidases classified into family 20 of glycosyl hydrolases. The nagB gene was successfully expressed in Escherichia coli, and the recombinant protein hydrolyzed N-acetylchitooligomers from dimer to hexamer and produced monomer as a final product. Reverse transcription-mediated PCR (RT-PCR) analysis revealed that nagB was transcribed when SUWA-9 cells were grown in the presence of colloidal chitin. In the upstream of the nagB gene, three genes, coding for putative N-acetylglucosamine kinase, beta-glucosidase, and ATP-binding protein of ABC-type transporter, were identified, and these genes likely to constitute an operon.
KeywordMeSH Terms
102. Suarez  G, Sierra  JC, Sha  J, Wang  S, Erova  TE, Fadl  AA, Foltz  SM, Horneman  AJ, Chopra  AK,     ( 2008 )

Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila.

Microbial pathogenesis 44 (4)
PMID : 18037263  :   DOI  :   10.1016/j.micpath.2007.10.005     PMC  :   PMC2430056    
Abstract >>
Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.
KeywordMeSH Terms
Multigene Family
103. Moura  A, Henriques  I, Ribeiro  R, Correia  A,     ( 2007 )

Prevalence and characterization of integrons from bacteria isolated from a slaughterhouse wastewater treatment plant.

The Journal of antimicrobial chemotherapy 60 (6)
PMID : 17913715  :   DOI  :   10.1093/jac/dkm340    
Abstract >>
To investigate the presence and distribution of integron-carrying bacteria from a slaughterhouse wastewater treatment plant (WWTP). Enterobacteriaceae and aeromonads were isolated at different stages of the wastewater treatment process and screened for the presence of integrase genes by dot-blot hybridization. Integrase-positive strains were characterized in terms of phylogenetic affiliation, genetic content of integrons and antimicrobial resistance profiles. Plasmid location of some integrons was established by Southern-blot hybridization. Strains containing integron-carrying plasmids were selected for mating experiments. Integrase genes were present in all samples, including the final effluent. The global prevalence was determined to be 35%, higher than in other aquatic environments. Forty-two integrase-positive isolates were further characterized. Nine distinct cassette arrays were found, containing genes encoding resistance to beta-lactams (bla(OXA-30)), aminoglycosides (aadA1, aadA2, aadA13, aadB), streptothricin (sat1, sat2), trimethoprim (dfrA1, dfrA12), a putative esterase (estX) and a protein with unknown function (orfF). Gene cassette arrays aadA1, dfrAI-aadA1 and estX-sat2-aadA1 were common to aeromonads and Enterobacteriaceae. The class 2 integron containing an estX-sat2-aadA1 cassette array was detected for the first time in Aeromonas sp. Nearly 12% (5 out of 43) of intI genes were located in plasmids. intI genes from isolates MM.1.3 and MM.1.5 were successfully conjugated into Escherichia coli at frequencies of 3.79 x 10(-5) and 5.46 x 10(-5) per recipient cell, respectively. Our data support the hypothesis that WWTPs constitute a potential hot spot for horizontal gene transfer and for selection of antimicrobial resistance genes among aquatic bacteria. Moreover, water discharges represent a possible risk for dissemination of undesirable genetic traits.
KeywordMeSH Terms
Abattoirs
Gene Transfer, Horizontal
Water Microbiology
104. Chang  YC, Shih  DY, Wang  JY, Yang  SS,     ( 2007 )

Molecular characterization of class 1 integrons and antimicrobial resistance in Aeromonas strains from foodborne outbreak-suspect samples and environmental sources in Taiwan.

Diagnostic microbiology and infectious disease 59 (2)
PMID : 17908616  :   DOI  :   10.1016/j.diagmicrobio.2007.04.007    
Abstract >>
One hundred thirty-three Aeromonas spp. isolates were examined for multiple antibiotic resistance phenotypes and prevalence of class 1 integron sequences. Twenty-four (18.0%) of these isolates contained class 1 integron. Seven different class 1 integrons were found among 24strains, with a total of 10 different gene cassettes encoding for resistance to trimethoprim (dfr12 and dfr2d), aminoglycosides (aadA1 and aadA2), beta-lactam antibiotics (oxa2), chloramphenicol (catB3 and catB8), quaternary ammonium amines (qacE2), and 2 ORFs (orfD and orfF) with unknown function. Rate of antibiotic resistance was different between integron-positive and integron-negative strains. Trimethoprim and trimethoprim-sulphamethoxazole resistances were commonly associated with integron, and all of integron-positive isolates were multiple resistant to more than 3 agents. Resistance to as many as 10 antimicrobial agents were observed in integron-positive strains. Several cassette arrays of class 1 integrons identified in this study were not previously reported in Aeromonas strains. This study demonstrates the wide distribution of class 1 integron in Aeromonas spp. isolated from foodborne outbreak-suspect samples and environmental sources in Taiwan.
KeywordMeSH Terms
Disease Outbreaks
Environmental Microbiology
105. Sha  J, Wang  SF, Suarez  G, Sierra  JC, Fadl  AA, Erova  TE, Foltz  SM, Khajanchi  BK, Silver  A, Graf  J, Schein  CH, Chopra  AK,     ( 2007 )

Further characterization of a type III secretion system (T3SS) and of a new effector protein from a clinical isolate of Aeromonas hydrophila--part I.

Microbial pathogenesis 43 (4)
PMID : 17644303  :   DOI  :   10.1016/j.micpath.2007.05.002    
Abstract >>
A type III secretion system (T3SS)-associated cytotoxin, AexT, with ADP-ribosyltransferase activity and homology to Pseudomonas aeruginosa bifuncational toxins ExoT/S, was recently identified from a fish pathogen Aeromonas salmonicida. In this study, we reported the molecular characterization of an aexT-like toxin gene (designated as aexU) from a diarrheal isolate SSU of A. hydrophila. The aexU gene was 1539bp in length and encoded a protein of 512 amino acid (aa) residues. The NH(2)-terminus of AexU (aa residues 1-231) exhibited a 67% homology with the NH(2)-terminus of AexT from A. salmonicida. Importantly, its COOH-terminus (aa residues 232-512) had no homology with any known functional proteins in the database; however, the full-length AexU retained ADP-ribosyltransferase activity. The expression and subsequent secretion of AexU was T3SS dependent, as inactivation of the ascV gene that codes for an inner-membrane component of the T3SS channel from the wild-type (WT) bacterium, blocked translocation of AexU in HT-29 human colonic epithelial cells. We provided evidence that inactivation of acrV and axsE genes (homologs of lcrV and exsE in Yersinia species and P. aeruginosa, respectively) from A. hydrophila SSU, altered expression and/or secretion of AexU. We deleted an aexU gene from the WT, as well as from the DeltaaopB mutant, of A. hydrophila, generating a single knockout (DeltaaexU) and a double knockout mutant, DeltaaopB/DeltaaexU. Increased phagocytosis was observed in RAW264.7 murine macrophages infected with the DeltaaopB/DeltaaexU mutant, as compared to macrophages when infected with the parental DeltaaopB strain. Further, mice infected with the DeltaaexU mutant had a 60% survival rate, compared to animals infected with the WT or the DeltaaexU-complemented strain that caused 90-100% of the animals to die at a 2-3 LD(50s) dose. Immunization of mice with the recombinant AexU protected them from subsequent lethal challenge dose by the WT bacterium. Finally, we detected specific anti-AexU antibodies in the sera of mice that survived challenge by the WT bacterium, which may indicate that AexU plays an important role in the pathogenesis of Aeromonas infections.
KeywordMeSH Terms
106. Zhang  D, Xu  DH, Qiu  J, Rasmussen-Ivey  CR, Liles  MR, Beck  BH,     ( 2017 )

Chitin degradation and utilization by virulent Aeromonas hydrophila strain ML10-51K.

Archives of microbiology 199 (4)
PMID : 28032191  :   DOI  :   10.1007/s00203-016-1326-1    
Abstract >>
Virulent Aeromonas hydrophila (vAh) is one of the most important bacterial pathogens that causes persistent outbreaks of motile Aeromonas septicemia in warm-water fishes. The survivability of this pathogen in aquatic environments is of great concern. The aim of this study was to determine the capability of the vAh strain ML10-51K to degrade and utilize chitin. Genome-wide analysis revealed that ML10-51K encodes a suite of proteins for chitin metabolism. Assays in vitro showed that four chitinases, one chitobiase and one chitin-binding protein were secreted extracellularly and participated in chitin degradation. ML10-51K was shown to be able to use not only N-acetylglucosamine and colloidal chitin but also chitin flakes as sole carbon sources for growth. This study indicates that ML10-51K is a highly chitinolytic bacterium and suggests that the capability of effective chitin utilization could enable the bacterium to attain high densities when abundant chitin is available in aquatic niches.
KeywordMeSH Terms
Chitin degradation
Chitin utilization
Chitinase
Chitinolytic enzymes
Virulent Aeromonas hydrophila
Chitin degradation
Chitin utilization
Chitinase
Chitinolytic enzymes
Virulent Aeromonas hydrophila
107. Zhao  H, Wei  H, Liu  X, Yao  Z, Xu  M, Wei  D, Wang  J, Wang  X, Chen  GQ,     ( 2016 )

Structural Insights on PHA Binding Protein PhaP from Aeromonas hydrophila.

Scientific reports 6 (N/A)
PMID : 28009010  :   DOI  :   10.1038/srep39424     PMC  :   PMC5180188    
Abstract >>
Phasins or PhaPs are a group of amphiphilic proteins that are found attached to the surface of microbial polyhydroxyalkanoate (PHA) granules. They have both structural and regulatory functions and can affect intracellular PHA accumulation and mediate protein folding. The molecular basis for the diverse functions of the PhaPs has not been fully understood due to the lack of the structural knowledge. Here we report the structural and biochemical studies of the PhaP cloned from Aeromonas hydrophila (PhaPAh), which is utilized in protein and tissue engineering. The crystal structure of PhaPAh was revealed to be a tetramer with 8 �\-helices adopting a coiled-coil structure. Each monomer has a hydrophobic and a hydrophilic surface, rendering the surfactant properties of the PhaPAh monomer. Based on the crystal structure, we predicted three key amino acid residues and obtained mutants with enhanced stability and improved emulsification properties. The first PhaP crystal structure, as reported in this study, is an important step towards a mechanistic understanding of how PHA is formed in vivo and why PhaP has such unique surfactant properties. At the same time, it will facilitate the study of other PhaP members that may have significant biotechnological potential as bio-surfactants and amphipathic coatings.
KeywordMeSH Terms
108. Wen  Y, Pu  X, Zheng  W, Hu  G,     ( 2016 )

High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

PloS one 11 (7)
PMID : 27427763  :   DOI  :   10.1371/journal.pone.0159418     PMC  :   PMC4948828    
Abstract >>
Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR) genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4%) were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2) and 32 isolates (17.0%) were positive for aac(6')-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6')-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05). In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05). All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp) and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6')-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.
KeywordMeSH Terms
Conjugation, Genetic
Water Microbiology
109. Khor  WC, Puah  SM, Tan  JA, Puthucheary  SD, Chua  KH,     ( 2015 )

Phenotypic and Genetic Diversity of Aeromonas Species Isolated from Fresh Water Lakes in Malaysia.

PloS one 10 (12)
PMID : 26710336  :   DOI  :   10.1371/journal.pone.0145933     PMC  :   PMC4692508    
Abstract >>
Gram-negative bacilli of the genus Aeromonas are primarily inhabitants of the aquatic environment. Humans acquire this organism from a wide range of food and water sources as well as during aquatic recreational activities. In the present study, the diversity and distribution of Aeromonas species from freshwater lakes in Malaysia was investigated using glycerophospholipid-cholesterol acyltransferase (GCAT) and RNA polymerase sigma-factor (rpoD) genes for speciation. A total of 122 possible Aeromonas strains were isolated and confirmed to genus level using the API20E system. The clonality of the isolates was investigated using ERIC-PCR and 20 duplicate isolates were excluded from the study. The specific GCAT-PCR identified all isolates as belonging to the genus Aeromonas, in agreement with the biochemical identification. A phylogenetic tree was constructed using the rpoD gene sequence and all 102 isolates were identified as: A. veronii 43%, A. jandaei 37%, A. hydrophila 6%, A. caviae 4%, A. salmonicida 2%, A. media 2%, A. allosaccharophila 1%, A. dhakensis 1% and Aeromonas spp. 4%. Twelve virulence genes were present in the following proportions--exu 96%, ser 93%, aer 87%, fla 83%, enolase 70%, ela 62%, act 54%, aexT 33%, lip 16%, dam 16%, alt 8% and ast 4%, and at least 2 of these genes were present in all 102 strains. The ascV, aexU and hlyA genes were not detected among the isolates. A. hydrophila was the main species containing virulence genes alt and ast either present alone or in combination. It is possible that different mechanisms may be used by each genospecies to demonstrate virulence. In summary, with the use of GCAT and rpoD genes, unambiguous identification of Aeromonas species is possible and provides valuable data on the phylogenetic diversity of the organism.
KeywordMeSH Terms
110. Huang  L, Qin  Y, Yan  Q, Lin  G, Huang  L, Huang  B, Huang  W,     ( 2015 )

MinD plays an important role in Aeromonas hydrophila adherence to Anguilla japonica mucus.

Gene 565 (2)
PMID : 25881868  :   DOI  :   10.1016/j.gene.2015.04.031    
Abstract >>
For a better understanding of the genitic regulation of the adhesion of Aeromonas hydrophila, a mini-Tn10 transposon mutagenesis system was introduced to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOF/Km) and the recipient A. hydrophila strain W. Out of 332 individual colonies, 7 mutants with significantly attenuated adhesion ability were selected. The DNA sequence flanking the mini-Tn10 transposon inserted in the mutant strain M45 was amplified by TAIL-PCR. The results showed that an ORF of approximately 870 bp (GenBank accession number KP271930) of the mutant strain M45 was inserted by mini-Tn10. This ORF putatively encodes a deduced 289 amino acid protein that displays the highest identity (100%) with the CobQ/CobB/MinD/ParA family protein of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type W, the mutant strain M45 and the complemented strain C45 were investigated. The results indicated that mutation of MinD gene in A. hydrophila could lead to abnormal cell division and reduce the ability of adhesion, biofilm formation and bacterial motility.
KeywordMeSH Terms
Adhesion
Aeromonas hydrophila
Biofilm formation
Cell division
MinD
Motility
Adhesion
Aeromonas hydrophila
Biofilm formation
Cell division
MinD
Motility
111. Merino  S, Canals  R, Knirel  YA, Tomás  JM,     ( 2015 )

Molecular and chemical analysis of the lipopolysaccharide from Aeromonas hydrophila strain AH-1 (Serotype O11).

Marine drugs 13 (4)
PMID : 25874921  :   DOI  :   10.3390/md13042233     PMC  :   PMC4413209    
Abstract >>
A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described; these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wbO11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waaO11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.
KeywordMeSH Terms
112. Nguyen  VS, Jobichen  C, Tan  KW, Tan  YW, Chan  SL, Ramesh  K, Yuan  Y, Hong  Y, Seetharaman  J, Leung  KY, Sivaraman  J, Mok  YK,     ( 2015 )

Structure of AcrH-AopB Chaperone-Translocator Complex Reveals a Role for Membrane Hairpins in Type III Secretion System Translocon Assembly.

Structure (London, England : 1993) 23 (11)
PMID : 26439768  :   DOI  :   10.1016/j.str.2015.08.014    
Abstract >>
Type III secretion systems (T3SSs) are adopted by pathogenic bacteria for the transport of effector proteins into host cells through the translocon pore composed of major and minor translocator proteins. Both translocators require a dedicated chaperone for solubility. Despite tremendous efforts in the past, structural information regarding the chaperone-translocator complex and the topology of the translocon pore have remained elusive. Here, we report the crystal structure of the major translocator, AopB, from Aeromonas hydrophila AH-1 in complex with its chaperone, AcrH. Overall, the structure revealed unique interactions between the various interfaces of AopB and AcrH, with the N-terminal "molecular anchor" of AopB crossing into the "N-terminal arm" of AcrH. AopB adopts a novel fold, and its transmembrane regions form two pairs of helical hairpins. From these structural studies and associated cellular assays, we deduced the topology of the assembled T3SS translocon; both termini remain extracellular after membrane insertion.
KeywordMeSH Terms
113. 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
114. Qin  YX, Yan  QP, Mao  XX, Chen  Z, Su  YQ,     ( 2014 )

Role of MshQ in MSHA pili biosynthesis and biofilm formation of Aeromonas hydrophila.

Genetics and molecular research : GMR 13 (4)
PMID : 25366789  :   DOI  :   10.4238/2014.October.31.13    
Abstract >>
Biofilm formation of pathogen bacterium is currently one of the most widely studied topics; however, little is known regarding pathogen bacteria biofilms in aquaculture. Aeromonas hydrophila is a representative species of the genus Aeromonas, which has been recognized as a common pathogen, is associated with many diseases in aquatic animals, and causes significant mortality. The objectives of this study are i) to confirm that A. hydrophila can form biofilms on abiotic substrates and construct a biofilm growth curve for this bacterium; ii) to identify the genes that play crucial roles in A. hydrophila biofilm formation. The biofilm growth curve of A. hydrophila was constructed using a crystal violet assay, which showed that biofilm formation for this bacterium is a dynamic process. Next, a mutant library of pathogenic A. hydrophila B11 was constructed using the mini-Tn10 transposon mutagenesis system. A total of 861 mutants were screened, and 5 mutants were stably deficient in biofilm formation. Molecular analysis of the mutant B112 revealed that the open reading frame that encodes the protein MshQ was disrupted. Comparison of biological characteristics including growth, motility, and adhesion between the mutant B112 and the wild-type strain B11 suggested that MshQ is necessary for mannose-sensitive hemagglutinin pilus biosynthesis of A. hydrophila, and that these pili play crucial roles in A.hydrophila adherence to a solid surface during the early stages of biofilm formation.
KeywordMeSH Terms
115. Pang  M, Jiang  J, Xie  X, Wu  Y, Dong  Y, Kwok  AH, Zhang  W, Yao  H, Lu  C, Leung  FC, Liu  Y,     ( 2015 )

Novel insights into the pathogenicity of epidemic Aeromonas hydrophila ST251 clones from comparative genomics.

Scientific reports 5 (N/A)
PMID : 26014286  :   DOI  :   10.1038/srep09833     PMC  :   PMC4444815    
Abstract >>
Outbreaks in fish of motile Aeromonad septicemia (MAS) caused by Aeromonas hydrophila have caused a great concern worldwide. Here, for the first time, we provide two complete genomes of epidemic A. hydrophila strains isolated in China. To gain an insight into the pathogenicity of epidemic A. hydrophila, we performed comparative genomic analyses of five epidemic strains belonging to sequence type (ST) 251, together with the environmental strain ATCC 7966(T). We found that the known virulence factors, including a type III secretion system, a type VI secretion system and lateral flagella, are not required for the high virulence of the ST251 clonal group. Additionally, our work identifies three utilization pathways for myo-inositol, sialic acid and L-fucose providing clues regarding the factors that underlie the epidemic and virulent nature of ST251 A. hydrophila. Based on the geographical distribution and biological resources of the ST251 clonal group, we conclude that ST251 is a high-risk clonal group of A. hydrophila which may be responsible for the MAS outbreaks in China and the southeastern United States.
KeywordMeSH Terms
Genome, Bacterial
116. Gobius  KS, Pemberton  JM,     ( 1988 )

Molecular cloning, characterization, and nucleotide sequence of an extracellular amylase gene from Aeromonas hydrophila.

Journal of bacteriology 170 (3)
PMID : 2449422  :   DOI  :   10.1128/jb.170.3.1325-1332.1988    
Abstract >>
The structural gene for excreted amylase from Aeromonas hydrophila JMP636 has been cloned within a 2.1-kilobase SmaI fragment of DNA. The amylase gene is transcribed from its own promoter in Escherichia coli, producing a gene product of Mr 49,000. The amylase gene product is secreted to the periplasm of E. coli; however, it is not excreted. Nucleotide sequencing revealed an open reading frame of 1,392 base pairs corresponding to a protein of 464 amino acid residues. A potential signal peptide of 21 amino acid residues is present at the NH2 terminal of the predicted protein. Three regions of homology with other procaryotic and eucaryotic alpha-amylases were detected within the predicted amino acid sequence.
KeywordMeSH Terms
117. Merino  S, Fulton  KM, Twine  SM, Wilhelms  M, Molero  R, Tomás  JM,     ( 2014 )

Aeromonas hydrophila flagella glycosylation: involvement of a lipid carrier.

PloS one 9 (2)
PMID : 24586923  :   DOI  :   10.1371/journal.pone.0089630     PMC  :   PMC3931799    
Abstract >>
Polar flagellin proteins from Aeromonas hydrophila strain AH-3 (serotype O34) were found to be O-glycosylated with a heterogeneous glycan. Mutants unable to produce WecP or Gne enzymes showed altered motility, and the study of their polar flagellin glycosylation showed that the patterns of glycosylation differed from that observed with wild type polar flagellin. This suggested the involvement of a lipid carrier in glycosylation. A gene coding for an enzyme linking sugar to a lipid carrier was identified in strain AH-3 (WecX) and subsequent mutation abolished completely motility, flagella production by EM, and flagellin glycosylation. This is the first report of a lipid carrier involved in flagella O-glycosylation. A molecular model has been proposed. The results obtained suggested that the N-acetylhexosamines are N-acetylgalactosamines and that the heptasaccharide is completely independent of the O34-antigen lipopolysaccharide. Furthermore, by comparing the mutants with differing degrees of polar flagellin glycosylation, we established their importance in A. hydrophila flagella formation and motility.
KeywordMeSH Terms
Protein Processing, Post-Translational
118. Persson  S, Al-Shuweli  S, Yapici  S, Jensen  JN, Olsen  KE,     ( 2015 )

Identification of clinical aeromonas species by rpoB and gyrB sequencing and development of a multiplex PCR method for detection of Aeromonas hydrophila, A. caviae, A. veronii, and A. media.

Journal of clinical microbiology 53 (2)
PMID : 25411168  :   DOI  :   10.1128/JCM.01963-14     PMC  :   PMC4298543    
Abstract >>
Conventional identification of Aeromonas species based on biochemical methods is challenged by the heterogeneous nature of the species. Here, we present a new multiplex PCR method directed toward the gyrB and rpoB genes that identifies four Aeromonas species, A. hydrophila, A. media, A. veronii, and A. caviae, and we describe the application of this method on a Danish strain collection.
KeywordMeSH Terms
119. Bottoni  C, Marcoccia  F, Compagnoni  C, Colapietro  M, Sabatini  A, Celenza  G, Segatore  B, Maturo  MG, Amicosante  G, Perilli  M,     ( 2015 )

Identification of New Natural CphA Metallo-�]-Lactamases CphA4 and CphA5 in Aeromonas veronii and Aeromonas hydrophila Isolates from Municipal Sewage in Central Italy.

Antimicrobial agents and chemotherapy 59 (8)
PMID : 25987617  :   DOI  :   10.1128/AAC.00628-15     PMC  :   PMC4505251    
Abstract >>
Two new natural CphA metallo-�]-lactamases, the CphA4 and CphA5 enzymes, were identified in water samples from municipal sewage in central Italy. Compared to CphA, the CphA4 and CphA5 enzymes showed numerous point mutations. These enzymes have a narrow spectrum of substrates focused on carbapenems only. CphA5 showed kcat values about 40-, 12-, and 97-fold higher than those observed for CphA4 versus imipenem, ertapenem, and biapenem, respectively.
KeywordMeSH Terms
120. Qin  Y, Lin  G, Chen  W, Huang  B, Huang  W, Yan  Q,     ( 2014 )

Flagellar motility contributes to the invasion and survival of Aeromonas hydrophila in Anguilla japonica macrophages.

Fish & shellfish immunology 39 (2)
PMID : 24859591  :   DOI  :   10.1016/j.fsi.2014.05.016    
Abstract >>
The interaction between pathogenic bacteria and the host phagocytes is complicated. It is generally believed that only obligate intracellular pathogens can invade and survive in host phagocytes. In this study, we revealed that the pathogenic Aeromonas hydrophila B11 can also invade and survive in the macrophages of its host Anguilla japonica in vitro. To further investigate the mechanisms of A. hydrophila invasion and survival in host macrophages, a mini-Tn10 transposon mutagenesis system was used to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOFKm) and the recipient A. hydrophila B11. Out of 465 individual colonies, 13 mutants impaired in survival within macrophages were selected, and the mutant BM116 was the most seriously impaired strain. Molecular analysis showed that an ORF of approximately 1335 bp (GenBank accession numbers JQ974982) of the mutant BM116 was inserted by mini-Tn10. This ORF putatively encodes a deduced 445 amino acids protein that displays the highest identity (99.6%) with the flagellar hook protein FlgE of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type B11, the mutant B116 and the complemented strain were investigated. The results reveal that the flagella of the mutant BM116 was absent and that these mutant bacteria exhibited defective motility, adhesion, and invasion and survival in host macrophages when compared with the wild type and the complemented strain. These findings indicate that flgE is required for flagellum biogenesis in A. hydrophila and that flagellar motility is required for A. hydrophila invasion and survival in the macrophages of its host. Our findings provide an important new understanding of the nonintracellular pathogenic bacteria invasion and survival in host phagocytes and the interactions between the pathogens and their host.
KeywordMeSH Terms
Aeromonas hydrophila
Flagella
Phagocytes
Survival
Aeromonas hydrophila
Flagella
Phagocytes
Survival
Anguilla
121. Hossain  MJ, Waldbieser  GC, Sun  D, Capps  NK, Hemstreet  WB, Carlisle  K, Griffin  MJ, Khoo  L, Goodwin  AE, Sonstegard  TS, Schroeder  S, Hayden  K, Newton  JC, Terhune  JS, Liles  MR,     ( 2013 )

Implication of lateral genetic transfer in the emergence of Aeromonas hydrophila isolates of epidemic outbreaks in channel catfish.

PloS one 8 (11)
PMID : 24278351  :   DOI  :   10.1371/journal.pone.0080943     PMC  :   PMC3835674    
Abstract >>
To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains.
KeywordMeSH Terms
Disease Outbreaks
Gene Transfer, Horizontal
122. Deng  YT, Wu  YL, Tan  AP, Huang  YP, Jiang  L, Xue  HJ, Wang  WL, Luo  L, Zhao  F,     ( 2014 )

Analysis of antimicrobial resistance genes in Aeromonas spp. isolated from cultured freshwater animals in China.

Microbial drug resistance (Larchmont, N.Y.) 20 (4)
PMID : 24350737  :   DOI  :   10.1089/mdr.2013.0068    
Abstract >>
The development of resistance to antimicrobials used in aquatic animals is an increasing concern for aquaculture and public health. To monitor the occurrence of antimicrobial resistance and resistance genes in Aeromonas, a total of 106 isolates were collected from cultured freshwater animals in China from 1995 to 2012. Antimicrobial susceptibilities were determined by the disk diffusion method. The highest resistance percentage occurred with ampicillin, rifampin, streptomycin, and nalidixic acid. Most strains were sensitive to fluoroquinolones, doxycycline, cefotaxime, chloramphenicol, and amikacin. The isolates from turtle samples had the highest levels of resistance to 11 of the 12 tested antimicrobials when compared with those from fish or shrimp. Polymerase chain reaction and DNA sequence results showed that all trimethoprim/sulfamethoxazole-resistant strains contained sul1, and 37.0% were positive for tetA in tetracycline-resistant strains. ant(3?)-Ia was identified in 13 (24.5%) streptomycin-resistant strains. Plasmid-borne quinolone resistance genes were detected in five Aeromonas hydrophila (4.7%), two of which carried qnrS2, while the other three strains harbored aac(6')-Ib-cr. Two cefotaxime-resistant A. hydrophila were positive for bla(TEM-1) and bla(CTX-M-3). To our knowledge, this is the first report characterizing antimicrobial resistance in Aeromonas isolated from cultured freshwater animals in China, and providing resistance information of pathogen in Chinese aquaculture.
KeywordMeSH Terms
Genes, Bacterial
123. Hossain  MJ, Sun  D, McGarey  DJ, Wrenn  S, Alexander  LM, Martino  ME, Xing  Y, Terhune  JS, Liles  MR,     ( 2014 )

An Asian origin of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish.

mBio 5 (3)
PMID : 24895303  :   DOI  :   10.1128/mBio.00848-14     PMC  :   PMC4049099    
Abstract >>
Since 2009, catfish farming in the southeastern United States has been severely impacted by a highly virulent and clonal population of Aeromonas hydrophila causing motile Aeromonas septicemia (MAS) in catfish. The possible origin of this newly emerged highly virulent A. hydrophila strain is unknown. In this study, we show using whole-genome sequencing and comparative genomics that A. hydrophila isolates from diseased grass carp in China and catfish in the United States have highly similar genomes. Our phylogenomic analyses suggest that U.S. catfish isolates emerged from A. hydrophila populations of Asian origin. Furthermore, we identified an A. hydrophila strain isolated in 2004 from a diseased catfish in Mississippi, prior to the onset of the major epidemic outbreaks in Alabama starting in 2009, with genomic characteristics that are intermediate between those of the Asian and Alabama fish isolates. Investigation of A. hydrophila strain virulence demonstrated that the isolate from the U.S. catfish epidemic is significantly more virulent to both channel catfish and grass carp than is the Chinese carp isolate. This study implicates the importation of fish or fishery products into the United States as the source of highly virulent A. hydrophila that has caused severe epidemic outbreaks in United States-farmed catfish and further demonstrates the potential for invasive animal species to disseminate bacterial pathogens worldwide. Catfish aquaculture farming in the southeastern United States has been severely affected by the emergence of virulent Aeromonas hydrophila responsible for epidemic disease outbreaks, resulting in the death of over 10 million pounds of catfish. Because the origin of this newly emerged A. hydrophila strain is unknown, this study used a comparative genomics approach to conduct a phylogenomic analysis of A. hydrophila isolates obtained from the United States and Asia. Our results suggest that the virulent isolates from United States-farmed catfish have a recent common ancestor with A. hydrophila isolates from diseased Asian carp. We have also observed that an Asian carp isolate, like recent U.S. catfish isolates, is virulent in catfish. The results from this study suggest that the highly virulent U.S. epidemic isolates emerged from an Asian source and provide another example of the threat that invasive species pose in the dissemination of bacterial pathogens.
KeywordMeSH Terms
124. Vega-Sánchez  V, Latif-Eugenín  F, Soriano-Vargas  E, Beaz-Hidalgo  R, Figueras  MJ, Aguilera-Arreola  MG, Castro-Escarpulli  G,     ( 2014 )

Re-identification of Aeromonas isolates from rainbow trout and incidence of class 1 integron and �]-lactamase genes.

Veterinary microbiology 172 (3��4��)
PMID : 25008317  :   DOI  :   10.1016/j.vetmic.2014.06.012    
Abstract >>
Forty-eight Aeromonas isolates from rainbow trout previously identified by the 16S rDNA-RFLP technique were re-identified using 2 housekeeping genes (gyrB and rpoD). After sequencing the prevalences of the species were A. veronii (29.2%), A. bestiarum (20.8%), A. hydrophila (16.7%), A. sobria (10.4%), A. media (8.3%), A. popoffii (6.2%), A. allosaccharophila (2.1%), A. caviae (2.1%), A. salmonicida (2.1%) and one isolate (2.1%) belongs to a candidate new species "Aeromonas lusitana". Coincident identification results to the 16S rDNA-RFLP technique were only obtained for 68.8% of the isolates. PCR amplification of the enterobacterial repetitive intergenic consensus (ERIC-PCR) indicated that the 48 isolates belonged to 33 different ERIC genotypes. Several genotypes were isolated from different farms and organs in the same fish, indicating a systemic dissemination of the bacteria. The presence of genes (blaIMP, blaCphA/IMIS, blaTEM, blaSHV and intI1) that encode extended-spectrum beta-lactamases (ESBLs), metallo-beta-lactamases (MBLs) and class 1 integrons were studied by PCR. Only 39.6% (19/48) of the strains showed the presence of one or more resistance genes. The gene blaCphA/IMIS was detected in 29.2% of the isolates, followed by the intI1 (6.2%) and blaSHV (4.2%) genes. The variable region of class 1 integrons of the 3 positive isolates was sequenced revealing the presence of the gene cassette aadA1 (aminoglycoside transferase) that plays a role in streptomycin/spectinomycin resistance.
KeywordMeSH Terms
ESBLs
Integrons
Aeromonas
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Aeromonas
ESBLs
Integrons
Rainbow trout
gyrB
rpoD
Oncorhynchus mykiss
125. Furmanek-Blaszk  B,     ( N/A )

Phenotypic and molecular characteristics of an Aeromonas hydrophila strain isolated from the River Nile.

Microbiological research 169 (7��8��)
PMID : 24315209  :   DOI  :   10.1016/j.micres.2013.11.001    
Abstract >>
Aeromonas hydrophila, an inhabitant of aquatic ecosystems found in most parts of the world, has considerable virulence potential. The polymerase chain reaction technique was used to assay for the presence of five virulence factor genes: haemolytic toxins aerA and ahh1, elastase ahyB, the enterotoxin act, and the polar flagella flaA/flaB in the A. hydrophila strain isolated from the River Nile. Drug screening showed high levels of resistance to �]-lactam antibiotics and tetracycline. Slime production was determined by the Congo red agar plate test. The isolate produced two restriction enzymes named AehI and AehII which are isoschizomers of XhoI and StuI respectively. The complete nucleotide sequence of the cryptic plasmid pAhy2.5 (2524 bp) from this strain was determined. Sequence analysis revealed the presence of two open reading frames (ORFs) encoding putative proteins. The protein coded by ORF1 is homologous with Rep proteins of plasmids belonging to the pC194 family, which are known to replicate by the rolling-circle mechanism. The putative double-strand origin of replication and a region with palindromic sequences that could function as a single-strand origin were detected in pAhy2.5.
KeywordMeSH Terms
Antimicrobial resistance
Plasmid
Virulence properties
Antimicrobial resistance
Plasmid
Virulence properties
126. Yadav  SK, Sahoo  PK, Dixit  A,     ( 2014 )

Characterization of immune response elicited by the recombinant outer membrane protein OmpF of Aeromonas hydrophila, a potential vaccine candidate in murine model.

Molecular biology reports 41 (3)
PMID : 24435974  :   DOI  :   10.1007/s11033-014-3033-9    
Abstract >>
Porins, the outer membrane proteins of gram negative bacteria, perform vital roles in bacterial survival and virulence, such as nutrient transportation across the membrane as well as adhesion to host cells during infection. The outer membrane proteins, OmpF and OmpC, are part of a two-component regulatory system, essential for the maintenance of solute concentrations in the cytoplasmic milieu of bacteria, and are thus considered vital for bacterial survival. Exposed on the surface of gram-negative bacteria, these channel proteins are highly immunogenic and can thus be exploited as vaccine candidates. In the present study, we have cloned, characterized, and expressed outer membrane protein OmpF of Aeromonas hydrophila, a major fish pathogen and also known to cause severe infections in humans. The cloned ompF gene of A. hydrophila consisting of an open reading frame corresponding to mature OmpF was expressed and purified from the heterologous host, E. coli. High level of expression resulted in recovery of ~120 mg/L of the purified rOmpF at shake flask level. Polyclonal antisera raised against the recombinant OmpF showed a very high endpoint titer (>1:80,000) and were able to specifically agglutinate live A. hydrophila. Further, anti-OmpF antisera cross-reacted with the cell lysates of various Aeromonas isolates, suggesting that anti-rOmpF antibodies can be used to identify different A. hydrophila isolates in infected conditions. Antibody isotyping, cytokine ELISA, and ELISPOT assay indicated predominantly Th1 type of immune response. The recombinant OmpF reported in the present study thus has the potential to be used as a vaccine candidate against A. hydrophila.
KeywordMeSH Terms
127. Pridgeon  JW, Yildirim-Aksoy  M, Klesius  PH, Kojima  K, Mobley  JA, Srivastava  KK, Reddy  PG,     ( 2013 )

Identification of gyrB and rpoB gene mutations and differentially expressed proteins between a novobiocin-resistant Aeromonas hydrophila catfish vaccine strain and its virulent parent strain.

Veterinary microbiology 166 (3��4��)
PMID : 23968889  :   DOI  :   10.1016/j.vetmic.2013.07.025    
Abstract >>
A total of 10 and 13 missense mutations were found in the deduced gyrB and rpoB proteins, respectively, between avirulent AH11NOVO vaccine strain and its virulent parent strain AH11P. SDS-PAGE revealed that six proteins bands were significantly over-expressed in AH11NOVO whereas five bands were significantly over-expressed in AH11P. Mass spectrometry identified seven proteins from the over-expressed AH11NOVO gel bands and five proteins from the over-expressed AH11P gel bands. QPCR confirmed that all 12 genes corresponding to the proteins identified by mass spectrometry were significantly over-expressed in AH11NOVO or AH11P. When AH11NOVO proteins were subjected to Western blot analysis, 13 protein bands exhibited significantly stronger reactivity with hyper-immune catfish sera. Fifteen proteins were identified from immunogenic protein bands, including six (formate acetyltransferase, chaperone htpG, transketolase, ATP synthase subunit alpha, asparagine-tRNA ligase, and serine hydroxymethyltransferase) that were over-expressed in AH11NOVO proteins and three (elongation factor G, class II fructose-bisphosphate aldolase, and a putative uncharacterized 23 kDa protein) that were over-expressed in AH11P. In addition, the following six proteins were also identified from the immunogenic protein bands: pyruvate dehydrogenase E1 component, ATP synthase subunit beta, ribose-phosphate pyrophosphokinase, glyceraldehyde-3-phosphate dehydrogenase, 50S ribosomal L10, and 50S ribosomal L15. Our results might provide insights on how to develop novel efficacious vaccine against Aeromonas hydrophila infection.
KeywordMeSH Terms
Aeromonas hydrophila
Immunogen
Mass spectrometry
Virulence factor
Aeromonas hydrophila
Immunogen
Mass spectrometry
Virulence factor
128. Martino  ME, Fasolato  L, Montemurro  F, Novelli  E, Cardazzo  B,     ( 2014 )

Aeromonas spp.: ubiquitous or specialized bugs?

Environmental microbiology 16 (4)
PMID : 23919504  :   DOI  :   10.1111/1462-2920.12215    
Abstract >>
The genus Aeromonas comprises ubiquitous bacteria that are known to play several roles in the environment. These bacteria were first described as fish pathogens, but their presence was documented in other reservoirs, such as animals and humans. Today, these bacteria are described as emerging pathogens, but their effective role in human pathogenicity is still controversial. In addition, their taxonomy is heavily debated, as species distinction is often difficult to achieve. To study the interspecies relationships and to investigate their connection with the environment, a multilocus sequence typing scheme previously developed for Aeromonas spp. was applied to 258 strains, and the genetic data were analysed by population software. Sampling was a fundamental step, including several of the main sources of Aeromonas: fish, food products and human cases of disease. The objective was to characterize the isolates and to find potential associations among them according to the following: species, sharing of virulence factors, source and adaptation to a specific habitat. The strains were characterized and demonstrated exceptionally high nucleotide variability in the Aeromonas genus. Among the sampled sources, different species distributions were found, highlighting the occurrence of adaptation processes towards specific habitats.
KeywordMeSH Terms
129. Merino  S, Bouamama  L, Knirel  YA, Senchenkova  SN, Regué  M, Tomás  JM,     ( 2012 )

Aeromonas surface glucan attached through the O-antigen ligase represents a new way to obtain UDP-glucose.

PloS one 7 (5)
PMID : 22563467  :   DOI  :   10.1371/journal.pone.0035707     PMC  :   PMC3341381    
Abstract >>
We previously reported that A. hydrophila GalU mutants were still able to produce UDP-glucose introduced as a glucose residue in their lipopolysaccharide core. In this study, we found the unique origin of this UDP-glucose from a branched �\-glucan surface polysaccharide. This glucan, surface attached through the O-antigen ligase (WaaL), is common to the mesophilic Aeromonas strains tested. The Aeromonas glucan is produced by the action of the glycogen synthase (GlgA) and the UDP-Glc pyrophosphorylase (GlgC), the latter wrongly indicated as an ADP-Glc pyrophosphorylase in the Aeromonas genomes available. The Aeromonas glycogen synthase is able to react with UDP or ADP-glucose, which is not the case of E. coli glycogen synthase only reacting with ADP-glucose. The Aeromonas surface glucan has a role enhancing biofilm formation. Finally, for the first time to our knowledge, a clear preference on behalf of bacterial survival and pathogenesis is observed when choosing to produce one or other surface saccharide molecules to produce (lipopolysaccharide core or glucan).
KeywordMeSH Terms
130. Maravi?  A, Sko?ibuši?  M, Samani?  I, Fredotovi?  Z, Cvjetan  S, Jutroni?  M, Puizina  J,     ( 2013 )

Aeromonas spp. simultaneously harbouring bla(CTX-M-15), bla(SHV-12), bla(PER-1) and bla(FOX-2), in wild-growing Mediterranean mussel (Mytilus galloprovincialis) from Adriatic Sea, Croatia.

International journal of food microbiology 166 (2)
PMID : 23973842  :   DOI  :   10.1016/j.ijfoodmicro.2013.07.010    
Abstract >>
Aeromonas species are becoming renowned as emerging pathogens by increasingly giving rise to a wide spectrum of food and waterborne infections in humans. Another worrisome feature of aeromonads is the growing frequency of antibiotic resistance as a consequence of their prominent diversity in terms of resistance determinants. This study aimed at determining the antimicrobial resistance pattern, prevalence and characterization of acquired �]-lactamases, including extended-spectrum-�]-lactamases (ESBLs) and AmpC cephalosporinases, as well as the presence of class 1 and 2 integrons, in Aeromonas isolates from wild-growing Mediterranean mussel (Mytilus galloprovincialis) of the eastern coast of Adriatic Sea, Croatia. Isolates were tested for susceptibility to 16 antibiotics and �]-lactam/�]-lactamase inhibitor combinations. Cephalosporin-resistant isolates were further screened by PCR for genes encoding AmpC (bla(FOX), bla(CMY), bla(MOX), bla(LAT), bla(BIL), bla(DHA), bla(ACC), bla(MIR), bla(ACT)), ESBLs (bla(TEM), bla(SHV), bla(CTX-M), bla(PER), bla(VEB), bla(GES/IBC), bla(OXA)) and integrases (intI1, intI2, intI3). Location of bla genes was characterized by plasmid DNA fingerprinting and Southern blot hybridization. Plasmids carrying ESBL genes were investigated for transferability by conjugation and PCR-based replicon typed. Out of 147 Aeromonas isolates recovered, 30 (20%) demonstrated multiple resistance profile, with co-resistance most frequently detected against penicillins, piperacillin/sulbactam and tetracycline. ESBL-encoding genes were detected in 21 (13 Aeromonas caviae and 8 Aeromonas hydrophila) isolates, with bla(CTX-M-15) gene identified in 19 and bla(SHV-12) in 12 isolates. Among them, 10 isolates simultaneously harboured bla(CTX-M-15) and bla(SHV-12), while 3 isolates additionally carried an AmpC �]-lactamase bla(FOX-2) gene. bla(PER-1) gene was identified in a single isolate also harbouring the bla(CTX-M-15) gene. While bla(SHV-12) was chromosomally encoded, bla(CTX-M-15) was located on conjugative IncFIB-type plasmids of ~40 kb in A. caviae isolates. IntI1 and intI2 genes were detected in 57.1% and 33.3% of ESBL-producing isolates. To the best our knowledge, this is the first report of environmental A. caviae isolates producing CTX-M-15, and isolation of SHV-12-producing A. hydrophila and A. caviae strains worldwide. This is also believed to be the first report of the FOX-2, CTX-M-15 and SHV-12 simultaneous production in aeromonads, highlighting both the potential risk for human health, and a role of these foodborne pathogens as reservoirs of resistance determinants in coastal marine environment.
KeywordMeSH Terms
Aeromonads
AmpC β-lactamase
Antibiotic resistance
ESBL
Marine environment
Mussels
Aeromonads
AmpC β-lactamase
Antibiotic resistance
ESBL
Marine environment
Mussels
Aeromonads
AmpC β-lactamase
Antibiotic resistance
ESBL
Marine environment
Mussels
Aeromonads
AmpC β-lactamase
Antibiotic resistance
ESBL
Marine environment
Mussels
131. Degiacomi  MT, Iacovache  I, Pernot  L, Chami  M, Kudryashev  M, Stahlberg  H, van der Goot  FG, Dal Peraro  M,     ( 2013 )

Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism.

Nature chemical biology 9 (10)
PMID : 23912165  :   DOI  :   10.1038/nchembio.1312    
Abstract >>
Aerolysin is the founding member of a superfamily of �]-pore-forming toxins whose pore structure is unknown. We have combined X-ray crystallography, cryo-EM, molecular dynamics and computational modeling to determine the structures of aerolysin mutants in their monomeric and heptameric forms, trapped at various stages of the pore formation process. A dynamic modeling approach based on swarm intelligence was applied, whereby the intrinsic flexibility of aerolysin extracted from new X-ray structures was used to fully exploit the cryo-EM spatial restraints. Using this integrated strategy, we obtained a radically new arrangement of the prepore conformation and a near-atomistic structure of the aerolysin pore, which is fully consistent with all of the biochemical data available so far. Upon transition from the prepore to pore, the aerolysin heptamer shows a unique concerted swirling movement, accompanied by a vertical collapse of the complex, ultimately leading to the insertion of a transmembrane �]-barrel.
KeywordMeSH Terms
132. Park  SC, Chai  JY, Shin  SP, Jun  JW,     ( 2012 )

First description of ColE-type plasmid in Aeromonas spp. carrying quinolone resistance (qnrS2) gene.

Letters in applied microbiology 55 (4)
PMID : 22862417  :   DOI  :   10.1111/j.1472-765X.2012.03293.x    
Abstract >>
Isolation and full sequence analysis of ColE-type plasmid, which carries the qnrS2 gene. Quinolone resistance (qnrS2) gene-carrying plasmids were isolated from Aeromonas sobria and Aeromonas hydrophila strains, and plasmid sequencing was achieved by a primer-walking approach. The total sizes of these plasmids (pAQ2-1 and pAQ2-2) were 6900 bp and 6903 bp, respectively, and they were 99�P1% identical to each other. The genes (oriV and repA) for plasmid replication were organized similar to the corresponding genes in the ColE2-type plasmids, pAsa3 and pAsa1, isolated from Aeromonas salmonicida subsp. salmonicida, but the gene (mobA) for mobilization was homologue to ColE1-type plasmid (pAsa2) from Aer. salmonicida subsp. salmonicida. Additionally, the qnrS2 gene was part of a mobile insertion cassette element in the plasmid. Two plasmids were assumed to be the same plasmid, and this identification of a plasmid-mediated qnrS2 gene from the two different strains underlines a possible diffusion of these resistance determinants in an aquaculture system. This is the first finding of the ColE-type plasmid carrying the qnrS2 gene.
KeywordMeSH Terms
133. Takahashi  E, Kobayashi  H, Yamanaka  H, Nakanishi  M, Tateishi  A, Abe  T, Arimoto  S, Negishi  T, Okamoto  K,     ( 2013 )

Analysis of carboxy terminal domain of metalloprotease of elastolytic Aeromonas hydrophila.

Biological & pharmaceutical bulletin 36 (7)
PMID : 23811566  :   DOI  :   10.1248/bpb.b13-00161    
Abstract >>
We examined the ability of Aeromonas hydrophila to lyse elastin. Eight of 13 strains showed elastolytic activity on agar medium containing elastin and 5 strains did not. In order to examine the involvement of the metalloprotease of A. hydrophila (AMP) in elastolytic activity, we made the amp-deletion mutant strain from an elastolytic strain. The elastolytic activity of the strain decreased with this deletion. The analysis of AMP released into the culture supernatant showed that AMP appeared outside of the cell as the intermediate consisting of a mature domain and carboxy terminal (C-terminal) propeptide domain. Further analysis showed that the intermediate has the ability to lyse elastin and that loss of the C-terminal domain causes loss of the elastolytic activity of the intermediate. We then determined the nucleotide sequence of the amps of all strains used in this study. Phylogenetic analysis revealed that these AMPs were divided into three groups. The AMPs from elastolytic strains belong to group I or group II, and AMPs from non-elastolytic strains belong to group III. The distance between group I and group II is small, but group III is located separately from groups I and II. Comparison of the amino acid residues of the C-terminal domain revealed that there are 13 amino acid residues specific to the C-terminal domain of group III. This indicates that the conformation of the C-terminal propeptide domain formed by these specific amino acid residues is important for AMP to express elastolytic activity.
KeywordMeSH Terms
134. Roger  F, Marchandin  H, Jumas-Bilak  E, Kodjo  A, N/A  N/A, Lamy  B,     ( 2012 )

Multilocus genetics to reconstruct aeromonad evolution.

BMC microbiology 12 (N/A)
PMID : 22545815  :   DOI  :   10.1186/1471-2180-12-62     PMC  :   PMC3487998    
Abstract >>
Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or disease specialized" while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters."
KeywordMeSH Terms
Environmental Microbiology
Evolution, Molecular
Genetic Variation
135. Tacão  M, Correia  A, Henriques  I,     ( 2012 )

Resistance to broad-spectrum antibiotics in aquatic systems: anthropogenic activities modulate the dissemination of bla(CTX-M)-like genes.

Applied and environmental microbiology 78 (12)
PMID : 22492443  :   DOI  :   10.1128/AEM.00359-12     PMC  :   PMC3370516    
Abstract >>
We compared the resistomes within polluted and unpolluted rivers, focusing on extended-spectrum beta-lactamase (ESBL) genes, in particular bla(CTX-M). Twelve rivers from a Portuguese hydrographic basin were sampled. Physicochemical and microbiological parameters of water quality were determined, and the results showed that 9 rivers were classified as unpolluted (UP) and that 3 were classified as polluted (P). Of the 225 cefotaxime-resistant strains isolated, 39 were identified as ESBL-producing strains, with 18 carrying a bla(CTX-M) gene (15 from P and 3 from UP rivers). Analysis of CTX-M nucleotide sequences showed that 17 isolates produced CTX-M from group 1 (CTX-M-1, -3, -15, and -32) and 1 CTX-M that belonged to group 9 (CTX-M-14). A genetic environment study revealed the presence of different genetic elements previously described for clinical strains. ISEcp1 was found in the upstream regions of all isolates examined. Culture-independent bla(CTX-M)-like libraries were comprised of 16 CTX-M gene variants, with 14 types in the P library and 4 types in UP library, varying from 68% to 99% similarity between them. Besides the much lower level of diversity among CTX-M-like genes from UP sites, the majority were similar to chromosomal ESBLs such as bla(RAHN-1). The results demonstrate that the occurrence and diversity of bla(CTX-M) genes are clearly different between polluted and unpolluted lotic ecosystems; these findings favor the hypothesis that natural environments are reservoirs of resistant bacteria and resistance genes, where anthropogenic-driven selective pressures may be contributing to the persistence and dissemination of genes usually relevant in clinical environments.
KeywordMeSH Terms
Drug Resistance, Bacterial
Water Microbiology
136. Pan  ZH, Lu  CP,     ( 2012 )

Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China.

Letters in applied microbiology 55 (3)
PMID : 22725694  :   DOI  :   10.1111/j.1472-765X.2012.03281.x    
Abstract >>
To investigate the species distribution in Aeromonas isolates from diseased fish, healthy controls and water environment in China; to evaluate the frequency of the aerolysin (aer), cytotonic enterotoxin (alt), cytotoxic enterotoxin (act), temperature-sensitive protease (eprCAI) and serine protease (ahp) genes in Aeromonas isolates; and to determine the potential pathogenicity of these isolates. Two hundred and two Aeromonas isolates from diseased fish (n = 42), healthy fish (n = 120) and water environment (n = 40) in China were identified to species levels based on sequencing of the housekeeping gene gyrB, while the distribution of five virulence factors, including aer, alt, act, eprCAI and ahp, was investigated by PCR. Aeromonas veronii (25/42; 60%) and Aeromonas hydrophila (14/42; 33%) were the species most commonly isolated from diseased fish, while Aer. veronii was the most common species in healthy fish (90/120; 75%) and water samples (25/40; 62�P5%). All the five virulence genes were present in 9% (19/202), among which 10 strains were from diseased fish and nine were identified as Aer. hydrophila. For the strains carrying five virulence genes, the average 50% lethal doses (LD(50s)) of strains from diseased fish were lower when compared with the strains from healthy fish and water environment. Aeromonas veronii is the most common species, but no significant difference exists in the isolates obtained from diseased fish and from healthy fish. However, Aer. hydrophila isolates were significantly more frequent from diseased fish than from healthy fish. aer+alt+act+eprCAI+ ahp+ was more frequent virulence genotype in Aeromonas isolates from diseased fish than from healthy fish and water environment, and the aer+alt+act+eprCAI+ahp+ isolates were more virulent to zebrafish comparing to the other genetic profiles. Aeromonas species in aquatic environments are various and have considerable virulence potential, and therefore, there is a need for more careful and intensive epidemiology studies.
KeywordMeSH Terms
Water Microbiology
137. Hilton  S, Buckley  JT, Kay  CM,     ( 1990 )

Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis.

Biochemistry 29 (38)
PMID : 2271578  :   DOI  :   10.1021/bi00490a026    
Abstract >>
A fatty acyltransferase with a reaction mechanism similar to that of mammalian lecithin: cholesterol acyltransferase has been purified from culture supernatants of a mutant Aeromonas salmonicida containing the cloned Aeromonas hydrophila structural gene. Typically, more than 35 mg of protein were isolated from 2 L of culture supernatant. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein corresponded to predictions based on the sequence of the gene, indicating that the signal sequence had been correctly removed during export but that no further processing had occurred. Analysis of the far-UV circular dichroic (CD) spectrum of the enzyme showed that it consists of 31% alpha-helix, 21% beta-sheet, and 16% beta-turn, with 12% of aperiodic form. Treatment of the purified protein with a variety of proteases resulted in nicking near the C-terminus. This led to an increase in enzyme activity against lipids in erythrocyte membranes and increased rate of hydrolysis of p-nitrophenyl butyrate. Activation was accompanied by a change in the CD spectrum and a change in its aggregation state. The trypsin cut site was located between the two cysteines in the enzyme. Evidence is presented that the cysteines are joined by a disulfide bond and therefore cannot participate in acyl transfer. This may distinguish the microbial enzyme from lecithin:cholesterol acyltransferase. This is the second extracellular A. hydrophila protein that we have shown can be activated by proteolysis after it is released.
KeywordMeSH Terms
138. Yeh  HY, Klesius  PH,     ( 2011 )

Over-expression, purification and immune responses to Aeromonas hydrophila AL09-73 flagellar proteins.

Fish & shellfish immunology 31 (6)
PMID : 21963857  :   DOI  :   10.1016/j.fsi.2011.09.016    
Abstract >>
Aeromonas hydrophila is ubiquitous in aquatic environments worldwide and causes many diseases in fish as well as human. Recent outbreaks of aeromonad diseases in channel catfish prompted us to investigate catfish immune responses during infection of A. hydrophila. In this communication, we report to amplify, over-express, purify and characterize 19 A. hydrophila flagellar proteins. All recombinant proteins were confirmed by nucleotide sequencing of expression plasmids, SDS-PAGE analysis and His tag Western blot of induced proteins. Our preliminary result also showed that the purified recombinant FlgK protein reacted strongly to sera from experimentally infected catfish, suggesting that this protein has potential for a novel target for vaccine development. It is also anticipated that these recombinant proteins will provide us with very useful tools to investigate host immune response to this microorganism.
KeywordMeSH Terms
Ictaluridae
139. Majumdar  T, Das  B, Bhadra  RK, Dam  B, Mazumder  S,     ( 2011 )

Complete nucleotide sequence of a quinolone resistance gene (qnrS2) carrying plasmid of Aeromonas hydrophila isolated from fish.

Plasmid 66 (2)
PMID : 21664930  :   DOI  :   10.1016/j.plasmid.2011.05.001    
Abstract >>
Aeromonas hydrophila strain AO1 isolated from an infected fish was found to be resistant to several quinolones. A plasmid isolated from the strain AO1, termed pBRST7.6, was cloned and sequenced and shown to be 7621bp in length with a GC content of 60%. Further analysis confirmed that it contained a gene with 100% identity to qnrS2 genes described in plasmids associated with other Aeromonas species, the product of which usually confers increased resistance to quinolones. The plasmid backbone contained a replication initiation module (repA repC) belonging to the IncQ-family and two genes (mobC and mobB), the products of which are putatively involved in plasmid mobilization. Putative iteron-based origin of replication and characteristic oriT like sequences were also present in the plasmid. The result suggests that Aeromonas spp. carrying plasmids with quinolone resistance genes are potential reservoirs of antimicrobial resistance determinants in the environment.
KeywordMeSH Terms
Plasmids
140. Han  JE, Kim  JH, Cheresca  CH, Shin  SP, Jun  JW, Chai  JY, Han  SY, Park  SC,     ( 2012 )

First description of the qnrS-like (qnrS5) gene and analysis of quinolone resistance-determining regions in motile Aeromonas spp. from diseased fish and water.

Research in microbiology 163 (1)
PMID : 22024339  :   DOI  :   10.1016/j.resmic.2011.09.001    
Abstract >>
Antimicrobial resistance patterns in a collection of 33 motile Aeromonas species were described in this study. Quinolone has been frequently employed for treatment of Aeromonas-related diseases, and prolonged use of antimicrobial compounds has led to development of resistant strains. In a sample of diseased fish and environmental water, we evaluated nalidixic acid (n = 19) and ciprofloxacin (n = 4) resistance via minimum inhibitory concentration (MIC) assays and the genetic basis was also investigated. Among the isolated Aeromonas spp., 17 strains encoded for chromosomal mutations of quinolone resistance-determining regions (QRDRs) in gyrA, 11 strains encoded for mutations of QRDRs in parC, 1 strain harbored plasmid-mediated quinolone resistance (PMQR) qnrS1-like gene and 4 strains harbored the PMQR qnrS2 gene. In particular, the new variant (qnrS1-like) differed from qnrS1 by 6 amino acid substitutions at positions 5 (Asn(5)��Arg(5)), 120 (Ser(120)��Thr(120)), 148 (Asn(148)��His(148)), 206 (Leu(206)��Glu(206)), 207 (Ile(207)�� Leu(207)), and 216 (Tyr(216)��Phe(216)), and the gene was designated qnrS5. These resistant strains may function as reservoirs of quinolone resistance.
KeywordMeSH Terms
Drug Resistance, Bacterial
141. Han  JE, Kim  JH, Choresca  CH, Shin  SP, Jun  JW, Chai  JY, Park  SC,     ( 2012 )

A small IncQ-type plasmid carrying the quinolone resistance (qnrS2) gene from Aeromonas hydrophila.

Letters in applied microbiology 54 (4)
PMID : 22260532  :   DOI  :   10.1111/j.1472-765X.2012.03208.x    
Abstract >>
To investigate the qnrS2 gene encoded by a plasmid obtained from Aeromonas hydrophila. To investigate the full-length sequence of the plasmid carrying qnrS2 (plasmid designated pAHH04) from the strain SNUFPC-A10, the full-length coding sequence of the qnrS region was first amplified. The remaining part of the plasmid was read outwards from this region. The plasmid pAHH04 contained the repC, repA, mobA and mobC genes, and its total size was 7191 bp with a G+C content of 60%. This study describes the full-length sequence of a plasmid carrying the qnrS2 gene from Aer. hydrophila. The plasmid pAHH04 carried plasmid replication and mobilization genes from IncQ-type plasmids. The isolated qnrS2 gene encoded by a plasmid from an Aer. hydrophila strain is of significant importance because it emphasizes the problem of antibiotic resistance as well as the ability of the determinants to spread among the different bacterial species that impact human health.
KeywordMeSH Terms
Plasmids
142. Khajanchi  BK, Kozlova  EV, Sha  J, Popov  VL, Chopra  AK,     ( 2012 )

The two-component QseBC signalling system regulates in vitro and in vivo virulence of Aeromonas hydrophila.

Microbiology (Reading, England) 158 (Pt 1)
PMID : 21998161  :   DOI  :   10.1099/mic.0.051805-0     PMC  :   PMC3352359    
Abstract >>
We recently demonstrated that the N-acyl-homoserine lactone [autoinducer (AI)-1] and LuxS (AI-2)-based quorum-sensing (QS) systems exerted positive and negative regulation, respectively, on the virulence of a diarrhoeal isolate SSU of Aeromonas hydrophila. However, the role of a newly identified, two-component-based QseBC QS system in the regulation of bacterial virulence in general is not well understood, with only a limited number of studies showing its function in bacterial pathogenesis. In this report, we identified and characterized the QseBC QS system in A. hydrophila SSU and found that, as was the case with enterohaemorrhagic Escherichia coli, the open reading frames for the qseB (the response regulator) and qseC (the sensor histidine kinase) genes overlapped by 4 bp at the ATGA motif. Our data provide evidence that deletion of the qseB gene from A. hydrophila resulted in attenuation of bacterial virulence in a septicaemic mouse model of infection and diminished swimming and swarming motility, and the mutant bacteria formed denser biofilms compared with those from the parental strain of A. hydrophila. The decrease in the virulence of the A. hydrophila �GqseB mutant correlated with reduced production of protease and the cytotoxic enterotoxin, which has associated haemolytic activity. The swimming and swarming motility, haemolytic activity, protease production and biofilm formation were restored in the qseBC-complemented strain to a level similar to that of the wild-type A. hydrophila SSU. Our study is the first, to our knowledge, to report a functional QseBC QS system in A. hydrophila which may be linked to AI-1 and AI-2 QS systems in modulating bacterial virulence, possibly through the cyclic diguanosine monophosphate.
KeywordMeSH Terms
Quorum Sensing
143. Vilches  S, Jiménez  N, Merino  S, Tomás  JM,     ( 2012 )

The Aeromonas dsbA mutation decreased their virulence by triggering type III secretion system but not flagella production.

Microbial pathogenesis 52 (2)
PMID : 22198000  :   DOI  :   10.1016/j.micpath.2011.10.006    
Abstract >>
Pathogenesis of Aeromonas species have been reported to be associated with virulence factors such as lipopolysaccharides (LPS), bacterial toxins, bacterial secretion systems, flagella, and other surface molecules. Dsb (Disulfide bond) proteins play an important role in catalyzing disulfide bond formation in proteins within the periplasmic space. An A. hydrophila dsbA mutant with attenuated virulence using Dictyostelium amoebae as an alternative host model was identified. The attenuated virulence was tested in other animal models (by intraperitoneal injection in fish and mice) and was correlated with the presence of a defective type III secretion system for the first time in non enteric bacteria. The dsbA mutation was shown in several enteric bacteria to involve the outer membrane secretin. The defect in Aeromonas also seems to involve the outer membrane secretin homologue named AscC. However, unlike what happen in Escherichia coli, no changes in motility or flagella expression were observed for A. hydrophila dsbA mutants. The loss of E. coli motility caused by deletion of dsbA is likely due to defective disulfide bond formation in FlgI, a component of the flagella. No disulfide bond formation in FlgI homologues in Aeromonas flagella biogenesis, either polar or lateral, could be expected according to their amino acid residues sequences.
KeywordMeSH Terms
Mutation
144.     ( 1997 )

Cloning and characterization of two immunophilin-like genes, ilpA and fkpA, on a single 3.9-kilobase fragment of Aeromonas hydrophila genomic DNA.

Journal of bacteriology 179 (11)
PMID : 9171380  :   DOI  :   10.1128/jb.179.11.3397-3403.1997     PMC  :   PMC179128    
Abstract >>
Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a previous study (C. Y. F. Wong, G. Mayrhofer, M. W. Heuzenroeder, H. M. Atkinson, D. M. Quinn, and R. L. P. Flower, FEMS Immunol. Med. Microbiol. 15:233-241, 1996) to protect mice against lethal infection by this organism. In this study, colony blot analysis of an A. hydrophila genomic library using antiserum to A. hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been described previously in aeromonads. The nucleotide sequence of a 3.9-kb fragment derived from this cosmid which expressed the 30-kDa protein revealed two potential open reading frames (ORFs) with homology to known immunophilin proteins. ORF1 encoded a 212-amino-acid protein (molecular mass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of Escherichia coli. ORF1 was subsequently designated ilpA (immunophilin-like protein). ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa. The inferred amino acid sequence showed 46% identity with the sequence of the FkpA protein of E. coli and 40% identity with the sequence of the macrophage infectivity potentiator (Mip) protein of Legionella pneumophila. ORF3 was designated fkpA (FK506 binding protein) by analogy with the E. coli FkpA protein. Expression of the FkpA protein was confirmed by Western blot (immunoblot) analysis, which detected a 30-kDa protein, with antiserum to the Mip protein of Legionella longbeachae and a specific antiserum to anA. hydrophila 30-kDa membrane protein. PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested. A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase. This suggests that either the fkpA gene is not essential in the virulence of A. hydrophila under these conditions or there are other genes in A. hydrophila coding for proteins with similar functions.
KeywordMeSH Terms
Genes, Bacterial
Genome, Bacterial
Immunophilins
Peptidylprolyl Isomerase
145.     ( 1997 )

Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules.

Journal of bacteriology 179 (17)
PMID : 9286976  :   DOI  :   10.1128/jb.179.17.5271-5281.1997     PMC  :   PMC179392    
Abstract >>
Spent culture supernatants from both Aeromonas hydrophila and Aeromonas salmonicida activate a range of biosensors responsive to N-acylhomoserine lactones (AHLs). The genes for a quorum sensing signal generator and a response regulator were cloned from each Aeromonas species and termed ahyRI and asaRI, respectively. Protein sequence homology analysis places the gene products within the growing family of LuxRI homologs. ahyR and asaR are transcribed divergently from ahyI and asaI, respectively, and in both Aeromonas species, the genes downstream have been identified by DNA sequence and PCR analysis. Downstream of both ahyI and asaI is a gene with close homology to iciA, an inhibitor of chromosome replication in Escherichia coli, a finding which implies that in Aeromonas, cell division may be linked to quorum sensing. The major signal molecule synthesized via both AhyI and AsaI was purified from spent culture supernatants and identified as N-(butanoyl)-L-homoserine lactone (BHL) by thin-layer chromatography, high-pressure liquid chromatography analysis, and mass spectrometry. In addition, a second, minor AHL, N-hexanoyl-L-homoserine lactone, was identified. Transcriptional reporter studies with ahyI::luxCDABE fusions indicate that AhyR and BHL are both required for ahyI transcription. For A. salmonicida, although the addition of exogenous BHL gives only a small stimulation of the production of serine protease with comparison to the control culture, the incorporation of a longer-chain AHL, N-(3-oxodecanoyl)-L-homoserine lactone, reduced the final level (by approximately 50%) and delayed the appearance (from an A650 of 0.9 in the control to an A650 of 1.2 in the test) of protease in the culture supernatant. These data add A. hydrophila and A. salmonicida to the growing family of gram-negative bacteria now known to control gene expression through quorum sensing.
KeywordMeSH Terms
Escherichia coli Proteins
Repressor Proteins
Trans-Activators
146.     ( 1997 )

Cloning and sequence analysis of the gene (eprA1) encoding an extracellular protease from Aeromonas hydrophila.

Gene 199 (1��2��)
PMID : 9358060  :   DOI  :   10.1016/s0378-1119(97)00371-5    
Abstract >>
A gene (eprA1) encoding the extracellular protease of Aeromonas hydrophila AH1 has been cloned and sequenced. Nucleotide sequence analysis of eprA1 predicted a single open reading frame (ORF) of 1038 bp encoding a 346 amino acid (aa) polypeptide, with a potential 21-aa signal peptide. When the eprA1 gene was expressed in minicells, one major band of approx. 37 kDa was identified, while protease activity staining experiments identified a caseinolytic band of approx. 29 kDa determined by SDS-PAGE analysis of the minicells. The deduced C-terminal aa region (Arg-290 to Gly-313) showed sequence homology to partial C-terminal sequences of other zinc metalloproteases including Penicillium citrinum metalloprotease (PlnC), Aspergillus oryzae metalloprotease (NpII), Aspergillus flavus metalloprotease (MepA), and Aspergillus fumigatus metalloprotease (Mep20), particularly with respect to zinc-binding residues.
KeywordMeSH Terms
Bacterial Proteins
147.     ( 1997 )

Molecular analysis and expression of the extracellular lipase of Aeromonas hydrophila MCC-2.

Microbiology (Reading, England) 143 (Pt 3) (N/A)
PMID : 9084164  :   DOI  :   10.1099/00221287-143-3-803    
Abstract >>
The structural gene encoding the extracellular lipase of Aeromonas hydrophila MCC-2 was cloned and found to be expressed in Escherichia coli using its own promoter. When the cloned gene (lip) was expressed in E. coli minicells, an 80 kDa protein was identified. Subcellular fractionation of E. coli carrying the lip gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79-9 kDa protein with an estimated pl of 10.36. The deduced protein contains two putative signal peptide cleavage sites: one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [3H]palmitate was incorporated into the Lip protein when expressed in E. coli. The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from A. hydrophila strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both A. hydrophila and recombinant E. coli. In hydrolysis of p-nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C10 to C12 for ester hydrolysis and C8 to C10 for triacylglycerol hydrolysis.
KeywordMeSH Terms
Gene Expression Regulation, Bacterial
Genes, Bacterial
148.     ( 1996 )

Comparative analysis of the cya locus in enterobacteria and related gram-negative facultative anaerobes.

Biochimie 78 (4)
PMID : 8874804  :   DOI  :   10.1016/0300-9084(96)82192-4    
Abstract >>
Comparison of the cya loci (cya codes for adenylyl cyclase (AC)) from a variety of phylogenetically divergent facultative anaerobic Gram-negative bacteria reveals conserved sequence features. The entire locus structure in enterobacteria is preserved, including two major promoters (a conserved cya strong promoter, P2, and a divergent promoter for a heme biosynthetic operon, hemCD) present in the upstream region of the cya gene. The region between hemC and cya is much longer in Proteus mirabilis than in other enterobacteria, and lacks the P1 upstream cya promoter. In Aeromonas hydrophila the cya promoter (the strong P2 promoter in E coli) is preserved, including a putative GATC methylation site situated immediately downstream from the -10 box. Each cya frame analyzed uses TTG as the translation start codon and is preceded by an unusual ribosome binding site. This suggests that a lower translation efficiency of the cya transcript could be the result of some selection pressure. This has been substantiated by in vitro mutagenesis and by selection of up mutations which all map at the cya ribosome binding site. In enterobacteria the cyaY frame is the only conserved reading frame downstream of cya, with the orientation opposite to that of cya. This organization is not preserved in Aeromonas. Experiments involving fusions with the lacZ gene demonstrated that cyaY is expressed. Finally, comparison of the different polypeptide sequences of ACs permits discussion of important features of the catalytic and regulatory centers of the protein.
KeywordMeSH Terms
149.     ( 1996 )

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion.

Molecular microbiology 19 (4)
PMID : 8820654  :   DOI  :   10.1046/j.1365-2958.1996.431958.x    
Abstract >>
Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.
KeywordMeSH Terms
Fimbriae Proteins
Genes, Bacterial
Oxidoreductases
150.     ( 1996 )

Cloning, sequencing, and characterization of the nucH gene encoding an extracellular nuclease from Aeromonas hydrophila JMP636.

Journal of bacteriology 178 (13)
PMID : 8682799  :   DOI  :   10.1128/jb.178.13.3926-3933.1996     PMC  :   PMC232655    
Abstract >>
An Escherichia coli clone expressing activity on DNase agar was obtained by cloning chromosomal DNA of Aeromonas hydrophila JMP636 into plasmid pUC19. Examination (of the clone's nuclease activity on a sodium dodecyl sulfate (SDS)-polyacrylamide gel containing DNA as a substrate revealed an activity band at approximately 100 kDa. Subsequently, subcloning localized the gene, designated nucH, to a 3.6-kb DNA fragment (pJP9521). Southern blotting of the nucH gene against chromosomal DNA of JMP636 confirmed that it had originated from this strain and demonstrated that it was present in a single copy, although additional faint bands were also detected. Analysis of the subclone using in vivo transcription and translation revealed only a single polypeptide of approximately 110 kDa. Sequencing of pJP9521 predicted an open reading frame of 3,213 bp encoding a protein of 1,070 amino acids and having a molecular mass of 114 kDa. Comparison of the deduced nucleotide sequence and the NucH predicted protein sequence with relevant databases indicated that no known homologs have previously been identified. A signal sequence was predicted from these data, and cellular fractionation of a nucH clone in E. coli indicated that the protein was able to be processed to the periplasm. An activity similar in size was detected in an extracellular protein sample of JMP636, while inactivation of the nucH gene resulted in loss of this activity band. By native SDS-polyacrylamide gel electrophoresis, NucH substrate specificity, cofactor requirements, and sensitivity to denaturing agents were assessed.
KeywordMeSH Terms
151.     ( 1996 )

Study of the intergenic exeF-exeG region and its application as a simple preliminary test for Aeromonas spp.

FEMS microbiology letters 137 (1)
PMID : 8935655  :   DOI  :   10.1111/j.1574-6968.1996.tb08079.x    
Abstract >>
The exeF-exeG intergenic regions from different hybridization groups (HG) of Aeromonas were studied by PCR amplification using a single pair of primers. Six main classes of PCR products were identified according to size: 360 bp, 320 bp, 280 bp, 230-240 bp, 220 bp and 160 bp. Direct sequencing of the PCR products indicated that the shorter intergenic regions had probably originated from deletion of DNA segments between direct repeats. Correlation of certain PCR products with Aeromonas caviae (HG4), A. caviae (HG5), A. veronii (HG8) and A. salmonicida (HG3) was revealed. The PCR reaction was also shown to be generally specific for Aeromonas spp. Thus, the usefulness of this rapid, single colony-based PCR test for both identification and preliminary differentiation of Aeromonas spp. is demonstrated.
KeywordMeSH Terms
152.     ( 1995 )

Protonation of histidine-132 promotes oligomerization of the channel-forming toxin aerolysin.

Biochemistry 34 (50)
PMID : 8845373  :   DOI  :   10.1021/bi00050a028    
Abstract >>
Aerolysin is a bacterial toxin that binds to a receptor on eukaryotic cells and oligomerizes to form stable, SDS-resistant, noncovalent oligomers that insert into the plasma membrane and produce well-defined channels. Little is known about the mechanisms controlling this process. Here we show that the protonation of a single histidine is required for oligomerization of aerolysin in solution. First we have investigated the effect of pH on the activity of aerolysin. The toxin's ability to disrupt human erythrocytes declined as the pH increased above 7.4. Experiments with receptor-free planar lipid bilayers demonstrated that the rate at which aerolysin formed channels also decreased with increasing pH, although the conductance of preexisting channels was not affected. The reduction in the rate of channel formation was shown to be due to a decrease in the toxin's ability to oligomerize. Our data indicate that the pH effect on activity is due to the deprotonation of a single residue rather than a global effect of pH on the protein. In agreement with our previous site-directed mutagenesis studies, His-132 is most likely to be the target of this pH effect. This conclusion was reinforced by the fact that we could shift the pH dependence of the activity to lower pH values by mutating Asp-139, a residue less than 3 A away from His-132 and likely to contribute to the usually high pKa of this histidine.
KeywordMeSH Terms
153.     ( 1993 )

Isolation and analysis of eight exe genes and their involvement in extracellular protein secretion and outer membrane assembly in Aeromonas hydrophila.

Journal of bacteriology 175 (20)
PMID : 8407845  :   DOI  :   10.1128/jb.175.20.6695-6703.1993     PMC  :   PMC206782    
Abstract >>
The exeE gene of Aeromonas hydrophila has been shown to be required for the secretion of most if not all of the extracellular proteins produced by this bacterium. In addition, an exeE::Tn5-751 insertion mutant of A. hydrophila was found to be deficient in the amounts of a number of its major outer membrane proteins (B. Jiang and S. P. Howard, J. Bacteriol. 173:1241-1249, 1991). The exeE gene and the exeF gene were previously isolated as part of a fragment which complemented this mutant. In this study, we have isolated and sequenced a further eight exe genes, exeG through exeN, which constitute the 3' end of the exe operon. These genes have a high degree of similarity with the extracellular secretion operons of a number of different gram-negative bacteria. Marker exchange mutagenesis was used to insert kanamycin resistance cassettes into three different regions of the exe operon. The phenotypes of these mutants showed that in A. hydrophila this operon is required not only for extracellular protein secretion but also for normal assembly of the outer membrane, in particular with respect to the quantities of the major porins. Five of the Exe proteins contain type IV prepilin signal sequences, although the prepilin peptidase gene does not appear to form part of the exe operon. Limited processing of the ExeG protein was observed when it was expressed in Escherichia coli, and this processing was greatly accelerated in the presence of the prepilin peptidase of Pseudomonas aeruginosa.
KeywordMeSH Terms
Genes, Bacterial
154.     ( 1996 )

Molecular and biochemical characterization of a heat-labile cytotonic enterotoxin from Aeromonas hydrophila.

Microbial pathogenesis 21 (5)
PMID : 8938643  :   DOI  :   10.1006/mpat.1996.0068    
Abstract >>
We report herein the DNA sequence analysis of the heat-labile cytotonic enterotoxin gene (alt) from Aeromonas hydrophila and the biological function of the purified hyperproduced toxin (Alt). One large open-reading frame (ORF), comprised of 1104 bp, was detected at positions 804 to 1907 bp on a 4.0-kb Sa/l DNA fragment from Aeromonas. This ORF encodes for a protein having 368 amino acids (aa) with a computed molecular weight of 38 kDa. The aa sequence of the first 15 NH2-terminal residues of the mature native Alt from A. hydrophila matched with the DNA-derived aa sequence of the alt gene expressed in E. coli starting at position 19, which was leucine. The first 18aa residues of the Alt represented a putative signal sequence with alanine at its carboxy terminus. A BLAST search of the entire database showed 45-51% identity of the Alt, starting at position 158 with the carboxy half of the phospholipase C (PLC) and lipase from A. hydrophila; however, the purified Alt had no lipase/PLC activity. The alt gene was hyperexpressed using gene fusion expression vector systems, and the recombinant Alt exhibited a size of 35-40 kDa. The pure recombinant Alt elongated Chinese hamster ovary cells and elicited fluid secretion in rats ligated intestinal loops, indicating its enterotoxicity. Immunization of mice with recombinant Alt resulted in a reduced fluid secretory response when challenged with Aeromonas. The biological activity of the recombinant Alt in E. coli was about 10-fold less, compared to native Alt from Aeromonas, indicating differential processing of the toxin. The antibodies to native Alt neutralized the biological activity of the recombinant toxin, and these antibodies reacted with the same specificity to the native and recombinant Alt in immunoblots. The role of cyclic adenosine monophosphate and prostaglandins in causing a fluid secretory response by Alt also was demonstrated.
KeywordMeSH Terms
Bacterial Toxins
Enterotoxins
155.     ( 1993 )

Cloning, expression, and sequence analysis of a cytolytic enterotoxin gene from Aeromonas hydrophila.

Canadian journal of microbiology 39 (5)
PMID : 8330262  :   DOI  :   10.1139/m93-073    
Abstract >>
The structural gene and regulatory element for a cytolytic enterotoxin of a diarrheal isolate, SSU, of Aeromonas hydrophila was cloned and its DNA sequence was determined. A complementary, mixed synthetic oligonucleotide based on the first 10 NH2-terminal amino acid residues of the Aeromonas cytolytic enterotoxin was used as a probe to screen a genomic library constructed in bacteriophage EMBL3. Cell lysates of Escherichia coli (lambda CH4), containing the cytolytic enterotoxin gene, lysed rabbit red blood cells and destroyed Chinese hamster ovary cells, caused fluid secretion in rat ileal loops, and were lethal to mice when injected intravenously. All biological activities associated with the cytolytic enterotoxin were neutralized by rabbit homologous polyclonal antibodies. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent Western blot analysis of the cell lysate of E. coli (lambda CH4) revealed a protein band of approximately 52 kDa, using antisera to the cytolytic enterotoxin or antibodies generated against a synthetic peptide to the toxin. DNA sequence analysis of a 2.8-kb SalI-BamHI fragment revealed the presence of one large open reading frame (1479 bp) that would encode a protein of 54.5 kDa, a precursor form of the cytolytic enterotoxin, with a 23 amino acid leader peptide. Despite a significant amount of homology at the DNA and amino acid levels between our cytolytic enterotoxin and two aerolysins of Aeromonas species, variation in the restriction maps of these three toxin genes was prominent. Likewise, considerable divergence in DNA sequence was observed upstream of the structural genes for the reported aerolysins and our cytolytic enterotoxin, suggesting that these structurally similar toxin molecules may be regulated differently. Finally, our data showed that the cytolytic enterotoxin from a diarrheal isolate, SSU, of A. hydrophila exhibited characteristics that were unique compared with those of the reported aerolysins.
KeywordMeSH Terms
Bacterial Proteins
156.     ( 1993 )

Prolyl endopeptidase from Aeromonas hydrophila: cloning, sequencing, and expression of the enzyme gene, and characterization of the expressed enzyme.

Journal of biochemistry 113 (6)
PMID : 8370677  :   DOI  :   10.1093/oxfordjournals.jbchem.a124120    
Abstract >>
A strain of Aeromonas hydrophila was found to show prolyl endopeptidase activity. The enzyme gene was cloned and expressed in Escherichia coli JM83. A 12 kbp EcoRI fragment containing the enzyme gene was subcloned at the HincII site of pUC19 to construct plasmid pAPEP-3 with a 3.5 kbp insert. E. coli JM83 transformed with this plasmid showed about 100-fold higher activity than the parent Aeromonas. Analysis of the nucleotide sequence of the insert revealed that the mature enzyme-encoding sequence starts just after the ATG initiation codon of the open reading frame. The enzyme was a single polypeptide composed of 689 amino acid residues with a molecular weight of 76,383. It showed properties very similar to those of Flavobacterium prolyl endopeptidase, except that the isoelectric point was 5.5. The amino acid sequence was 56 and 41% homologous to those of Flavobacterium and porcine brain prolyl endopeptidases, respectively. From a survey of sequence homology with other members of the prolyl endopeptidase family, the amino acid residues involved in the catalytic triad were deduced to be Ser-537, His-656, and Asp-512 (or Asp-621).
KeywordMeSH Terms
Genes, Bacterial
157.     ( 1994 )

Influence of active site and tyrosine modification on the secretion and activity of the Aeromonas hydrophila lipase/acyltransferase.

The Journal of biological chemistry 269 (3)
PMID : 8294469  :  
Abstract >>
Aeromonas sp. secrete a lipase/acyltransferase that shares several properties with the mammalian plasma enzyme lecithin:cholesterol acyltransferase. Reaction of the enzyme with tetranitromethane led to modification of 2 tyrosines and a nearly 80% decline in enzyme activity. Replacing Tyr230 with Phe altered the activity of the enzyme in the same way as did treatment with tetranitromethane. Unlike the wild type enzyme, which preferentially hydrolyzes the 2-position acyl chain of phosphatidylcholine, the Y230F mutant enzyme did not discriminate between the 1- and 2-positions of the phospholipid. Tyr230 may be necessary to correctly position phospholipid substrates at the active site. Several amino acids around the active site Ser16 of the lipase were also changed. Replacing Ser18 with Gly, bringing the enzyme's sequence into line with the "lipase consensus sequence," resulted in reduced secretion of the protein and complete loss of activity. Changing this serine to Val led to an inactive protein that was not secreted at all. Substituting Phe13 in the hydrophobic region of the consensus sequence with Ser also prevented secretion, although the mutant protein appeared to be active. The Aeromonas lipase may represent a distinct group of lipolytic enzymes which have a novel active site structure.
KeywordMeSH Terms
Tyrosine
158.     ( 1995 )

Purification and characterization of a CHO cell-elongating toxin produced by Aeromonas hydrophila.

Microbial pathogenesis 19 (1)
PMID : 8559035  :   DOI  :   10.1006/mpat.1995.0039    
Abstract >>
A Chinese hamster ovary (CHO) cell-elongating toxin produced by Aeromonas hydrophila was purified from cell-free supernatant fluids by ammonium sulfate precipitation and fast protein liquid chromatography. The purified toxin had an isolelectric point (pl) of 3.7 and a molecular weight of 70,000 in a single band on isoelectric focusing (IEF) gels and SDS-PAGE gels, respectively. The N-terminal sequence, amino acids 1-20, and the amino acid content were determined from Western blots of the 70 kDa band. No homology with any known microbial toxin was found. CHO cell activity was not neutralized by antiserum to cholera toxin (anti-CT), and the toxin did not react with anti-CT on Western blots. The toxin did not increase cyclic AMP, cyclic GMP, or prostaglandin E2 levels in CHO cells. No cytotoxic activity was observed. Intragastric administration of purified toxin (5 x 10(4) and 5 x 10(8) CHO cell units) induced intestinal fluid accumulation in infant mice. These results suggest that this toxin may be a novel cytotonic toxin distinct from previously described toxins produced by A. hydrophila or A. sobria.
KeywordMeSH Terms
159.     ( 1993 )

Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from an Aeromonas hydrophila human isolate.

Applied and environmental microbiology 59 (8)
PMID : 8368830  :   PMC  :   PMC182299    
Abstract >>
A structural gene which codes for an extracellular lipase (EC 3.1.1.3) in Aeromonas hydrophila H3, which was isolated from a female hospitalized patient, was cloned in Escherichia coli by using pBR322 as a vector. Lipase purified from both A. hydrophila culture supernatant and the periplasmic fluids of E. coli containing the lip determinant in the original clone (plasmid pLA2) showed an M(r) of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the M(r) determined by Sephacryl S-200 chromatography. Regarding substrate specificity, the optimum chain lengths for the acyl moiety were C6 for ester hydrolysis and C6 and C8 for triacylglycerol hydrolysis. Sequence analysis showed a major open reading frame of 2,052 bp, which predicts a polypeptide with an M(r) of 71,804. The polypeptide was found to contain an amino acid sequence (V-H-F-L-G-H-S-L-G-A) which is highly preserved among lipases.
KeywordMeSH Terms
Genes, Bacterial
160.     ( 1993 )

Cloning and nucleotide sequence of an extracellular alpha-amylase gene from Aeromonas hydrophila MCC-1.

Journal of general microbiology 139 (12)
PMID : 8126440  :   DOI  :   10.1099/00221287-139-12-3215    
Abstract >>
A gene encoding the extracellular alpha-amylase of Aeromonas hydrophila MCC-1 was cloned and expressed using its own promoter on the recombinant plasmid pCA101. Subcellular fractionation of Escherichia coli JA221 carrying pCA101 revealed that approximately 60% of the amylase activity was localized in the periplasmic space. The extracellular amylase was purified to homogeneity, identified as an alpha-type and its amino-terminal sequence was determined. Nucleotide sequence analysis predicted a 443 amino acid ORF and 24 amino acids at the amino terminus of the sequence that are not found in the secreted protein. This 24 amino acid sequence has many of the characteristics common to known signal peptides. The predicted amino acid sequence has considerable similarity with mammalian, invertebrate and Streptomycete alpha-amylases. Most of the amino acid residues that are involved in catalytic activity, substrate binding and calcium binding in several alpha-amylases were also present in A. hydrophila alpha-amylase at the corresponding positions.
KeywordMeSH Terms
Genes, Bacterial
161.     ( 1994 )

Isolation and characterization of a second exe operon required for extracellular protein secretion in Aeromonas hydrophila.

Journal of bacteriology 176 (22)
PMID : 7961440  :   DOI  :   10.1128/jb.176.22.6819-6826.1994     PMC  :   PMC197049    
Abstract >>
Strain C5.84 is a Tn5-751 insertion mutant of Aeromonas hydrophila which is unable to secrete extracellular proteins, instead accumulating them in the periplasm (B. Jiang and S.P. Howard, J. Bacteriol. 173:1241-1249, 1991). A 3.5-kb BglII fragment which complements this mutation was isolated from the chromosome of the parent strain. Analysis of this fragment revealed an operon-like structure with two complete genes, exeA and exeB, a functional promoter 5' to the exeA gene, and a 13-bp inverted repeat immediately 3' to the exeB gene. Although the transposon had inserted in exeA, provision of a wild-type copy of this gene alone in trans did not restore competence for export to C5.84. Complementation required the presence of both exeA and exeB, and marker exchange mutagenesis confirmed the requirement for both gene products for secretion. In vitro expression as well as analysis of the deduced amino acid sequence of ExeA indicated that it is a hydrophilic 60-kDa protein with a consensus ATP binding site. ExeB is a 25-kDa basic protein which shares limited homology with PulB, a protein of unknown function associated with the maltose regulon of Klebsiella oxytoca, and OutB, a protein which has been shown to be required for efficient secretion in Erwinia chrysanthemi. The hydrophilic character of these proteins and preliminary localization studies suggested that they are anchored to the inner membrane. These results demonstrate the involvement of a second operon encoding a putative ATP-binding protein in the secretion of extracellular proteins from gram-negative bacteria and further suggest that the cytoplasmic compartment may play a greater role in protein translocation across the outer membrane from the periplasm than previously thought.
KeywordMeSH Terms
Membrane Transport Proteins
162.     ( 1993 )

Purification and characterization of a novel zinc-proteinase from cultures of Aeromonas hydrophila.

The Journal of biological chemistry 268 (12)
PMID : 8473348  :  
Abstract >>
While searching for an enzyme capable of breaking epsilon-(gamma-Glu)-Lys isopeptide bonds cross-linking protein chains, we purified a metallo-proteinase which mimics the action of an isopeptidase on the gamma-chain dimers of cross-linked fibrin. The enzyme is present in the growth medium of the bacterium Aeromonas hydrophila, isolated from the intestinal tract of the leech Hirudo medicinalis. It is a 19-kDa protein which specifically hydrolyzes the Gly-Ala peptide bond within the Gly-Gly-Ala sequence, located near the cross-link site in the gamma-chain dimer of fibrin. Substrate specificity studies with a number of synthetic peptides suggest that the enzyme prefers Gly-Gly or acetyl-Gly in the P2 and P1 positions, respectively (Schecter, I., and Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162). Nonpolar amino acid residues seem to be favored in the P1' and P2' positions. The enzyme contains one atom of zinc and is inhibited by 1,10-phenanthroline, but not by EDTA. Iodoacetate, leupeptin, diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, pepstatin, and alpha 2-macroglobulin have no effect on enzyme activity. Disulfide reducing reagents, such as dithiothreitol or 2-mercaptoethanol, inactivate the enzyme completely. The partial amino-terminal sequence shows 46% identity with a zinc metallo-proteinase from a strain of Lysobacter enzymogenes and 69% identity with the LasA protein from Pseudomonas aeruginosa.
KeywordMeSH Terms
163. Nemec  A, Haywood-Farmer  A, Mackie  GA,     ( 1995 )

Conserved amino acid residues in the primary structure of ribosomal protein S20 from selected gram-negative bacteria.

Biochimica et biophysica acta 1263 (2)
PMID : 7640306  :   DOI  :   10.1016/0167-4781(95)00100-u    
Abstract >>
Partial DNA sequences encoding ribosomal protein S20 have been determined for a number of Gram-negative bacteria including three species of Aeromonas, Klebsiella pneumoniae, Proteus mirabilis, and Salmonella typhimurium and compared to the known sequence from Escherichia coli. The derived amino acid sequence is well conserved, particularly in the C-terminal one-third of S20. In contrast, there is much less conservation of the nucleotide sequence or potential secondary structure in the corresponding mRNA.
KeywordMeSH Terms
164. Favre  D, Ngai  PK, Timmis  KN,     ( 1993 )

Relatedness of a periplasmic, broad-specificity RNase from Aeromonas hydrophila to RNase I of Escherichia coli and to a family of eukaryotic RNases.

Journal of bacteriology 175 (12)
PMID : 7685334  :   DOI  :   10.1128/jb.175.12.3710-3722.1993     PMC  :   PMC204786    
Abstract >>
The isolation, sequencing, and characterization of a periplasmic RNase gene from Aeromonas hydrophila AH1133 is described. Following subcloning of the gene on a 2.7-kb PstI fragment, its direction of transcription and approximate location were determined. Analysis of the nucleotide sequence reveals that the gene is 645 bp long, coding for 215 amino acid residues with a total molecular weight of 24,215. A typical leader sequence is present at the beginning of the corresponding protein. Computer analysis revealed strong local similarities to Escherichia coli RNase I and to the active site of a family of eukaryotic RNases. Expression studies indicate that the RNase natural promoter functions poorly in E. coli. In this organism, the enzyme is mainly localized in the cytoplasm and periplasm, although high levels of expression lead to significant release into the extracellular medium. Functional and physical characterizations further indicate that the periplasmic and cytoplasmic enzymes of A. hydrophila are likely to be the counterparts of E. coli RNase I and its cytoplasmic form RNase I*: as for the E. coli enzymes, the A. hydrophila RNase forms have similar sizes and show broad specificity, and the periplasmic form is more active towards natural polymer RNA than its cytoplasmic counterpart. Both forms are relatively thermosensitive and are reversibly inactivated by up to 0.6% sodium dodecyl sulfate. Southern hybridization revealed homology to E. coli K-12 and Shigella sp. genomic DNA, a finding which correlates with the presence of secreted RNases in these organisms. In contrast, species of phylogenetically closer genera, such as Vibrio and Plesiomonas, did not hybridize to the A. hydrophila RNase gene.
KeywordMeSH Terms
165.     ( 1994 )

Maltose transport in Aeromonas hydrophila: purification, biochemical characterization and partial protein sequence analysis of a periplasmic maltose-binding protein.

Microbiology (Reading, England) 140 (Pt 4) (N/A)
PMID : 8012611  :   DOI  :   10.1099/00221287-140-4-945    
Abstract >>
A clinical isolate of Aeromonas hydrophila was demonstrated to transport [14C]maltose with similar kinetics to enteric bacteria (Km: 0.3 microM; Vmax: 22 nmol min-1 per 10(9) cells). The uptake of [14C]maltose was completely inhibited in the presence of unlabelled maltose or maltodextrins, whereas other mono- and disaccharides, such as glucose, galactose, sucrose, lactose or melibiose, had no effect. A protein with an apparent molecular mass of 39 kDa (maltose-binding protein; MBP) was identified in osmotic-shock fluid of maltose-grown cells by SDS-gel electrophoresis, and was purified to homogeneity by either amylose affinity chromatography or ion-exchange chromatography. Equilibrium dialysis experiments revealed the ability of the purified protein to bind [14C]maltose with high affinity (KD = 1.6 microM). Unlabelled maltose and maltodextrins competed for the binding site. In a reconstitution experiment, A. hydrophila MBP poorly restored the transport activity of a binding-protein-deficient Escherichia coli (delta malE) mutant. N-terminal sequence analyses of the purified native protein and of peptides generated by cleavage with CNBr and subsequently separated by HPLC revealed about 56% identical amino acid residues, as compared to enterobacterial MBPs. We conclude that maltose is transported into A. hydrophila via a binding-protein-dependent transport system.
KeywordMeSH Terms
ATP-Binding Cassette Transporters
Escherichia coli Proteins
Monosaccharide Transport Proteins
Periplasmic Binding Proteins
166. Thomas  SR, Trust  TJ,     ( 1995 )

Tyrosine phosphorylation of the tetragonal paracrystalline array of Aeromonas hydrophila: molecular cloning and high-level expression of the S-layer protein gene.

Journal of molecular biology 245 (5)
PMID : 7844827  :   DOI  :   10.1006/jmbi.1994.0047    
Abstract >>
High virulence strains of the fish pathogenic bacterium Aeromonas hydrophila produce a tetragonally arranged paracrystalline surface protein array (S-layer). The gene (ahsA) encoding the S-protein subunit of A. hydrophila TF7 was cloned into lambda EMBL 3, and sub-cloned into pUC 18. Transformation into Escherichia coli led to stable high-level expression of full-size S-protein under the control of its native promoter. The DNA sequence revealed a 1406 base-pair open reading frame encoding a protein consisting of a 19 amino acid residue signal peptide, and a 448 residue 45,400 Da molecular mass mature protein with a predicted isoelectric point (pI) of 6.72 compared with the measured M(r) of 52,000 and pI of 4.6. This suggested that the S-protein was post-translationally modified and in vivo cell labelling with [32P]orthophosphate, acid phosphatase digestion of S-protein, ascending thin-layer chromatography of partially acid hydrolysed S-protein and Western blot analysis with monoclonal anti-phosphotyrosine antibody showed that the S-protein of strain TF7 contained phosphotyrosine. S-proteins produced by the other strains of motile aeromonads tested also reacted with this anti-phosphotyrosine antibody. Cell fractionation studies employing plasmid-encoded ahsA showed that in A. hydrophila the S-protein subunits were secreted across the outer membrane by the native S-protein secretion pathway, while in E. coli and A. salmonicida the cloned A. hydrophila S-protein inserted into the outer membrane of the foreign host. These findings indicate that the process employed to translocate Aeromonas S-proteins across the outer membrane is highly specific.
KeywordMeSH Terms
Bacterial Outer Membrane Proteins
167. Ingham  AB, Pemberton  JM,     ( 1995 )

A lipase of Aeromonas hydrophila showing nonhemolytic phospholipase C activity.

Current microbiology 31 (1)
PMID : 7767226  :  
Abstract >>
Extracellular lipase activity detected on tributyrin agar has been identified in a cosmid clone, JM3084, constructed from the chromosome of Aeromonas hydrophila and vector pHC79. This lipase, named apl-1, also exhibits nonhemolytic phospholipase C activity on lecithin and p-nitrophenylphosphorylcholine. Subcloning of the cosmid JMP3084 with partial Sau3a1 digestion localized the lipase gene to a 3.4-kb DNA fragment. Southern blot analysis shows the gene apl-1 to exist in single copy on the A. hydrophila chromosome. Expression of apl-1 in the pT7 system identified a single protein of molecular weight 70 kDa. Nucleotide sequencing of apl-1 has identified an open reading frame of 2055 bases predicting a protein of 73 kDa. The presence of an amino terminal signal sequence of 18 amino acids accounts for this molecular weight disparity. Further analysis of the lipase amino acid sequence revealed the presence of a classical serine active lipase site (Gly-X-Ser-X-Gly) located between residues 561 and 570. The A. hydrophila chromosomal copy of apl-1 has been inactivated by use of the mutagenesis vector pJP5603, resulting in the complete removal of phospholipase C activity and lowered levels of lipase activity detected on tributyrin agar.
KeywordMeSH Terms
168. Thomas  SR, Trust  TJ,     ( 1995 )

A specific PulD homolog is required for the secretion of paracrystalline surface array subunits in Aeromonas hydrophila.

Journal of bacteriology 177 (14)
PMID : 7608063  :   DOI  :   10.1128/jb.177.14.3932-3939.1995     PMC  :   PMC177120    
Abstract >>
Aeromonas hydrophila is an important pathogen of fish, and its high-virulence strains display a two-dimensional paracrystalline layer (S-layer) on their outermost surfaces. The nucleotide sequence of a 4.1-kb region located 700 bp upstream of the A. hydrophila TF7 S-layer protein gene (ahsA) has been determined. A sequence analysis of the region revealed the presence of three complete open reading frames ending in a gene encoding a 79.8-kDa polypeptide that shows high homology to the PulD family of secretion proteins. The sequenced region displays both organizational and sequence homology to the Xanthomonas campestris pv. campestris Xps secretory system. Insertional inactivation of the spsD (S-protein secretion D) gene showed that the loss of expression of the PulD homolog coincided with the localization of the S-protein in the periplasm and the loss of the S-layer from the surface of the bacterium. However, the secretion of the enzymes hemolysin, amylase, and protease was unaffected in the mutant with the nonfunctional spsD gene, as was the export of flagella and fimbrial proteins. Southern blot analysis showed that the spsD gene was not conserved among all strains of S-protein-producing A. hydrophila or Aeromonas veronii biotype sobria. Use of the promoterless chloramphenicol acetyltransferase gene showed that unlike pulD and its homologs, spsD contains its own promoter. A. hydrophila has been shown to contain the exe operon, which is responsible for the secretion of a number of extracellular enzymes in this bacterium. A fragment of DNA was generated from the exeD gene of A. hydrophilia Ah65 by PCR and was subsequently used in hybridization studies to probe the chromosome of A. hydrophila TF7. The presence of an exeD homolog in A. hydrophila TF7 was found; therefore, the spsD gene encodes a second pulD homolog that displays a high specificity for the secretion of the S-protein. This gene appears to be part of a second terminal branch of the general secretory pathway in A. hydrophila.
KeywordMeSH Terms
169. Parker  MW, Buckley  JT, Postma  JP, Tucker  AD, Leonard  K, Pattus  F, Tsernoglou  D,     ( 1994 )

Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states.

Nature 367 (6460)
PMID : 7510043  :   DOI  :   10.1038/367292a0    
Abstract >>
Aerolysin is chiefly responsible for the pathogenicity of Aeromonas hydrophila, a bacterium associated with diarrhoeal diseases and deep wound infections. Like many other microbial toxins, the protein changes in a multistep process from a completely water-soluble form to produce a transmembrane channel that destroys sensitive cells by breaking their permeability barriers. Here we describe the structure of proaerolysin determined by X-ray crystallography at 2.8 A resolution. The protoxin (M(r) 52,000) adopts a novel protein fold. Images of an aerolysin oligomer derived from electron microscopy have assisted in constructing a model of the membrane channel and have led to the proposal of a scheme to account for insertion of the protein into lipid bilayers to form ion channels.
KeywordMeSH Terms
170. Thornton  J, Howard  SP, Buckley  JT,     ( 1988 )

Molecular cloning of a phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Sequence homologies with lecithin-cholesterol acyltransferase and other lipases.

Biochimica et biophysica acta 959 (2)
PMID : 3280033  :   DOI  :   10.1016/0005-2760(88)90026-4    
Abstract >>
We have determined the nucleotide sequence of a gene encoding Aeromonas hydrophila phospholipid-cholesterol acyltransferase, an enzyme which shares many properties with mammalian lecithin:cholesterol acyltransferase. The derived amino acid sequence of the protein contains two regions which are homologous to the proposed active sites and binding sites of the plasma acyltransferase and to similar sequences in other interfacially acting lipolytic enzymes. The amino terminus is preceded by a typical 18 amino acid signal sequence. The protein, which is released into the culture supernatant by Aeromonas hydrophila, is confined to the periplasm of Escherichia coli.
KeywordMeSH Terms
171. Hossain  S, Dahanayake  PS, De Silva  BCJ, Wickramanayake  MVKS, Wimalasena  SHMP, Heo  GJ,     ( 2019 )

Multidrug resistant Aeromonas spp. isolated from zebrafish (Danio rerio): antibiogram, antimicrobial resistance genes and class 1 integron gene cassettes.

Letters in applied microbiology 68 (5)
PMID : 30790321  :   DOI  :   10.1111/lam.13138    
Abstract >>
Aeromonas spp. are Gram-negative opportunistic bacteria which have been commonly associated with fish diseases. In this study, antibiogram, antimicrobial resistance genes and integrons of 43 zebrafish-borne Aeromonas spp. were studied. The isolates were identified as six Aeromonas species (A. veronii biovar veronii (n = 26), A. veronii biovar sobria (n = 3), A. hydrophila (n = 8), A. caviae (n = 3), A. enteropelogenes (n = 2) and A. dhakensis (n = 1)). Antibiogram of the isolates indicated that most of them were resistant to amoxicillin (100�P00%), nalidixic acid (100�P00%), oxytetracycline (100�P00%), ampicillin (93�P02%), tetracycline (74�P42%), rifampicin (67�P44%) and imipenem (65�P15%). Multiple antimicrobial resistance (MAR) index values ranged from 0�P19-0�P44 to 90�P70% isolates showed multidrug resistance. PCR of antimicrobial resistance genes revealed that the tetracycline resistance gene (tetA) was the most predominant (67�P44%) among the isolates. The qnrS (53�P49%), tetB (30�P23%), tetE (30�P23%), qnrB (23�P26%) and aac(6')-Ib-cr (4�P65%) genes were also detected. Class 1 integrase (IntI1) gene was found in 46�P51% of the isolates. Two types of class 1 integron gene cassette profiles (qacG-aadA6-qacG and drfA1) were identified. The results showed that zebrafish-borne aeromonads can harbour different types of antimicrobial resistance genes and class 1 integrons. SIGNIFICANCE AND IMPACT OF THE STUDY: Aeromonas spp. are important pathogens found in diverse environments. Antimicrobial resistance genes and integrons of ornamental fish-borne Aeromonas spp. are not well studied. The antibiogram, antimicrobial resistance genes and class 1 integrons of Aeromonas spp. isolated from zebrafish were characterized for the first time in Korea. The prevalence of tetracycline resistance genes, plasmid-mediated quinolone resistance genes and class 1 integron gene cassettes were observed among the isolates. The qacG-aadA6-qacG gene cassette was identified for the first time in Aeromonas spp. The results suggest that the wise use of antimicrobials is necessary for the better management of the ornamental fish.
KeywordMeSH Terms
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
Aeromonas spp.
antibiogram
antimicrobial resistance genes
class 1 integron gene cassettes
zebrafish
172. Howard  SP, Garland  WJ, Green  MJ, Buckley  JT,     ( 1987 )

Nucleotide sequence of the gene for the hole-forming toxin aerolysin of Aeromonas hydrophila.

Journal of bacteriology 169 (6)
PMID : 3584074  :   DOI  :   10.1128/jb.169.6.2869-2871.1987     PMC  :   PMC212202    
Abstract >>
The gene for the hole-forming toxin aerolysin from Aeromonas hydrophila was sequenced. Although most of the sequence seems unrelated to that of Staphylococcus aureus alpha-toxin, both proteins are very hydrophilic, and they each contain a nearly identical string of 10 amino acids.
KeywordMeSH Terms
Genes, Bacterial
173. Stumpf  AK, Vortmann  M, Dirks-Hofmeister  ME, Moerschbacher  BM, Philipp  B,     ( 2019 )

Identification of a novel chitinase from Aeromonas hydrophila AH-1N for the degradation of chitin within fungal mycelium.

FEMS microbiology letters 366 (1)
PMID : 30596975  :   DOI  :   10.1093/femsle/fny294    
Abstract >>
Defined organic waste products are ideal and sustainable secondary feedstocks for production organisms in microbial biotechnology. Chitin from mycelia of fungal fermentation processes represents a homogeneous and constantly available waste product that can, however, not be utilised by typical bacterial production strains. Therefore, enzymes that degrade chitin within fungal mycelia have to be identified and expressed in production organisms. In this study, chitin-degrading bacteria were enriched and isolated from lake water with mycelia of Aspergillus tubingensis as sole organic growth substrate. This approach yielded solely strains of Aeromonas hydrophila. Comparison of the isolated strains with other A. hydrophila strains regarding their chitinolytic activities on fungal mycelia identified strain AH-1N as the best enzyme producer. From this strain, a chitinase (EC:3.2.1.14) was identified by peptide mass fingerprinting. Heterologous expression of the respective gene combined with mass spectrometry showed that the purified enzyme was capable of releasing chitobiose from fungal mycelia with a higher yield than a well-described chitinase from Serratia marcescens. Expression of the newly identified chitinase in biotechnological production strains could be the first step for making fungal mycelium accessible as a secondary feedstock. Additionally, the enrichment strategy proved to be feasible for identifying strains able to degrade fungal chitin.
KeywordMeSH Terms
174. De Silva  BCJ, Hossain  S, Dahanayake  PS, Heo  GJ,     ( 2019 )

Aeromonas spp. from marketed Yesso scallop (Patinopecten yessoensis): molecular characterization, phylogenetic analysis, virulence properties and antimicrobial susceptibility.

Journal of applied microbiology 126 (1)
PMID : 30218592  :   DOI  :   10.1111/jam.14106    
Abstract >>
Yesso scallop (Patinopecten yessoensis) is a popular seafood in Korea. Aeromonas spp., well-known pathogenic bacteria, has been reported in some molluscan shellfish, but it has not been studied in scallops so far. Therefore, we aimed to isolate, identify and characterize the Aeromonas spp. isolated from marketed Yesso scallops to estimate their potential risk to public health. Thirty-two Aeromonas spp. including A. hydrophila (n = 13), A. salmonicida (n = 11), A. media (n = 3), A. caviae (n = 2), A. veronii (n = 2) and A. enteropelogenes (n = 1) were isolated from 105 marketed scallops and tested for phenotypic pathogenicity, virulence genes and antimicrobial susceptibility. Mean total bacterial count of scallop meat was 1�P34 �� 104 CFU per gram. Slime production and lipase tests were positive in 97% of the isolates while DNase, protease, gelatinase, phospholipase and haemolysis were shown by 88, 88, 81, 88 and 72% of the isolates respectively. Eleven virulence genes were detected among Aeromonas spp. (act (75%), alt (59%), ast (47%), aerA (78%), lip (59%), ahyB (94%), ser (75%), hlyA (75%), fla (64%), gcat (84%) and ascV (23%)), and exu was negative in all isolates. Aeromonas hydrophila and A. salmonicida harboured ?7 virulence genes and positive for enterotoxin genes, act, alt and ast. All the isolates were multidrug resistant and 100% resistant to ampicillin, colistin, vancomycin and cephalothin. Also, 30, 31, 20, 21, 29, 24, 27 and 27 of the isolates were resistant to piperacillin, clindamycin, erythromycin, nalidixic acid, imipenem, meropenem, trimethoprim-sulfamethoxazole and rifampicin respectively. It is obvious with our results that the Aeromonas spp. isolated from Yesso scallops are highly virulent and potentially pathogenic, whereas the multidrug resistance further expedite their importance. To our knowledge, this is the first study reporting Aeromonas spp. in scallop. This implies that not only the common varieties like oysters, but other bivalves can also harbour potentially pathogenic aeromonads which may have impacts on consumer health.
KeywordMeSH Terms
gyrB
Aeromonas spp.
antimicrobial resistance
prevalence
scallop
virulence
gyrB
Aeromonas spp.
antimicrobial resistance
prevalence
scallop
virulence
gyrB
Aeromonas spp.
antimicrobial resistance
prevalence
scallop
virulence
gyrB
Aeromonas spp.
antimicrobial resistance
prevalence
scallop
virulence
Aeromonas
175. Zhang  M, Yan  Q, Mao  L, Wang  S, Huang  L, Xu  X, Qin  Y,     ( 2018 )

KatG plays an important role in Aeromonas hydrophila survival in fish macrophages and escape for further infection.

Gene 672 (N/A)
PMID : 29906530  :   DOI  :   10.1016/j.gene.2018.06.029    
Abstract >>
The success of the pathogenic bacteria is partly attributable to their ability to thwart host innate immune responses, which includes resisting the antimicrobial functions of macrophages. And reactive oxygen species (ROS) is one of the most effective antimicrobial components of macrophages to kill invading bacteria. Our previous studies found that Aeromonas hydrophila can survive in fish macrophages, which suggested that this bacterium might take fish macrophages as their shelters to resist drug killings and other immune damage. But how A. hydrophila survive in host macrophages remains unknown. Since KatG has been reported to have not only catalase activity but also peroxidase and peroxynitritase activity, the amino acid sequence and protein structure of KatG was analyzed in this study, the function of KatG in A. hydrophila survival in and escape from host macrophages was also carried out. The bioinformatics analysis displayed that KatG of A. hydrophila B11 showed >93% homologous to that of KatG in other Aeromonas. KatG of A. hydrophila was stable silenced by shRNA and RT-qPCR confirmed the expression of KatG in KatG-RNAi was significantly reduced. The survival rate of intracellular KatG-RNAi decreased by 80% compared to that of the wild type strain B11, while the intracellular ROS level of the macrophages that phagocytosed KatG-RNAi increased 65.9% when compared to that of the macrophages phagocytosed wild-type strain. The immune escape rate of A. hydrophila decreased by 85% when the expression of KatG was inhibited. These results indicated that (1) The amino acid sequence and protein structure of KatG of A. hydrophila is conserved; (2) KatG helped A. hydrophila to survive in fish macrophages by eliminating the harm of intracellular H2O2 and inhibiting intracellular ROS levels increased; (3) A small portion of intracellular A. hydrophila could escape from host macrophages for further infection, in this process KatG also played important role.
KeywordMeSH Terms
Aeromonas hydrophila
Escape
Intracellular survival
KatG
RNAi
Reactive oxygen species
Aeromonas hydrophila
Escape
Intracellular survival
KatG
RNAi
Reactive oxygen species
176. Ma  S, Sun  C, Hulth  A, Li  J, Nilsson  LE, Zhou  Y, Börjesson  S, Bi  Z, Bi  Z, Sun  Q, Wang  Y,     ( 2018 )

Mobile colistin resistance gene mcr-5 in porcine Aeromonas hydrophila.

The Journal of antimicrobial chemotherapy 73 (7)
PMID : 29659855  :   DOI  :   10.1093/jac/dky110     PMC  :   PMC6005042    
Abstract >>
To characterize the mobile colistin resistance gene mcr-5 in Aeromonas hydrophila from backyard pigs in rural areas of China. Pig faecal samples from 194 households were directly tested for the presence of mcr-5 by PCR assay and the phenotypic antimicrobial susceptibility profiles of the mcr-5-positive isolates were determined using the broth dilution method. The genomic location and transferability of mcr-5 were analysed by S1-PFGE with Southern blotting and DNA hybridization, and natural transformation, respectively. One strain isolated from an mcr-5-positive sample was subjected to WGS and the stability of the mcr-5-harbouring plasmid over successive generations was examined by subculturing. One mcr-5-positive A. hydrophila isolate showing resistance, with a colistin MIC of 4 mg/L, was isolated from a backyard pig faecal sample. mcr-5 was located on a 7915 bp plasmid designated pI064-2, which could naturally transform into a colistin-susceptible A. hydrophila strain of porcine origin and mediated colistin resistance in both the original isolate and its transformants. The plasmid backbone (3790 bp) of pI064-2 showed 81% nucleotide sequence identity to the corresponding region of the ColE2-type plasmid pAsa1 from Aeromonas salmonicida, while similar replication primases are widely distributed among aeromonads, Enterobacteriaceae and Pseudomonas species. To the best of our knowledge, this is the first identification of the novel colistin resistance gene mcr-5 in an A. hydrophila isolate from the faeces of a backyard pig. mcr-5 is expected to be able to disseminate among different bacterial species and genera.
KeywordMeSH Terms
177. Howard  SP, Buckley  JT,     ( 1986 )

Molecular cloning and expression in Escherichia coli of the structural gene for the hemolytic toxin aerolysin from Aeromonas hydrophila.

Molecular & general genetics : MGG 204 (2)
PMID : 3020368  :   DOI  :   10.1007/bf00425512    
Abstract >>
The structural gene for the hemolytic toxin aerolysin has been cloned into the plasmid vectors pBR322 and pEMBL8+. The gene was localized on the hybrid plasmids by analysis of plasmids generated by transposon mutagenesis. The sequence of the first 683 bases of an insert in pEMBL8+ was determined and shown to encode the amino terminus of the protein as well as a typical signal sequence of 23 amino acids. Aerolysin is produced by E. coli cells containing the cloned aerolysin gene and it is processed normally by removal of the signal sequence, however it is not released from the cell. The protein appears to be translocated across the inner membrane of E. coli as its signal sequence is removed and the processed protein can be released by osmotic shock.
KeywordMeSH Terms
Cloning, Molecular
Genes
Genes, Bacterial
178. Liu  J, Xie  L, Zhao  D, Yang  T, Hu  Y, Sun  Z, Yu  X,     ( 2019 )

A fatal diarrhoea outbreak in farm-raised Deinagkistrodon acutus in China is newly linked to potentially zoonotic Aeromonas hydrophila.

Transboundary and emerging diseases 66 (1)
PMID : 30222905  :   DOI  :   10.1111/tbed.13020    
Abstract >>
Deinagkistrodon acutus is a venomous pit viper commonly used in traditional Chinese medicine; farming these snakes has become a major industry. In 2017, an outbreak of fatal diarrhoea among farm-raised D. acutus in Hunan Province caused the deaths of 5,600 snakes within 3 weeks. We isolated a brand-new sequence type of Aeromonas hydrophila (ST516) from lesions and confirmed that this bacterium was the causal agent of the outbreak. Snakes infected with the bacterium in the laboratory showed similar clinical symptoms to those of snakes in the original outbreak. We also tested bacterial virulence in Kunming mice to examine the likelihood of zoonosis. Isolates were pathogenic to mice, causing diarrhoea within 4 hr post-challenge, which indicates that the bacterium can potentially infect mammals. Environmental analysis showed that polluted spring water likely caused the diarrhoea in snakes. This study is the first to report on a large-scale outbreak of fatal diarrhoea in farm-raised snakes, originating in a pathogen that can infect mammals. These results should raise awareness regarding potential anthropozoonosis among poikilotherms, mammals, and humans; appropriate prevention or control methods should be developed.
KeywordMeSH Terms
Aeromonas hydrophila
Deinagkistrodon acutus
diarrhoea
traditional Chinese medicine
zoonosis
Aeromonas hydrophila
Deinagkistrodon acutus
diarrhoea
traditional Chinese medicine
zoonosis
Crotalinae
179. Amos  GCA, Ploumakis  S, Zhang  L, Hawkey  PM, Gaze  WH, Wellington  EMH,     ( 2018 )

The widespread dissemination of integrons throughout bacterial communities in a riverine system.

The ISME journal 12 (3)
PMID : 29374269  :   DOI  :   10.1038/s41396-017-0030-8     PMC  :   PMC5864220    
Abstract >>
Anthropogenic inputs increase levels of antimicrobial resistance (AMR) in the environment, however, it is unknown how these inputs create this observed increase, and if anthropogenic sources impact AMR in environmental bacteria. The aim of this study was to characterise the role of waste water treatment plants (WWTPs) in the dissemination of class 1 integrons (CL1s) in the riverine environment. Using sample sites from upstream and downstream of a WWTP, we demonstrate through isolation and culture-independent analysis that WWTP effluent significantly increases both CL1 abundance and antibiotic resistance in the riverine environment. Characterisation of CL1-bearing isolates revealed that CL1s were distributed across a diverse range of bacteria, with identical complex genetic resistance determinants isolated from both human-associated and common environmental bacteria across connected sites. Over half of sequenced CL1s lacked the 3'-conserved sequence ('atypical' CL1s); surprisingly, bacteria carrying atypical CL1s were on average resistant to more antibiotics than bacteria carrying 3'-CS CL1s. Quaternary ammonium compound (QAC) resistance genes were observed across 75% of sequenced CL1 gene cassette arrays. Chemical data analysis indicated high levels of boron (a detergent marker) downstream of the WWTP. Subsequent phenotypic screening of CL1-bearing isolates demonstrated that ~90% were resistant to QAC detergents, with in vitro experiments demonstrating that QACs could solely select for the transfer of clinical antibiotic resistance genes to a naive Escherichia coli recipient. In conclusion, this study highlights the significant impact of WWTPs on environmental AMR, and demonstrates the widespread carriage of clinically important resistance determinants by environmentally associated bacteria.
KeywordMeSH Terms
Gene Transfer, Horizontal
Integrons
180. Hoel  S, Vadstein  O, Jakobsen  AN,     ( 2017 )

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi.

Frontiers in microbiology 8 (N/A)
PMID : 28596762  :   DOI  :   10.3389/fmicb.2017.00931     PMC  :   PMC5442234    
Abstract >>
Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). �]-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7-3.4%. The average gyrB sequence similarity between all species was 93%, demonstrating its acceptable taxonomic resolution. The presence of multiple species of potential pathogenic Aeromonas in fresh retail sushi raises new food safety issues related to the increased consumption of ready-to-eat food composed of raw ingredients.
KeywordMeSH Terms
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
Aeromonas spp.
food safety
gyrB
ready-to-eat food
sushi
virulence factors
181.     ( 2013 )

Molecular cloning and heterologous expression of laccase from Aeromonas hydrophila NIU01 in Escherichia coli with parameters optimization in production.

Applied biochemistry and biotechnology 169 (7)
PMID : 23423657  :   DOI  :   10.1007/s12010-013-0128-z    
Abstract >>
Prior studies disclosed that Aeromonas hydrophila NIU01 was a biodecolorization and bioelectricity bacterium which was isolated from a cross-strait of Taiwan. However, enzymatic function, laccase, involved in this strain had never been reported. This first attempt is to explore its laccase activity, the molecular cloning and heterologous recombinant expression in Escherichia coli. A full-length novel gene of 1,647 bp, LacA, encoding of 549 amino acids was successfully cloned by polymerase chain reaction. The recombinant pET-15b(+)-NIU-LacA expression was compared in different E. coli strains. By applying Taguchi's L9 in culture optimization, the soluble laccase increased to 22.7 %, in which the conditions were obtained at 22 �XC with initial shaking speed at 200 rpm, addition of lactose of 0.2 mM and CuSO4 of 0.5 mM to the medium, and shaking off while cell mass reached to OD(600nm) of 1.5. NIU-LacA was strongly inhibited by chloride ion. The optimal temperature was 60 �XC and the optimum pH for ABTS (2,2'-azino-bis (3-ethylbenzthiazolinesulfonic acid) and 2,6-DMP (2,6-dimethoxyphenol) were pH 2.1 and pH 7.5 which enzymatic activity was 274.6 and 44.8 U/L, respectively. Further study in structural modeling of NIU-LacA showed the C terminal domain was the major variance in the three most closely A. hydrophila strains.
KeywordMeSH Terms
182.     ( 2013 )

Biochemical and molecular characterization of the novobiocin and rifampicin resistant Aeromonas hydrophila vaccine strain AL09-71N+R compared to its virulent parent strain AL09-71.

Veterinary microbiology 165 (3��4��)
PMID : 23608477  :   DOI  :   10.1016/j.vetmic.2013.03.018    
Abstract >>
To understand the fitness cost of novobiocin- and rifampicin-resistance in an attenuated Aeromonas hydrophiila vaccine strain AL09-71 N+R compared to its virulent parent strain AL09-71, colony size, cell size, cell proliferation rate, chemotactic response, and the ability to invade catfish gill cells of the two strains were compared. Our results revealed that: (1) the cell size and the colony size of AL09-71 N+R was significantly (P<0.05) smaller than that of AL09-71; (2) the proliferation rate of AL09-71 N+R was significantly (P<0.05) slower than that of AL09-71; (3) AL09-71 N+R had a significantly (P<0.05) lower chemotactic response to catfish mucus than that of AL09-71; 4) the ability of AL09-71 N+R to invade catfish gill cells was significantly (P<0.05) lower than that of AL09-71. To understand whether target site mutation might play a role in antibiotic resistance, novobiocin's target site DNA gyrase subunit B gyrB and rifampicin's target site RNA polymerase subunit B rpoB were sequenced from the two strains. Our results revealed the following five mutations: (1) two missense mutations (CGC to ATC resulting in arginine/R to serine/S; TAC to TGC resulting in tyrosine/Y to cysteine/C) between AL09-71 gyrB and AL09-71 N+R gyrB; (2) three missense mutations (GAC to AAC resulting in aspartic acid/D to asparagine/N; CTG to CCG resulting in leucine/L to proline/P; CTG to CCG resulting in leucine/L to proline/P) between AL09-71 rpoB and AL09-71 N+R rpoB. To determine whether any unique DNA sequences were present in AL09-71 but absent in AL09-71 N+R, PCR-select bacterial genome subtractive hybridization was performed. Of 96 clones selected from the subtractive genomic DNA library, 32 sequences were found. None of the 32 sequences was confirmed to be present in AL09-71 but absent in AL09-71 N+R. At the transcription level, 29 of the 32 genes were found to be expressed greater than 10-fold in AL09-71 N+R compared to that in AL09-71.
KeywordMeSH Terms
183. Han  JE, Kim  JH, Choresca  CH, Shin  SP, Jun  JW, Chai  JY, Park  SC,     ( 2012 )

Prevalence of tet gene and complete genome sequencing of tet gene-encoded plasmid (pAHH01) isolated from Aeromonas species in South Korea.

Journal of applied microbiology 112 (4)
PMID : 22309714  :   DOI  :   10.1111/j.1365-2672.2012.05237.x    
Abstract >>
KeywordMeSH Terms
Plasmids
Tetracycline Resistance
184.     ( 2013 )

Evaluation of the roles played by Hcp and VgrG type 6 secretion system effectors in Aeromonas hydrophila SSU pathogenesis.

Microbiology (Reading, England) 159 (Pt 6)
PMID : 23519162  :   DOI  :   10.1099/mic.0.063495-0     PMC  :   PMC3709694    
Abstract >>
Aeromonas hydrophila, a Gram-negative bacterium, is an emerging human pathogen equipped with both a type 3 and a type 6 secretion system (T6SS). In this study, we evaluated the roles played by paralogous T6SS effector proteins, hemolysin co-regulated proteins (Hcp-1 and -2) and valine glycine repeat G (VgrG-1, -2 and -3) protein family members in A. hydrophila SSU pathogenesis by generating various combinations of deletion mutants of the their genes. In addition to their predicted roles as structural components and effector proteins of the T6SS, our data clearly demonstrated that paralogues of Hcp and VgrG also influenced bacterial motility, protease production and biofilm formation. Surprisingly, there was limited to no observed functional redundancy among and/or between the aforementioned T6SS effector paralogues in multiple assays. Our data indicated that Hcp and VgrG paralogues located within the T6SS cluster were more involved in forming T6SS structures, while the primary roles of Hcp-1 and VgrG-1, located outside of the T6SS cluster, were as T6SS effectors. In terms of influence on bacterial physiology, Hcp-1, but not Hcp-2, influenced bacterial motility and protease production, and in its absence, increases in both of the aforementioned activities were observed. Likewise, VgrG-1 played a major role in regulating bacterial protease production, while VgrG-2 and VgrG-3 were critical in regulating bacterial motility and biofilm formation. In an intraperitoneal murine model of infection, all Hcp and VgrG paralogues were required for optimal bacterial virulence and dissemination to mouse peripheral organs. Importantly, the observed phenotypic alterations of the T6SS mutants could be fully complemented. Taking these results together, we have further established the roles played by the two known T6SS effectors of A. hydrophila by defining their contributions to T6SS function and virulence in both in vitro and in vivo models of infection.
KeywordMeSH Terms
185.     ( 2013 )

Characterization of Aeromonas strains isolated from Indian foods using rpoD gene sequencing and whole cell protein analysis.

World journal of microbiology & biotechnology 29 (4)
PMID : 23255059  :   DOI  :   10.1007/s11274-012-1212-1    
Abstract >>
Aeromonas are responsible for causing gastroenteritis and extra-intestinal infections in humans. Twenty-two Aeromonas strains isolated from different food sources were re-identified up to species level using rpoD gene sequence analysis. Biochemical tests and 16S rRNA gene sequencing were insufficient to identify Aeromonas till species level. However, incorporation of additional biochemical tests lead to correct identification of 95.5 % strains up to species level. The 16S rRNA gene sequencing was useful to identify Aeromonas isolates at the genus level only. Sequences of the rpoD gene showed greater discriminatory power than 16S rRNA gene and provided conclusive discrimination of the strains for which the phenotypic species identification was uncertain. All these 22 strains were accurately identified up to species level by rpoD gene as A. salmonicida (6), A. veronii bv. veronii (4), A. caviae (3), A. hydrophila (2), A. veronii bv. sobria (2), A. jandaei (1), A. trota (1), A. sobria (1), A. allosaccharophila (1) and A. bivalvium (1). All these strains were also characterized using whole cell protein (WCP) analysis by gradient SDS-PAGE and showed different whole cell protein (WCP) profile [22-28 polypeptide bands (~10 to >97 kDa)], indicating high genetic diversity. The present work emphasizes the use of molecular methods such as rpoD gene sequencing along with comprehensive biochemical tests for the rapid and accurate identification of Aeromonas isolates till species level. The WCP profile can be subsequently used to characterize Aeromonas isolates below species level.
KeywordMeSH Terms
Food Microbiology
186.     ( 2012 )

High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

PloS one 7 (9)
PMID : 22984449  :   DOI  :   10.1371/journal.pone.0043879     PMC  :   PMC3440374    
Abstract >>
We recently characterized a specific inorganic triphosphatase (PPPase) from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i)) is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i) but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i) in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i), which could be cytotoxic because of its high affinity for Ca(2+), thereby interfering with Ca(2+) signaling.
KeywordMeSH Terms
187.     ( 2013 )

Comparative sequence analysis of a multidrug-resistant plasmid from Aeromonas hydrophila.

Antimicrobial agents and chemotherapy 57 (1)
PMID : 23070174  :   DOI  :   10.1128/AAC.01239-12     PMC  :   PMC3535917    
Abstract >>
Aeromonas hydrophila is a pathogenic bacterium that has been implicated in fish, animal, and human disease. Recently, a multidrug resistance (MDR) plasmid, pR148, was isolated from A. hydrophila obtained from a tilapia (Oreochromis niloticus) farm in Thailand. pR148 is a 165,906-bp circular plasmid containing 147 coding regions showing highest similarity to pNDM-1_Dok1, an MDR plasmid isolated from a human pathogen. pR148 was also very similar to other IncA/C plasmids isolated from humans, animals, food, and fish. pR148 contains a mercuric resistance operon and encodes the complete set of genes for the type 4 secretion system. pR148 encodes a Tn21 type transposon. This transposon contains the drug resistance genes qacH, bla(OXA-10), aadA1, and sul1 in a class 1 integron; tetA and tetR in transposon Tn1721; and catA2 and a duplicate sul1 in a locus showing 100% similarity to IncU plasmids isolated from fish. The bla(OXA-10) and aadA1 genes showed 100% similarity to those from the Acinetobacter baumannii AYE genome. The similarity of pR148 to a human pathogen-derived plasmid indicates that the plasmids were either transferred between different genera or that they are derived from a common origin. Previous studies have shown that IncA/C plasmids retain a conserved backbone, while the accessory region points to lateral gene transfer. These observations point out the dangers of indiscriminate use of antibiotics in humans and in animals and the necessity of understanding how drug resistance determinants are disseminated and transferred.
KeywordMeSH Terms
Genes, Bacterial
188.     ( 1990 )

Purification and characterization of inducible beta-lactamases in Aeromonas spp.

Antimicrobial agents and chemotherapy 34 (1)
PMID : 2327760  :   DOI  :   10.1128/aac.34.1.44     PMC  :   PMC171518    
Abstract >>
beta-Lactamases from Aeromonas hydrophila and A. sobria were purified and characterized. Both species produced beta-lactamases that were inducible by either cefoxitin or imipenem. These species were resistant to ampicillin and cephalothin but not imipenem. Isoelectric focusing of sonic extracts revealed one band at pI 8.0 and a second band at pI 7.0 for A. hydrophila. Likewise, A. sobria produced two bands, one at pI 8.4 and the other at pI 7.0. Two enzymes from each species were separated by flatbed electrofocusing gel and purified to homogeneity. The molecular weight of the pI 7.0 enzyme (A1) from both species was estimated to be 42,500, whereas the pI 8.0 (A2h) and 8.4 (A2s) enzymes of A. hydrophila and A. sobria had molecular weights of 31,500 and 35,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The relative Vmax values for cephalothin, penicillin, and imipenem for these enzymes indicated that A1 was primarily a cephalosporinase while A2h and A2s were penicillinases highly active against carbapenems. A1 was susceptible to inhibition by cloxacillin, while the A2 enzymes were inhibited by clavulanic acid and EDTA and required zinc for activity. Thus, there appear to be two distinct inducible beta-lactamases in A. hydrophila and A. sobria that play an important role in the beta-lactam resistance of these species.
KeywordMeSH Terms
189.     ( 2012 )

Phylogenetic diversity, antibiotic resistance and virulence traits of Aeromonas spp. from untreated waters for human consumption.

International journal of food microbiology 159 (3)
PMID : 23107502  :   DOI  :   10.1016/j.ijfoodmicro.2012.09.008    
Abstract >>
It is well known that water constitutes an important contamination route for microorganisms. This is especially true for Aeromonas which are widespread in untreated and treated waters. In this study, Portuguese untreated waters not regularly monitored were screened for the presence and diversity of aeromonads. A total of 206 isolates were discriminated by RAPD-PCR and 80 distinct strains were identified by gyrB based phylogenetic analysis. The most frequently detected species were Aeromonas hydrophila, Aeromonas bestiarum and Aeromonas media. The antibiotic susceptibility profile of these strains was determined and showed a typical profile of the genus. Nonetheless, the percentage of resistant strains to tetracycline, chloramphenicol and/or trimethoprim/sulfamethoxazole was lower than that reported for clinical isolates and isolates recovered from aquacultures and other environments historically subjected to antibiotic contamination. This suggests that the existence of such pressures in those environments selects for resistant Aeromonas. A similar trend for integron presence was found. Genes coding for CphA and TEM, and tet(A), (E), (C) or (D) genes were found in 28%, 1%, and 10% of the strains, respectively. 10% of the strains contained an integron. Variable regions of seven class 1 integrons and one class 2 integron were characterised. Furthermore, strains displayed virulence related phenotypes such as extracellular lipolytic and proteolytic activities as well as aerolysin related genes (43% of strains). The ascV and aexT genes were found in 16% and 3% of strains respectively and, in some cases, concomitantly in the same specimen. This study shows that diverse Aeromonas spp. presenting distinct antibiotic resistance features and putative virulence traits are frequently present in waters for human and animal consumption in Portugal. Genes associated to antibiotic resistance and microbial virulence previously identified in organisms with human health significance were detected in these aeromonads, suggesting that these waters may act as a pivotal route for infections.
KeywordMeSH Terms
Biodiversity
Phylogeny
Water Microbiology
Water Quality
190.     ( 2012 )

Characterization of an actin-targeting ADP-ribosyltransferase from Aeromonas hydrophila.

The Journal of biological chemistry 287 (44)
PMID : 22969084  :   DOI  :   10.1074/jbc.M112.397612     PMC  :   PMC3481304    
Abstract >>
The mono-ADP-ribosyltransferase (mART) toxins are contributing factors to a number of human diseases, including cholera, diphtheria, traveler's diarrhea, and whooping cough. VahC is a cytotoxic, actin-targeting mART from Aeromonas hydrophila PPD134/91. This bacterium is implicated primarily in diseases among freshwater fish species but also contributes to gastrointestinal and extraintestinal infections in humans. VahC was shown to ADP-ribosylate Arg-177 of actin, and the kinetic parameters were K(m)(NAD(+)) = 6 �gM, K(m)(actin) = 24 �gM, and k(cat) = 22 s(-1). VahC activity caused depolymerization of actin filaments, which induced caspase-mediated apoptosis in HeLa Tet-Off cells. Alanine-scanning mutagenesis of predicted catalytic residues showed the predicted loss of in vitro mART activity and cytotoxicity. Bioinformatic and kinetic analysis also identified three residues in the active site loop that were critical for the catalytic mechanism. A 1.9 ? crystal structure supported the proposed roles of these residues and their conserved nature among toxin homologues. Several small molecules were characterized as inhibitors of in vitro VahC mART activity and suramin was the best inhibitor (IC(50) = 20 �gM). Inhibitor activity was also characterized against two other actin-targeting mART toxins. Notably, these inhibitors represent the first report of broad spectrum inhibition of actin-targeting mART toxins.
KeywordMeSH Terms
191.     ( 2012 )

Evaluation of two outer membrane proteins, Aha1 and OmpW of Aeromonas hydrophila as vaccine candidate for common carp.

Veterinary immunology and immunopathology 149 (3��4��)
PMID : 22917476  :   DOI  :   10.1016/j.vetimm.2012.07.013    
Abstract >>
Aeromonas hydrophila is an important fish pathogen responsible for huge economic losses in aquaculture sector. The bacterial outer membrane proteins (OMPs), especially adhesins play a key role in the virulence of the bacteria and are considered potential vaccine candidates. We evaluated the immunogenicity of two important outer membrane proteins namely Aha1 and OmpW of A. hydrophila. These proteins were over-expressed in Escherichia coli, purified and used for the vaccination of common carp. Sequence analysis predicted that, Aha1 and OmpW are adhesins and antigenic. Common carp immunized with recombinant Aha1 and OmpW proteins showed significant antibody production and a relative percentage survival of 52 and 71 respectively indicating their protective efficacy against A. hydrophila infection.
KeywordMeSH Terms
Carps
192.     ( 1998 )

Aeromonas hydrophila adenylyl cyclase 2: a new class of adenylyl cyclases with thermophilic properties and sequence similarities to proteins from hyperthermophilic archaebacteria.

Journal of bacteriology 180 (13)
PMID : 9642185  :   PMC  :   PMC107287    
Abstract >>
Complementation of an Escherichia coli cya mutant with a genomic library from Aeromonas hydrophila allowed isolation of clones containing two different cya genes. Whereas one of these genes (cyaA) coded for an adenylyl cyclase (AC1) belonging to the previously described class I adenylyl cyclases (ACs), the second one (cyaB) coded for a protein (AC2) that did not match any previously characterized protein when compared to protein sequence databases. In particular, it did not align with any of members of the three known classes of ACs. The purified AC2 enzyme exhibited remarkable biochemical characteristics, namely, an optimum activity at a high temperature (65 degrees C) and at an alkalinic pH (9.5). In order to investigate the functions of both cyclases in A. hydrophila, each gene was inactivated in the chromosome and the resulting mutant strains were examined for physiological alterations. It was shown that, in contrast to cyaA, the cyaB gene was not expressed under usual laboratory growth conditions. However, introduction of a plasmid harboring the cyaB gene in a cyaA mutant, as well as in a cyaA cyaB mutant, allowed cyclic AMP production. AC2 is the first member of a new class of previously unrecognized ACs, and to date, no functional counterpart has been demonstrated in other organisms. However, scanning databases revealed a significant similarity between AC2 and the gene product of three hyperthermophilic archaebacteria: Methanobacterium thermoautotrophicum, Archaeglobus fulgidus, and Methanococcus jannaschii. The possibility of a gene transfer between such phylogenetically divergent bacteria is discussed.
KeywordMeSH Terms
Chromosome Mapping
193.     ( 1998 )

Structure of CARB-4 and AER-1 carbenicillin-hydrolyzing beta-lactamases.

Antimicrobial agents and chemotherapy 42 (8)
PMID : 9687391  :   PMC  :   PMC105717    
Abstract >>
We determined the nucleotide sequences of blaCARB-4 encoding CARB-4 and deduced a polypeptide of 288 amino acids. The gene was characterized as a variant of group 2c carbenicillin-hydrolyzing beta-lactamases such as PSE-4, PSE-1, and CARB-3. The level of DNA homology between the bla genes for these beta-lactamases varied from 98.7 to 99.9%, while that between these genes and blaCARB-4 encoding CARB-4 was 86.3%. The blaCARB-4 gene was acquired from some other source because it has a G+C content of 39.1%, compared to a G+C content of 67% for typical Pseudomonas aeruginosa genes. DNA sequencing revealed that blaAER-1 shared 60.8% DNA identity with blaPSE-3 encoding PSE-3. The deduced AER-1 beta-lactamase peptide was compared to class A, B, C, and D enzymes and had 57.6% identity with PSE-3, including an STHK tetrad at the active site. For CARB-4 and AER-1, conserved canonical amino acid boxes typical of class A beta-lactamases were identified in a multiple alignment. Analysis of the DNA sequences flanking blaCARB-4 and blaAER-1 confirmed the importance of gene cassettes acquired via integrons in bla gene distribution.
KeywordMeSH Terms
194.     ( 1998 )

Glycosylphosphatidylinositol anchors of membrane glycoproteins are binding determinants for the channel-forming toxin aerolysin.

The Journal of biological chemistry 273 (4)
PMID : 9442081  :   DOI  :   10.1074/jbc.273.4.2355    
Abstract >>
Cells that are sensitive to the channel-forming toxin aerolysin contain surface glycoproteins that bind the toxin with high affinity. Here we show that a common feature of aerolysin receptors is the presence of a glycosylphosphatidylinositol anchor, and we present evidence that the anchor itself is an essential part of the toxin binding determinant. The glycosylphosphatidylinositol (GPI)-anchored T-lymphocyte protein Thy-1 is an example of a protein that acts as an aerolysin receptor. This protein retained its ability to bind aerolysin when it was expressed in Chinese hamster ovary cells, but could not bind the toxin when expressed in Escherichia coli, where the GPI anchor is absent. An unrelated GPI-anchored protein, the variant surface glycoprotein of trypanosomes, was shown to bind aerolysin with similar affinity to Thy-1, and this binding ability was significantly reduced when the anchor was removed chemically. Cathepsin D, a protein with no affinity for aerolysin, was converted to an aerolysin binding form when it was expressed as a GPI-anchored hybrid in COS cells. Not all GPI-anchored proteins bind aerolysin. In some cases this may be due to differences in the structure of the anchor itself. Thus the GPI-anchored proteins procyclin of Trypanosoma congolense and gp63 of Leishmania major did not bind aerolysin, but when gp63 was expressed with a mammalian GPI anchor in Chinese hamster ovary cells, it bound the toxin.
KeywordMeSH Terms
195.     ( 1998 )

Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model.

Microbiology (Reading, England) 144 (Pt 2) (N/A)
PMID : 9493366  :   DOI  :   10.1099/00221287-144-2-291    
Abstract >>
The contribution of two unrelated Aeromonas hydrophila beta-haemolytic toxins to virulence was assessed in a suckling mouse model. The first haemolysin gene, isolated from an A. hydrophila A6 cosmid bank, encoded a potential gene product of 621 amino acids and a predicted molecular size of 69.0 kDa. The inferred amino acid sequence showed 89% identity to the AHH1 haemolysin of A. hydrophila ATCC 7966, and 51% identity to the HlyA haemolysin of Vibrio cholerae EI Tor strain O17. The second haemolysin gene (designated aerA), which encodes aerolysin, a pore-forming toxin, was partially cloned by PCR for the purpose of mutant construction. This PCR product was a 1040 bp fragment from the C-terminal region of aerA. It is proposed that the 69.0 kDa V. cholerae-HlyA-like haemolysin gene be termed hlyA to contrast with the aerA terminology for the aerolysin. A suicide vector was used to inactivate both the hlyA and aerA genes in A. hydrophila A6. When assessed in the suckling mouse model, only the hlyA aerA double mutant showed a statistically significant reduction in virulence--a 20-fold change in LD50 (Scheffe test, P < 0.05). Cytotoxicity to buffalo green monkey kidney cell monolayers and haemolysis on horse blood agar were eliminated only in the hlyA aerA double mutants. This is the first report of cloning and mutagenesis of two unrelated haemolytic toxin genes in the same strain of a mesophilic aeromonad. For A. hydrophila, a two-toxin model provides a more complete explanation of virulence.
KeywordMeSH Terms
196.     ( 1997 )

Detection and speciation of bacteria through PCR using universal major cold-shock protein primer oligomers.

Journal of industrial microbiology & biotechnology 19 (4)
PMID : 9439003  :  
Abstract >>
The detection of bacteria using PCR is a well-established diagnostic technique. However, conventional PCR requires the use of DNA primer oligomers that are specific to the target organism and, as a consequence, a sample can only be tested for the presence of that specific target. A significant advantage would be to probe a sample for the presence of any bacteria, followed by identification. To achieve this it is necessary to identify a DNA sequence common to all bacteria. Here we demonstrate that such a sequence may be that encoding the major cold-shock proteins. Using two universal PCR primer oligomers from conserved regions of these gene homologues, we have amplified a 200 base-pair DNA sequence from more than 30 diverse Gram-positive and Gram-negative bacteria, including representatives from the genera Aeromonas, Bacillus, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Listeria, Pediococcus, Photobacterium, Proteus, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Sequence analysis of the amplified products confirmed a high level of DNA homology. Significantly, however, there are sufficient nucleotide variations to allow the unique allocation of each amplified sequence to its parental bacterium.
KeywordMeSH Terms
DNA Primers

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