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

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.     

Binding of laminin and fibronectin by the trypsin-resistant major structural domain of the crystalline virulence surface array protein of Aeromonas salmonicida.

The surface of Aeromonas salmonicida is covered by a tetragonal paracrystalline array (A-layer) composed of a single protein (A-protein, Mr = 50,778). This array is a virulence factor. Cells containing A-layer and isolated A-layer sheets specifically bound laminin and fibronectin with high affinity. Binding by cells was inactivated by selective removal of A-layer at pH 2.2, and neither isogenic A-layer-deficient A. salmonicida mutants nor tetragonal paracrystalline array producing Aeromonas hydrophila and Aeromonas sobria strains bound either matrix protein. Laminin binding was by a single class of high affinity interactions (cell Kd = 1.52 nM), whereas fibronectin bound via two classes of interactions, one being similar to that of laminin (cell Class 2 interaction Kd = 6.6 nM). This interaction with both proteins was partly hydrophobic. The Class 1 fibronectin interaction was of lower affinity (cell Kd = 218 nM) and distinct. Purified A-protein inhibited binding of both matrix proteins to A-layer, and trypsin cleavage localized the matrix-protein binding region to the N-terminal major trypsin-resistant structural domain of A-protein. Monoclonal antibody inhibition studies showed that A-protein was folded such that Fabs of only one of two antibodies with epitopes mapping C-terminal to this trypsin-resistant peptide was capable of blocking binding.     

Structure of the tetragonal surface virulence array protein and gene of Aeromonas salmonicida

The paracrystalline surface protein array of the pathogenic bacterium Aeromonas salmonicida is a primary virulence factor with novel binding capabilities. The species-specific structural gene (vapA) for this array protein (A-protein) was cloned into lambda gt11 but was unstable when expressed in Escherichia coli, undergoing an 816-base pair deletion due to a 21-base pair direct repeat within the gene. However, the gene was stable in cosmid pLA2917 as long as expression was poor. A-protein was located in the cytoplasmic, inner membrane and periplasmic fractions in E. coli. The DNA sequence revealed a 1,506-base pair open reading frame encoding a protein consisting of a 21-amino acid signal peptide, and a 481-residue 50,778 molecular weight protein containing considerable secondary structure. When assembled into a paracrystalline protein array on Aeromonas the cell surface A-protein was totally refractile to cleavage by trypsin, but became trypsin sensitive when disassembled. Trypsin cleavage of the isolated protein provided evidence that both the NH2- and COOH-terminal regions form distinct structural domains, consistent with three-dimensional ultrastructural evidence. The NH2-terminal 274-residue domain remained refractile to trypsin activity. This segment connects by a trypsin and CNBr-sensitive 78-residue linker region to a COOH-terminal 129-residue fragment which could apparently refold into a partially trypsin-resistant structure after cleavage at residue 323.     

Biochemical and functional characterization of UDP-galactose 4-epimerase from Aeromonas hydrophila

Bacteria of genus Aeromonas, responsible for a variety of pathological conditions in humans and fish, are ubiquitous waterborne bacteria. Aeromonas produces several virulent factors including a complex of lipopolysaccharide and surface array protein, involved in colonization. UDP-galactose 4-epimerase (GalE) catalyzes the production of UDP-galactose, a precursor for lipopolysaccharide biosynthesis, and thus is an important drug target. GalE exhibits interspecies variation and heterogeneity at its structural and functional level and therefore, the differences between the GalE of the host and the pathogen can be exploited for drug designing. In the present study, we report biochemical and functional characterization of the recombinant GalE of Aeromonas hydrophila. Unlike GalE reported from all other species, the purified recombinant GalE of A. hydrophila was found to exist as a monomer. This is the first report of UDP-galactose 4-epimerase from any species being a monomer. The molecular mass of the 6xHis-rGalE was determined to be 38271.477 (m/z). The 6xHis-rGalE with a K(m) of 0.5 mM for UDP-galactose exhibited optimum activity at 37 degrees C and pH 8-9. Spectrofluorimetric and CD analysis confirmed that the thermal inactivation was due to structural changes and not due to the NAD-dissociation. A relatively more ordered structure of the enzyme at pH 8 and 9 as compared to that at pH 6 or 7 suggests a key role of the electrostatic interactions in maintaining its native tertiary structure.     

Biochemical characterization and site-directed mutational analysis of the double chitin-binding domain from chitinase 92 of Aeromonas hydrophila JP101

Chitinase 92 from Aeromonas hydrophila JP101 contains C-terminal repeated chitin-binding domains (ChBDs) which were named ChBD(CI) and ChBD(CII) and classified into family 5 carbohydrate-binding modules on the basis of sequence. In this work, we constructed single and double ChBD by use of the pET system, which expressed as isolated ChBD(CII) or ChBD(CICII). Polysaccharide-binding studies revealed that ChBD(CICII) not only bound to chitin, but also to other insoluble polysaccharides such as cellulose (Avicel) and xylan. In comparison with ChBD(CII), the binding affinities of ChBD(CICII) are about 10- and 12-fold greater toward colloidal and powdered chitin, indicating that a cooperative interaction exists between ChBD(CI) and ChBD(CII). In order to investigate the roles of the highly conserved aromatic amino acids in the interaction of ChBD(CICII) and chitin, we have performed site-directed mutagenesis. The data showed that W773A, W792A, Y796A and W797A mutant proteins exhibited a much weaker affinity for chitin than wild-type protein, suggesting that these residues play important roles in chitin binding.     

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

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.     

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

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)).     

Enumeration and characterization of Aeromonas hydrophila and Aeromonas caviae isolated from grocery store produce.

Starch-ampicillin agar was used to quantitatively isolate Aeromonas sp. from retail grocery store produce. All produce sampled, including parsley, spinach, celery, alfalfa sprouts, broccoli, and lettuce, contained Aeromonas sp. In most instances, the count of Aeromonas sp. increased 10- to 1,000-fold during 2 weeks of storage at 5 degrees C. Eleven (92%) of 12 kinds of produce yielded cytotoxic Aeromonas sp. Identification as Aeromonas hydrophila was the strongest indicator of cytotoxicity, and all 29 (100%) A. hydrophila isolates and 1 (6%) of 16 A. caviae isolates were cytotoxic. Twenty-seven (90%) of 30 cytotoxic Aeromonas sp. strains produced hemolysins. Strong correlations were also noted between ability to produce cytotoxin and positive Voges-Proskauer, lysine decarboxylase, and sorbitol fermentation reactions. It appears that grocery store produce is a potentially significant source of cytotoxic Aeromonas sp. and should be considered in the epidemiology of A. hydrophila gastroenteritis.     

Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein.

The surface protein composition of members of a serogroup of Aeromonas hydrophila which exhibit high virulence for fish was examined. Treatment of whole cells of representative strain A. hydrophila TF7 with 0.2 M glycine buffer (pH 4.0) resulted in the release of sheets of a tetragonal surface protein array. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis showed that this sheet material was composed primarily of a protein of apparent molecular weight 52,000 (52K protein). A 52K protein was also the predominant protein in glycine extracts of other members of the high-virulence serogroup. Immunoblotting with antiserum raised against formalinized whole cells of A. hydrophila TF7 showed the 52K S-layer protein to be the major surface protein antigen, and impermeant Sulfo-NHS-Biotin cell surface labeling showed that the 52K S-layer protein was the only protein accessible to the Sulfo-NHS-Biotin label and effectively masked underlying outer membrane (OM) proteins. In its native surface conformation the 52K S-layer protein was only weakly reactive with a lactoperoxidase 125I surface iodination procedure. A UV-induced rough lipopolysaccharide (LPS) mutant of TF7 was found to produce an intact S layer, but a deep rough LPS mutant was unable to maintain an array on the cell surface and excreted the S-layer protein into the growth medium, indicating that a minimum LPS oligosaccharide size was required for A. hydrophila S-layer anchoring. The 52K S-layer protein exhibited hear-dependent SDS-solubilization behavior when associated with OM, but was fully solubilized at all temperatures after removal from the OM, indicating a strong interaction of the S layer with the underlying OM. The native S layer was permeable to 125I in the lactoperoxidase radiolabeling procedure, and two major OM proteins of molecular weights 30,000 and 48,000 were iodinated. The 48K species was a peptidoglycan-associated, transmembrane protein which exhibited heat-modifiable SDS solubilization behaviour characteristic of a porin protein. A 50K major peptidoglycan-associated OM protein which was not radiolabeled exhibited similar SDS heat modification characteristics and possibly represents a second porin protein.     

Biochemical and immunological characterization of the cell surface of the fish pathogenic bacterium Aeromonas salmonicida

The identification of lipopolysaccharide as periodic acid-Schiff positive material, present in the membrane fraction of the fish pathogenic Gram-negative bacterium Aeromonas salmonicida, analyzed by SDS-polyacrylamide gel electrophoresis, is shown. Such analysis has revealed several periodic acid-Schiff positive bands and many membrane proteins among which a pathogenicity-related Mr 54000 protein as a constituent of an additional surface layer outside the outer membrane (Evenberg et al., (1982) Biochim. Biophys. Acta 684, 241-248). The latter protein, designated as additional cell envelope protein or ACE protein, has been purified and characterized in our laboratory (Evenberg and Lugtenberg, (1982) Biochim. Biophys. Acta 684, 249-254). Most strains produce both high and low molecular weight lipopolysaccharide species, presumably corresponding with the presence and (virtual) absence, respectively, of an O-antigenic chain. The property to produce high molecular weight lipopolysaccharide can be lost upon subculturing in laboratory growth media and such is greatly enhanced by the prior loss of the ability to produce ACE protein. Lipopolysaccharide and ACE protein were identified as the major antigens. A new polysaccharide-like antigen, designated as PS-antigen, was detected. Moreover, immunological indications for the presence of a lipoprotein in A. salmonicida are described. The surface localization of the antigens was determined by testing whether preadsorption of antisera by intact cells decreased the binding of IgG to these antigens, or decreased the ability of the sera to agglutinate cells. According to these criteria lipopolysaccharide, ACE protein and PS-antigen are the major surface-located antigens. Material cross-reactive with lipopolysaccharide, ACE protein and PS-antigen has been found in a large number of strains. Several lines of evidence indicate the presence of interactions between ACE protein and lipopolysaccharide. Based on these results a molecular model of the cell envelope of virulent A. salmonicida is presented.     

嗜水气单胞菌若干毒力因子同时缺失的突变株的构建及特性分析

以携带质粒pAM12 0 (Tcr Tn916 )的大肠杆菌CG12 0株为供体菌 ,采用滤膜接合法与受体菌嗜水气单胞菌J_1株 (cfzr)进行接合转移 ,在含Tc和cfz选择平板上进行筛选。共获接合转移菌落 380 0个 ,其接合频率为 3× 10 - 5(按供体细胞计算 )。任取 38个接合子 ,提取基因组DNA ,以嗜水气单胞菌特异性 16SrDNA引物进行PCR扩增 ,所有接合子均阳性。为证明Tn916确实插入基因组 ,以四环素基因 (tet)引物进行PCR扩增 ,结果所有抗性接合子均扩增出一条特异条带。与亲本J_1株相比 ,所有接合子的主要毒力因子如蛋白酶、溶血素、DNA酶和淀粉酶等均不表达 ,对小鼠失去致病力 ,其LD50 大于 10 9CFU。接合子连传 10次后 ,四环素抗性消失 ,但毒力未恢复 ,说明通过转座子Tn916的插入可获得稳定的无毒嗜水气单胞菌突变株。Tn916引起嗜水气单胞菌毒力性状改变的机制有待研究 ,推测可能与该菌染色体上存在Tn916的热点或毒力岛有关。     

Molecular cloning and characterization of an extracellular protease gene from Aeromonas hydrophila.

A structural gene which codes for an extracellular protease in Aeromonas hydrophilia SO2/2 and D13 was cloned in Escherichia coli C600-1 by using pBR322 as a vector. The gene codes for a temperature-stable protease with a molecular mass of approximately 38,000 daltons. The protein was secreted to the periplasm of E. coli C600-1 and purified by osmotic shock. Cloned protease (P3) was identical in molecular mass and properties to the one purified from A. hydrophila SO2/2 culture supernatant as an extracellular product.     

Isolation and biochemical characterization of the S-layer protein from a pathogenic Aeromonas hydrophila strain.

The regular surface protein array (S layer) present on Aeromonas hydrophila TF7 is composed of a single species of protein of apparent molecular weight 52,000. This protein was extracted from whole cells by treatment with 0.2 M glycine hydrochloride (pH 3.0). The protein was purified to homogeneity by ion-exchange chromatography and reverse-phase high-performance liquid chromatography. Amino acid composition analysis showed that the protein contained 520 residues per molecule, 41% of which were hydrophobic. Cysteine was absent. A pI of 4.6 was determined for the protein, and only a single isoelectric form was detected. The purified protein displayed the hydrophobic characteristic of binding to octyl-Sepharose gels, but the salt aggregation test showed that it did not confer hydrophobicity to the cell surface when present as an intact S layer. The molecule aggregated strongly in aqueous solution as determined by sedimentation equilibrium studies. Circular dichroism spectra showed that the S-layer protein was composed of a large amount of beta-sheet (approximately 44%), a limited amount of alpha-helix (19%), and 12% beta-turn, with the remainder of the molecule being aperiodic. No significant difference in secondary structure content was measured in the presence of the metal chelator EDTA. The N-terminal amino acid sequence was determined for the first 30 residues. No sequence homology with other S-layer proteins was found.     

Porphyrin binding by the surface array virulence protein of Aeromonas salmonicida.

Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.     

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

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.     

Prevalence and distribution of Aeromonas hydrophila in the United States.

The abundance of Aeromonas hydrophila was measured in 147 natural aquatic habitats in 30 states and Puerto Rico. Viable cell counts were used to estimate density at all sites by using Rimler-Shotts medium, a differential presumptive medium for A. hydrophila. Temperature, pH, conductivity, salinity, and turbidity were measured simultaneously with water sample collection. The density of A. hydrophila was higher in lotic than in lentic systems. Saline systems had higher densities of A. hydrophila than did freshwater systems. A. hydrophila could not be isolated from extremely saline, thermal, or polluted waters, even though it was found over wide ranges of salinity, conductivity, temperature, pH, and turbidity. Of the water quality parameters measured, only conductivity was significantly regressed with density of A. hydrophila.     

Purification and characterization of the N-terminal domain of ExeA: a novel ATPase involved in the type II secretion pathway of Aeromonas hydrophila

Aeromonas hydrophila secretes a number of degradative enzymes and toxins into the external milieu via the type II secretory pathway or secreton. ExeA is an essential component of this system and is necessary for the localization and/or multimerization of the secretin ExeD. ExeA contains two sequence motifs characteristic of the Walker superfamily of ATPases. Previous examination of substitution derivatives altered in these motifs suggested that ATP binding or hydrolysis is required for ExeAB complex formation and subsequent secretion function. To directly examine ExeA function, the N-terminal cytoplasmic domain of ExeA with the addition of a C-terminal hexahistidine tag (cytExeA) was overproduced in Escherichia coli and purified by metal chelate affinity and anion-exchange chromatographic techniques. Purified preparations of cytExeA exhibited ATPase activity in the presence of several divalent cations, Mg2+ being the preferred cation, with an optimum reaction temperature of approximately 37 to 42 degrees C and an optimum pH of 7 to 8. cytExeA exhibited an apparent K(m) for Mg-ATP of 0.22 mM and a V(max) of 0.72 nmol min(-1) mg(-1) of protein. cytExeA displayed low specificity for nucleoside triphosphate substrates and was significantly inhibited by F-type ATPase inhibitors. Gel filtration analyses of cytExeA, ExeA, and ExeAB indicated that ExeA dimerizes and forms a very large complex with ExeB. These findings support a model whereby ExeAB utilizes energy derived from ATP hydrolysis to facilitate the correct localization and multimerization of the ExeD secretin.     

Dynamics of Aeromonas hydrophila, Aeromonas sobria, and Aeromonas caviae in a sewage treatment pond.

The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at all stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic.     

Prevalence and distribution of Aeromonas hydrophila in the United States.

The abundance of Aeromonas hydrophila was measured in 147 natural aquatic habitats in 30 states and Puerto Rico. Viable cell counts were used to estimate density at all sites by using Rimler-Shotts medium, a differential presumptive medium for A. hydrophila. Temperature, pH, conductivity, salinity, and turbidity were measured simultaneously with water sample collection. The density of A. hydrophila was higher in lotic than in lentic systems. Saline systems had higher densities of A. hydrophila than did freshwater systems. A. hydrophila could not be isolated from extremely saline, thermal, or polluted waters, even though it was found over wide ranges of salinity, conductivity, temperature, pH, and turbidity. Of the water quality parameters measured, only conductivity was significantly regressed with density of A. hydrophila.     

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.