Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes.

DNA methylation is widespread amongst eukaryotes and prokaryotes to modulate gene expression and confer viral resistance. 5-Methylcytosine (m5C) methylation has been described in genomes of a large fraction of bacterial species as part of restriction-modification systems, each composed of a methyltransferase and cognate restriction enzyme. Methylases are site-specific and target sequences vary across organisms. High-throughput methods, such as bisulfite-sequencing can identify m5C at base resolution but require specialized library preparations and single molecule, real-time (SMRT) sequencing usually misses m5C. Here, we present a new method called RIMS-seq (rapid identification of methylase specificity) to simultaneously sequence bacterial genomes and determine m5C methylase specificities using a simple experimental protocol that closely resembles the DNA-seq protocol for Illumina. Importantly, the resulting sequencing quality is identical to DNA-seq, enabling RIMS-seq to substitute standard sequencing of bacterial genomes. Applied to bacteria and synthetic mixed communities, RIMS-seq reveals new methylase specificities, supporting routine study of m5C methylation while sequencing new genomes.

Profile of protein lysine propionylation in Aeromonas hydrophila and its role in enzymatic regulation.

Protein lysine propionylation (Kpr) modification is a novel post-translational modification (PTM) of prokaryotic cells that was recently discovered; however, it is not clear how this modification regulates bacterial life. In this study, the protein Kpr modification profile in Aeromonas hydrophila was identified by high specificity antibody-based affinity enrichment combined with high resolution LC MS/MS. A total of 98 lysine-propionylated peptides with 59 Kpr proteins were identified, most of which were associated with energy metabolism, transcription and translation processes. To further understand the role of Kpr modified proteins, the K168 site on malate dehydrogenase (MDH) and K608 site on acetyl-coenzyme A synthetase (AcsA) were subjected to site-directed mutation to arginine (R) and glutamine (Q) to simulate deacylation and propionylation, respectively. Subsequent measurement of the enzymatic activity showed that the K168 site of Kpr modification on MDH may negatively regulate the MDH enzymatic activity while also affecting the survival of mdh derivatives when using glucose as the carbon source, whereas Kpr modification of K608 of AcsA does not. Overall, the results of this study indicate that protein Kpr modification plays an important role in bacterial biological functions, especially those involved in the activity of metabolic enzymes.

Structural insights into acyl-ACP selective recognition by the Aeromonas hydrophila AHL synthase AhyI.

BACKGROUND: Aeromonas hydrophila is a gram-negative bacterium and the major causative agent of the fish disease motile aeromonad septicemia (MAS). It uses N-acyl-homoserine lactone (AHL) quorum sensing signals to coordinate biofilm formation, motility, and virulence gene expression. The AHL signaling pathway is therefore considered to be a therapeutic target against pathogenic A. hydrophila infection. In A. hydrophila, AHL autoinducers biosynthesis are specifically catalyzed by an ACP-dependent AHL synthase AhyI using the precursors SAM and acyl-ACP. Our previously reported AhyI was heterologously expressed in E. coli, which showed the production characteristics of medium-long chain AHLs. This contradicted the prevailing understanding that AhyI was only a short-chain C4/C6-HSL synthase. RESULTS: In this study, six linear acyl-ACP proteins with C-terminal his-tags were synthesized in Vibrio harveyi AasS using fatty acids and E. coli produced active holo-ACP proteins, and in vitro biosynthetic assays of six AHL molecules and kinetic studies of recombinant AhyI with a panel of four linear acyl-ACPs were performed. UPLC-MS/MS analyses indicated that AhyI can synthesize short-, medium- and long-chain AHLs from SAM and corresponding linear acyl-ACP substrates. Kinetic parameters measured using a DCPIP colorimetric assay, showed that there was a notable decrease in catalytic efficiency with acyl-chain lengths above C6, and hyperbolic or sigmoidal responses in rate curves were observed for varying acyl-donor substrates. Primary sequence alignment of the six representative AHL synthases offers insights into the structural basis for their specific acyl substrate preference. To further understand the acyl chain length preference of AhyI for linear acyl-ACP, we performed a structural comparison of three ACP-dependent LuxI homologs (TofI, BmaI1 and AhyI) and identified three key hydrophobic residues (I67, F125 and L157) which confer AhyI to selectively recognize native C4/C6-ACP substrates. These predictions were further supported by a computational Ala mutation assay. CONCLUSIONS: In this study, we have redefined AhyI as a multiple short- to long-chain AHL synthase which uses C4/C6-ACP as native acyl substrates and longer acyl-ACPs (C8 ~ C14) as non-native ones. We also theorized that the key residues in AhyI would likely drive acyl-ACP selective recognition.

Prevalence of Potentially Pathogenic Antibiotic-Resistant Aeromonas spp. in Treated Urban Wastewater Effluents versus Recipient Riverine Populations: a 3-Year Comparative Study.

Antibiotic resistance continues to be an emerging threat both in clinical and environmental settings. Among the many causes, the impact of postchlorinated human wastewater on antibiotic resistance has not been well studied. Our study compared antibiotic susceptibility among Aeromonas spp. in postchlorinated effluents to that of the recipient riverine populations for three consecutive years against 12 antibiotics. Aeromonas veronii and Aeromonas hydrophila predominated among both aquatic environments, although greater species diversity was evident in treated wastewater. Overall, treated wastewater contained a higher prevalence of nalidixic acid-, trimethoprim-sulfamethoxazole (SXT)-, and tetracycline-resistant isolates, as well as multidrug-resistant (MDR) isolates compared to upstream surface water. After selecting for tetracycline-resistant strains, 34.8% of wastewater isolates compared to 8.3% of surface water isolates were multidrug resistant, with nalidixic acid, streptomycin, and SXT being the most common. Among tetracycline-resistant isolates, efflux pump genes tetE and tetA were the most prevalent, though stronger resistance correlated with tetA. Over 50% of river and treated wastewater isolates exhibited cytotoxicity that was significantly correlated with serine protease activity, suggesting many MDR strains from effluent have the potential to be pathogenic. These findings highlight that conventionally treated wastewater remains a reservoir of resistant, potentially pathogenic bacterial populations being introduced into aquatic systems that could pose a threat to both the environment and public health.IMPORTANCE Aeromonads are Gram-negative, asporogenous rod-shaped bacteria that are autochthonous in fresh and brackish waters. Their pathogenic nature in poikilotherms and mammals, including humans, pose serious environmental and public health concerns especially with rising levels of antibiotic resistance. Wastewater treatment facilities serve as major reservoirs for the dissemination of antibiotic resistance genes (ARGs) and resistant bacterial populations and are, thus, a potential major contributor to resistant populations in aquatic ecosystems. However, few longitudinal studies exist analyzing resistance among human wastewater effluents and their recipient aquatic environments. In this study, considering their ubiquitous nature in aquatic environments, we used Aeromonas spp. as bacterial indicators of environmental antimicrobial resistance, comparing it to that in postchlorinated wastewater effluents over 3 years. Furthermore, we assessed the potential of these resistant populations to be pathogenic, thus elaborating on their potential public health threat.

An Enzymatic Flow-Based Preparative Route to Vidarabine.

The bi-enzymatic synthesis of the antiviral drug vidarabine (arabinosyladenine, ara-A), catalyzed by uridine phosphorylase from Clostridium perfringens (CpUP) and a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), was re-designed under continuous-flow conditions. Glyoxyl-agarose and EziGTM1 (Opal) were used as immobilization carriers for carrying out this preparative biotransformation. Upon setting-up reaction parameters (substrate concentration and molar ratio, temperature, pressure, residence time), 1 g of vidarabine was obtained in 55% isolated yield and >99% purity by simply running the flow reactor for 1 week and then collecting (by filtration) the nucleoside precipitated out of the exiting flow. Taking into account the substrate specificity of CpUP and AhPNP, the results obtained pave the way to the use of the CpUP/AhPNP-based bioreactor for the preparation of other purine nucleosides.

Recognition and interaction of CphA from Aeromonas hydrophila with imipenem and biapenem by spectroscopic analysis in combination with molecular docking.

The molecular recognition and interaction of CphA from Aeromonas hydrophila with imipenem (Imip) and biapenem (Biap) were studied by means of the combined use of fluorescence spectra and molecular docking. The results showed that both the fluorescence quenching of CphA by Imip and Biap were caused through the combined dynamic and static quenching, and the latter was dominating in the process; the microenvironment and conformational of CphA were altered upon the addition of Imip and Biap from synchronous and three-dimensional fluorescence. The binding of CphA with Imip or Biap caused a conformational change in the loop of CphA, and through the conformational change, the loop opened the binding pocket of CphA to allow for an induced fit of the newly introduced ligand. In the binding of CphA with Imip, the whole molecule entered into the active pocket of CphA. The binding was driven by enthalpy change, and the binding force between them was mainly hydrogen bonding and Van der Waals force; whereas in the binding of CphA with Biap, only the beta-lactam ring of Biap entered into the binding pocket of CphA while the side chain was located outside the active pocket. The binding was driven by the enthalpy change and entropy change together, and the binding force between them was mainly electrostatic interaction. This study provided an insight into the recognition and binding of CphA with antibiotics, which may be helpful for designing new substrate for beta-lactamase and developing new antibiotics resistant to superbugs.

Knockout of alanine racemase gene attenuates the pathogenicity of Aeromonas hydrophila.

BACKGROUND: Aeromonas hydrophila is an opportunistic pathogen of poikilothermic and homoeothermic animals, including humans. In the present study, we described the role of Alanine racemase (alr-2) in the virulence of A. hydrophila using an alr-2 knockout mutant (A.H.Δalr). RESULTS: In mouse and common carp models, the survival of animals challenged with A.H.Δalr was significantly increased compared with the wild-type (WT), and the mutant was also impaired in its ability to replicate in the organs and blood of infected mice and fish. The A.H.Δalr significantly increased phagocytosis by macrophages of the mice and fish. These attenuation effects of alr-2 could be complemented by the addition of D-alanine to the A.H.Δalr strain. The histopathology results indicated that the extent of tissue injury in the WT-infected animals was more severe than in the A.H.Δalr-infected groups. The expression of 9 virulence genes was significantly down-regulated, and 3 outer membrane genes were significantly up-regulated in A.H.Δalr. CONCLUSIONS: Our data suggest that alr-2 is essential for the virulence of A. hydrophila. Our findings suggested alanine racemase could be applied in the development of new antibiotics against A. hydrophila.

The role of sodA and sodB in Aeromonas hydrophila resisting oxidative damage to survive in fish macrophages and escape for further infection.

Several bacteria have been defined as extracellular pathogens; however, in recent years, it has been confirmed that they have the ability to survive and escape the attack of host phagocytes, thus causing further infection. Previous studies have shown that Aeromonas hydrophila could survive in fish macrophages; however, the mechanism remains unknown. In this study, sodA and sodB of the strain A. hydrophila B11 were stable silenced by shRNA. The survival rates of intracellular sodA-RNAi and sodB-RNAi decreased by 91.8% and 74.9% and the immune escape rates decreased by about 32% and 92% respectively. At the same time, reactive oxygen species (ROS) in fish macrophages that phagocytosed sodA-RNAi and sodB-RNAi increased by 40% and 32.6%, respectively, compared to those of macrophages that phagocytosed the wild-type strain. Compared to sodA, the expression of sodB predominates in A. hydrophila without oxidative stress; however, when exposed to oxidative stress, the magnitude of up-regulation of sodA expression is significantly higher than that of sodB. With increased of methyl viologen concentration, the survival rates of sodA-RNAi and sodB-RNAi were significantly decreased. The expressions of sodA and sodB did not affect the growth of A. hydrophila without oxidative stress, but the inhibition of sodA and sodB expression led to a slight decrease in bacterial growth under oxidative stress. These results indicated that (1) sodA and sodB play an important role in the process of bacterial resistance to ROS damage in host phagocytic cells, allowing them to survive or even escape fish macrophages; (2) the sodB expression was dominant in A. hydrophila without oxidative stress, the sodA expression was up-regulated more significantly under oxidative stress, and sodA and sodB contributed equally to the process of bacterial resistance to ROS; (3) sodA and sodB complement each other and cooperate in the process of intracellular survival of bacteria to protect against ROS damage.

Aeromonas hydrophila biofilm, exoprotease, and quorum sensing responses to co-cultivation with diverse foodborne pathogens and food spoilage bacteria on crab surfaces.

The effects of dual species interactions on biofilm formation by Aeromonas hydrophila in the presence of Pseudomonas aeruginosa, Pseudomonas fluorescens, Pectobacterium carotovorum, Salmonella Typhimurium, and Listeria monocytogenes were examined. High-performance liquid chromatography and liquid-chromatography-mass spectrometry were performed to identify N-acyl homoserine lactone (AHL) molecules secreted by monocultures and dual cultures grown in crab broth. Field emission scanning electron microscopy was performed to observe attachment and biofilm formation. P. aeruginosa and P. fluorescens inhibited biofilm formation by A. hydrophila on the crab surface, without affecting their own biofilm-forming abilities. Dual biofilms of S. Typhimurium, L. monocytogenes, or P. carotovorum did not affect A. hydrophila biofilm formation. Exoprotease, AHL, and AI-2 levels were significantly reduced in dual cultures of P. aeruginosa and P. fluorescens with A. hydrophila, supporting the relationship between quorum sensing and biofilm formation. Dual-species biofilms were studied in their natural environment and in the laboratory.

Degradation and decolourization potential of an ligninolytic enzyme producing Aeromonas hydrophila for crystal violet dye and its phytotoxicity evaluation.

This study deals the biodegradation of crystal violet dye by a ligninolytic enzyme producing bacterium isolated from textile wastewater that was characterized and identified as Aeromonas hydrophila based on the 16 S rRNA gene sequence analysis. The degradation of crystal violet dye was studied under different environmental and nutritional conditions, and results showed that the isolated bacterium was effective to decolourize 99% crystal violet dye at pH 7 and temperature 35 °C in presence of sucrose and yeast extract as C and N source, respectively. This bacterium also produced lignin peroxidase and laccase enzyme, which were characterized by the SDS-PAGE analysis and found to have the molecular weight of ~ 40 and ~ 60 kDa, respectively. Further, the GC-MS analysis showed that CV dye was biotransformed into phenol, 2, 6-bis (1,1-dimethylethyl), 2',6'-dihydroxyacetophenone and benzene by the isolated bacterium and the toxicity of CV dye was reduced upto a significant level as it showed 60%, 56.67% and 46.67% inhibition in seed germination. But, after the bacterial degradation/decolourization, it showed only 43.33%, 36.67% and 16.67% inhibition in seed germination after 24, 48 and 72 h, respectively. Thus, this study concluded that the isolated bacterium has high potential for the degradation/decolourization of CV dye as well to reduce its toxicity upto a significant level.

Structural and Functional Analyses of Periplasmic 5'-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase from Aeromonas hydrophila.

The Gram-negative, rod-shaped bacterium Aeromonas hydrophila has two multifunctional 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) enzymes, MtaN-1 and MtaN-2, that differ from those in other bacteria. These proteins are essential for several metabolic pathways, including biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing. To gain insight into how these two proteins function, we determined four high-resolution crystal structures of MtaN-1 in its apo form and in complex with the substrates S-adenosyl-l-homocysteine, 5'-methylthioadenosine, and 5'-deoxyadenosine. We found that the domain structures were generally similar, although slight differences were evident. The crystal structure demonstrates that AhMtaN-1 has an extension of the binding pocket and revealed that a tryptophan in the active site (Trp199) may play a major role in substrate binding, unlike in other MTAN proteins. Mutation of the Trp199 residue completely abolished the enzyme activity. Trp199 was identified as an active site residue that is essential for catalysis. Furthermore, biochemical characterization of AhMtaN-1 and AhMtaN-2 demonstrated that AhMtaN-1 exhibits inherent trypsin resistance that is higher than that of AhMtaN-2. Additionally, the thermally unfolded AhMtaN-2 protein is capable of refolding into active forms, whereas the thermally unfolded AhMtaN-1 protein does not have this ability. Examining the different biochemical characteristics related to the functional roles of AhMtaN-1 and AhMtaN-2 would be interesting. Indeed, the biochemical characterization of these structural features would provide a structural basis for the design of new antibiotics against A. hydrophila.

Selection and characterization of alanine racemase inhibitors against Aeromonas hydrophila.

BACKGROUND: Combining experimental and computational screening methods has been of keen interest in drug discovery. In the present study, we developed an efficient screening method that has been used to screen 2100 small-molecule compounds for alanine racemase Alr-2 inhibitors. RESULTS: We identified ten novel non-substrate Alr-2 inhibitors, of which patulin, homogentisic acid, and hydroquinone were active against Aeromonas hydrophila. The compounds were found to be capable of inhibiting Alr-2 to different extents with 50% inhibitory concentrations (IC50) ranging from 6.6 to 17.7 µM. These compounds inhibited the growth of A. hydrophila with minimal inhibitory concentrations (MICs) ranging from 20 to 120 µg/ml. These compounds have no activity on horseradish peroxidase and D-amino acid oxidase at a concentration of 50 µM. The MTT assay revealed that homogentisic acid and hydroquinone have minimal cytotoxicity against mammalian cells. The kinetic studies indicated a competitive inhibition of homogentisic acid against Alr-2 with an inhibition constant (K i) of 51.7 µM, while hydroquinone was a noncompetitive inhibitor with a K i of 212 µM. Molecular docking studies suggested that homogentisic acid binds to the active site of racemase, while hydroquinone lies near the active center of alanine racemase. CONCLUSIONS: Our findings suggested that combining experimental and computational methods could be used for an efficient, large-scale screening of alanine racemase inhibitors against A. hydrophila that could be applied in the development of new antibiotics against A. hydrophila.

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

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.

Detection and Whole-Genome Sequencing of Carbapenemase-Producing Aeromonas hydrophila Isolates from Routine Perirectal Surveillance Culture.

Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a 6-week period and were without epidemiological links. Detection of the blaKPC-2 gene in one isolate prompted inclusion of non-Enterobacteriaceae in our surveillance culture workup. Whole-genome sequencing confirmed that the isolates were unrelated and provided data for Aeromonas reference genomes.

Quantitative proteomic analysis of cell envelope preparations under iron starvation stress in Aeromonas hydrophila.

BACKGROUND: Iron homeostasis is an essential process over the entire lives of both hosts and bacterial pathogens, and also plays roles in many other metabolic functions. Currently, knowledge is limited on the iron scavenging mechanism of the cell envelope in the aquatic pathogen, Aeromonas hydrophila. To understand the iron homeostasis mechanism in A. hydrophila, a dimethyl labelling based quantitative proteomics method was used to compare the differential expression of cell envelope proteins under iron starvation. RESULTS: A total of 542 cell envelope proteins were identified by LC-MS/MS, with 66 down-regulated and 104 up-regulated proteins. Bioinformatics analysis showed that outer membrane siderophores, heme and iron receptors, periplasmic iron binding proteins, inner membrane ABC transporters and H(+)-ATP synthase subunits increased in abundance while iron-cluster proteins, electron transport chain and redox proteins were down-regulated. Further q-PCR validation, in vivo addition of exogenous metabolites, and an enzyme inhibition assay revealed that redox, the energy generation process, and ATP synthase elevated the susceptibility of A. hydrophila to iron starvation. CONCLUSIONS: Our study demonstrates that the redox and energy generation process, and ATP synthase in A. hydrophila may play critical roles in iron acquisition under conditions of iron-stress. An understanding of the iron scavenging mechanism may be helpful for the development of strategies for preventing and treating A. hydrophila infection.

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

Two new natural CphA metallo-ß-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.

Theoretical studies of the hydrolysis of antibiotics catalyzed by a metallo-ß-lactamase.

In this paper, hybrid QM/MM molecular dynamics (MD) simulations have been performed to explore the mechanisms of hydrolysis of two antibiotics, Imipenen (IMI), an antibiotic belonging to the subgroup of carbapenems, and the Cefotaxime (CEF), a third-generation cephalosporin antibiotic, in the active site of a mono-nuclear ß-lactamase, CphA from Aeromonas hydrophila. Significant different transition state structures are obtained for the hydrolysis of both antibiotics: while the TS of the CEF is an ionic species with negative charge on nitrogen, the IMI TS presents a tetrahedral-like character with negative charge on oxygen atom of the carbonyl group of the lactam ring. Thus, dramatic conformational changes can take place in the cavity of CphA to accommodate different substrates, which would be the origin of its substrate promiscuity. Since CphA shows only activity against carbapenem antibiotic, this study sheds some light into the origin of the selectivity of the different MbL and, as a consequence, into the discovery of specific and potent MßL inhibitors against a broad spectrum of bacterial pathogens. We have finally probed that a re-parametrization of semiempirical methods should be done to properly describe the behavior the metal cation in active site, Zn(2+), when used in QM/MM calculations.

Aeromonas piscicola AH-3 expresses an extracellular collagenase with cytotoxic properties.

UNLABELLED: The aim of this study was to investigate the presence and the phenotypic expression of a gene coding for a putative collagenase. This gene (AHA_0517) was identified in Aeromonas hydrophila ATCC 7966 genome and named colAh. We constructed and characterized an Aeromonas piscicola AH-3::colAh knockout mutant. Collagenolytic activity of the wild-type and mutant strains was determined, demonstrating that colAh encodes for a collagenase. ColAh-collagen interaction was assayed by Far-Western blot, and cytopathic effects were investigated in Vero cells. We demonstrated that ColAh is a gluzincin metallopeptidase (approx. 100 kDa), able to cleave and physically interact with collagen, that contributes for Aeromonas collagenolytic activity and cytotoxicity. ColAh possess the consensus HEXXH sequence and a glutamic acid as the third zinc binding positioned downstream the HEXXH motif, but has low sequence similarity and distinct domain architecture to the well-known clostridial collagenases. In addition, these results highlight the importance of exploring new microbial collagenases that may have significant relevance for the health and biotechnological industries. SIGNIFICANCE AND IMPACT OF THE STUDY: Collagenases play a central role in processes where collagen digestion is needed, for example host invasion by pathogenic micro-organisms. We identified a new collagenase from Aeromonas using an integrated in silico/in vitro strategy. This enzyme is able to bind and cleave collagen, contributes for AH-3 cytotoxicity and shares low similarity with known bacterial collagenases. This is the first report of an enzyme belonging to the gluzincin subfamily of the M9 family of peptidases in Aeromonas. This study increases the current knowledge on collagenolytic enzymes bringing new perspectives for biotechnology/medical purposes.

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

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.

Characterization of the Vibrio cholerae VolA surface-exposed lipoprotein lysophospholipase.

Bacterial lipases play important roles in bacterial metabolism and environmental response. Our laboratory recently discovered that a novel lipoprotein lysophospholipase, VolA, localizes on the surface of the Gram-negative aquatic pathogen Vibrio cholerae. VolA functions to cleave exogenous lysophosphatidylcholine, freeing the fatty acid moiety for use by V. cholerae. This fatty acid is transported into the cell and can be used as a nutrient and, more importantly, as a way to alter the membrane architecture via incorporation into the phospholipid biosynthesis pathway. There are few examples of Gram-negative, surface-exposed lipoproteins, and VolA is unique, as it has a previously undercharacterized function in V. cholerae membrane remodeling. Herein, we report the biochemical characterization of VolA. We show that VolA is a canonical lipoprotein via mass spectrometry analysis and demonstrate the in vitro activity of VolA under a variety of conditions. Additionally, we show that VolA contains a conserved Gly-Xaa-Ser-Xaa-Gly motif typical of lipases. Interestingly, we report the observation of VolA homologs in other aquatic pathogens. An Aeromonas hydrophila VolA homolog complements a V. cholerae VolA mutant in growth on lysophosphatidylcholine as the sole carbon source and in enzymatic assays. These results support the idea that the lipase activity of surface-exposed VolA likely contributes to the success of V. cholerae, improving the overall adaptation and survival of the organism in different environments.