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.

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.

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.

Insights into the Inhibition of Aeromonas hydrophila d-Alanine-d-Alanine Ligase by Integration of Kinetics and Structural Analysis.

Aeromonas hydrophila, a pathogenic bacterium, is harmful to humans, domestic animals, and fishes and, moreover, of public health concern due to the emergence of multiple drug-resistant strains. The cell wall has been discovered as a novel and efficient drug target against bacteria, and d-alanine-d-alanine ligase (Ddl) is considered as an essential enzyme in bacterial cell wall biosynthesis. Herein, we studied the A. hydrophila HBNUAh01 Ddl (AhDdl) enzyme activity and kinetics and determined the crystal structure of AhDdl/d-Ala complex at 2.7 Å resolution. An enzymatic assay showed that AhDdl exhibited higher affinity to ATP (Km: 54.1 ± 9.1 µM) compared to d-alanine (Km: 1.01 ± 0.19 mM). The kinetic studies indicated a competitive inhibition of AhDdl by d-cycloserine (DCS), with an inhibition constant (Ki) of 120 µM and the 50% inhibitory concentrations (IC50) value of 0.5 mM. Meanwhile, structural analysis indicated that the AhDdl/d-Ala complex structure adopted a semi-closed conformation form, and the active site was extremely conserved. Noteworthy is that the substrate d-Ala occupied the second d-Ala position, not the first d-Ala position. These results provided more insights for understanding the details of the catalytic mechanism and resources for the development of novel drugs against the diseases caused by A. hydrophila.

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.

Identification of a Novel N-Acyl Homoserine Lactone Synthase, AhyI, in Aeromonas hydrophila and Structural Basis for Its Substrate Specificity.

In the Gram-negative bacterium Aeromonas hydrophila, N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS) influences pathogenicity, protein secretion, and motility. However, the catalytic mechanism of AHL biosynthesis and the structural basis and substrate specificity for AhyI members remain unclear. In this study, we cloned the ahyI gene from the isolate A. hydrophila HX-3, and the overexpressed AhyI protein was confirmed to produce six types of AHLs by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis, contrasting with previous reports that AhyI only produces N-butanoyl-l-homoserine lactone (C4-HSL) and N-hexanoyl-l-homoserine lactone (C6-HSL). The results of an in vitro biosynthetic assay showed that purified AhyI can catalyze the formation of C4-HSL using S-adenosyl-l-methionine (SAM) and butyryl-acyl carrier protein (ACP) as substrates and indicated that the fatty acyl substrate used in AhyI-mediated AHL synthesis is derived from acyl-ACP rather than acyl-CoA. The kinetic data of AhyI using butyryl-ACP as an acyl substrate indicated that the catalytic efficiency of the A. hydrophila HX-3 AhyI enzyme is within an order of magnitude compared to other LuxI homologues. In this study, for the first time, the tertiary structural modeling results of AhyI and those of molecular docking and structural and functional analyses showed the importance of several crucial residues, as well as the secondary structure with respect to acylation. A Phe125-Phe152 clamp grasps the terminal methyl group to assist in stabilizing the long acyl chains in a putative binding pocket. The stacking interactions within a strong hydrophobic environment, a hydrogen-bonding network, and a ß bulge presumably stabilize the ACP acyl chain for the attack of the SAM α-amine toward the thioester carbon, offering a relatively reasonable explanation for how AhyI can synthesize AHLs with diverse acyl-chain lengths. Moreover, Trp34 participates in forming the binding pocket for C4-ACP and becomes ordered upon SAM binding, providing a good basis for catalysis. The novel finding that AhyI can produce both short- and long-chain AHLs enhances current knowledge regarding the variety of AHLs produced by this enzyme. These structural data are expected to serve as a molecular rationale for AHL synthesis by AhyI.

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.

Potential KPC-2 carbapenemase reservoir of environmental Aeromonas hydrophila and Aeromonas caviae isolates from the effluent of an urban wastewater treatment plant in Japan.

Aeromonas hydrophila and Aeromonas caviae adapt to saline water environments and are the most predominant Aeromonas species isolated from estuaries. Here, we isolated antimicrobial-resistant (AMR) Aeromonas strains (A. hydrophila GSH8-2 and A. caviae GSH8M-1) carrying the carabapenemase blaKPC-2 gene from a wastewater treatment plant (WWTP) effluent in Tokyo Bay (Japan) and determined their complete genome sequences. GSH8-2 and GSH8M-1 were classified as newly assigned sequence types ST558 and ST13, suggesting no supportive evidence of clonal dissemination. The strains appear to have acquired blaKPC-2 -positive IncP-6-relative plasmids (pGSH8-2 and pGSH8M-1-2) that share a common backbone with plasmids in Aeromonas sp. ASNIH3 isolated from hospital wastewater in the United States, A. hydrophila WCHAH045096 isolated from sewage in China, other clinical isolates (Klebsiella, Enterobacter and Escherichia coli), and wastewater isolates (Citrobacter, Pseudomonas and other Aeromonas spp.). In addition to blaKPC-2 , pGSH8M-1-2 carries an IS26-mediated composite transposon including a macrolide resistance gene, mph(A). Although Aeromonas species are opportunistic pathogens, they could serve as potential environmental reservoir bacteria for carbapenemase and AMR genes. AMR monitoring from WWTP effluents will contribute to the detection of ongoing AMR dissemination in the environment and might provide an early warning of potential dissemination in clinical settings and communities.

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.

Application of docking and active site analysis for enzyme linked biodegradation of textile dyes.

Growth of textile industries led to production of enormous dye varieties. These textile dyes are largely used, chemically stable and easy to synthesize. But they are recalcitrant and persist as less biodegradable pollutants when discharged into waterbodies. Potential use of enzyme-linked bioremediation of textile dyes will control their toxicity in waterbodies. Bioinformatics and Molecular docking tool provides an insight into remediation mechanism by predicting susceptibility of dye degradation using oxidoreductive enzymes. In this study, six dyes, Reactive Red F3B, Remazol Red RGB, Joyfix Red RB, Joyfix Yellow MR, Remazol Blue RGB and Turquoise CL-5B of azo, anthraquinone and phthalocyanine molecular class were identified as potential targets for degradation by laccase and azoreductase of Aeromonas hydrophila in addition to Lysinibacillus sphaericus through in silico docking tool BioSolveIT-FlexX. Azoreductase breaks azo bonds by ping-pong mechanism whereas laccase decolorizes dyes by free radical mechanism which is not specific in nature. Results were analyzed based on parameters like stability, catalytic action and selectivity for enzyme-dye interactions. Amino acids of enzymes interacted with several dyes substantiating variations in active site for enzyme-ligand binding affinity. This suggests the role of enzymes in decolorizing an extensive variety of textile dyes, thereby, aiding in understanding the enzyme mechanisms in Bioremediation.

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.

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

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.

A carbapenem-resistant clinical isolate of Aeromonas hydrophila in Japan harbouring an acquired gene encoding GES-24 ß-lactamase.

Several species of Aeromonas produce the enzyme CphA metallo-ß-lactamase. This study describes an isolate of Aeromonas hydrophila harbouring an acquired gene encoding the carbapenemase GES-24. This isolate was obtained from an inpatient in Okinawa, Japan, with no apparent record of travelling overseas. The minimum inhibitory concentrations of carbapenems against this isolate were 8 µg ml-1 for imipenem and 16 µg ml-1 for meropenem. Recombinant GES-24 hydrolyzed all of the tested ß-lactams, including imipenem and meropenem. The genomic environment surrounding blaGES-24 was intI1-blaGES-24-aac(6')-IIc-qacEdelta1-sulI-orfX-tetR-tetE. This is the first report of A. hydrophila producing a GES-type carbapenemase.

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

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.

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.

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.

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.

1,2,4-Triazole-3-thione Compounds as Inhibitors of Dizinc Metallo-ß-lactamases.

Metallo-ß-lactamases (MBLs) cause resistance of Gram-negative bacteria to ß-lactam antibiotics and are of serious concern, because they can inactivate the last-resort carbapenems and because MBL inhibitors of clinical value are still lacking. We previously identified the original binding mode of 4-amino-2,4-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione (compound IIIA) within the dizinc active site of the L1 MBL. Herein we present the crystallographic structure of a complex of L1 with the corresponding non-amino compound IIIB (1,2-dihydro-5-(2-methylphenyl)-3H-1,2,4-triazole-3-thione). Unexpectedly, the binding mode of IIIB was similar but reverse to that of IIIA. The 3 D structures suggested that the triazole-thione scaffold was suitable to bind to the catalytic site of dizinc metalloenzymes. On the basis of these results, we synthesized 54 analogues of IIIA or IIIB. Nineteen showed IC50 values in the micromolar range toward at least one of five representative MBLs (i.e., L1, VIM-4, VIM-2, NDM-1, and IMP-1). Five of these exhibited a significant inhibition of at least four enzymes, including NDM-1, VIM-2, and IMP-1. Active compounds mainly featured either halogen or bulky bicyclic aryl substituents. Finally, some compounds were also tested on several microbial dinuclear zinc-dependent hydrolases belonging to the MBL-fold superfamily (i.e., endonucleases and glyoxalase II) to explore their activity toward structurally similar but functionally distinct enzymes. Whereas the bacterial tRNases were not inhibited, the best IC50 values toward plasmodial glyoxalase II were in the 10 µm range.