Purification and Characterization of the Lecithin-Dependent Thermolabile Hemolysin Vhe1 from the Vibrio sp. Strain MA3 Associated with Mass Mortality of Pearl Oyster (Pinctada fucata).

In a previous study, we isolated a Vibrio sp. strain MA3 and its virulence factor, a hemolysin encoded by vhe1. This strain is associated with mass mortalities of the pearl oyster Pinctada fucata. In the present study, the vhe1 gene from strain MA3 was cloned and its encoded product was purified and characterized. Our results show that the vhe1 gene encodes a protein of 417 amino acids with an estimated molecular mass of 47.2 kDa and a pI of 5.14. The deduced protein, Vhe1, was found to contain the conserved amino acid sequence (GDSL motif) of the hydrolase/esterase superfamily and five conserved blocks characteristic of SGNH hydrolases. A BLAST homology search indicated that Vhe1 belongs the lecithin-dependent hemolysin/thermolabile hemolysin (LDH/TLH) family. In activity analyses, the optimal temperature for both the hemolytic and phospholipase activities of Vhe1 was 50 °C. Vhe1 hemolytic activity and phospholipase activity were highest at pH 8.5 and pH 8.0, respectively. However, both enzymatic activities sharply decreased at high temperature (> 50 °C) and pH < 7.0. Compared with previously reported hemolysins, Vhe1 appeared to be more thermal- and pH-labile. Both its hemolytic activity and phospholipase activity were significantly inhibited by CuCl2, CdCl2, ZnCl2, and NiCl2, and slightly inhibited by MnCl2 and CoCl2. Vhe1 showed higher phospholipase activity toward medium-chain fatty acids (C8-C12) than toward shorter- and longer-chain fatty acids. These results accumulate knowledge about the LDH/TLH of V. alginolyticus, which detailed characterization has not been reported, and contribute to solving of the mass mortality of pearl oyster.

Comparative proteomic profiling reveals specific adaption of Vibrio anguillarum to oxidative stress, iron deprivation and humoral components of innate immunity.

The gram-negative bacterium Vibrio (Listonella) anguillarum (VA) is the causative agent of vibriosis, a terminal hemorrhagic septicemia affecting the aquacultural industry across the globe. In the current study we used label-free quantitative proteomics to investigate how VA adapts to conditions that mimic defined aspects of vibriosis-related stress such as exposure to oxidative stress (H2O2), exposure to humoral factors of innate immunity through incubation with Atlantic salmon serum, and iron deprivation upon supplementation of 2,2'-dipyridyl (DIP) to the growth medium. We also investigated how regulation of virulence factors may be governed by the VA growth phase and availability of nutrients. All experimental conditions explored revealed stress-specific proteomic adaption of VA and only nine proteins were found to be commonly regulated in all conditions. A general observation made for all stress-related conditions was regulation of multiple metabolic pathways. Notably, iron deprivation and exposure to Atlantic salmon serum evoked upregulation of iron acquisition mechanisms. The findings made in the present study represent a source of potential virulence determinants that can be of use in the search for means to understand vibriosis. SIGNIFICANCE: Vibriosis in fish and shellfish caused by V. anguillarum (VA) is responsible for large economic losses in the aquaculture sector across the globe. However, not much is known about the defense mechanism of this pathogen to percept and adapt to the imposed stresses during infection. Analyzing the response of VA to multiple host-related physiochemical stresses, the quantitative proteomic analysis of the present study indicates modulation of several virulence determinants and key defense networks of this pathogen. Our findings provide a theoretical basis to enhance our understanding of VA pathogenesis and can be employed to improve current intervention strategies to control vibriosis in aquaculture.

Characteristics of the iron uptake-related process of a pathogenic Vibrio splendidus strain associated with massive mortalities of the sea cucumber Apostichopus japonicus.

Under low iron bioavailability environment, many bacteria acquire iron for growth and survival through siderophore-mediated iron acquisition systems. However, until now, little research on the growth, siderophore production and siderophore receptors of Vibrio splendidus Vs under iron limited conditions has been reported. In our present study, V. splendidus Vs could survive in media supplemented with 160 µM 2,2'-dipyridyl (DIP), but 74.5% of the growth was suppressed at 48 h, while the siderophore production of V. splendidus Vs increased by 35.9%. As the OD600 of V. splendidus Vs decreased when the concentration of DIP was increased from 40 to 80 µM, the siderophore production of V. splendidus Vs increased from 34.0% to 43.4% at 24 h, and it was further determined to be a hydroxamate siderophore. To explore the potential siderophore receptors anchored on the outer membrane of V. splendidus Vs, outer membrane proteins from cells grown with and without 80 µM DIP were extracted and the differentially expressed proteins were identified by SDS-PAGE and MALDI-TOF/TOF MS. Five proteins, aerobactin siderophore receptor IutA, enterobactin receptor protein FepA, ATP synthase subunit A, ATP synthase subunit B and the ATP synthase F0F1 subunit beta were identified. Real-time reverse transcriptase PCR showed that mRNA levels of iutA, fepA, atpA, atpB and atpß-F0F1 were upregulated 271.5-, 15.1-, 1.1-, 2.5- and 67.9-fold respectively, after 6 h in cells treated with 80 µM DIP. In addition, the promoters of the siderophore receptor genes of iutA and fepA had apparent ferric uptake regulator (Fur) binding sites. Combined with the simultaneous production of both the hydroxamate siderophore and its corresponding aerobactin siderophore receptor IutA, these results suggested that there might be a hydroxamate siderophore-IutA mediated iron uptake pathway in V. splendidus Vs.

Speeding up bioproduction of selenium nanoparticles by using Vibrio natriegens as microbial factory.

Selenium and selenium nanoparticles (SeNPs) are extensively used in biomedicine, electronics and some other industrial applications. The bioproduction of SeNPs is gaining interest as a green method to manufacture these biotechnologically relevant products. Several microorganisms have been used for the production of SeNPs either under aerobic or anaerobic conditions. Vibrio natriegens is a non-pathogenic fast-growing bacterium, easily cultured in different carbon sources and that has recently been engineered for easy genetic manipulation in the laboratory. Here we report that V. natriegens was able to perfectly grow aerobically in the presence of selenite concentrations up to 15 mM with a significant survival still observed at concentrations as high as 100 mM selenite. Electron microscopy and X-ray spectroscopy analyses demonstrate that V. natriegens cells growing aerobically in selenite-containing LB medium at 30 °C produced spherical electron-dense SeNPs whose size ranged from 100-400 nm. Selenite reduction just started at the beginning of the exponential growth phase and the release of SeNPs was observed after cell lysis. Remarkably, V. natriegens produced SeNPs faster than other described microorganisms that were proposed as model bioreactors for SeNPs production. Thus, the fast-growing V. natriegens bacterium becomes a suitable biocatalyst for bioremediation of selenite and for speeding-up the eco-friendly synthesis of SeNPs.

Autoinducer-2 detection among commensal oral streptococci is dependent on pH and boric acid.

Autoinducer-2, considered a universal signaling molecule, is produced by many species of bacteria; including oral strains. Structurally, autoinducer-2 can exist bound to boron (borated autoinducer-2). Functionally, autoinducer-2 has been linked to important bacterial processes such as virulence and biofilm formation. In order to test production of autoinducer-2 by a given bacterial strain, a bioassay using marine bioluminescent bacteria Vibrio harveyi as a reporter for autoinducer-2 has been designed. We hypothesize that pH adjustment and addition of boron are required for optimal bioluminescence and accurate autoinducer-2 detection. Using this reporter strain we tested autoinducer-2 activity from two oral commensal species, Streptococcus gordonii DL1 and Streptococcus oralis 34. Spent broth was collected and adjusted to pH 7.5 and supplemented with boric acid prior to measuring autoinducer- 2 activity. Results show that low pH inhibits bioluminescence of the reporter strain, but pH 7.5 allows for bioluminescence induction and proper readings of autoinducer-2 activity. Addition of boric acid also has a positive effect on bioluminescence allowing for a more sensitive detection of autoinducer-2 activity. Our data suggests that although autoinducer-2 is present in spent broth, low pH and/or low levels of boric acid become an obstacle for proper autoinducer-2 detection. For proper autoinducer-2 detection, we propose a protocol using this bioassay to include pH adjustment and boric acid addition to spent broth. Studies on autoinducer-2 activity in several bacteria species represent an important area of study as this universal signaling molecule is involved in critical bacterial phenotypes such as virulence and biofilm formation.

Biosurfactant production by hydrocarbon-degrading Brevibacterium and Vibrio isolates from the sea pen Pteroeides spinosum (Ellis, 1764).

Among filter-feeders, pennatulids are the most complex and polymorphic members of the cnidarian class Anthozoa. They display a wide distribution throughout all the oceans, constituting a significant component of the sessile megafauna from intertidal to abyssal depths. In this study, a total of 118 bacterial isolates from enrichment cultures, carried out with homogenates of the sea pen Pteroeides spinosum (Ellis, 1764), were screened for hydrocarbon utilization by using the 2,6-dichlorophenol indophenol assay. Among them, 83 hydrocarbon-oxidizing isolates were analyzed for biosurfactant production by standard screening tests (i.e., emulsifying activity, E24 detection, surface tension measurement, microplate assay). The 16S rRNA gene sequencing revealed the affiliation of the most promising isolates to the genera Brevibacterium and Vibrio. Biosurfactant production resulted strongly affected by salinity and temperature conditions, and occurred in the presence of diesel oil and/or crude oil, whereas no production was observed when isolates were grown on tetradecane. The strains resulted able to create stable emulsions, thus suggesting the production of biosurfactants. Further analyses revealed a glycolipidic nature of the biosurfactant extracted from Vibrio sp. PBN295, a genus that has been only recently reported as biosurfactant producer. Results suggest that pennatulids could represent a novel source for the isolation of hydrocarbon-oxidizing bacteria with potential in biosurfactant production.

Inhibition of marine Vibrio sp. by pyoverdine from Pseudomonas aeruginosa PA1.

Siderophores are low-molecular-weight chemicals that are secreted by many microorganisms to chelate iron from the external environment in order to facilitate their growth and diverse metabolisms. In this study, a fluorescent siderophore, pyoverdine, secreted by Pseudomonas aeruginosa PA1 was purified by affinity chromatography using Cu-sepharose. Pyoverdine was determined to have a molecular mass of 1333.54 Da, as determined by MALDI-TOF/TOF, and belong to type I pyoverdine, as determined by PCR analysis of its corresponding outer membrane ferri-pyoverdine receptor. Pyoverdine showed different degrees of inhibitory effects on the growth of marine Vibrio sp. strains. It was also shown that the biofilm developed by Vibrio parahaemolyticus WzW1 and Wz2121 and Vibrio cyclitrophicus HS12 was significantly reduced, alone with the repressed growth in the presence of pyoverdine. Siderophore production was determined in the strains of Vibrio sp. in response to the pyoverdine-induced iron-limited conditions. The siderophore production of most Vibrio sp. was up-regulated, with the exception of the bacteria that produced little siderophore. Furthermore, Apostichopus japonicus cultured in pyoverdine pretreated seawater showed a relative percent of survival of 89% when they were challenged by Vibrio splendidus. Our results demonstrated that pyoverdine may be a promising agent that could be potentially applied to treat vibriosis.

Novel bioemulsifier produced by a Paenibacillus strain isolated from crude oil.

BACKGROUND: Surface active compounds produced by microorganisms are attracting a pronounced interest due to their potential advantages over their synthetic counterparts, and to the fact that they could replace some of the synthetics in many environmental and industrial applications. RESULTS: Bioemulsifier production by a Paenibacillus sp. strain isolated from crude oil was studied. The bioemulsifier was produced using sucrose with and without adding hydrocarbons (paraffin or crude oil) under aerobic and anaerobic conditions at 40°C. It formed stable emulsions with several hydrocarbons and its emulsifying ability was not affected by exposure to high salinities (up to 300 g/l), high temperatures (100°C-121°C) or a wide range of pH values (2-13). In addition, it presented low toxicity and high biodegradability when compared with chemical surfactants. A preliminary chemical characterization by Fourier Transform Infrared Spectroscopy (FT-IR), proton and carbon nuclear magnetic resonance (1H NMR and 13C CP-MAS NMR) and size exclusion chromatography indicated that the bioemulsifier is a low molecular weight oligosaccharide-lipid complex. CONCLUSION: The production of a low molecular weight bioemulsifier by a novel Paenibacillus strain isolated from crude oil was reported. To the best of our knowledge, bioemulsifier production by Paenibacillus strains has not been previously reported. The features of this novel bioemulsifier make it an interesting biotechnological product for many environmental and industrial applications. Graphical Abstract Novel bioemulsifier from Paenibacillus sp.

Changes in bacterial community metabolism and composition during the degradation of dissolved organic matter from the jellyfish Aurelia aurita in a Mediterranean coastal lagoon.

Spatial increases and temporal shifts in outbreaks of gelatinous plankton have been observed over the past several decades in many estuarine and coastal ecosystems. The effects of these blooms on marine ecosystem functioning and particularly on the dynamics of the heterotrophic bacteria are still unclear. The response of the bacterial community from a Mediterranean coastal lagoon to the addition of dissolved organic matter (DOM) from the jellyfish Aurelia aurita, corresponding to an enrichment of dissolved organic carbon (DOC) by 1.4, was assessed for 22 days in microcosms (8 l). The high bioavailability of this material led to (i) a rapid mineralization of the DOC and dissolved organic nitrogen from the jellyfish and (ii) the accumulation of high concentrations of ammonium and orthophosphate in the water column. DOM from jellyfish greatly stimulated heterotrophic prokaryotic production and respiration rates during the first 2 days; then, these activities showed a continuous decay until reaching those measured in the control microcosms (lagoon water only) at the end of the experiment. Bacterial growth efficiency remained below 20%, indicating that most of the DOM was respired and a minor part was channeled to biomass production. Changes in bacterial diversity were assessed by tag pyrosequencing of partial bacterial 16S rRNA genes, DNA fingerprints, and a cultivation approach. While bacterial diversity in control microcosms showed little changes during the experiment, the addition of DOM from the jellyfish induced a rapid growth of Pseudoalteromonas and Vibrio species that were isolated. After 9 days, the bacterial community was dominated by Bacteroidetes, which appeared more adapted to metabolize high-molecular-weight DOM. At the end of the experiment, the bacterial community shifted toward a higher proportion of Alphaproteobacteria. Resilience of the bacterial community after the addition of DOM from the jellyfish was higher for metabolic functions than diversity, suggesting that jellyfish blooms can induce durable changes in the bacterial community structure in coastal lagoons.

Iron shortage and bile salts play a major role in the expression of ompK gene in Vibrio anguillarum.

The outer membrane protein K, OmpK first identified in Vibrio parahaemolyticus has been shown to be a receptor for a broad host range vibriophage KVP40 infecting members of the Vibrionaceae. In the study, the effect of culture conditions on the expression of ompK in V. anguillarum was studied using real-time PCR. The expression increased significantly in the presence of bile salts and iron chelating agent 2, 2' bipyridine, suggesting a role for this protein in bile resistance and also in iron acquisition by V. anguillarum. OmpK induction by iron limitation and the presence of bile salts was reconfirmed by western blot technique after growing the cells in trypticase soy broth supplemented with bile salts, blood and 2, 2' bipyridine. We surmise that the expression of OmpK protein of V. anguillarum is bile salt and iron chelating agent-dependent.

[Identification of Vibrio metschnikovii from Homarus americanus and its changes in membrane fatty acid composition in response to low temperature].

OBJECTIVE: Bacterial strain F5-1 isolated from the Homarus americanus was characterized and its changes in membrane fatty acid composition in response to low temperature were also studied. METHODS: The physiological and biochemical characteristics were carried out by using VITEK 2 compact automated microbiology system. The 16S rRNA gene was sequenced and subjected to phylogenetic analysis. Fatty acids were detected by gas chromatography-mass spectrometry (GC-MS). RESULTS: Strain F5-1 was Gram-negative and susceptible to the vibriostatic agent O/129. Strain F5-1 was resistant to Penicillin. The isolated strain exhibited the highest levels of 99% probability to Vibrio metschnikovii based on the conventional physiological test. The sequence analysis of 16S rRNA gene of F5-1 isolation and comparison with that of other related vibrios showed that F5-1 was very close to V. metschnikovii (GenBank No. HQ658055). The similarity was 99%. The major fatty acids were C12:0, C14:0, C16:0 and C16:1 (n-7). Palmitoleic acid was the dominant unsaturated fatty acids. The major change in fatty acid composition occurred in response to low temperature, with an increase in palmitoleic acid from 34% to 40%. CONCLUSION: Bacterial strain F5-1 isolated from Homarus americanus was identified as V. metschnikovii and was sensitive to multiple drugs. The fatty acid composition of F5-1 was different from V. metschnikovii isolated from a drinking water reservoir near Vladivostok City in the Russia Far East. Results of this study indicated that environmental conditions allowed modulation of the fatty acid composition of V. metschnikovii.

Bifidobacterium spp. influences the production of autoinducer-2 and biofilm formation by Escherichia coli O157:H7.

The effect of Bifidobacterium spp. on the production of quorum-sensing (QS) signals and biofilm formation by enterohemorrhagic Escherichia coli (EHEC) O157:H7 was investigated. In an AI-2 bioassay, cell extracts of Bifidobacterium longum ATCC 15707 resulted in a 98-fold reduction in AI-2 activity in EHEC O157:H7 as well as in the Vibrio harveyi reporter strain, even though they did not inhibit the growth of EHEC O157:H7. In addition, they resulted in a 36% reduction in biofilm formation by the organism. Consistently, the virulence of EHEC O157:H7 was significantly attenuated by the presence of cell extracts of B. longum ATCC 15707 in the Caenorhabditis elegans nematode in vivo model. By a proteome analysis using two dimensional electrophoresis (2-DE), we determined that seven proteins including formation of iron-sulfur protein (NifU), thiol:disulfide interchange protein (DsbA), and flagellar P-ring protein (FlgI) were differentially regulated in the EHEC O157:H7 when supplemented with cell extracts of B. longum ATCC 15707. Taken together, these findings propose a novel function of a dairy adjunct in repressing the virulence of EHEC O157:H7.

Secondary structure of antisense RNAß, an internal transcriptional terminator of the plasmid-encoded iron transport-biosynthesis operon of Vibrio anguillarum.

RNAß affects the transcription process of the iron transport-biosynthesis operon encoded in the pJM1 plasmid of Vibrio anguillarum at a stem-loop structure located in the intergenic region between the fatA and angR genes. The net result is a higher level of the fatD, fatC, fatB, and fatA moiety as compared with the longer transcript encoding those genes as well as the angR and angT genes. In this work we report the secondary structure of RNAß determined by treatment with single and double strand specific ribonucleases as well as lead acetate followed by sequencing. The generated in vitro structural data indicated that three of the four previously described loops are in agreement with the original model, however, the alteration of loop IV as well as several other structural differences in the overall shape of the molecule led to the necessity of creating a new in silico model. Using the sites of mutations in the various loops we modeled the change in the RNAß secondary structure induced by those mutations. Mutations of loops III and IV to their complementary bases alter the overall structure of the RNAß significantly and increase its function while mutations in loops I and II have the opposite effect, the structure is unchanged but the activity of RNAß decreases. This indicates that loops I and II are necessary for interaction with the target mRNA. It is possible that the structural rearrangement introduced by mutations in loops III and IV promote activity and binding in loops I and II through reducing steric hindrance or increased binding to the target. This result also indicates that the exact relative positions of the critical loops are unimportant for activity.

Structural and phylogenetic analyses of the GP42 transglutaminase from Phytophthora sojae reveal an evolutionary relationship between oomycetes and marine Vibrio bacteria.

Transglutaminases (TGases) are ubiquitous enzymes that catalyze selective cross-linking between protein-bound glutamine and lysine residues; the resulting isopeptide bond confers high resistance to proteolysis. Phytophthora sojae, a pathogen of soybean, secretes a Ca(2+)-dependent TGase (GP42) that is activating defense responses in both host and non-host plants. A GP42 fragment of 13 amino acids, termed Pep-13, was shown to be absolutely indispensable for both TGase and elicitor activity. GP42 does not share significant primary sequence similarity with known TGases from mammals or bacteria. This suggests that GP42 has evolved novel structural and catalytic features to support enzymatic activity. We have solved the crystal structure of the catalytically inactive point mutant GP42 (C290S) at 2.95 Å resolution and identified residues involved in catalysis by mutational analysis. The protein comprises three domains that assemble into an elongated structure. Although GP42 has no structural homolog, its core region displays significant similarity to the catalytic core of the Mac-1 cysteine protease from Group A Streptococcus, a member of the papain-like superfamily of cysteine proteases. Proteins that are taxonomically related to GP42 are only present in plant pathogenic oomycetes belonging to the order of the Peronosporales (e.g. Phytophthora, Hyaloperonospora, and Pythium spp.) and in marine Vibrio bacteria. This suggests that a lateral gene transfer event may have occurred between bacteria and oomycetes. Our results offer a basis to design and use highly specific inhibitors of the GP42-like TGase family that may impair the growth of important oomycete and bacterial pathogens.

Biodegradable and biocompatible biomaterial, polyhydroxybutyrate, produced by an indigenous Vibrio sp. BM-1 isolated from marine environment.

Polyhydroxybutyrate (PHB) is one of the polyhydroxyalkanoates (PHAs) which has biodegradable and biocompatible properties. They are adopted in the biomedical field, in, for example, medical implants and drug delivery carriers. This study seeks to promote the production of PHB by Vibrio sp. BM-1, isolated from a marine environment by improving constituents of medium and implementing an appropriate fermentation strategy. This study successfully developed a glycerol-yeast extract-tryptone (GYT) medium that can facilitate the growth of Vibrio sp. BM-1 and lead to the production of 1.4 g/L PHB at 20 h cultivation. This study also shows that 1.57 g/L PHB concentration and 16% PHB content were achieved, respectively, when Vibrio sp. BM-1 was cultivated with MS-GYT medium (mineral salts-supplemented GYT medium) for 12 h. Both cell dry weight (CDW) and residual CDW remained constant at around 8.2 g/L and 8.0 g/L after the 12 h of cultivation, until the end of the experiment. However, both 16% of PHB content and 1.57 g/L of PHB production decreased rapidly to 3% and 0.25 g/L, respectively from 12 h of cultivation to 40 h of cultivation. The results suggest that the secretion of PHB depolymerase that might be caused by the addition of mineral salts reduced PHB after 12 h of cultivation. However, work will be done to explain the effect of adding mineral salts on the production of PHB by Vibrio sp. BM-1 in the near future.

Expression of Vibrio harveyi acyl-ACP synthetase allows efficient entry of exogenous fatty acids into the Escherichia coli fatty acid and lipid A synthetic pathways.

Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

Structure-based discovery and experimental verification of novel AI-2 quorum sensing inhibitors against Vibrio harveyi.

Quorum sensing has been implicated in the control of pathologically relevant bacterial behavior such as secretion of virulence factors, biofilm formation, sporulation, and swarming motility. The AI-2 quorum sensing pathway is found in both gram-positive and gram-negative bacteria. Therefore, antagonizing AI-2 quorum sensing is a possible approach to modifying bacterial behaviour. However, efforts in developing inhibitors of AI-2-mediated quorum sensing are especially lacking. High-throughput virtual screening using the V. harveyi LuxP crystal structure identified two compounds that were found to antagonize AI-2-mediated quorum sensing in V. harveyi without cytotoxicity. The sulfone functionality of these inhibitors was identified as critical to their ability to mimic the natural ligand in their interactions with Arg 215 and Arg 310 of the active site.

Isolation and characterization of mucous exopolysaccharide (EPS) produced by Vibrio furnissii strain VB0S3.

Marine bacterial strains were isolated from coastal regions of Goa and screened for the strains that produce the highest amount of mucous exopolysaccharide (EPS). Our screening resulted in the identification of the strain Vibrio furnissii VB0S3 (hereafter called VB0S3), as it produced the highest EPS in batch cultures during the late logarithmic growth phase. The isolate was identified as VB0S3 based on morphological and biochemical properties. Growth and EPS production were studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The exopolymer was recovered from the culture supernatant by using three volumes of cold ethanol precipitation and dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, and proteins. Fourier-transform infrared (FT-IR) spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide, and the EPS also possessed good emulsification activity. The gas chromatographic analysis of an alditol-acetate derivatized sample of EPS revealed that it was mainly composed of galactose and glucose. Minor components found were mannose, rhamnose, fucose, ribose, arabinose, and xylose. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like exopolysaccharide. This is the first report of exopolysaccharide characterization that describes the isolation and characterization of an EPS expressed by Vibrio furnissii strain VB0S3. The results of the study contribute significantly and go a long way towards an understanding of the correlation between growth and EPS production, chemical composition, and industrial applications of the exopolysaccharide in environmental biotechnology and bioremediation.

Environmental stress determines the quality of bacterial lysate and its utilization efficiency in a simple microbial loop.

Heterotrophic bacteria provide the critical link in the microbial loop by converting dissolved organic matter (DOM) into particulate form. In this study, DOM was prepared from recently isolated estuarine bacterial strain Vibrio sp. (DSM14379) grown at different salinities [0.2%, 0.5%, 3%, 5%, or 10% (w/v)], washed, concentrated, and lysed by autoclaving. The corresponding lysate-containing media were designated LM(0.2), LM(0.5), LM(3), LM(5), and LM(10). Vibrio sp. cells grown at different salinities had similar C/N/P ratios, but different C/S ratios, different trace element composition, and different 2D gel electrophoresis protein profiles. Pseudoalteromonas sp. (DSM06238) isolated from a similar environment was able to grow on all lysates, and its biomass production was dependent on lysate type. The highest growth rate and biomass production of Pseudoalteromonas sp. at saturation lysate concentrations were observed in LM(3). The biomass production at saturation lysate concentrations was about 3-fold higher as compared to LM(0.2) and LM(10). The initial respiration rate, intracellular adenosine triphosphate (ATP) levels, and (3)H-Leu and (3)H-TdR incorporation rates were lowest in LM(3). On the other hand, in LM(0.2) or LM(10) lysates the situation was reversed, the growth rates and biomass production were lowest, whereas (3)H-Leu and (3)H-TdR incorporation, respiration rates, as well as ATP levels, were highest. These results imply uncoupling of catabolism from growth in either high- or low-salinity lysates. The results also suggest that differences in organic carbon quality generated during Vibrio sp. growth at different NaCl concentrations were propagated through the simple microbial loop, which may have important ecological implications for higher trophic levels that depend on microbial grazing.

Isolation and characterization of exopolysaccharide produced by Vibrio harveyi strain VB23.

AIMS: The aim of the study was to isolate and characterize exopolysaccharide (EPS) produced by Vibrio harveyi strain VB23. METHODS AND RESULTS: Growth and EPS production by V. harveyi strain VB23, was studied in mineral salts medium supplemented with NaCl (1.5%) and glucose (0.2%). The rate of EPS production in batch cultures was highest during the late log phase of growth when compared with stationary growth phase. The exopolymer was recovered from the culture supernatant by using a cold ethanol precipitation-dialysis procedure. Chemical analyses of EPS revealed that it is primarily composed of neutral sugars, uronic acids, proteins and sulfates. The purified EPS revealed prominent functional reactive groups, such as hydroxyl, carboxylic and amides, which correspond to a typical heteropolymeric polysaccharide and the EPS, also possessed good emulsification activity. The gas chromatographic analysis of an alditol acetate-derivatized sample of EPS revealed that it is composed primarily of galactose and glucose. Minor components found were rhamnose, fucose, ribose, arabinose, xylose and mannose. CONCLUSIONS: The EPS produced by V. harveyi strain VB23 is a heteropolysaccharide possessing good emulsification activity. EPS was readily isolated from culture supernatants, which suggests that the EPS was a slime-like EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of EPS characterization in luminous V. harveyi bacteria, which describes the isolation and characterization of an EPS expressed by V. harveyi. The results of the study contributes significantly towards an understanding of the chemical composition and applications of the EPS in environmental biotechnology and bioremediation.