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.

Metabolic engineering of Vibrio natriegens for anaerobic succinate production.

The biotechnological production of succinate bears serious potential to fully replace existing petrochemical approaches in the future. In order to establish an economically viable bioprocess, obtaining high titre, yield and productivity is of central importance. In this study, we present a straightforward engineering approach for anaerobic succinate production with Vibrio natriegens, consisting of essential metabolic engineering and optimization of process conditions. The final producer strain V. natriegens Δlldh Δdldh Δpfl Δald Δdns::pycCg (Succ1) yielded 1.46 mol of succinate per mol of glucose under anaerobic conditions (85% of the theoretical maximum) and revealed a particularly high biomass-specific succinate production rate of 1.33 gSucc gCDW -1 h-1 compared with well-established production systems. By applying carbon and redox balancing, we determined the intracellular flux distribution and show that under the tested conditions the reductive TCA as well as the oxidative TCA/glyoxylate pathway contributed to succinate formation. In a zero-growth bioprocess using minimal medium devoid of complex additives and expensive supplements, we obtained a final titre of 60.4 gSucc l-1 with a maximum productivity of 20.8 gSucc l-1 h-1 and an overall volumetric productivity of 8.6 gSucc l-1 h-1 during the 7 h fermentation. The key performance indicators (titre, yield and productivity) of this first engineering approach in V. natriegens are encouraging and compete with costly tailored microbial production systems.

Patterns and drivers of Vibrio isolates phylogenetic diversity in the Beibu Gulf, China.

Members of the genus Vibrio are ubiquitous in aquatic environments and can be found either in a culturable or a viable but nonculturable (VBNC) state. Despite widespread concerns as to how to define the occurrence and dynamics of Vibrio populations by culture-independent approaches, further physiological research and relevant biotechnological developments will require the isolation and cultivation of the microbes from various environments. The present work provides data and perspectives on our understanding of culturable Vibrio community structure and diversity in the Beibu Gulf. Finally, we isolated 1,037 strains of Vibrio from 45 samples and identified 18 different species. Vibrio alginolyticus, V. cyclitrophicus, V. tasmaniensis, V. brasiliensis, and V. splendidus were the dominant species that had regional distribution characteristics. The correlation between the quantitative distribution and community structure of culturable Vibrio and environmental factors varied with the Vibrio species and geographical locations. Among them, salinity, nitrogen, and phosphorus were the main factors affecting the diversity of culturable Vibrio. These results help to fill a knowledge gap on Vibrio diversity and provide data for predicting and controlling pathogenic Vibrio outbreaks in the Beibu Gulf.

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.

Dietary ß-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota.

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, ß-glucan (MacroGard(®)) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast ß-1,3/1,6-glucan in form of MacroGard(®) at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of ß-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard(®) fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by ß-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1ß was observed. We conclude that the administration of MacroGard(®) induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

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.

Comparative biochemical characterization of three exolytic oligoalginate lyases from Vibrio splendidus reveals complementary substrate scope, temperature, and pH adaptations.

Marine microbes use alginate lyases to degrade and catabolize alginate, a major cell wall matrix polysaccharide of brown seaweeds. Microbes frequently contain multiple, apparently redundant alginate lyases, raising the question of whether these enzymes have complementary functions. We report here on the molecular cloning and functional characterization of three exo-type oligoalginate lyases (OalA, OalB, and OalC) from Vibrio splendidus 12B01 (12B01), a marine bacterioplankton species. OalA was most active at 16°C, had a pH optimum of 6.5, and displayed activities toward poly-ß-d-mannuronate [poly(M)] and poly-α-l-guluronate [poly(G)], indicating that it is a bifunctional enzyme. OalB and OalC were most active at 30 and 35°C, had pH optima of 7.0 and 7.5, and degraded poly(M·G) and poly(M), respectively. Detailed kinetic analyses of oligoalginate lyases with poly(G), poly(M), and poly(M·G) and sodium alginate as substrates demonstrated that OalA and OalC preferred poly(M), whereas OalB preferred poly(M·G). The catalytic efficiency (kcat/Km) of OalA against poly(M) increased with decreasing size of the substrate. OalA showed kcat/Km from 2,130 mg(-1) ml s(-1) for the trisaccharide to 224 mg(-1) ml s(-1) for larger oligomers of ∼50 residues, and 50.5 mg(-1) ml s(-1) for high-molecular-weight alginate. Although OalA was most active on the trisaccharide, OalB and OalC preferred dimers. Taken together, our results indicate that these three Oals have complementary substrate scopes and temperature and pH adaptations.

[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.

Co-evolutionary dynamics of the bacteria Vibrio sp. CV1 and phages V1G, V1P1, and V1P2: implications for phage therapy.

Bacterial infections are the second largest cause of mortality in shrimp hatcheries. Among them, bacteria from the genus Vibrio constitute a major threat. As the use of antibiotics may be ineffective and banned from the food sector, alternatives are required. Historically, phage therapy, which is the use of bacteriophages, is thought to be a promising option to fight against bacterial infections. However, as for antibiotics, resistance can be rapidly developed. Since the emergence of resistance is highly undesirable, a formal characterization of the dynamics of its acquisition is mandatory. Here, we explored the co-evolutionary dynamics of resistance between the bacteria Vibrio sp. CV1 and the phages V1G, V1P1, and V1P2. Single-phage treatments as well as a cocktail composed of the three phages were considered. We found that in the presence of a single phage, bacteria rapidly evolved resistance, and the phages decreased their infectivity, suggesting that monotherapy may be an inefficient treatment to fight against Vibrio infections in shrimp hatcheries. On the contrary, the use of a phage cocktail considerably delayed the evolution of resistance and sustained phage infectivity for periods in which shrimp larvae are most susceptible to bacterial infections, suggesting the simultaneous use of multiple phages as a serious strategy for the control of vibriosis. These findings are very promising in terms of their consequences to different industrial and medical scenarios where bacterial infections are present.

Prevention of quorum-sensing-mediated biofilm development and virulence factors production in Vibrio spp. by curcumin.

The increasing occurrence of disease outbreaks caused by Vibrio spp. and the emergence of antibiotic resistance has led to a growing interest in finding alternative strategies to prevent vibriosis. Since the pathogenicity of vibrios is controlled in part by quorum-sensing (QS) system, interfering with this mechanism would prevent the pathogenicity of vibrios without developing resistance. Hence, a non-toxic phytochemical curcumin from Curcuma longa was assessed for its potential in reducing the production of QS-dependent virulence factors in Vibrio spp. The obtained results evidenced 88% reduction in bioluminescence of Vibrio harveyi by curcumin. Further, curcumin exhibited a significant inhibition in alginate, exopolysaccharides, motility, biofilm development and other virulence factors production in Vibrio parahaemolyticus, Vibrio vulnificus and V. harveyi. In in vivo analysis, curcumin enhanced the survival rate of Artemia nauplii up to 67% against V. harveyi infection by attenuating its QS-mediated virulence.

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.

Autonomous bioluminescent expression of the bacterial luciferase gene cassette (lux) in a mammalian cell line.

BACKGROUND: The bacterial luciferase (lux) gene cassette consists of five genes (luxCDABE) whose protein products synergistically generate bioluminescent light signals exclusive of supplementary substrate additions or exogenous manipulations. Historically expressible only in prokaryotes, the lux operon was re-synthesized through a process of multi-bicistronic, codon-optimization to demonstrate for the first time self-directed bioluminescence emission in a mammalian HEK293 cell line in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Autonomous in vitro light production was shown to be 12-fold greater than the observable background associated with untransfected control cells. The availability of reduced riboflavin phosphate (FMNH(2)) was identified as the limiting bioluminescence substrate in the mammalian cell environment even after the addition of a constitutively expressed flavin reductase gene (frp) from Vibrio harveyi. FMNH(2) supplementation led to a 151-fold increase in bioluminescence in cells expressing mammalian codon-optimized luxCDE and frp genes. When injected subcutaneously into nude mice, in vivo optical imaging permitted near instantaneous light detection that persisted independently for the 60 min length of the assay with negligible background. CONCLUSIONS/SIGNIFICANCE: The speed, longevity, and self-sufficiency of lux expression in the mammalian cellular environment provides a viable and powerful alternative for real-time target visualization not currently offered by existing bioluminescent and fluorescent imaging technologies.

Evaluation of mutagenic and antimutagenic properties of some bioactive xanthone derivatives using Vibrio harveyi test.

AIMS: Drug safety evaluation plays an important role in the early phase of drug development, especially in the preclinical identification of compounds' biological activity. The Vibrio harveyi assay was used to assess mutagenic and antimutagenic activity of some aminoalkanolic derivatives of xanthone (1-5), which were synthesized and evaluated for their anticonvulsant and hemodynamic activities. METHODS AND RESULTS: A novel V. harveyi assay was used to assess mutagenic and antimutagenic activity of derivatives of xanthone 1-5. Two V. harveyi strains were used: BB7 (natural isolate) and BB7M (BB7 derivative containing mucA and mucB genes on a plasmid pAB91273, products of these genes enhance error-prone DNA repair). According to the results obtained, the most beneficial mutagenic and antimutagenic profiles were observed for compounds 2 and 3. A modification of the chemical structure of compound 2 by the replacement of the hydroxy group by a chloride improved considerably the antimutagenic activity of the compound. Thus, antimutagenic potency reached a maximum with the presence of tertiary amine and chloride atom in the side chain. CONCLUSIONS: Among the newly synthesized aminoalkanolic derivatives of xanthone with potential anticonvulsant properties, there are some compounds exhibiting in vitro antimutagenic activity. In addition, it appears that the V. harveyi assay can be applied for primary mutagenicity and antimutagenicity assessment of compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained preliminary mutagenicity and antimutagenicity results encourage further search in the group of amino derivatives of xanthone as the potential antiepileptic drugs also presenting some antimutagenic potential. Furthermore, V. harveyi test may be a useful tool for compounds safety evaluation.

Mechanism of drug resistance in a clinical isolate of Vibrio fluvialis: involvement of multiple plasmids and integrons.

The role of mobile genetic elements in imparting multiple drug resistance to a clinical isolate of Vibrio fluvialis (BD146) was investigated. This isolate showed complete or intermediate resistance to all of the 14 antibiotics tested. Polymerase chain reaction (PCR) revealed the presence of a class 1 integron and the absence of the SXT element in this isolate. The strain harboured a 7.5 kb plasmid and a very low copy number plasmid of unknown molecular size. Transformation of Escherichia coli with plasmid(s) from BD146 generated two kinds of transformants, one that harboured both of these plasmids and the other that harboured only the low copy number plasmid. PCR and antibiogram analysis indicated the association of the class 1 integron with the low copy number plasmid, which also conferred all the transferable resistance traits except trimethoprim to the parent strain. A BLAST search with the sequence of the 7.5kb plasmid showed that it was 99% identical to plasmid pVN84 from Vibrio cholerae O1 in Vietnam, indicating that these two plasmids are probably one and the same. To the best of our knowledge, this is the first report of horizontal transfer of a plasmid between V. fluvialis and V. cholerae.

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.

Comparative genomics of the KdgR regulon in Erwinia chrysanthemi 3937 and other gamma-proteobacteria.

In the plant-pathogenic enterobacterium Erwinia chrysanthemi, almost all known genes involved in pectin catabolism are controlled by the transcriptional regulator KdgR. In this study, the comparative genomics approach was used to analyse the KdgR regulon in completely sequenced genomes of eight enterobacteria, including Erw. chrysanthemi, and two Vibrio species. Application of a signal recognition procedure complemented by operon structure and protein sequence analysis allowed identification of new candidate genes of the KdgR regulon. Most of these genes were found to be controlled by the cAMP-receptor protein, a global regulator of catabolic genes. At the next step, regulation of these genes in Erw. chrysanthemi was experimentally verified using in vivo transcriptional fusions and an attempt was made to clarify the functional role of the predicted genes in pectin catabolism. Interestingly, it was found that the KdgR protein, previously known as a repressor, positively regulates expression of two new members of the regulon, phosphoenolpyruvate synthase gene ppsA and an adjacent gene, ydiA, of unknown function. Other predicted regulon members, namely chmX, dhfX, gntB, pykF, spiX, sotA, tpfX, yeeO and yjgK, were found to be subject to classical negative regulation by KdgR. Possible roles of newly identified members of the Erw. chrysanthemi KdgR regulon, chmX, dhfX, gntDBMNAC, spiX, tpfX, ydiA, yeeO, ygjV and yjgK, in pectin catabolism are discussed. Finally, complete reconstruction of the KdgR regulons in various gamma-proteobacteria yielded a metabolic map reflecting a globally conserved pathway for the catabolism of pectin and its derivatives with variability in transport and enzymic capabilities among species. In particular, possible non-orthologous substitutes of isomerase KduI and a new oligogalacturonide transporter in the Vibrio species were detected.

Hazard assessment of a simulated oil spill on intertidal areas of the St. Lawrence River with SPMD-TOX.

Phytoremediation in a simulated crude oil spill was studied with a "minimalistic" approach. The SPMD-TOX paradigm-a miniature passive sorptive device to collect and concentrate chemicals and microscale tests to detect toxicity-was used to monitor over time the bioavailability and potential toxicity of an oil spill. A simulated crude oil spill was initiated on an intertidal freshwater grass-wetland along the St. Lawrence River southwest of Quebec City, Quebec, Canada. Several phytoremediation treatments were investigated; to dissipate and ameliorate the spill, treatments included nutrient amendments with inorganic nitrogen sources (ammonium nitrate and sodium nitrate) and phosphate (super triple phosphate) with and without cut plants, with natural attenuation (no phytoremedial treatment) as a control. Sequestered oil residues were bioavailable in all oil-treated plots in Weeks 1 and 2. Interestingly, the samples were colored and fluoresced under ultraviolet light. In addition, microscale tests showed that sequestered residues were acutely toxic and genotoxic, as well as that they induced hepatic P(450) enzymes. Analysis of these data suggested that polycyclic aromatic hydrocarbons were among the bioavailable residues sequestered. In addition, these findings suggested that the toxic bioavailable fractions of the oil spill and degradation products dissipated rapidly over time because after the second week the water column contained no oil or detectable degradation products in this riverine intertidal wetland. SPMD-TOX revealed no evidence of bioavailable oil products in Weeks 4, 6, 8, and 12. All phytoremediation efforts appeared to be ineffective in changing either the dissipation rate or the ability to ameliorate the oil toxicity. SPMD-TOX analysis of the water columns from these riverine experimental plots profiled the occurrence, dissipation, and influence of phytoremediation on the bioavailability and toxicity of oil products (parent or degradation products).

Production and mechanism of secretion of interleukin-1beta from the marine fish gilthead seabream.

Mammalian interleukin-1beta (IL-1beta) is a secretory cytokine lacking a signal peptide, which does not follow the classical endoplasmic reticulum to Golgi pathway of secretion. Its post-translational processing by IL-1beta-converting enzyme (ICE) and subsequent release from activated macrophages requires ATP acting on P2X7 receptors. Little information is available on the production and release of fish IL-1beta, but the IL-1beta gene sequences reported to date lack a conserved ICE recognition site. We show for the first time that lipopolysaccharide (LPS)/macrophage-activating factor/bacterial DNA (VaDNA)-primed immune cells of the marine fish gilthead seabream (Sparus aurata) accumulate intracellular IL-1beta as a approximately 30 kDa polypeptide (proIL-1beta). The combination of LPS and VaDNA was found to be synergistic, suggesting that each ligand is recognized by a different pattern recognition receptor. More importantly, addition of extracellular ATP does not promote IL-1beta secretion by immune cells and fails to induce phosphatidylserine flip. In contrast, gilthead seabream SAF-1 fibroblasts shed microvesicles containing a 22 kDa IL-1beta form within 30 min of activation with ATP. Notably, the post-translational processing of IL-1beta by SAF-1 cells is abrogated by a specific ICE inhibitor.