Bacterial consortium proteomics under 4‐chlorosalicylate carbon‐limiting conditions

In this study, the stable consortium composed by Pseudomonas reinekei strain MT1 and Achromobacter xylosoxidans strain MT3 (cell numbers in proportion 9:1) was under investigation to reveal bacterial interactions that take place under severe nutrient‐limiting conditions. The analysis of steady states in continuous cultures was carried out at the proteome, metabolic profile, and population dynamic levels. Carbon‐limiting studies showed a higher metabolic versatility in the community through upregulation of parallel catabolic enzymes (salicylate 5‐hydroxylase and 17‐fold on 2‐keto‐4‐pentenoate hydratase) indicating a possible alternative carbon routing in the upper degradation pathway highlighting the effect of minor proportions of strain MT3 over the major consortia component strain MT1 with a significant change in the expression levels of the enzymes of the mainly induced biodegradation pathway such as salicylate 1‐hydroxylase and catechol 1,2‐dioxygenase together with important changes in the outer membrane composition of P. reinekei MT1 under different culture conditions. The study has demonstrated the importance of the outer membrane as a sensing/response protective barrier caused by interspecies interactions highlighting the role of the major outer membrane proteins OprF and porin D in P. reinekei sp. MT1 under the culture conditions tested.     

Role of the protease in the permeability enhancement by Vibrio vulnificus

The protease produced by Vibrio vulnificus enhances vascular permeability through histamine release from mast cells and activation of the plasma kallikrein-kinin system which generates bradykinin when injected into the dorsal skin. V. vulnificus living cells also enhanced vascular permeability within a few hours after the injection into the dorsal skin. The permeability-enhancing activity of living cells was greatly reduced by addition of soybean trypsin inhibitor, a specific inhibitor for plasma kallikrein-kinin system, or anti-protease IgG. Two protease-deficient mutants induced by nitrosoguanidine treatment had only one-tenth permeability-enhancing activity of a wild-type strain. These results indicate that V. vulnificus elaborates the protease in vivo and that the protease elaborated enhances vascular permeability through release of chemical mediators such as histamine and bradykinin and forms edema.     

Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds

Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron-tetra(4-sulfonatophenyl)porphyrin (Fe-TPPS), as well as from a natural heme, protoheme. This assimilation of iron bound to TPPS was demonstrated to be a common property of V. vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources. Strain L-180 could also utilize Fe-TCPP, but not Fe-TMPyP, as a sole iron source. TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe-TPPS. When inoculated into human serum supplemented with Fe-TCPP, L-180 could grow only in the presence of a protease from the same bacterium. In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge. Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V. vulnificus.     

Siderophore production and outer membrane proteins of selected Vibrio vulnificus strains under conditions of iron limitation

Abstract The tonB gene product is necessary for the energy-dependent transport of ferric chelates and vitamin B₁₂ across the Escherichia coli outer membrane. When carried on multicopy plasmids, the cloned tonB gene complemented tonB hosts, restoring transport of ferri-siderophone complexes and vitamin B₁₂, and susceptibility to the group B colicins and phage ф80. The levels of these activities were all markedly lower than when the tonB ⁺ gene was present in single copy. This depression of TonB function occurred even when the chromosome carried the normal tonB ⁺ allele, but plasmids carrying only a portion of the tonB gene, including the 5'-regulatory region, were not inhibitory.     

FERONIA receptor kinase interacts with S‐adenosylmethionine synthetase and suppresses S‐adenosylmethionine production and ethylene biosynthesis in Arabidopsis

Environmental inputs such as stress can modulate plant cell metabolism, but the detailed mechanism remains unclear. We report here that FERONIA (FER), a plasma membrane receptor‐like kinase, may negatively regulate the S‐adenosylmethionine (SAM) synthesis by interacting with two S‐adenosylmethionine synthases (SAM1 and SAM2). SAM participates in ethylene, nicotianamine and polyamine biosynthetic pathways and provides the methyl group for protein and DNA methylation reactions. The Arabidopsis fer mutants contained a higher level of SAM and ethylene in plant tissues and displayed a dwarf phenotype. Such phenotype in the fer mutants was mimicked by over‐expressing the S‐adenosylmethionine synthetase in transgenic plants, whereas sam1/2 double mutant showed an opposite phenotype. We propose that FER receptor kinase, in response to environmental stress and plant hormones such as auxin and BR, interacts with SAM synthases and down‐regulates ethylene biosynthesis.     

Novel host‐specific iron acquisition system in the zoonotic pathogen Vibrio vulnificus

Vibrio vulnificus is a marine bacterium associated with human and fish (mainly farmed eels) diseases globally known as vibriosis. The ability to infect and overcome eel innate immunity relies on a virulence plasmid (pVvbt2) specific for biotype 2 (Bt2) strains. In the present study, we demonstrated that pVvbt2 encodes a host‐specific iron acquisition system that depends on an outer membrane receptor for eel transferrin called Vep20. The inactivation of vep20 did not affect either bacterial growth in human plasma or virulence for mice, while bacterial growth in eel blood/plasma was abolished and virulence for eels was significantly impaired. Furthermore, vep20 is an iron‐regulated gene overexpressed in eel blood during artificially induced vibriosis both in vitro and in vivo. Interestingly, homologues to vep20 were identified in the transferable plasmids of two fish pathogen species of broad‐host range, Vibrio harveyi (pVh1) and Photobacterium damselae subsp. damselae (pPHDD1). These data suggest that Vep20 belongs to a new family of plasmid‐encoded fish‐specific transferrin receptors, and the acquisition of these plasmids through horizontal gene transfer is likely positively selected in the fish‐farming environment. Moreover, we propose Ftbp (fish transferrin binding proteins) as a formal name for this family of proteins.     

Antacid Increases Survival of Vibrio vulnificus and Vibrio vulnificus Phage in a Gastrointestinal Model

Viable counts of three strains of Vibrio vulnificus and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37°C. V. vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5). Viable V. vulnificus cells delivered from the gastric compartment during the first 30 min of exposure reached 10⁶ to 10⁸ CFU/ml in the intestinal compartment after 9 h (pH 7.0). Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5). Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0). When the gastric compartment contained antacid V. vulnificus counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 10⁷ to 10⁹ CFU/ml. Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h. Presence of antacid in the gastric compartment of the model greatly increased the ability of both V. vulnificus and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness.     

Antacid increases survival of Vibrio vulnificus and Vibrio vulnificus phage in a gastrointestinal model.

Viable counts of three strains of Vibrio vulnificus and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37 degrees C. V. vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5). Viable V. vulnificus cells delivered from the gastric compartment during the first 30 min of exposure reached 10(6) to 10(8) CFU/ml in the intestinal compartment after 9 h (pH 7.0). Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5). Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0). When the gastric compartment contained antacid V. vulnificus counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 10(7) to 10(9) CFU/ml. Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h. Presence of antacid in the gastric compartment of the model greatly increased the ability of both V. vulnificus and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness.     

Role of Iron in Human Serum Resistance of the Clinical and Environmental Vibrio vulnificus Genotypes

We recently reported a simple PCR procedure that targets a sequence variation of the virulence-correlated gene locus vcg. It was found that 90% of all clinical isolates possessed the vcgC sequence variant, while 93% of all environmental isolates possessed the vcgE sequence variant. Here we report that the clinical genotype of Vibrio vulnificus is significantly better able to survive in human serum than is the environmental genotype. The presence of a siderophore-encoding gene, viuB, influenced serum survivability among all isolates of V. vulnificus tested. Those strains positive for viuB (all C-type strains but very few E-type strains) showed greater serum survivability than those lacking viuB (most E-type strains). The addition of iron (in the form of ferric ammonium citrate) to human serum restored the survival of E-type strains lacking viuB to levels not significantly different from those of C-type and E-type strains that possess viuB. These findings suggest that viuB may dictate serum survival in both C- and E-type strains of V. vulnificus and may explain why some strains (C- and E-type strains) are pathogenic and others (predominately E-type strains) are not. Additionally, C-type strains exhibited a cross-protective response against human serum, not exhibited by E-type strains, after incubation under nutrient and osmotic downshift conditions that mimicked estuarine waters. This suggests that the nutrient/osmotic environment may influence the survival of V. vulnificus following entry into the human body, leading to selection of the C genotype over the E genotype.     

Identification of a flavin‐containing S‐oxygenating monooxygenase involved in alliin biosynthesis in garlic

S‐Alk(en)yl‐l ‐cysteine sulfoxides are cysteine‐derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S‐alk‐(en)yl‐l ‐cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin‐containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S‐oxygenation reaction in the biosynthesis of S‐allyl‐l ‐cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S‐oxygenation of S‐allyl‐l ‐cysteine to nearly exclusively yield (R_CS_S)‐S‐allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S‐oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin‐containing monooxygenases. AsFMO1 preferred S‐allyl‐l ‐cysteine to γ‐glutamyl‐S‐allyl‐l ‐cysteine as the S‐oxygenation substrate, suggesting that in garlic, the S‐oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre‐emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S‐allyl‐l ‐cysteine S‐oxygenase, and contributes to the production of alliin both through the conversion of stored γ‐glutamyl‐S‐allyl‐l ‐cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves.     

Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV under varied limiting conditions and an insight into the biosynthetic genes involved

Aims:  A new strain of Bacillus, Bacillus cereus SPV, was found to be capable of using a wide range of carbon sources for the production of polyhydroxyalkanoates (PHAs) ( Valappil et al. 2007b ). Limiting nutrient in the culture conditions is crucial for PHA production. In this study, B.   cereus SPV was grown in different culture conditions with limitation of potassium, nitrogen, sulphur and phosphorous to establish the impact of nutritional limitation on PHA production.
Methods and Results:  The PHA yields obtained were found to be 13·4, 38, 13·15 and 33·33% dcw for potassium, nitrogen, sulphur and phosphorus limitations, respectively. Gas chromatography–mass spectrometry analysis of the isolated polymers showed the presence of P(3HB) under nitrogen, sulphur and phosphate-limiting conditions and P(3HB-3HV) copolymer under potassium limiting conditions. This ability of B. cereus SPV to accumulate different PHA monomers from structurally unrelated carbon sources led to an interest in the molecular analysis of PHA biosynthesis in this organism. To achieve this, PCR was used to identify the polyhydroxyalkanoate biosynthetic genes in B. cereus SPV.
Conclusion:  Sequence analysis of the PCR products from B. cereus SPV revealed the sequence of the putative biosynthetic genes, and possible regions involved in substrate binding.
The nucleotide sequence reported in this paper is in the GenBank nucleotide sequence database under accession number DQ486135 .
Significance and Impact of the Study:  This is the first report comparing the capability of B. cereus SPV to produce PHAs under different culture conditions of potassium, nitrogen, sulfur and phosphate limitations. The results in this study suggest the unique ability of B. cereus SPV to supply both 3HB and 3HV monomers from a structurally unrelated carbon source, glucose.     

Integrative genome‐scale metabolic analysis of Vibrio vulnificus for drug targeting and discovery

Although the genomes of many microbial pathogens have been studied to help identify effective drug targets and novel drugs, such efforts have not yet reached full fruition. In this study, we report a systems biological approach that efficiently utilizes genomic information for drug targeting and discovery, and apply this approach to the opportunistic pathogen Vibrio vulnificus CMCP6. First, we partially re‐sequenced and fully re‐annotated the V. vulnificus CMCP6 genome, and accordingly reconstructed its genome‐scale metabolic network, VvuMBEL943. The validated network model was employed to systematically predict drug targets using the concept of metabolite essentiality, along with additional filtering criteria. Target genes encoding enzymes that interact with the five essential metabolites finally selected were experimentally validated. These five essential metabolites are critical to the survival of the cell, and hence were used to guide the cost‐effective selection of chemical analogs, which were then screened for antimicrobial activity in a whole‐cell assay. This approach is expected to help fill the existing gap between genomics and drug discovery.     

Epidemiology and pathogenesis of Vibrio vulnificus

Vibrio vulnificus is capable of causing severe and often fatal infections in susceptible individuals. It causes two distinct disease syndromes, a primary septicemia and necrotizing wound infections. This review discusses the interaction of environmental conditions, host factors, and bacterial virulence determinants that contribute to the epidemiology and pathogenesis of V. vulnificus.