Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 101 - 4 Desulfovibrio putealis sp . nov., a novel sulfate-reducing bacterium isolated from a deep subsurface aquifer; Basso O et al.; A novel sulfate-reducing bacterium was isolated from a well that collected water from a deep aquifer at a depth of 430 m in the Paris Basin, France . The strain, designated B7-43(T), was made up of vibrioid cells that were motile by means of a single polar flagellum . Cells contained desulfoviridin . In the presence of sulfate, the following substrates were used as energy and carbon sources: lactate, pyruvate, malate, fumarate, ethanol, butanol, acetate/H(2) and glycine . Sulfite and thiosulfate were also used as electron acceptors in the presence of lactate . In the absence of electron acceptors, pyruvate, malate and fumarate were fermented . Optimal growth was obtained in 1 g NaCl l(-1) and at pH 7 . On the basis of 16S rRNA gene sequence analysis, the isolate was most closely related to members of the genus Desulfovibrio (90 % similarity) . It is thus proposed that strain B7-43(T) (=DSM 16056(T)=ATCC BAA-905(T)) represents a novel species within this genus, Desulfovibrio putealis sp . nov. Int J Syst Evol Microbiol, 2005 Jan, 55(Pt 1), 57 - 66 Application of sliding-window discretization and minimization of stochastic complexity for the analysis of fAFLP genotyping fingerprint patterns of Vibrionaceae; Dawyndt P et al.; Minimization of stochastic complexity (SC) was used as a method for classification of genotypic fingerprints . The method was applied to fluorescent amplified fragment length polymorphism (fAFLP) fingerprint patterns of 507 Vibrionaceae representatives . As the current BinClass implementation of the optimization algorithm for classification only works on binary vectors, the original fingerprints were discretized in a preliminary step using the sliding-window band-matching method, in order to maximally preserve the information content of the original band patterns . The novel classification generated using the BinClass software package was subjected to an in-depth comparison with a hierarchical classification of the same dataset, in order to acknowledge the applicability of the new classification method as a more objective algorithm for the classification of genotyping fingerprint patterns . Recent DNA-DNA hybridization and 16S rRNA gene sequence experiments proved that the classification based on SC-minimization forms separate clusters that contain the fAFLP patterns for all representatives of the species Enterovibrio norvegicus, Vibrio fortis, Vibrio diazotrophicus or Vibrio campbellii, while previous hierarchical cluster analysis had suggested more heterogeneity within the fAFLP patterns by splitting the representatives of the above-mentioned species into multiple distant clusters . As a result, the new classification methodology has highlighted some previously unseen relationships within the biodiversity of the family Vibrionaceae. Proc Natl Acad Sci U S A . 2005 Jan 14; {Epub ahead of print} Seasonal epidemics of cholera inversely correlate with the prevalence of environmental cholera phages; Faruque SM et al.; The relationship among (i) the local incidence of cholera, (ii) the prevalence in the aquatic environment of Vibrio cholerae, and (iii) bacterial viruses that attack potentially virulent O1 and O139 serogroup strains of this organism (cholera phages) was studied in Dhaka, Bangladesh . Over nearly a 3-year period, we found that significantly more environmental water samples contained either a phage or a phage-susceptible V . cholerae strain than both (P < 0.00001) . The number of cholera patients varied seasonally during this period and frequently coincided with the presence of pathogenic V . cholerae strains in water samples that otherwise lacked detectable cholera phages . Interepidemic periods were characterized by water samples containing cholera phages but no viable bacteria . Our data support the conclusion that cholera phages can influence cholera seasonality and may also play a role in emergence of new V . cholerae pandemic serogroups or clones. Dis Aquat Organ, 2004 Nov 23, 62(1-2), 65 - 74 Two vibrio splendidus related strains collaborate to kill Crassostrea gigas: taxonomy and host alterations; Gay M et al.; For several years, strains phenotypically related to Vibrio splendidus have been associated with mortality outbreaks of molluscs . A former study on Crassostrea gigas demonstrated the genetic diversity of V . splendidus strains associated with diseased animals . Another study suggested that different strains may act in an additive/synergistic way leading to higher C . gigas mortality rates . Here, a strain pair (31+32) was characterised at taxonomic and virulence levels . Using a polyphasic approach, these strains were confirmed to be V . splendidus-related, without a clear discrimination between V . kanaloae and V . pomeroyi since hybridisation rates with both these strains were above 70 % . Following experimental infection of C . gigas by injection in the adductor muscle or in the pallial cavity, the host alterations induced were described . After injection of strains 31 and/or 32, bacteria were localised at the periphery of the muscle and induced extensive lesions of the translucent part of the adductor muscle . Muscle alterations were of 3 kinds: (1) presence of isolated rounded muscular fibres containing non-homogenous granular material and surrounded by a translucent halo; (2) presence of non-homogenous granular material in the cytoplasm of entire muscle bands; (3) affection of wide muscle areas with extremely condensed muscle fibres . Infiltration associated with these lesions was notably absent in the vast majority of the individuals. Environ Toxicol Chem, 2004 Dec, 23(12), 2941 - 9 Comparative toxicity of oil, dispersant, and oil plus dispersant to several marine species; Fuller C et al.; Dispersants are a preapproved chemical response agent for oil spills off portions of the U.S . coastline, including the Texas-Louisiana coast . However, questions persist regarding potential environmental risks of dispersant applications in nearshore regions (within three nautical miles of the shoreline) that support dense populations of marine organisms and are prone to spills resulting from human activities . To address these questions, a study was conducted to evaluate the relative toxicity of test media prepared with dispersant, weathered crude oil, and weathered crude oil plus dispersant . Two fish species, Cyprinodon variegatus and Menidia beryllina, and one shrimp species, Americamysis bahia (formerly Mysidopsis bahia), were used to evaluate the relative toxicity of the different media under declining and continuous exposure regimes . Microbial toxicity was evaluated using the luminescent bacteria Vibrio fisheri . The data suggested that oil media prepared with a chemical dispersant was equal to or less toxic than the oil-only test medium . Data also indicated that continuous exposures to the test media were generally more toxic than declining exposures . The toxicity of unweathered crude oil with and without dispersant was also evaluated using Menidia beryllina under declining exposure conditions . Unweathered oil-only media were dominated by soluble hydrocarbon fractions and found to be more toxic than weathered oil-only media in which colloidal oil fractions dominated . Total concentrations of petroleum hydrocarbons in oil-plus-dispersant media prepared with weathered and unweathered crude oil were both dominated by colloidal oil and showed no significant difference in toxicity . Analysis of the toxicity data suggests that the observed toxicity was a function of the soluble crude oil components and not the colloidal oil. Environ Toxicol Chem, 2004 Dec, 23(12), 2837 - 43 Assessment of the toxicity of triasulfuron and its photoproducts using aquatic organisms; Vulliet E et al.; The toxicological effects of the sulfonylurea herbicide triasulfuron and its photoproducts were assessed on four aquatic organisms . Toxicity varied with tested organism and with triasulfuron irradiation time . Triasulfuron and its photoproducts had no significant effects on the crustacean (Cladocera) Daphnia magna (causing 50% effective concentration {EC50} {48 h} = 49 +/- 1 mg/L) and the marine bacteria Vibrio fischeri (EC50 {30 min} > 100 mg/L) . In contrast, primary producers (the duckweed Lemna minor, the microalgae Pseudokirchneriella subcapitata, and Chlorella vulgaris) were very sensitive to triasulfuron (EC50s < 11 microg/L) . For these organisms, triasulfuron photoproducts were less toxic than the parent compound but the residual toxicity observed still represented a potential environmental hazard. Arch Microbiol . 2005 Jan 13; {Epub ahead of print} Oxaloacetate decarboxylase of Vibrio cholerae: purification, characterization, and expression of the genes in Escherichia coli; Dahinden P et al.; The oxaloacetate decarboxylase (OAD) Na(+) pump consists of subunits alpha, beta, and gamma, which are expressed from an oadGAB gene cluster present in various anaerobic bacteria . Vibrio cholerae has two copies of oad genes, which are termed oad-1 and oad-2 . The oad-2 genes are part of the citrate fermentation operon, while the oad-1 genes are flanked by genes encoding products not involved in a catabolic pathway . The gene sequences of oad-1 and oad-2 of V . cholerae strain O395-N1 were determined . The apparent frameshift in the published sequence of the oadA-2 gene from V . cholerae El Tor N16961 was not present in strain O395-N1 . Upon anaerobic growth of V . cholerae on citrate, exclusively the oad-2 genes are expressed . OAD was isolated from these cells by monomeric avidin-Sepharose affinity chromatography . The enzyme was of higher specific activity than that from Klebsiella pneumoniae and was significantly more stable . Decarboxylase activity was Na(+) dependent, and the activation profile showed strong cooperativity with a Hill coefficient n(H)=1.8 . Oxalate and oxomalonate inhibited the enzyme with half-maximal concentrations of 10 muM and 200 muM, respectively . After reconstitution into proteoliposomes, the enzyme acted as a Na(+) pump . With size-exclusion chromatography, the enzyme eluted in a symmetrical peak at a retention volume corresponding to an apparent molecular mass of approximately 570 kDa, suggesting a tetrameric structure for OAD-2 . The two oad gene clusters were heterologously expressed in Escherichia coli, and the decarboxylases were isolated from the host cells. J Biol Chem . 2005 Jan 12; {Epub ahead of print} Crystal structure of the virulence gene activator AphA from vibrio cholerae reveals it is a novel member of the winged helix transcription factor superfamily; De Silva RS et al.; AphA is a member of a new and largely uncharacterized family of transcriptional activators that is required for initiating virulence gene expression in Vibrio cholerae, the causative agent of the frequently fatal epidemic diarrheal disease, cholera . AphA activates transcription by an unusual mechanism that appears to involve a direct interaction with the LysR-type regulator AphB at the tcpPH promoter . As a first step toward understanding the molecular basis for tcpPH activation by AphA and AphB, we have determined the crystal structure of AphA to 2.2 A resolution . AphA is a dimer with an N-terminal winged helix DNA binding domain that is architecturally similar to that of the MarR family of transcriptional regulators . Unlike this family, however, AphA has a unique C-terminal antiparallel coiled coil domain that serves as its primary dimerization interface . AphA monomers would be highly unstable by themselves, and form a linked topology, requiring the protein to partially unfold in order to form the dimer . The structure of AphA also provides insights into how it cooperates with AphB to activate transcription, most likely by forming a heterotetrameric complex at the tcpPH promoter. Can J Microbiol, 2004 Nov, 50(11), 911 - 22 Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish; Panicker G et al.; In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus . Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates . The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V . vulnificus . No amplification of other Vibrio spp . or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method . The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 103 V . vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate . This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples . A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method . Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons . The rapid, sensitive, and specific detection of clinically important pathogenic V . vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen. Can J Microbiol, 2004 Oct, 50(10), 827 - 834 Pandemic strains of O3:K6 Vibrio parahaemolyticus in the aquatic environment of Bangladesh; Islam MS et al.; A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V . parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe . Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene . Examination by PCR confirmed that the 5 strains were V . parahamolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains . All 5 strains gave positive Kanagawa phenomenon reaction with characteristic &beta;-haemolysis on Wagatsuma agar medium . Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon . However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6) . The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone . Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns . Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains . This is the first report on the isolation of pandemic O3:K6 strains of V . parahaemolyticus from the aquatic environment of Bangladesh. Am J Trop Med Hyg, 2004 Dec, 71(6), 822 - 7 A cholera epidemic among the nicobarese tribe of nancowry, andaman, and nicobar, India; Sugunan AP et al.; Cholera has not been reported from the Andaman and Nicobar Islands in India . In October 2002, an outbreak of diarrhea occurred among the Nicobarese tribe of the Nancowry group of islands . The outbreak affected 16 of the 45 inhabited villages of three islands with an attack rate of 12.8% and a case fatality ratio of 1.3% . Vibrio cholerae O1 El Tor was isolated from 18 of the 67 patients tested . A study conducted in one of the villages indicated that the outbreak was started there by a person who traveled to a nearby village where an outbreak was occurring . No specific water source could be identified as the source of infection because persons consuming water from all wells were affected . Water samples from 55 sources were tested and 38 of them were contaminated with Escherichia coli . The possible sources of V . cholerae are effluents from ships or poachers from neighboring countries where cholera is endemic. Aquat Toxicol, 2005 Jan 26, 71(2), 183 - 92 Epub 2004 Dec 29. Ecotoxicological evaluation of the additive butylated hydroxyanisole using a battery with six model systems and eighteen endpoints; Jos A et al.; The occurrence and fate of additives in the aquatic environment is an emerging issue in environmental chemistry . This paper describes the ecotoxicological effects of the commonly used additive butylated hydroxyanisole (BHA) using a test battery, comprising of several different organisms and in vitro test systems, representing a proportion of the different trophic levels . The most sensitive system to BHA was the inhibition of bioluminescence in Vibriofischeri bacteria, which resulted in an acute low observed adverse effect concentration (LOAEC) of 0.28muM . The next most sensitive system was the immobilization of the cladoceran Daphniamagna followed by: the inhibition of the growth of the unicellular alga Chlorellavulgaris; the endpoints evaluated in Vero (mammalian) cells (total protein content, LDH activity, neutral red uptake and MTT metabolization), mitotic index and root growth inhibition in the terrestrial plant Allium cepa, and finally, the endpoints used on the RTG-2 salmonid fish cell line (neutral red uptake, total protein content, MTS metabolization, lactate dehydrogenase leakage and activity, and glucose-6-phosphate dehydrogenase activity) . Morphological alterations in RTG-2 cells were also assessed and these included loss of cells, induction of cellular pleomorphism, hydropic degeneration and induction of apoptosis at high concentrations . The results from this study also indicated that micronuclei were not induced in A.cepa exposed to BHA . The differences in sensitivity for the diverse systems that were used (EC(50) ranged from 1.2 to >500muM) suggest the importance for a test battery approach in the evaluation of the ecological consequences of chemicals . According to the results, the levels of BHA reported in industrial wastewater would elicite adverse effects in the environment . This, coupled with its potential to bioaccumulate, makes BHA a pollutant of concern not only for acute exposures, but also for the long-term. Appl Environ Microbiol, 2005 Jan, 71(1), 98 - 104 Isolation of Vibrio alginolyticus and Vibrio splendidus from Aquacultured Carpet Shell Clam (Ruditapes decussatus) Larvae Associated with Mass Mortalities; Gomez-Leon J et al.; Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain . Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events . Vibrio splendidus biovar II, in addition to V . alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event . The larval mortality rates for these events were 62 and 73%, respectively . Mortality was also detected in spat . To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality . The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene . In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion . The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams . V . alginolyticus TA15 and V . splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms . The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100 degrees C for 10 min) . Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15 . ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria. Zh Mikrobiol Epidemiol Immunobiol, 2004 Nov-Dec, (6), 3 - 6 {Adhesive and other properties of vibrio cholerae tcp+ ctx- isolated from environmental objects in the Rostov region in 2002} {Severe sepsis with organ failure in necrotizing fasciitis of the right leg caused by infection with Vibrio vulnificus} Vecer J, Kubatova H, Hanek P, Esnerova A, Klazarova H, Lochmann. Interni klinika FN Motol, PrahaVibrio vulnificus is a human pathogen which can cause the septicemia and necrosis of the soft tissue, especially fasciitis . The occurrence is most common in the summer, the source of infection can be sea water or the sand on the beaches, however the infection after eating seafood was described as well . The patient with predominant liver dysfunction are more often affected . The clinical course is characterized by severe sepsis, and massive skin lesion . The mortality more than 60% is reported . This case report describes the course of disease in 64 year old patient, who has spent his vacation in Bulgaria . After return he was admitted with severe sepsis with multiorgan failure and despite the intensive therapy and high amputation of the limb which was performed, the patient died. J Clin Microbiol, 2005 Jan, 43(1), 514 - 5 Vibrio fluvialis peritonitis in a patient receiving continuous ambulatory peritoneal dialysis; Ratnaraja N et al.; We describe a case of peritonitis due to Vibrio fluvialis in a patient receiving continuous ambulatory peritoneal dialysis; we believe the case to be associated with the consumption of poorly prepared seafood . This was shown to be an important but rare cause of recurrent infection in our patient. Microbiology, 2005 Jan, 151(Pt 1), 311 - 22 Molecular evolution of Vibrio pathogenicity island-2 (VPI-2): mosaic structure among Vibrio cholerae and Vibrio mimicus natural isolates; Jermyn WS et al.; Vibrio cholerae is a Gram-negative rod that inhabits the aquatic environment and is the aetiological agent of cholera, a disease that is endemic in much of Southern Asia . The 57.3 kb Vibrio pathogenicity island-2 (VPI-2) is confined predominantly to toxigenic V . cholerae O1 and O139 serogroup isolates and encodes 52 ORFs (VC1758 to VC1809), which include homologues of an integrase (VC1758), a restriction modification system, a sialic acid metabolism gene cluster (VC1773-VC1783), a neuraminidase (VC1784) and a gene cluster that shows homology to Mu phage . In this study, a 14.1 kb region of VPI-2 comprising ORFs VC1773 to VC1787 was identified by PCR and Southern blot analyses in all 17 Vibrio mimicus isolates examined . The VPI-2 region in V . mimicus was inserted adjacent to a serine tRNA similar to VPI-2 in V . cholerae . In 11 of the 17 V . mimicus isolates examined, an additional 5.3 kb region encoding VC1758 and VC1804 to VC1809 was present adjacent to VC1787 . The evolutionary history of VPI-2 was reconstructed by comparative analysis of the nanH (VC1784) gene tree with the species gene tree, deduced from the housekeeping gene malate dehydrogenase (mdh), among V . cholerae and V . mimicus isolates . Both gene trees showed an overall congruence; on both gene trees V . cholerae O1 and O139 serogroup isolates clustered together, whereas non-O1/non-O139 serogroup isolates formed separate divergent branches with similar clustering of strains within the branches . One exception was noted: on the mdh gene tree, V . mimicus sequences formed a distinct divergent lineage from V . cholerae sequences; however, on the nanH gene tree, V . mimicus clustered with V . cholerae non-O1/non-O139 isolates, suggesting horizontal transfer of this region between these species. J Bacteriol, 2005 Jan, 187(2), 778 - 84 Deletion Analysis of the Carboxyl-Terminal Region of the PomB Component of the Vibrio alginolyticus Polar Flagellar Motor; Yakushi T et al.; The stator of the sodium-driven flagellar motor of Vibrio alginolyticus is a membrane protein complex composed of four PomA and two PomB subunits . PomB has a peptidoglycan-binding motif in the C-terminal region . In this study, four kinds of PomB deletions in the C terminus were constructed . None of the deletion proteins restored motility of the DeltapomB strain . The PomA protein was coisolated with all of the PomB derivatives under detergent-solubilized conditions . Homotypic disulfide cross-linking of all of the deletion derivatives through naturally occurring Cys residues was detected . We conclude that the C-terminal region of PomB is essential for motor function but not for oligomerization of PomB with itself or PomA. J Bacteriol, 2005 Jan, 187(2), 752 - 7 Vibrios commonly possess two chromosomes; Okada K et al.; The prevalence of the two-chromosome configuration was investigated in 34 species of vibrios and closely related species . Pulsed-field gel electrophoresis of undigested genomic DNA suggested that vibrios commonly have two chromosomes . The size of the large chromosome is predominantly within a narrow range (3.0 to 3.3 Mb), whereas the size of the small chromosome varies considerably among the vibrios (0.8 to 2.4 Mb) . This fact suggests that the structure of the small chromosome is more flexible than that of the large chromosome during the evolution of vibrios. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2005 Jan, 21(1), 57 - 9 {Cloning and sequence analysis of variable region gene of anti-idiotype monoclonal antibody against vibrio alginolyticus.}; Fu JF et al.; AIM: To clone and sequence V(H) and V(L) genes of anti-idiotype monoclonal antibody (mAb) against vibrio alginolyticuslz . METHODS: Total RNA was extracted from hybridoma cell AL1 secreting mAb against vibrio alginolyticus and cDNA was amplified by RT-PCR . Then the cDNA was inserted into PMD18-T vector and its sequence was analyzed . RESULTS: The V(H) gene contained 369 bp and encoded 123 amino acid residues; the V(L) gene contained 339 bp and encoded 113 amino acid residues . There were four FRs, three CDRs and two characteristic cysteine residues in the V(H) and V(L) genes, respectively . CONCLUSION: The successful cloning of the V(H) and V(L) genes of anti-idiotype mAb against vibrio alginolyticus provides a sound basis for construction of gene-engineering vaccine of the anti-idiotype mAb against vibrio alginolyticus. Biochemistry, 2005 Jan 11, 44(1), 261 - 7 Redox Potential and Equilibria in the Reductive Half-Reaction of Vibrio harveyi NADPH-FMN Oxidoreductase; Lei B et al.; Vibrio harveyi NADPH:FMN oxidoreductase P (FRP(Vh)) is a homodimeric enzyme having a bound FMN per enzyme monomer . The bound FMN functions as a cofactor of FRP(Vh) in transferring reducing equivalents from NADPH to a flavin substrate in the absence of V . harveyi luciferase but as a substrate for FRP(Vh) in the luciferase-coupled bioluminescent reaction . As part of an integral plan to elucidate the regulation of functional coupling between FRP(Vh) and luciferase, this study was carried out to characterize the equilibrium bindings, reductive potential, and the reversibility of the reduction of the bound FMN in the reductive half-reaction of FRP(Vh) . Results indicate that, in addition to NADPH binding, NADP(+) also bound to FRP(Vh) in either the oxidized (K(d) 180 muM) or reduced (K(d) 230 muM) form . By titrations with NADP(+) and NADPH and by an isotope exchange experiment, the reduction of the bound FMN by NADPH was found to be readily reversible (K(eq) = 0.8) . Hence, the reduction of FRP(Vh)-bound FMN is not the committed step in coupling the NADPH oxidation to bioluminescence . To our knowledge, such an aspect of flavin reductase catalysis has only been clearly established for FRP(Vh) . Although the reductive potentials and some other properties of a R203A variant of FRP(Vh) and an NADH/NADPH-utilizing flavin reductase from Vibrio fischeri are quite similar to that of the wild-type FRP(Vh), the reversal of the reduction of bound FMN was not detected for either of these two enzymes. J Formos Med Assoc, 2004 Dec, 103(12), 935 - 8 Bacteremic necrotizing fasciitis caused by Vibrio cholerae serogroup O56 in a patient with liver cirrhosis; Cheng NC et al.; ABSTRACT: Bacteremic necrotizing fasciitis caused by non-O1 Vibrio cholerae has rarely been reported . We describe a case of necrotizing fasciitis of the bilateral lower extremities in a 68-year-old man with liver cirrhosis and diabetes mellitus . Cultures of blood and the debrided tissue all yielded V . cholerae serogroup non-O1 (O56) . Despite extensive radical debridement and antibiotic treatment with ceftazidime and doxycycline, the patient died on the 12th hospital day due to multiple organ failure . The present case is the first report of necrotizing fasciitis and bacteremia caused by V . cholerae serogroup O56. DNA Seq, 2004 Oct-Dec, 15(5-6), 344 - 50 Cloning, Sequence Analysis and Expression of Gene alyVI Encoding Alginate Lyase from Marine Bacterium Vibrio sp . QY101; Han F et al.; The gene (alyVI) encoding an alginate lyase of marine bacterium Vibrio sp . QY101, which was isolated from a decaying thallus of Laminaria, was cloned using a strategy of combined degenerate PCR and long range-inverse PCR (LR-IPCR), then sequenced and expressed in Escherichia coli . Gene alyVI was composed of a 1014 bp open reading frame (ORF) encoding 338 amino acid residues . The calculated molecular mass of alyVI product is 38.4 kDa, but a signal peptide is cleaved off, leaving a mature protein of 34 kDa . AlyVI was purified from culture supernatants to electrophoretic homogeneity using affinity chromatography . AlyVI was most active at pH 7.5 and 40 degrees C in the presence of 1 mM ZnCl(2) . A nine-amino-acid consensus region (YXRESLREM), which was only found in polyguluronate lyases, was also observed in the amino-terminal region of AlyVI . However, AlyVI could degrade both M block and G block . These results indicate that a novel alginate lyase-encoding gene has been cloned. FEMS Microbiol Lett, 2005 Jan 15, 242(2), 241 - 7 A variant type of Vibrio cholerae SXT element in a multidrug-resistant strain of Vibrio fluvialis; Ahmed AM et al.; Vibrio fluvialis strain H-08942 was isolated from an infant aged 6 months who was suffering from cholera-like diarrhea in India . This strain showed the typical multidrug-resistance phenotype of an SXT element . It was resistant to sulfamethoxazole (Su), trimethoprim (Tm), chloramphenicol (Cm) and streptomycin (Sm), in addition to other antibiotics such as ampicillin (Am), furazolidone (Fz), nalidixic acid (Na), and gentamicin (Gm) . The SXT element is a Vibrio cholerae-derived integrative and conjugative element (ICE) that has also been referred to as a conjugative transposon . Our goal was to find a relationship between these resistant phenotypes and the presence of the SXT element in this unique strain . By using PCR, we detected the antibiotic resistance genes, the integrase gene and the attP attachment site of SXT element . Cloning and DNA sequencing results showed that both the SXT integrase gene and its attP site of V . fluvialis were similar but not identical to those of V . cholerae . The SXT integrase gene of V . fluvialis has a 99% identity to that of V . cholerae, and the attP site of SXT of V . fluvialis is variant and shorter (641 bp) than that of V . cholerae (785 bp) . It was possible for the SXT of V . fluvialis to be transferred by conjugation to a laboratory strain of Escherichia coli . Here, we report the detection of a variant SXT element in species other than V . cholerae, with molecular characterization and analysis of its integrase gene and its attP site. FEMS Microbiol Lett, 2005 Jan 15, 242(2), 221 - 5 Asymmetric swimming pattern of Vibrio alginolyticus cells with single polar flagella; Kudo S et al.; The swimming pattern of bacteria with single polar flagella has usually been described as "run and reverse" . We observed the swimming traces of monotrichously flagellated Vibrio alginolyticus cells and examined the relationship between the swimming pattern and the sense of progress . Swimming in regions other than a solid surface was confirmed to be linear run and reverse . Near a solid surface, the traces consisted of "run and arc"; the cells were found to curve sharply during backward swimming, while they progressed linearly during forward swimming . The "run and arc" swimming pattern may play an important role in the chemotaxis strategy of marine bacteria at solid surfaces. Chemosphere, 2005 Feb, 58(5), 551 - 7 Study on the toxicity of binary equitoxic mixtures of metals using the luminescent bacteria Vibrio fischeri as a biological target; Fulladosa E et al.; Results from two mathematical approaches to predict the toxicity of all the possible binary equitoxic mixtures of Co, Cd, Cu, Zn and Pb were compared to the observed toxicity of these mixtures to Vibrio fischeri bacteria . Combined effect of the metals was found to be antagonistic for Co-Cd, Cd-Zn, Cd-Pb and Cu-Pb, synergistic for Co-Cu and Zn-Pb and merely additive in other cases, revealing a complex pattern of possible interactions . Besides, Cd appears much less toxic to the bacterial model than to animal cells . The synergistic effect of the Co-Cu combination and the strong lowering of Pb toxicity in the presence of Cd deserve much attention when establishing environmental safety regulations. Appl Microbiol Biotechnol . 2004 Dec 22; {Epub ahead of print} Rapid screening and dereplication of bacterial isolates from marine sponges of the Sula Ridge by Intact-Cell-MALDI-TOF mass spectrometry (ICM-MS); Dieckmann R et al.; Rapid grouping of bacterial isolates is critical in comprehensive microbial studies of environmental samples or screening programmes e.g . in unknown marine environments where large numbers of strains have to be isolated on different growth media . Sets of bacteria have been cultured from the marine sponges Isops phlegraei, Haliclona sp . 1, Phakellia ventilabrum and Plakortis sp . growing at a depth of about 300 m on the Sula Ridge close to the Norwegian coast . We employed Intact-Cell MALDI-TOF (ICM) mass spectrometry to achieve a rapid proteometric clustering of a subset of the strain collection including 456 isolates . Cluster analysis of mass spectra resolved the strains into 11 groups corresponding to species of Alteromonas (15), Bacillus (3), Colwellia (31), Erythrobacter (19), Marinobacter (14), Marinococcus (6), Pseudoalteromonas (297), Pseudomonas (56), Roseobacter (3), Sphingomonas (2) and Vibrio (10) as verified by 16 S rDNA analysis . A further discrimination into subgroups was demonstrated for different isolates from the genus Pseudoalteromonas . The approach described here permits the rapid identification of isolates for dereplication, and the selection of strains representing rare species for subsequent characterization. Mol Microbiol, 2005 Jan, 55(1), 125 - 36 Distinct segregation dynamics of the two Vibrio cholerae chromosomes; Fogel MA et al.; Summary The study of prokaryotic chromosome segregation has focused primarily on bacteria with single circular chromosomes . Little is known about segregation in bacteria with multipartite genomes . The human diarrhoeal pathogen Vibrio cholerae has two circular chromosomes of unequal sizes . Using static and time-lapse fluorescence microscopy, we visualized the localization and segregation of the origins of replication of the V . cholerae chromosomes . In all stages of the cell cycle, the two origins localized to distinct subcellular locations . In newborn cells, the origin of chromosome I (oriCI(vc)) was located near the cell pole while the origin of chromosome II (oriCII(vc)) was at the cell centre . Segregation of oriCI(vc) occurred asymmetrically from a polar position, with one duplicated origin traversing the length of the cell towards the opposite pole and the other remaining relatively fixed . In contrast, oriCII(vc) segregated later in the cell cycle than oriCI(vc) and the two duplicated oriCII(vc) regions repositioned to the new cell centres . DAPI staining of the nucleoid demonstrated that both origin regions were localized to the edge of the visible nucleoid and that oriCI(vc) foci were often associated with specific nucleoid substructures . The differences in localization and timing of segregation of oriCI(vc) and oriCII(vc) suggest that distinct mechanisms govern the segregation of the two V . cholerae chromosomes. Biochemistry, 2004 Dec 28, 43(51), 16193 - 202 The membrane-associated lipoprotein-9 GmpC from Staphylococcus aureus binds the dipeptide GlyMet via side chain interactions; Williams WA et al.; Bacterial dipeptide ABC transporters function to import a wide range of dipeptide substrates . This ability to transport a wide variety of dipeptides is conferred by the cognate substrate binding protein (SBP) of these transporters . SBPs bind dipeptides with little regard for their amino acid content . Here, we report the 1.7 A resolution structure of lipoprotein-9 (SA0422) of Staphylococcus aureus in complex with the dipeptide glycylmethionine . Experimental characterization of the subcellular location of the protein confirmed that SA0422 is an acylated, peripheral membrane protein . This is the first structure determined for an SBP of a Gram-positive dipeptide ABC transporter . Usually, binding of dipeptides occurs in a binding pocket that is largely hydrated and able to accommodate the side chains of several different amino acid residues . Unlike any other known SBP, lipoprotein-9 binds the side chains of the glycylmethionine dipeptide through very specific interactions . Lipoprotein-9 shares significant structural and sequence homology with the MetQ family of methionine SBP . Sequence comparisons between MetQ-like proteins and lipoprotein-9 suggest that the residues forming the tight interactions with the methionine side chains of the ligand are highly conserved between lipoprotein-9 and MetQ homologues, while the residues involved in coordinating the glycine residue are not . Modeling of the Vibrio cholerae MetQ and lipoprotein-9 binding pockets can account for lipoprotein-9 substrate specificity toward glycylmethionine . For this reason, we have designated lipoprotein-9 GmpC, for glycylmethionine binding protein. J Biotechnol, 2005 Jan 12, 115(1), 67 - 79 On-line multi-analyzer monitoring of biomass, glucose and acetate for growth rate control of a Vibrio cholerae fed-batch cultivation; Navratil M et al.; In situ near-infrared (NIR) spectroscopy and in-line electronic nose (EN) mapping were used to monitor and control a cholera-toxin producing Vibrio cholerae fed-batch cultivation carried out with a laboratory method as well as with a production method . Prediction models for biomass, glucose and acetate using NIR spectroscopy were developed based on spectral identification and partial-least squares (PLS) regression resulting in high correlation to reference data (standard errors of prediction for biomass, glucose and acetate were 0.20gl(-1), 0.26gl(-1) and 0.28gl(-1)) . A compensation algorithm for aerated bioreactor disturbances was integrated in the model computation, which in particular improved the prediction by the biomass model . First, the NIR data were applied together with EN in-line data selected by principal component analysis (PCA) for generating a trajectory representation of the fed-batch cultivation . A correlation between the culture progression and EN signals was demonstrated, which proved to be beneficial in monitoring the culture quality . It was shown that a deviation from a normal cultivation behavior could easily be recognized and that the trajectory was able to alarm a bacterial contamination . Second, the NIR data indicated the potential of predicting the concentration of formed cholera toxin with a model prediction error of 0.020gl(-1) . Third, the on-line biomass prediction based on the NIR model was used to control the overflow metabolism acetate formation of the V . cholerae culture . The controller compared actual specific growth rate as estimated from the prediction with the critical acetate formation growth rate, and from that difference adjusted the glucose feed rate. Am J Orthop, 2004 Nov, 33(11), 568 - 71 Vibrio vulnificus infection of the upper extremity; Shah MA et al.; Vibrio vulnificus is a potentially limb- and life-threatening infection . This pathogen should be suspected in any patient with a rapidly progressive infection who has a history of saltwater contact . Although the infection may occur in otherwise healthy individuals, V vulnificus has a proclivity for patients with underlying chronic disease, particularly hepatic dysfunction . Prompt recognition and immediate treatment with antibiotics and possibly surgical debridement may prevent amputation or death. J Bacteriol, 2005 Jan, 187(1), 249 - 56 Molecular analysis of the Vibrio cholerae type II secretion ATPase EpsE; Camberg JL et al.; The type II secretion system is a macromolecular assembly that facilitates the extracellular translocation of folded proteins in gram-negative bacteria . EpsE, a member of this secretion system in Vibrio cholerae, contains a nucleotide-binding motif composed of Walker A and B boxes that are thought to participate in binding and hydrolysis of ATP and displays structural homology to other transport ATPases . Here we demonstrate that purified EpsE is an Mg2+-dependent ATPase and define optimal conditions for the hydrolysis reaction . EpsE displays concentration-dependent activity, which may suggest that the active form is oligomeric . Size exclusion chromatography showed that the majority of purified EpsE is monomeric; however, detailed analyses of specific activities obtained following gel filtration revealed the presence of a small population of active oligomers . We further report that EpsE binds zinc through a tetracysteine motif near its carboxyl terminus, yet metal displacement assays suggest that zinc is not required for catalysis . Previous studies describing interactions between EpsE and other components of the type II secretion pathway together with these data further support the hypothesis that EpsE functions to couple energy to the type II apparatus, thus enabling secretion. Eur J Clin Microbiol Infect Dis, 2004 Dec, 23(12), 912 - 5 Two cases of severe sepsis due to Vibrio vulnificus wound infection acquired in the Baltic Sea; Ruppert J et al.; Two severe cases of Vibrio vulnificus wound infection with secondary septicemia occurred during 1 week in August 2003 on the German island of Usedom in the southwestern Baltic Sea . In both cases, pre-existing wounds were inoculated by wading in contaminated sea water . One of the patients died from septic multiorgan failure . To the best of our knowledge, this is the first fatality due to a V . vulnificus infection to have occurred in Germany . Microbiological analysis revealed high concentrations of V . vulnificus in sea water along the coastline, following a period when water temperature exceeded 20 degrees C for more than 2 weeks. Mol Gen Mikrobiol Virusol, 2004, (4), 28 - 33 {Retrospective VNTR-analysis of genotypes of Vibrio cholerae 01 strains isolated, during the 7th cholera pandemic, in Rostov Region} {Evolution of the cholera agent genome} {No authors listed} "Mikrob" Russian Research Anti-Plague Institute, Saratov Studies of the genomic evolution of pathogenic bacteria became a priority research trend of modern molecular genetics . Vibrio cholerae, whose pathogenic properties are conditioned by the presence of virulence blocks of differing phylogenetic origins in 2 chromosomes, turned out to be a unique model object for studies of evolutionary transformations of genomes that are causative agents of extra dangerous infections . The molecular-and-genetic mechanisms underlying the change of biovariants and the emergence of a cholera agent of a new serogroup are in the focus of attention . Finally, the possibility that the new V . cholerae pathogenic clone originated due to horizontal genetic transfers and recombination phenomena is under discussion in the survey. Biochemistry, 2004 Dec 21, 43(50), 15975 - 82 Identity of the emitter in the bacterial luciferase luminescence reaction: binding and fluorescence quantum yield studies of 5-decyl-4a-hydroxy-4a,5-dihydroriboflavin-5'-phosphate as a model; Lei B et al.; The excited state of 4a-hydroxy-4a,5-dihydroFMN has been postulated to be the emitter in the bacterial bioluminescence reaction . However, while the bioluminescence quantum yield of the luciferase emitter is about 0.16, chemiluminescence and fluorescence quantum yields of earlier flavin models mimicking the luciferase emitter were no more than 10(-5) . To further examine the proposed chemical identity of the luciferase emitter, 5-decyl-4a-hydroxy-4a,5-dihydroFMN was prepared as a new flavin model . Both the wild-type Vibrio harveyi luciferase and a catalytically active alphaC106A mutant formed complexes with the flavin model at a 1:1 molar ratio with K(d) values at 2.4 and 1.2 microM, respectively . This flavin model inhibited the activity of both luciferases, suggesting that it was bound to the enzyme active center . While the free flavin model was itself only very weakly fluorescent, its binding to either luciferase species resulted in markedly enhanced fluorescence, peaking at 440 nm . The fluorescence quantum yields of 5-decyl-4a-hydroxy-4a,5-dihydroFMN bound to wild-type and alphaC106A luciferases were 0.08 and 0.05, respectively, which are about 50% of the respective emitter bioluminescence quantum yields of these two luciferases . The present findings clearly demonstrated that the luciferase active site was suitable for marked enhancement of fluorescence of 4a-hydroxyflavin and, hence, provides a strong support to the proposed identity of 4a-hydroxy-4a,5-dihydroFMN, in its exited state, as the luciferase emitter. Indian J Med Res, 2004 Nov, 120(5), 478 - 80 Re-emergence of El Tor vibrio in outbreak of cholera in & around Nagpur; Mishra M et al.; Contrary to earlier outbreaks of cholera due to Vibrio cholerae O139 during 1993 and its reemergence in 1998 in and around Nagpur and only sporadic episodes thereafter for next couple of years, a large outbreak was encountered between June and October 2003 . V . cholerae 01 El Tor were isolated in 198 cases, of which 152 were Ogawa, 3 Inaba, 4 Hikojima and 39 were non agglutinating (NAG) vibrios . No isolate of V . cholerae O139 was detected during the entire outbreak . The isolates were multi drug resistant to antibiotic susceptibility tests . This points to the resurgence of V . cholerae El Tor Ogawa causing outbreaks of cholera with a discernible increase in the incidence of multi drug resistant strains. J Med Microbiol, 2005 Jan, 54(1), 15 - 22 Depletion of lymphocytes, but not neutrophils, via apoptosis in a murine model of Vibrio vulnificus infection; Kashimoto T et al.; Vibrio vulnificus causes severe sepsis in humans . There are several reports about the relationship between host immunity and bacterial growth in V . vulnificus infection . However, the effect on leukocytes of V . vulnificus infection in vivo has not been elucidated . A murine model of V . vulnificus infection was used to investigate its effects on leukocytes in this study . Bacteria were recovered from the blood of mice 3 h after subcutaneous injection in the right lower flank . They were detected in 87.5 % (n = 7/8) of mice at 6 h, but this value decreased to 12.5 % (n = 1/8) at 12 h . In contrast, the number of lymphocytes in peripheral blood had already started to decrease at 3 h, and reached a minimum at 6-9 h post-inoculation . Typical DNA laddering, a hallmark of apoptosis, was also detected in thymocytes and splenocytes at 6 and 9 h, and showed a tendency to disappear by 12 h . Although the number of lymphocytes decreased in the model, the numbers of neutrophils did not . These results suggested that V . vulnificus has selective cytotoxicity for lymphocytes in peripheral blood in vivo, and the lymphocyte depletion was probably associated with apoptosis in vivo. Photochem Photobiol . 2004 Oct 1; {Epub ahead of print} Effects of Iodide on the Fluorescence and Activity of the Hydroperoxyflavin Intermediate of Vibrio harveyi Luciferase; Huang S et al.; The 4a-hydroperoxy-4a,5-dihydroFMN intermediate II of Vibrio harveyi luciferase is known to transform from a low-quantum- yield IIx to a high-quantum-yield (lambdamax 485 nm, uncorrected) IIy fluorescent species upon exposure to excitation light . Similar results were observed with II prepared from the alphaH44A luciferase mutant, which is very weak in bioluminescence activity . Because the rapid decay of the alphaH44A II, its true fluorescence was obscured by the more intense 520 nm fluorescence (uncorrected) from its decay product FMN . KI at 0.2 M was effective in quenching the FMN fluorescence, leaving the 485 nm fluorescence of II from both the wild-type and alphaH44A luciferase readily detectable . For both II species, the bound peroxyflavin species was well shielded from KI quenching . KI also enhanced the decay rates of both the wild- type and alphaH44A II . For alphaH44A, the transformation of IIx to IIy can be induced by KI in the dark and it is proposed to be a consequence of a luciferase conformational change . The wild- type II formed a bioluminescence-inactive complex with KI, resulting in two distinct decay time courses based on absorption changes and decreases of bioluminescence activity of II. DNA Cell Biol, 2004 Nov, 23(11), 723 - 41 Genetics of stress adaptation and virulence in toxigenic Vibrio cholerae; Faruque SM et al.; Vibrio cholerae, a Gram-negative bacterium belonging to the gamma-subdivision of the family Proteobacteriaceae is the etiologic agent of cholera, a devastating diarrheal disease which occurs frequently as epidemics . Any bacterial species encountering a broad spectrum of environments during the course of its life cycle is likely to develop complex regulatory systems and stress adaptation mechanisms to best survive in each environment encountered . Toxigenic V . cholerae, which has evolved from environmental nonpathogenic V . cholerae by acquisition of virulence genes, represents a paradigm for this process in that this organism naturally exists in an aquatic environment but infects human beings and cause cholera . The V . cholerae genome, which is comprised of two independent circular mega-replicons, carries the genetic determinants for the bacterium to survive both in an aquatic environment as well as in the human intestinal environment . Pathogenesis of V . cholerae involves coordinated expression of different sets of virulence associated genes, and the synergistic action of their gene products . Although the acquisition of major virulence genes and association between V . cholerae and its human host appears to be recent, and reflects a simple pathogenic strategy, the establishment of a productive infection involves the expression of many more genes that are crucial for survival and adaptation of the bacterium in the host, as well as for its onward transmission and epidemic spread . While a few of the virulence gene clusters involved directly with cholera pathogenesis have been characterized, the potential exists for identification of yet new genes which may influence the stress adaptation, pathogenesis, and epidemiological characteristics of V . cholerae . Coevolution of bacteria and mobile genetic elements (plasmids, transposons, pathogenicity islands, and phages) can determine environmental survival and pathogenic interactions between bacteria and their hosts . Besides horizontal gene transfer mediated by genetic elements and phages, the evolution of pathogenic V . cholerae involves a combination of selection mechanisms both in the host and in the environment . The occurrence of periodic epidemics of cholera in endemic areas appear to enhance this process. Dis Aquat Organ, 2004 Oct 21, 61(1-2), 169 - 74 Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing; Liu CH et al.; A gram-negative, rod-shaped bacterium identified as Vibrio alginolyticus was isolated from diseased Litopenaeus vannamei (also called Penaeus vannamei) in Taiwanese culture ponds . The diseased shrimp displayed poor growth, anorexia, inactivity, reddish pleural borders of antennae, uropods and telson, opaque and whitish musculature, and mortality . In histological preparations, melanized hemocytic granulomas were observed in the connective tissue around hemal sinuses together with hemocytic aggregation in necrotic musculature . Six isolates of Vibrio were collected from diseased shrimp at 3 farms, and these were evaluated for characteristics including morphology, physiology, biochemistry and sensitivity to antibiotics . The results indicated that the isolates belonged to a single species that grew in 1 to 8% NaCl, at 10 to 40 degrees C and on TCBS (thiosulfatecitrate-bile sucrose) agar, and that gave positive catalase, O/F (Oxidation/Fermentation), lysine decarboxylase, gelatinase and cytochrome-oxidase tests . Identification of CH003 (1 of 6 isolates) was confirmed by PCR assay for V . alginolyticus (expected amplicon 1486 bp) . The 16S rDNA sequence (GenBank accession number AY373027) gave 99.9% sequence identity to V . alginolyticus (GenBank accession number X74690) . The calculated 96 h LD50 dose of the isolated strain was 3.0 x 10(5) colony forming units (CFU) shrimp(-1) (6.6 x 10(4) CFU g(-1)). Dis Aquat Organ, 2004 Oct 21, 61(1-2), 123 - 35 Antimicrobial peptides discovered in the black tiger shrimp Penaeus monodon using the EST approach; Supungul P et al.; Two cDNA libraries were prepared from hemocytes of normal and Vibrio harveyi-challenged black tiger shrimp Penaeus monodon . A total of 1062 expressed sequence tag (EST) clones were sequenced unidirectionally . ESTs representing the antimicrobial peptide (AMP) homologues, antilipopolysaccharide factors (ALF), penaeidins and crustins were discovered . They predominated among immune-related genes, representing 29.2% and 64.0% of the normal and challenged libraries, respectively . Several types of each AMP homologue were found . Sequence alignments of ALF in P . monodon (ALFPm 1 to 5) implied possible alternative splicing of different exons at both NH2 and COOH-termini . Only one major type of penaeidin (penPm3) was found in P . monodon . In addition, crustin homologues (crusPms1 to 4) and a newly identified glycine-rich antibacterial peptide (GAMPPm1) were also isolated and characterized . Using RT-PCR analysis, expression of ALF, penaeidin and crustin transcripts was detected in various tissues but the main expression site was in hemocytes . Expression of these antimicrobial peptides in P . monodon subjected to V . harveyi challenge revealed a significant increase in expression of ALFPms (p < 0.05) but a decrease in expression of crustins and penaeidins. J Clin Microbiol, 2004 Dec, 42(12), 5854 - 6 Diverse CTX phages among toxigenic Vibrio cholerae O1 and O139 strains isolated between 1994 and 2002 in an area where cholera is endemic in Bangladesh; Nusrin S et al.; PCR surveillance of the rstR genes of CTX phages in Vibrio cholerae O1 and O139 showed no relationship between the incidence of disease and changes in the rstR but showed variations in their presence in O1 and O139 strains and the occurrence of multiple types in a few strains. Microbiology, 2004 Dec, 150(Pt 12), 4053 - 63 The Vibrio seventh pandemic island-II is a 26.9 kb genomic island present in Vibrio cholerae El Tor and O139 serogroup isolates that shows homology to a 43.4 kb genomic island in V . vulnificus; O'Shea YA et al.; Vibrio cholerae is the aetiological agent of the deadly diarrhoeal disease cholera . In this study the 7.5 kb Vibrio seventh pandemic island-II (VSP-II) that is unique to V . cholerae El Tor and O139 serogroups was analysed and it was found to be part of a novel 26.9 kb genomic island (GEI) encompassing VC0490-VC0516 . The low-GC-content VSP-II encompassed 24 predicted ORFs, including DNA repair and methyl-accepting chemotaxis proteins, a group of hypothetical proteins and a bacteriophage-like integrase adjacent to a tRNA gene . Interestingly, V . cholerae ORFs VC0493-VC0498, VC0504-VC0510 and VC0516, which encodes an integrase, were homologous to Vibrio vulnificus strain YJ016 ORFs VV0510-VV0516, VV0518-VV0525 and VV0560, which also encodes an integrase, respectively . Some ORFs showed amino acid identities greater than 90 % between the two species in these regions . In V . vulnificus strain YJ016, a 43.4 kb low-GC-content (43 %) GEI encompassing ORFs VV0509-VV0560 was identified and named V . vulnificus island-I (VVI-I) . The 52 ORFs of VVI-I included a phosphotransferase system gene cluster, genes required for sugar metabolism and transposase genes . There was synteny and homology between the 5' region of V . cholerae VSP-II and the 5' region of V . vulnificus VVI-I; however, VVI-I contained an additional 31.5 kb of DNA between VV0526 and VV0560 in strain YJ016 . A second V . vulnificus strain, CMCP6, did not contain the 43.4 kb VVI-I; in this strain two ORFs were found between the 5' and 3' flanking genes VV10636 and VV10632, showing 100 % identity to VV0508 and VV0561, respectively, which flank VVI-I. Microbiology, 2004 Dec, 150(Pt 12), 3969 - 77 Identification of residues in the Pseudomonas aeruginosa elastase propeptide required for chaperone and secretion activities; McIver KS et al.; An important virulence factor of the opportunistic human pathogen Pseudomonas aeruginosa is elastase, a secreted thermolysin-like neutral zinc-metalloprotease (TNP) . Elastase is synthesized as a larger precursor with an amino-terminal 18 kDa propeptide, and was the first TNP shown to require its propeptide as an intramolecular chaperone (IMC) for activity and secretion . This paper reports the analysis of the elastase propeptide to identify residues conserved among other TNP precursors that may be critical for its IMC function . The prosequences of TNP precursors from both Gram-negative (Vibrio species and Legionella species) and Gram-positive (Bacillus species) bacteria were found to show homology to the elastase propeptide . Two regions of conserved residues were observed: a hydrophilic region (ProM) found in the middle of the elastase propeptide and a more hydrophobic region (ProC) located proximal to the propeptide-processing site . To test whether such conserved motifs were important to function, single residue substitutions at eight conserved amino acids were introduced within the full-length pre-proelastase precursor by site-specific mutagenesis of lasB, the gene encoding elastase . Mutant lasB alleles were expressed from plasmids within a lasB-deleted P . aeruginosa strain, FRD740, and the effects of these propeptide alterations on elastase enzyme activity, processing, stability and accumulation inside and outside of the cell were examined . Within the ProM region, substitution at Arg74 resulted in a dramatic accumulation of proelastase in the cell, suggesting a secretion defect, and a dramatic reduction in supernatant elastolytic activity . Substitution at Asn68 adversely affected the amount of elastase in the culture supernatant, apparently as a result of the reduced stability of the mutated proelastase in the cell . Within the ProC region, mutations at Ile181 and Ala183 abolished the accumulation of a stable elastase molecule in the supernatant . Most mutations produced a phenotype consistent with a defect in protein folding and stability . Overall, the data from this preliminary study show that conserved residues within the elastase propeptide are essential for its function and begin to define the mechanisms of action of IMCs in the TNP family. J Bacteriol, 2004 Dec, 186(24), 8309 - 16 TcpH influences virulence gene expression in Vibrio cholerae by inhibiting degradation of the transcription activator TcpP; Beck NA et al.; Expression of toxT, the transcription activator of cholera toxin and pilus production in Vibrio cholerae, is the consequence of a complex cascade of regulatory events that culminates in activation of the toxT promoter by TcpP and ToxR, two membrane-localized transcription factors . Both are encoded in operons with genes whose products, TcpH and ToxS, which are also membrane localized, are hypothesized to control their activity . In this study we analyzed the role of TcpH in controlling TcpP function . We show that a mutant of V . cholerae lacking TcpH expressed virtually undetectable levels of TcpP, although tcpP mRNA levels remain unaffected . A time course experiment showed that levels of TcpP, expressed from a plasmid, are dramatically reduced over time without co-overexpression of TcpH . By contrast, deletion of toxS did not affect ToxR protein levels . A fusion protein in which the TcpP periplasmic domain is replaced with that of ToxR remains stable, suggesting that the periplasmic domain of TcpP is the target for degradation of the protein . Placement of the periplasmic domain of TcpP on ToxR, an otherwise stable protein, results in instability, providing further evidence for the hypothesis that the periplasmic domain of TcpP is a target for degradation . Consistent with this interpretation is our finding that derivatives of TcpP lacking a periplasmic domain are more stable in V . cholerae than are derivatives in which the periplasmic domain has been truncated . This work identifies at least one role for the periplasmic domain of TcpP, i.e., to act as a target for a protein degradation pathway that regulates TcpP levels . It also provides a rationale for why the V . cholerae tcpH mutant strain is avirulent . We hypothesize that regulator degradation may be an important mechanism for regulating virulence gene expression in V . cholerae. Appl Environ Microbiol, 2004 Dec, 70(12), 7481 - 6 Viable but nonculturable Vibrio cholerae O1 in the aquatic environment of Argentina; Binsztein N et al.; In Argentina, as in other countries of Latin America, cholera has occurred in an epidemic pattern . Vibrio cholerae O1 is native to the aquatic environment, and it occurs in both culturable and viable but nonculturable (VNC) forms, the latter during interepidemic periods . This is the first report of the presence of VNC V . cholerae O1 in the estuarine and marine waters of the Rio de la Plata and the Argentine shelf of the Atlantic Ocean, respectively . Employing immunofluorescence and PCR methods, we were able to detect reservoirs of V . cholerae O1 carrying the virulence-associated genes ctxA and tcpA . The VNC forms of V . cholerae O1 were identified in samples of water, phytoplankton, and zooplankton; the latter organisms were mainly the copepods Acartia tonsa, Diaptomus sp., Paracalanus crassirostris, and Paracalanus parvus . We found that under favorable conditions, the VNC form of V . cholerae can revert to the pathogenic, transmissible state . We concluded that V . cholerae O1 is a resident of Argentinean waters, as has been shown to be the case in other geographic regions of the world. Appl Environ Microbiol, 2004 Dec, 70(12), 7436 - 44 Detection of pathogenic Vibrio spp . in shellfish by using multiplex PCR and DNA microarrays; Panicker G et al.; This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish . Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V . cholerae, and with tlh, tdh, trh, and open reading frame 8 for V . parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated . For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer . Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye . Slides were imaged by using an arrayWoRx scanner . The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100% . However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate . Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline . Application of the DNA microarray methodology to natural oysters revealed the presence of V . vulnificus (100%) and V . parahaemolyticus (83%) . However, V . cholerae was not detected in natural oysters . An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers. Appl Environ Microbiol, 2004 Dec, 70(12), 7288 - 94 Seasonal incidence of autochthonous antagonistic Roseobacter spp . and Vibrionaceae strains in a turbot larva (Scophthalmus maximus) rearing system; Hjelm M et al.; Bacteria inhibitory to fish larval pathogenic bacteria were isolated from two turbot larva rearing farms over a 1-year period . Samples were taken from the rearing site, e.g., tank walls, water, and feed for larvae, and bacteria with antagonistic activity against Vibrio anguillarum were isolated using a replica plating assay . Approximately 19,000 colonies were replica plated from marine agar plates, and 341 strains were isolated from colonies causing clearing zones in a layer of V . anguillarum . When tested in a well diffusion agar assay, 173 strains retained the antibacterial activity against V . anguillarum and Vibrio splendidus . Biochemical tests identified 132 strains as Roseobacter spp . and 31 as Vibrionaceae strains . Partial sequencing of the 16S rRNA gene of three strains confirmed the identification as Roseobacter gallaeciensis . Roseobacter spp . were especially isolated in the spring and early summer months . Subtyping of the 132 Roseobacter spp . strains by randomly amplified polymorphic DNA with two primers revealed that the strains formed a very homogeneous group . Hence, it appears that the same subtype was present at both fish farms and persisted during the 1-year survey . This indicates either a common, regular source of the subtype or the possibility that a particular subtype has established itself in some areas of the fish farm . Thirty-one antagonists were identified as Vibrio spp., and 18 of these were V . anguillarum but not serotype O1 or O2 . Roseobacter spp . strains were, in particular, isolated from the larval tank walls, and it may be possible to establish an antagonistic, beneficial microflora in the rearing environment of turbot larvae and thereby limit survival of pathogenic bacteria. Appl Environ Microbiol, 2004 Dec, 70(12), 7024 - 32 Protocol for specific isolation of virulent strains of Vibrio vulnificus serovar E (biotype 2) from environmental samples; Sanjuan E et al.; The eel pathogen Vibrio vulnificus biotype 2 comprises at least three serovars, with serovar E being the only one involved in both epizootics of eel vibriosis and sporadic cases of human infections . The virulent strains of this serovar (VSE) have only been recovered from clinical (mainly eel tissue) sources . The main objective of this work was to design and validate a new protocol for VSE-specific isolation from environmental samples . The key element of the new protocol is the broth used for the first step (saline eel serum broth {SEB}), which contains eel serum as a nutritive and selective component . This approach takes advantage of the ability of VSE cells to grow in eel serum and thus to separate themselves from the pool of competitors . The growth yield in SEB after 8 h of incubation was 1,000 times higher for VSE strains than for their putative competitors (including biotype 1 strains of the species) . The selective and differential agar Vibrio vulnificus medium (VVM) was selected from five selective media for the second step because it gave the highest plating efficiency not only for the VSE group but also for other V . vulnificus groups, including biotype 3 . The entire protocol was validated by field studies, with alkaline peptone water plus VVM as a control . V . vulnificus was isolated by both protocols, but serovar E was only recovered by the new method described here . All selected serovar E isolates were identified as VSE since they were virulent for both eels and iron-overloaded mice and resisted the bactericidal action of eel and iron-overloaded human sera . In conclusion, this new protocol is a suitable method for the isolation of VSE strains from environmental samples and is recommended for epidemiological studies of the pathogenic serovar E. J Environ Monit, 2004 Dec, 6(12), 953 - 6 Epub 2004 Dec. The effect of temperature and NaCl concentration on the kinetic method of toxicity determination using Vibrio fischeri; Correia Faria E et al.; In this paper the effect of temperature and NaCl concentration on the kinetic method of toxicity determination using Vibrio fischeri was studied for 50 ppm Zn(2+) . This work shows that both NaCl concentration and temperature affect the kinetics of toxicity as well as the luminescence of the bacteria, and hence these are important factors that need to be considered in the development of a miniaturised portable instrument . Furthermore, this work shows that the conditions for which the kinetic test was most sensitive, i.e . exhibited the greatest response, were 23 degrees C and 2% NaCl . However, at these conditions small variations in temperature and NaCl concentration could lead to great errors in the results . Thus 12.5 degrees C and 2% NaCl are preferred as at these conditions the obtained results are more robust . Although at the latter conditions the toxicity rate constant was found to be 5.5 times less than that for 23 degrees C, the value is comparable to that obtained for 15 degrees C . From the data available it was also found that the temperature dependence of the toxicity rate fits the Arrhenius equation, in a behaviour similar to that of simpler chemical reactions. Res Microbiol, 2004 Dec, 155(10), 835 - 42 Proteomic analysis of salt-sensitive outer membrane proteins of Vibrio parahaemolyticus; Xu C et al.; Vibrio parahaemolyticus, a universal marine pathogen with available genome sequences, could be used as a bacterial model to clarify the various physiological phenomena of its native and host environments . In the present study, proteomic methodologies were applied to investigate the expression pattern of outer membrane proteins (OMPs) of V . parahaemolyticus at different NaCl concentrations . OmpW, OmpV, elongation factor TU and polar flagellin were determined to be osmoregulation-sensitive OMPs, among which OmpW and OmpV were reported to vary with changed NaCl concentrations in the pattern of osmolarity regulation . Therefore, our results not only expand our knowledge on osmoregulation-related proteins, but also provide a valuable strategy for the screening of salt-sensitive proteins. Biochem Biophys Res Commun, 2005 Jan 7, 326(1), 108 - 14 Equilibrium unfolding of an oligomeric protein involves formation of a multimeric intermediate state(s); Hsieh HC et al.; Superoxide dismutases (SODs) are important metalloenzymes which protect cells against oxidative stress by scavenging reactive superoxides . Missense mutations in SODs are known to lead to some familial cases of amyotrophic lateral sclerosis and several forms of cancers . In the present study, we investigate the guanidinium hydrochloride (GdnHCl)-induced equilibrium unfolding of apo-manganese superoxide dismutase (apo-MnSOD) isolated from Vibrio alginolyticus using a variety of biophysical techniques . GdnHCl-induced equilibrium unfolding of apo-MnSOD is non-cooperative and involves the accumulation of stable intermediate state(s) . Results of 1-anilino-8-naphthalene sulfonate binding experiments suggest that the equilibrium intermediate state(s) accumulates maximally in 1.5M GdnHCl . The intermediate state(s) appears to be obligatory and occurs both in the unfolding and refolding pathways . Size-exclusion chromatography and sedimentation velocity data reveal that the equilibrium intermediate state(s) is multimeric . To our knowledge, this is the first report of the identification of a multimeric intermediate in the unfolding pathway(s) of oligomeric proteins . The formation and dissociation of the multimeric intermediate state(s) appears to dictate the fate of the protein either to refold to its native conformation or misfold and form aggregates as observed in amyotrophic lateral sclerosis. Clin Diagn Virol, 1994 Oct, 2(6), 359 - 66 Molecular characterization of a human group A rotavirus isolated from an adult with severe dehydrating diarrhea and its relationship to strains concurrently circulating among children; Kaga E et al.; Background: While group A rotavirus is widely accepted as an important etiology of acute gastroenteritis in children, this agent rarely causes severe diarrhea in adults and, thus, is not considered by physicians to be an etiological agent for such diseases . Objectives: None of the reports describing such rare cases in adults has examined the causative strains genetically in detail . Study design: We determined the G type, the gene 4 genotype, the electropherotype, and the genomic RNA constellation (genogroup) of a group A rotavirus strain isolated from an adult with severe diarrhea . This patient, the first documented case of rotavirus diarrhea in adult in Japan, suffered from severe dehydrating diarrhea with 'rice-water' appearance, vomiting and little abdominal pain, presenting a clinical picture typical of cholera . Results: Rotavirus antigen and genomic RNA were detected in the stool but other enteric pathogens including Vibrio cholerae responsible for the disease were not isolated . Molecular characterization revealed that the patient was infected with a strain of the DS-1 genogroup with G2 and gene 4 genotype 4 which was circulating among children during the same period . Conclusions: The result that the same group A rotavirus strain was isolated from children and an adult underlines the necessity of examining rotavirus in adults with acute diarrhea. Fish Shellfish Immunol, 2005 Apr, 18(4), 297 - 310 Molecular cloning and characterisation of a pattern recognition molecule, lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP) from the white shrimp Litopenaeus vannamei; Cheng W et al.; A lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP) cDNA was cloned from the haemocyte and hepatopancreas of white shrimp Litopenaeus vannamei using oligonucleotide primers and RT-PCR . Both 3'- and 5'-regions were isolated by rapid amplification of cDNA end RACE method . Analysis of nucleotide sequence revealed that the cDNA clone has an open reading frame of 1101 bp encoding a protein of 367 amino acids including a 17 amino acid signal peptide . The calculated molecular mass of the mature proteins (350 amino acids) is 39.92 kDa with an estimated pI of 4.37 . Two putative integrin binding motifs (cell adhesion site), RGD (Arg-Gly-Asp) and a potential recognition motif for beta- (1-->3) linkage of polysaccharides were observed in the LGBP . Sequence comparison showed that LGBP deduced amino acid of L . vannamei has an overall similarity of 95%, 92% and 61% to that of blue shrimp Litopenaeus stylirostris LGBP, tiger shrimp Penaeus monodon BGBP and crayfish Pacifastacus leniusculus LGBP, respectively . Quantitative real-time RT-PCR analysis showed that LGBP transcript in haemocyte of L . vannamei increased in 3- and 6-h post Vibrio alginolyticus injection. Fish Shellfish Immunol, 2005 Apr, 18(4), 269 - 78 The immune response of white shrimp Litopenaeus vannamei and its susceptibility to Vibrio alginolyticus at different salinity levels; Wang LU et al.; White shrimp Litopenaeus vannamei held in 25 per thousand seawater were injected with TSB-grown Vibrio alginolyticus (1 x 10(4) cfu shrimp(-1)), and then transferred to 5, 15, 25 (control) and 35 per thousand . Over 24-96 h, the mortality of V . alginolyticus-injected shrimp held in 5 per thousand and 15 per thousand was significantly higher than that of shrimp held in 25 per thousand and 35 per thousand, and the mortality of V . alginolyticus-injected shrimp held in 5 per thousand was the highest . Shrimp held in 25 per thousand and then transferred to 5, 15, 25 (control) and 35 per thousand were examined for THC (total haemocyte count), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency to V . alginolyticus after 12-72 h . The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 and 15 per thousand after 12 h . It is concluded that the shrimp transferred from 25 per thousand to low salinity levels (5 and 15 per thousand) had reduced immune ability and decreased resistance against V . alginolyticus infection. Infect Immun, 2004 Dec, 72(12), 7326 - 9 Two tonB systems function in iron transport in Vibrio anguillarum, but only one is essential for virulence; Stork M et al.; We have identified two functional tonB systems in the marine fish pathogen Vibrio anguillarum, tonB1 and tonB2 . Each of the tonB genes is transcribed in an operon with the cognate exbB and exbD genes in response to iron limitation . Only tonB2 is essential for transport of ferric anguibactin and virulence. Protein Expr Purif, 2004 Dec, 38(2), 170 - 6 A method for the purification of Shiga-like toxin 1 subunit B using a commercially available galabiose-agarose resin; Tarrago-Trani MT et al.; We describe a procedure for the affinity purification of Shiga toxin 1 subunit B (SLTB) using a commercial galabiose-agarose resin . Recombinant SLTB was purified to 99% homogeneity in a single-step protocol, from the periplasmic extracts of Vibrio cholerae 0395 N1/pSBC54 . SDS-PAGE of the affinity purified SLTB showed one band of 8 kDa MW . SLTB purified by this procedure retained its chemical and biological activity as demonstrated by re-binding to the galabiose-agarose resin, and receptor-mediated binding and uptake in Vero cells . The galabiose-agarose resin could isolate roughly 1mg of SLTB/mL of gel . The resin was stable over 3 years and 500 cycles/year of usage . Hence, this method is a straightforward approach to the large-scale preparation of SLTB at a reasonable cost. Zh Mikrobiol Epidemiol Immunobiol, 2004 Sep-Oct, (5), 114 - 8 {Role of lipopolysaccharide in the action of complement on gram negative bacteria} {Hemolytic activity of Vibrio cholerae eltor and V . cholerae O139 toxigenic and nontoxigenic strains} {No authors listed} A total of 20 ctx- and 16 ctx+ V . cholerae eltor strains, 20 ctx- and 22 ctx+ V . cholerae O139 strains were under study . Hemolytic activity was tested in modified Greig test with sheep, guinea pig and rabbit red blood cells . The comparative study of the hemolytic properties of V . cholerae O1 and O139 under different conditions of cultivation demonstrated their capacity of lysing sheep red blood cells (SRBC) irrespective of the presence of toxigenic properties . A wider spectrum of lytic activity of ctx- strains in Greig test with respect to red blood cells of different animals and the capacity of lysing SRBC, most resistant to the action of toxin, may be due to a considerably greater content of Hly+ clones in their population. Zh Mikrobiol Epidemiol Immunobiol, 2004 Sep-Oct, (5), 12 - 6 {Role of lectin (hlyA) in the hemolytic and hemagglutinating activity of Vibrio cholerae}; Biochemical and virulence characterization of viable but nonculturable cells of Vibrio parahaemolyticus; Department of Microbiology, Soochow University, Taipei, Taiwan 111, Republic of China . wonghc@scu.edu.tw Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer . Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist . This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V . parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt-0.5% NaCl medium incubated at 4 degrees C . The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed . The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state . Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells . Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells . Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation . Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V . parahaemolyticus enters into the VBNC state. J Food Prot, 2004 Nov, 67(11), 2424 - 9 Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix; Kaufman GE et al.; This study examined the relationship between levels of total Vibrio parahaemolyticus found in oyster tissues and mantle fluid with the goal of using mantle fluid as a template matrix in a new quantitative real-time PCR assay targeting the thermolabile hemolysin (tlh) gene for the enumeration of total V . parahaemolyticus in oysters . Oysters were collected near Mobile Bay, Ala., in June, July, and September and tested immediately after collection and storage at 26 degrees C for 24 h . Initial experiments using DNA colony hybridization targeting tlh demonstrated that natural V . parahaemolyticus levels in the mantle fluid of individual oysters were strongly correlated (r = 0.85, P < 0.05) with the levels found in their tissues . When known quantities of cultured V . parahaemolyticus cells were added to real-time PCR reactions that contained mantle fluid and oyster tissue matrices separately pooled from multiple oysters, a strong linear correlation was observed between the real-time PCR cycle threshold and the log concentration of cells inoculated into each PCR reaction (mantle fluid: r = 0.98, P < 0.05; and oyster: r = 0.99, P < 0.05) . However, the mantle fluid exhibited less inhibition of the PCR amplification than the homogenized oyster tissue . Analysis of natural V . parahaemolyticus populations in mantle fluids using both colony hybridization and real-time PCR demonstrated a significant (P < 0.05) but reduced correlation (r = -0.48) between the two methods . Reductions in the efficiency of the real-time PCR that resulted from low population densities of V . parahaemolyticus and PCR inhibitors present in the mantle fluid of some oysters (with significant oyster-to-oyster variation) contributed to the reduction in correlation between the methods that was observed when testing natural V . parahaemolyticus populations . The V . parahaemolyticus-specific real-time PCR assay used for this study could estimate elevated V . parahaemolyticus levels in oyster mantle fluid within 1 h from sampling time. Arch Microbiol, 2005 Jan, 183(1), 37 - 44 Epub 2004 Nov 18. Gene cloning, expression and functional characterization of a phosphopantetheinyl transferase from Vibrio anguillarum serotype O1; Liu Q et al.; Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins from fatty acid synthetases (FASs) in primary metabolism and polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NRPSs) in secondary metabolism . Bacteria typically harbor one PPTase specific for carrier proteins of primary metabolism (ACPS-type PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism (Sfp-type PPTases) . Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, has been reported to be synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulent plasmid pJM1 from strain 775 of V . anguillarum serotype O1, and the PPTase, necessary for the activation of the anguibactin-NRPS, is therefore expected to lie on the pJM1 plasmid . In this work, a putative PPTase gene, angD, was first identified on pEIB1 plasmid (a pJM1-like plasmid) from a virulent strain MVM425 of V . anguillarum serotype O1 . A recombinant clone carrying complete angD was able to complement an Escherichia coli entD mutant deficient in Sfp-type PPTase . angD was overexpressed in E . coli and the resultant protein, AngD, was purified . Simultaneously, two carrier proteins involved in anguibactin-NRPS, ArCP and PCP, were overproduced in E . coli and purified . The purified AngD, PCP and ArCP were used to establish an in vitro enzyme reaction, and the PPTase activity of AngD was proved through HPLC analysis to detect the conversion of inactive carrier proteins to active carrier proteins in the reaction mixture . Co-expression of AngD with PCP or ArCP showed that AngD functioned well as a PPTase in vivo in E . coli, modifying PCP and ArCP completely. J Bacteriol, 2004 Dec, 186(23), 8137 - 43 Vibrio cholerae strains with mutations in an atypical type I secretion system accumulate RTX toxin intracellularly; Boardman BK et al.; This study shows that the Vibrio cholerae RTX toxin is secreted by a four-component type I secretion system (TISS) encoded by rtxB, rtxD, rtxE, and tolC . ATP-binding site mutations in both RtxB and RtxE blocked secretion, demonstrating that this atypical TISS requires two transport ATPases that may function as a heterodimer. Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2137 - 40 Vibrio crassostreae sp . nov., isolated from the haemolymph of oysters (Crassostrea gigas); Faury N et al.; Polyphasic analysis of five new Vibrio isolates originating from the haemolymph of diseased cultured oysters is described . The new isolates were closely related to Vibrio splendidus, having 98 % 16S rRNA gene sequence similarity . gyrB phylogenetic analysis, fluorescent amplified-fragment length polymorphism (FAFLP) fingerprinting and DNA-DNA hybridization experiments clearly showed that the new isolates form a tight genomic group that is different from the currently known Vibrio species . It is proposed to accommodate these isolates in a novel species, Vibrio crassostreae sp . nov . (type strain LGP 7(T)=LMG 22240(T)=CIP 108327(T)) . Phenotypic and chemotaxonomic features that differentiate V . crassostreae from other known Vibrio species include arginine dihydrolase, utilization and fermentation of various carbon sources, beta-galactosidase activity, NO(2) production and the presence of the fatty acids 14 : 0 iso and 16 : 0 iso. Science, 2004 Nov 12, 306(5699), 1186 - 8 Microbial factor-mediated development in a host-bacterial mutualism; Koropatnick TA et al.; Tracheal cytotoxin (TCT), a fragment of the bacterial surface molecule peptidoglycan (PGN), is the factor responsible for the extensive tissue damage characteristic of whooping cough and gonorrhea infections . Here, we report that Vibrio fischeri also releases TCT, which acts in synergy with lipopolysaccharide (LPS) to trigger tissue development in its mutualistic symbiosis with the squid Euprymna scolopes . As components of PGN and LPS have commonly been linked with pathogenesis in animals, these findings demonstrate that host interpretation of these bacterial signal molecules is context dependent . Therefore, such differences in interpretation can lead to either inflammation and disease or to the establishment of a mutually beneficial animal-microbe association. J Mol Biol, 2004 Nov 26, 344(3), 619 - 33 The structure of the cytoplasmic domain of EpsL, an inner membrane component of the type II secretion system of Vibrio cholerae: an unusual member of the actin-like ATPase superfamily; Abendroth J et al.; The type II secretion system (T2SS) is used by several Gram-negative bacteria for the secretion of hydrolytic enzymes and virulence factors across the outer membrane . In these secretion systems, a complex of 12-15 so-called "Gsp proteins" spans from a regulatory ATPase in the cytoplasm, via several signal or energy transducing proteins in the inner membrane and the pseudopilins in the periplasm, to the actual pore in the outer membrane . The human pathogen Vibrio cholerae employs such an assembly, called the Eps system, for the export of its major virulence factor, cholera toxin, from its periplasm into the lumen of the gastro-intestinal tract of the host . Here, we report the atomic structure of the major cytoplasmic domain of the inner membrane-spanning EpsL protein from V . cholerae . EpsL is the binding partner of the regulatory ATPase EpsE as well as of EpsM and pseudopilins, and is therefore a critical link between the cytoplasmic and the periplasmic part of the Eps-system . The 2.7A resolution structure was determined by a combination of Se-Met multiple anomalous dispersion (MAD) and multiple isomorphous replacement with anomalous scattering (MIRAS) phasing methods . The 28kDa cytoplasmic domain of EpsL (cyto-EpsL) consists of three beta-sheet-rich domains . With domains I and III similar to the RNaseH-fold, cyto-EpsL unexpectedly shows structural homology with the superfamily of actin-like ATPases . cyto-EpsL, however, is an unusual member of this superfamily as it misses the canonical actin domains 1B and 2B, which are common yet variable in this superfamily . Moreover, cyto-EpsL has an additional domain II, which has the topology of an SHS2-fold module . Within the superfamily this fold module has been observed only for domain 1C of the cell division protein FtsA, in which it mediates protein-protein interactions . This domain II displays great flexibility and contributes to a pronounced negatively charged canyon on the surface of cyto-EpsL . Functional data as well as structural homology and sequence conservation suggest that domain II interacts with EpsE, the major cytoplasmic binding partner of EpsL. Toxicon, 2004 Dec 15, 44(8), 887 - 93 Generation of active fragments from human zymogens in the brady kinin-generating cascade by extracellular proteases from Vibrio vulnificus and V . parahaemolyticus; Miyoshi S et al.; Vibrio vulnificus is an opportunistic human pathogen causing septicemia, and the infection is characterized by formation of the edematous skin lesions on limbs . This pathogenic species secretes a thermolysin-like metalloprotease as a virulence determinant . The metalloprotease was confirmed to activate human factor XII-plasma kallikrein-kinin cascade that results in liberation of bradykinin, a chemical mediator enhancing the vascular permeability, from high-molecular weight kininogen . Namely, the metalloprotease showed to generate active fragments by cleavage of Arg-Ile, Arg-Val or Gly-Leu peptide bond in human zymogens (plasma prekallikrein and factor XII) . In spite of induction of the sufficient vascular permeability-enhancing and edema-forming reaction in the guinea pig model, a serine protease from V . parahaemolyticus, a human pathogen causing primarily watery diarrhea, showed far less ability to activate and to cleave the human zymogens . These results in part may explain why only V . vulnificus often causes serious edematous skin damages in humans. Appl Environ Microbiol, 2004 Nov, 70(11), 6909 - 13 Detection and enumeration of Vibrio vulnificus in oysters from two estuaries along the southwest coast of India, using molecular methods; Parvathi A et al.; This study was conducted to understand the seasonal distribution of Vibrio vulnificus in oysters from two estuaries and the effect of environmental factors on the abundance of V . vulnificus in tropical waters . V . vulnificus was detected in 56.6% of the samples tested by colony hybridization with an alkaline phosphatase-labeled oligonucleotide probe (VV-AP), and the counts ranged from <10/g during the summer months to 10(3)/g in the monsoon season at both sites . The density of V . vulnificus appeared to be controlled more by salinity than by temperature . A nested PCR used in this study detected V . vulnificus in 85% of the samples following 18 h of enrichment in alkaline peptone water. Appl Environ Microbiol, 2004 Nov, 70(11), 6855 - 64 Relationship of Vibrio species infection and elevated temperatures to yellow blotch/band disease in Caribbean corals; Cervino JM et al.; The bacterial and temperature factors leading to yellow blotch/band disease (YBD), which affects the major reef-building Caribbean corals Montastrea spp., have been investigated . Groups of bacteria isolated from affected corals and inoculated onto healthy corals caused disease signs similar to those of YBD . The 16S rRNA genes from these bacteria were sequenced and found to correspond to four Vibrio spp . Elevating the water temperature notably increased the rate of spread of YBD on inoculated corals and induced greater coral mortality . YBD-infected corals held at elevated water temperatures had 50% lower zooxanthella densities, 80% lower division rates, and a 75% decrease in chlorophyll a and c2 pigments compared with controls . Histological sections indicated that the algal pyrenoid was fragmented into separate segments, along with a reconfiguration and swelling of the zooxanthellae, as well as vacuolization . YBD does not appear to produce the same physiological response formerly observed in corals undergoing temperature-related bleaching . Evidence indicates that YBD affects primarily the symbiotic algae rather than coral tissue. Appl Environ Microbiol, 2004 Nov, 70(11), 6401 - 6 Serologic and molecular characterization of Vibrio parahaemolyticus strains isolated from seawater and fish products of the Gulf of Mexico; Cabrera-Garcia ME et al.; The thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the main virulence factors of Vibrio parahaemolyticus . We isolated V . parahaemolyticus from seawater, fish, and oysters obtained from the Pueblo Viejo Lagoon in Veracruz, determined the serogroups, phenotypically and genotypically characterized TDH and TRH, and investigated the presence of the toxR gene . A total of 46 V . parahaemolyticus strains were isolated, and all of them amplified the 368-bp toxR gene fragment . The trh gene was not identified in any of the strains; 4 of the 46 strains were Kanagawa phenomenon (KP) positive and amplified the 251-bp tdh gene fragment . The most frequent serogroup was serogroup O3 . This is the first report of the presence of KP-positive tdh-positive environmental V . parahaemolyticus strains in Mexico. Cell Microbiol, 2004 Dec, 6(12), 1139 - 51 NO means 'yes' in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of a beneficial association; Davidson SK et al.; During colonization of the Euprymna scolopes light organ, symbiotic Vibrio fischeri cells aggregate in mucus secreted by a superficial ciliated host epithelium near the sites of eventual inoculation . Once aggregated, symbiont cells migrate through ducts into epithelium-lined crypts, where they form a persistent association with the host . In this study, we provide evidence that nitric oxide synthase (NOS) and its product nitric oxide (NO) are active during the colonization of host tissues by V . fischeri . NADPH-diaphorase staining and immunocytochemistry detected NOS, and the fluorochrome diaminofluorescein (DAF) detected its product NO in high concentrations in the epithelia of the superficial ciliated fields, ducts, and crypt antechambers . In addition, both NOS and NO were detected in vesicles within the secreted mucus where the symbionts aggregate . In the presence of NO scavengers, cells of a non-symbiotic Vibrio species formed unusually large aggregates outside of the light organ, but these bacteria did not colonize host tissues . In contrast, V . fischeri effectively colonized the crypts and irreversibly attenuated the NOS and NO signals in the ducts and crypt antechambers . These data provide evidence that NO production, a defense response of animal cells to bacterial pathogens, plays a role in the interactions between a host and its beneficial bacterial partner during the initiation of symbiotic colonization. J Bone Joint Surg Am, 2004 Nov, 86-A(11), 2497 - 502 Systemic Vibrio infection presenting as necrotizing fasciitis and sepsis . A series of thirteen cases; Tsai YH et al.; BACKGROUND: Vibrio species are an uncommon cause of necrotizing fasciitis and primary septicemia, which are likely to occur in patients with hepatic disease, diabetes mellitus, adrenal insufficiency, and immunocompromised conditions . These organisms are found in warm sea waters and are often present in raw oysters, shellfish, and other seafood . The purposes of the present report were to describe a series of patients who had this potentially lethal infection and to identify clinical features associated with a poor prognosis . METHODS: We retrospectively reviewed the records of thirteen patients (ten men and three women) who had necrotizing fasciitis and sepsis caused by Vibrio species . All patients had a history of contact with seawater or raw seafood . Eight patients had a hepatic disease such as hepatitis or cirrhosis of the liver, three had diabetes mellitus (without hepatic disease), and two had chronic renal or adrenal insufficiency (without hepatic disease) . RESULTS: Twelve patients underwent fasciotomy or limb amputation . Five patients (38%) died within two to six days after admission, and eight patients survived . Patients with a systolic blood pressure of < or =90 mm Hg and leukopenia in the emergency room had a significantly higher mortality rate (p < 0.05) . CONCLUSIONS: The diagnosis of Vibrio necrotizing fasciitis should be suspected when a patient has the appropriate clinical findings and a history of contact with seawater or raw seafood . The treatment should begin as early as possible, essentially when the patient has symptoms of sepsis . Although emergency fasciotomy or limb amputation did not reduce the mortality rate in this series, we consider such operations to be an important aspect of treatment. Bull Math Biol, 2004 Nov, 66(6), 1575 - 96 Sliding window discretization: a new method for multiple band matching of bacterial genotyping fingerprints; Austin B et al.; Microbiologists have traditionally applied hierarchical clustering algorithms as their mathematical tool of choice to unravel the taxonomic relationships between micro-organisms . However, the interpretation of such hierarchical classifications suffers from being subjective, in that a variety of ad hoc choices must be made during their construction . On the other hand, the application of more profound and objective mathematical methods--such as the minimization of stochastic complexity--for the classification of bacterial genotyping fingerprints data is hampered by the prerequisite that such methods only act upon vectorized data . In this paper we introduce a new method, coined sliding window discretization, for the transformation of genotypic fingerprint patterns into binary vector format . In the context of an extensive amplified fragment length polymorphism (AFLP) data set of 507 strains from the Vibrionaceae family that has previously been analysed, we demonstrate by comparison with a number of other discretization methods that this new discretization method results in minimal loss of the original information content captured in the banding patterns . Finally, we investigate the implications of the different discretization methods on the classification of bacterial genotyping fingerprints by minimization of stochastic complexity, as it is implemented in the BinClass software package for probabilistic clustering of binary vectors . The new taxonomic insights learned from the resulting classification of the AFLP patterns will prove the value of combining sliding window discretization with minimization of stochastic complexity, as an alternative classification algorithm for bacterial genotyping fingerprints. Mol Microbiol, 2004 Nov, 54(4), 935 - 47 Characterization of XerC- and XerD-dependent CTX phage integration in Vibrio cholerae; McLeod SM et al.; CTXphi is a filamentous bacteriophage that encodes cholera toxin and integrates site-specifically into the larger of the two Vibrio cholerae chromosomes . The CTXphi genome lacks an integrase; instead, its integration depends on the chromosome-encoded tyrosine recombinases XerC and XerD . During integration, recombination occurs between regions of homology in CTXphi and the V . cholerae chromosome . Here, we define the elements on the phage genome (attP) and bacterial chromosome (attB) required for CTXphi integration . attB is a short sequence composed of one binding site for XerC and XerD spanning the site of recombination . Together, XerC and XerD bind to two sites within attP . While one XerC/D binding site in attP spans the core recombination region, the other site is approximately 80 bp away . Although integration occurs at the core XerC/D binding site in attP, the second site is required for CTXphi integration, suggesting it performs an architectural role in the integration reaction . In vitro cleavage reactions showed that XerC and XerD are capable of cleaving attB and attP sequences; however, additional cellular processes such as DNA replication or Holliday junction resolution by a host resolvase may contribute to integration in vivo. Mol Microbiol, 2004 Nov, 54(4), 851 - 4 Smarter than the average phage; Blakely GW; The seventh cholera pandemic emerged in the poorer nations of the world towards the end of the 20th century and continues to kill thousands of people per year . The causative agent of cholera, the Gram-negative bacterium Vibrio cholera, is only pathogenic when it contains a lysogenic bacteriophage, CTXphi, that encodes the toxin responsible for inducing massive fluid loss from the human host . Site-specific integration of CTXphi into chromosome I of V . cholera occurs at a site, dif, that is normally required for resolution of chromosome dimers generated by homologous recombination . An article in this issue of Molecular Microbiology reports the analysis of interactions between two host encoded recombinases, XerC and XerD, and the recombination sites involved in lysogeny . Surprisingly, recombination between the CTXphi attP site and the chromosomal dif site requires additional recombinase binding sites, downstream from the positions of strand exchange, which might play an architectural role . The positions of strand cleavage also differ significantly between the two sites, suggesting a novel recombination mechanism that implicates additional host factors in resolution of the Holliday junction intermediate. Chemosphere, 2004 Dec, 57(11), 1725 - 32 Comparative toxic effects of formulated simazine on Vibrio fischeri and gilthead seabream (Sparus aurata L.) larvae; Arufe MI et al.; The use of Early Life Stage (ELS) tests is a useful tool in risk assessment . The purpose of this study was to compare the sensitivity of the seabream (Sparus aurata) larvae with the extensively used Microtox test on a commercial formulation containing simazine, an s-triazine herbicide . To this end, survival, growth and histopathological changes displayed by seabream yolk sac larvae exposed during 72 h post-hatching to nominal concentrations of the commercial preparation up to its saturating concentration in water, and bioluminescence of the marine bacteria Vibrio fischeri (Microtox) were studied . Survival of larvae after three days of exposure was significantly reduced in the highest (4.5 mg/l) concentration, but no effects on growth were found in any of the simazine treatments . The 72 h LC50 value for yolk sac larvae was estimated as 4.19 mg/l . Commercial grade simazine did not exert any significant toxicity to the marine bacterium V . fischeri at the concentrations tested. BMC Microbiol . 2004 Oct 29;4(1):42. Gel shift analysis of the empA promoter region in Vibrio anguillarum; Denkin SM et al.; BACKGROUND: The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus . Previously we have shown that there are significant differences in empA expression in two strains of V . anguillarum, M93Sm and NB10 . It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements . RESULTS: Two strains of V . anguillarum, M93Sm and NB10, were examined and compared for the presence of DNA regulatory proteins that bind to and control the empA promoter region . Gel mobility shift assays, using a digoxigenin (DIG)-labeled oligomer containing a lux box-like element and the promoter for empA, were done to demonstrate the presence of a DNA-binding protein . Protein extracts from NB10 cells incubated in Luria Bertani broth + 2% NaCl (LB20), nine salts solution + 200 microg/ml mucus (NSSM), 3M (marine minimal medium), or NSS resulted in a gel mobility shift . No gel mobility shift was seen when protein extracts from either LB20- or NSSM-grown M93Sm cells were mixed with the DIG-labeled empA oligomer . The azocasein assay detected protease activity in all incubation conditions for NB10 culture supernatants . In contrast, protease activity was detected in M93Sm culture supernatants only when incubated in NSSM . Since the luxR homologue in V . anguillarum, vanT, has been cloned, sequenced, and shown to be required for protease activity, we wanted to determine if vanT mutants of NB10 exhibit the same gel shift observed in the wild-type . Site-directed mutagenesis was used to create vanT mutants in V . anguillarum M93Sm and NB10 to test whether VanT is involved with the gel mobility shift . Both vanT mutants, M02 and NB02, did not produce protease activity in any conditions . However, protein extracts from NB02 incubated in each condition still exhibited a gel shift when mixed with the DIG-labeled empA oligomer . CONCLUSIONS: The data demonstrate that protein extracts of V . anguillarum NB10 cells contain a protein that binds to a 50 bp oligomer containing the empA promoter-lux box-like region . NB10 cells express empA during stationary phase in all growth conditions . The DNA binding protein is not present in M93Sm extracts . M93Sm cells express protease activity only when incubated at high cell density in fish gastrointestinal mucus . The gel shift observed with NB10 cells is not due to VanT binding . The data also suggest that the DNA binding protein is responsible for the less restrictive expression of empA in NB10 compared to M93Sm. Trop Doct, 2004 Oct, 34(4), 249 - 51 Seasonality and antimicrobial resistance pattern of Vibrio cholerae in a tertiary care hospital of North India; Mohanty S et al.; We retrospectively analysed the seasonal distribution of cholera and the antimicrobial resistance pattern of Vibrio cholerae isolates over a 5-year period from January 1998 to December 2002 . Of 3213 stool specimens processed from 3213 patients with acute watery diarrhoea during this period, 431 samples (13.4%) were found positive for V . cholerae . There were 423 V . cholerae O1 biotype E1 Tor, 2 V . cholerae O139 and six isolates of non-O1 non-O139 . The highest number of cholera cases occurred in May-June followed by July-August . Cases started appearing in April for all years except in the year 2002 when three cases occurred in the first week of March . A large number (90.25% strains) were resistant to at least one antibiotic. Mol Pharmacol . 2004 Oct 27; {Epub ahead of print} P-glycoprotein substrate binding domains are located at the transmembrane domain : transmembrane domain interfaces - A combined photoaffinity labeling - protein homology modeling approach; Pleban K et al.; P-glycoprotein (P-gp) is an energy dependent multidrug efflux pump conferring resistance to cancer-chemotherapy . Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy . In addition, P-glycoprotein plays an important role for early ADMET (absorption, distribution, metabolism, excretion and toxicity) profiling in drug development . A set of propafenone-type substrate photoaffinity ligands has been used in this study in conjunction with MALDI-TOF mass-spectrometry to define the substrate-binding-domain(s) of P-gp in more detail . Highest labeling was observed in transmembrane segments 3, 5, 8 and 11 . A homology model for P-gp was generated based on the dimeric crystal structure of Vibrio cholerae MsbA (Vc-MsbA), an essential lipid transporter . Subsequently, the labeling pattern was projected onto the 3D atomic detail model of P-gp to allow a visualization of the binding domain(s) . Labeling is predicted by the model to occur at the two TMD:TMD interfaces formed betwee