Appl Environ Microbiol, 2003 Oct, 69(10), 5928 - 34 Population dynamics of Vibrio fischeri during infection of Euprymna scolopes; McCann J et al.; The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes . Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations . To understand the population dynamics of V . fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored . When grown in culture, there was no detectable metabolic burden on V . fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V . fischeri genome . Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments . Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V . fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected . When three differentially marked strains of V . fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum . Further, these results indicated that, when exposed to low numbers of V . fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient. Appl Environ Microbiol, 2003 Oct, 69(10), 5919 - 27 Role of ectoine in Vibrio cholerae osmoadaptation; Pflughoeft KJ et al.; Vibrio cholerae is both an intestinal pathogen and a microbe in the estuarine community . To persist in the estuarine environment, V . cholerae must adjust to changes in ionic composition and osmolarity . These changes in the aquatic environment have been correlated with cholera epidemics . In this work, we study the response of V . cholerae to increases in environmental osmolarity . Optimal growth of V . cholerae in minimal medium requires supplementation with 200 mM NaCl and KCl . However, when the NaCl concentration is increased beyond 200 mM, a proportionate delay in growth is observed . During this delay in growth, osmotic equilibrium is reached by cytoplasmic accumulation of small, uncharged solutes that are compatible with growth . We show that synthesis of the compatible solute ectoine and transport of the compatible solute glycine betaine impact the length of the osmoadaptive growth delay . We also demonstrate that high-osmolarity-adapted V . cholerae displays a growth advantage when competed against unadapted cells in high-osmolarity medium . In contrast, low-osmolarity-adapted V . cholerae displays no growth advantage when competed against high-osmolarity-adapted cells in low-osmolarity medium . These results may have implications for V . cholerae population dynamics when seawater and freshwater and their attendant microbes mix. Berl Munch Tierarztl Wochenschr, 2003 Sep-Oct, 116(9-10), 396 - 400 Isolation of Vibrio vulnificus and atypical Vibrio from surface water of the Baltic Sea in Germany; Jores J et al.; From 1995 to 1997 several defined species like V . alginolyticus, V . anguillarum, V . cholerae (non O1 and non O139), V . mimicus, V . parahaemolyticus and V . vulnificus were isolated during a survey to determine the presence of V . vulnificus in the brackish water of the Baltic Sea in Germany . Moreover atypical Vibrio isolates were detected . Four isolates belonging to a group of atypical Vibrio and possibly representing a new species in the genus Vibrio were characterized in detail . All four strains were isolated from surface costal waters . Based on 16S rDNA sequence analysis they showed the highest relatedness to the species V . navarrensis and V . vulnificus . The strains did not harbor the species specific hemolysin gene vvhA from V . vulnificus as shown by PCR and hybridization experiments . Moreover, they differed in at least two biochemical parameters tested from the hitherto described Vibrio species . All these strains induced hemolysis on washed blood agar dishes and showed phase variations on Luria Bertani agar dishes . Because of the similarity to the eel pathogen V . vulnificus, we infected eels with one of the four atypical strains (CH-291), but no pathogenicity for eels could be detected . Furthermore, Vero cell tests with supernatants of bacterial cultures did not reveal secreted Vero cell cytotoxic compounds . This indicates a nonpathogenic nature of these strains. Emerg Infect Dis, 2003 Sep, 9(9), 1116 - 22 Reemergence of epidemic Vibrio cholerae O139, Bangladesh; Faruque SM et al.; During March and April 2002, a resurgence of Vibrio cholerae O139 occurred in Dhaka and adjoining areas of Bangladesh with an estimated 30,000 cases of cholera . Patients infected with O139 strains were much older than those infected with O1 strains (p<0.001) . The reemerged O139 strains belong to a single ribotype corresponding to one of two ribotypes that caused the initial O139 outbreak in 1993 . Unlike the strains of 1993, the recent strains are susceptible to trimethoprim, sulphamethoxazole, and streptomycin but resistant to nalidixic acid . The new O139 strains carry a copy of the Calcutta type CTX(Calc) prophage in addition to the CTX(ET) prophage carried by the previous strains . Thus, the O139 strains continue to evolve, and the adult population continues to be more susceptible to O139 cholera, which suggests a lack of adequate immunity against this serogroup . These findings emphasize the need for continuous monitoring of the new epidemic strains. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai), 2003 Oct, 35(10), 956 - 9 DNA sequencing of a plasmid with virulence from marine fish pathogen Vibrio anguillarum; Wu HZ et al.; DNA sequence of a plasmid pEIB1 associated with virulence from the marine fish pathogen Vibrio anguillarum was determined using the methods of restriction endonuclease digestion, subcloning, and primer walking . The whole length of obtained pEIB1 DNA sequence was 66 164 bp, and the overall G+C content of DNA sequence is 42.7% . This sequence encodes 44 open reading frames containing the genes of DNA replication, biosynthesis and regulation of the siderophore anguibactin and transport of ferric-anguibactin complexes. J Fish Dis, 2003 Aug, 26(8), 477 - 85 Synergistic effects of dietary iron and omega-3 fatty acid levels on survival of farmed Atlantic salmon, Salmo salar L., during natural outbreaks of furunculosis and cold water vibriosis; Rorvik KA et al.; The present study demonstrates that farmed Atlantic salmon, Salmo salar, health is positively and significantly affected by synergistic effects between very long-chain polyunsaturated fatty acids of the n-3 family eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA) and iron, where positive effects of high dietary levels of EPA/DHA are enhanced when combined with low levels of iron . Based on cumulative mortalities in the different experimental groups, relative percentage of survival (RPS) for the high EPA/DHA-low iron group was 70% during an outbreak of furunculosis and 96% during an outbreak of cold water vibriosis compared with the controls . A non-additive effect between EPA/DHA and iron was confirmed by statistical analyses that revealed a significant effect of EPA/DHA alone and an interaction of iron with EPA/DHA . Liver cell cultures treated with EPA/DHA revealed that the synergistic effect could be related to an EPA/DHA dependent regulation of mRNA for proteins important for transport (transferrin) and storage (ferritin) of iron in the salmon . In keeping with this finding, the transcriptional down-regulation of iron metabolism in vitro was reflected in decreased in vivo iron stores with increasing levels of dietary EPA/DHA . Hence, to avoid overloading of the iron transport/storage-systems resulting in increased susceptibility to bacterial infections, high levels of dietary EPA/DHA should be accompanied by low levels of dietary iron. Environ Microbiol, 2003 Oct, 5(10), 850 - 8 Persistence of adhesive properties in Vibrio cholerae after long-term exposure to sea water; Pruzzo C et al.; The effect of exposure to artificial sea water (ASW) on the ability of classical Vibrio cholerae O1 cells to interact with chitin-containing substrates and human intestinal cells was studied . Incubation of vibrios in ASW at 5 degrees C and 18 degrees C resulted in two kinds of cell responses: the viable but non-culturable (VBNC) state (i.e . <0.1 colony forming unit ml-1) at 5 degrees C, and starvation (i.e . maintenance of culturability of the population) at 18 degrees C . The latter remained rod shaped and, after 40 days' incubation, presented a 47-58% reduction in the number of cells attached to chitin, a 48-53% reduction in the number of bacteria adhering to copepods, and a 48-54% reduction in the number of bacteria adhering to human cultured intestinal cells, compared to control cells not suspended in ASW . Bacteria suspended in ASW at 5 degrees C became coccoid and, after 40 days, showed 34-42% fewer cells attached to chitin, 52-55% fewer adhering to copep-ods, and 45-48% fewer cells adhering to intestinal cell monolayers, compared to controls . Sarkosyl-insoluble membrane proteins that bind chitin particles were isolated and analysed by SDS-PAGE . After 40 days incubation in ASW at both 5 degrees C and 18 degrees C vibrios expressed chitin-binding ligands similar to bacteria harvested in the stationary growth phase . It is concluded that as vibrios do not lose adhesive properties after long-term exposure to ASW, it is important to include methods for VBNC bacteria when testing environmental and clinical samples for purposes of public health safety. Indian J Med Sci, 2003 Apr, 57(4), 155 - 7 Emergence and re-emergence of Vibrio cholerae 0139: an epidemiological study during 1993-2002 at Nagpur, Central India; Agrawal G et al.; The pattern of Vibrio cholerae 01 and 0139 isolates at Indira Gandhi Medical College and Mayo General Hospital, Nagpur from 1993 to 2002 is presented . Emergence of the novel serotype 0139 in 1993 was followed by periods of quiescence and re-emergence . For the first time after 1993, the 0139 isolates out numbered 01 isolates in 2001 . The peculiar epidemiological pattern is compared with other reports. Mol Microbiol, 2003 Oct, 50(1), 319 - 31 The Vibrio fischeri quorum-sensing systems ain and lux sequentially induce luminescence gene expression and are important for persistence in the squid host; Lupp C et al.; Bacterial quorum sensing using acyl-homoserine lactones (acyl-HSLs) as cell-density dependent signalling molecules is important for the transcriptional regulation of many genes essential in the establishment and the maintenance of bacteria-host associations . Vibrio fischeri, the symbiotic partner of the Hawaiian bobtail squid Euprymna scolopes, possesses two distinct acyl-HSL synthase proteins, LuxI and AinS . Whereas the cell density-dependent regulation of luminescence by the LuxI-produced signal is a well-described phenomenon, and its role in light organ symbiosis has been defined, little is known about the ain system . We have investigated the impact of the V . fischeri acyl-HSL synthase AinS on both luminescence and symbiotic colonization . Through phenotypic studies of V . fischeri mutants we have found that the AinS-signal is the predominant inducer of luminescence expression in culture, whereas the impact of the LuxI-signal is apparent only at the high cell densities occurring in symbiosis . Furthermore, our studies revealed that ainS regulates activities essential for successful colonization of E . scolopes, i.e . the V . fischeri ainS mutant failed to persist in the squid light organ . Mutational inactivation of the transcriptional regulator protein LuxO in the ainS mutant partially or completely reversed all the observed phenotypes, demonstrating that the AinS-signal regulates expression of downstream genes through the inactivation of LuxO . Taken together, our results suggest that the two quorum-sensing systems in V . fischeri, ain and lux, sequentially induce the expression of luminescence genes and possibly other colonization factors. J Mass Spectrom, 2003 Sep, 38(9), 924 - 30 Electron ionization mass spectrometric study of monomeric models of O-polysaccharides of Vibrio cholerae O:1, serotypes Ogawa and Inaba; Kovacik V et al.; Fragmentation mechanisms of electron ionization (EI) mass spectrometry of the title compounds have been elucidated by high-resolution (HR) mass spectrometric measurements of the elemental composition and measurements of the metastable transitions (B(2)/E, CID) . The experimental results were interpreted with the help of Mass Frontier 3.0 software, which aided the elucidation of fragmentation mechanisms and helped to deduce structures of the ions formed . Characteristic under the conditions of EI-MS measurement was the production of protonated adducts . Three distinct pathways observed include the formation of oxonium type ions, the conjugated transfer of electrons in the pyranose ring, and cleavage of the acylamide side chains . By applying the results obtained, the molecular mass, as well as the structures of both the saccharide and acylamide side chain involved in related substances, can be determined . Bioprocess Biosyst Eng, 2003 Jan, 25(4), 221 - 8 Epub 2002 Dec 05. Probing control of glucose feeding in Vibrio cholerae cultivations; de Mare L et al.; Infection with Vibrio cholerae is a significant problem in many developing countries . Cultivation of V . cholerae is used in production of cholera toxin B subunit, which is a component in a cholera vaccine . Fed-batch cultivations with V . cholerae in defined media have been conducted and reproducible results were obtained . A probing feeding strategy developed by Akesson for Escherichia coli cultivations has been tested . The strategy is working as well for V . cholerae as for E . coli in minimizing the amount of acetic acid formed and avoiding anaerobic conditions . At 2 h after the feed start most of the acetic acid accumulated during the batch phase is consumed . The resulting feed rate tends to be the highest possible with respect to the constraints from cell metabolism and mass transfer, thus maximizing productivity in terms of biomass . A cell dry weight of 20-23 g/l is obtained after 12 h of feeding. J Food Prot, 2003 Sep, 66(9), 1675 - 80 Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purified template DNA; Hara-Kudo Y et al.; PCR is an important method for the detection of thermostable direct hemolysin gene (tdh)-positive (pathogenic hemolysin-producing) strains of Vibrio parahaemolyticus in seafood because tdh-negative (nonpathogenic) V . parahaemolyticus strains often contaminate seafood and interfere with the direct isolation of tdh-positive V . parahaemolyticus . In this study, the use of PCR to detect the tdh gene of V . parahaemolyticus in various seafoods artificially contaminated with tdh-positive V . parahaemolyticus was examined . PCR was inhibited by substances in oysters, squid, mackerel, and yellowtail but not by cod, sea bream, scallop, short-necked clam, and shrimp . To improve detection, DNA was purified by either the silica membrane method, the glass fiber method, or the magnetic separation method, and the purified DNA was used as the PCR primer template . For all samples, the use of the silica membrane method and the glass fiber method increased detection sensitivity . The results of this study demonstrate that the use of properly purified template DNA for PCR markedly increases the effectiveness of the method in detecting pathogenic tdh-positive V . parahaemolyticus in contaminated seafood. Infect Immun, 2003 Oct, 71(10), 5583 - 9 The global regulator ArcA modulates expression of virulence factors in Vibrio cholerae; Sengupta N et al.; A Vibrio cholerae arcA mutant was constructed and used to examine the role of the global anaerobiosis response regulator ArcA in the expression of virulence factors in this important human pathogen . In V . cholerae, expression of the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) is regulated by the transcriptional activator ToxT . toxT expression, in turn, is controlled by the transmembrane DNA binding proteins ToxR and TcpP . In the V . cholerae arcA mutant, although ToxR and TcpP were unaffected, Northern blot and reverse transcription-PCR analyses indicated that the expression of toxT was significantly decreased with concomitant reduction in the expression of CT and TCP . CT and TCP expression was completely restored in the V . cholerae arcA mutant strain by expressing a cloned toxT gene in the mutant . These results suggest that ArcA functions as a positive regulator of toxT expression under both aerobic and anaerobic conditions, although as expected, the effect was more pronounced during anaerobic growth . This was reflected in a reduction of virulence of the V . cholerae arcA mutant strain in the infant mouse cholera model. Infect Immun, 2003 Oct, 71(10), 5498 - 504 Construction and evaluation of a safe, live, oral Vibrio cholerae vaccine candidate, IEM108; Liang W et al.; IEM101, a Vibrio cholerae O1 El Tor Ogawa strain naturally deficient in CTXPhi, was previously selected as a live cholera vaccine candidate . To make a better and safer vaccine that can induce protective immunity against both the bacteria and cholera toxin (CT), a new vaccine candidate, IEM108, was constructed by introducing a ctxB gene and an El Tor-derived rstR gene into IEM101 . The ctxB gene codes for the protective antigen CTB subunit, and the rstR gene mediates phage immunity . The stable expression of the two genes was managed by a chromosome-plasmid lethal balanced system based on the housekeeping gene thyA . Immunization studies indicate that IEM108 generates good immune responses against both the bacteria and CT . After a single-dose intraintestinal vaccination with 10(9) CFU of IEM108, both anti-CTB immunoglobulin G and vibriocidal antibodies were detected in the immunized-rabbit sera . However, only vibriocidal antibodies are detected in rabbits immunized with IEM101 . In addition, IEM108 but not IEM101 conferred full protection against the challenges of four wild-type toxigenic strains of V . cholerae O1 and 4 micro g of CT protein in a rabbit model . By introducing the rstR gene, the frequency of conjugative transfer of a recombinant El Tor-derived RS2 suicidal plasmid to IEM108 was decreased 100-fold compared to that for IEM101 . This indicated that the El Tor-derived rstR cloned in IEM108 was fully functional and could effectively inhibit the El Tor-derived CTXPhi from infecting IEM108 . Our results demonstrate that IEM108 is an efficient and safe live oral cholera vaccine candidate that induces antibacterial and antitoxic immunity and CTXPhi phage immunity. Int J Antimicrob Agents, 2003 Sep, 22(3), 200 - 4 A new dimer interface for an ABC transporter; Shilling RA et al.; The crystallization of MsbA, an ATP-binding cassette (ABC) transporter involved in the transport of Lipid A in Escherichia coli, provided a fascinating glimpse into the high-resolution structure of an ABC transporter at 4.8 A . The E . coli crystal structure of MsbA reveals a dimer . Although the structure of the MsbA monomer is consistent with the biochemistry of ABC transporters, including the human multidrug resistance P-glycoprotein, the interface between the monomers in the MsbA dimer may not reflect the biologically relevant interface . We considered the interface in a two-armed MsbA dimer, named spiral . Our findings indicate that (i) the spiral MsbA dimer may have biological relevance for ABC transporters that interact with lipophilic substrates, and (ii) the dimer interface observed in the crystal structure of E . coli MsbA represents a crystallization artefact . A comparison of the spiral MsbA dimer with the recently published structure of MsbA in Vibrio cholera is also described. Biochem J, 2004 Jan 1, 377(Pt 1), 225 - 32 Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio; Honda Y et al.; A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus . The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii . The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate {GlcNAc-1-P} and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase . In the synthetic reaction, the ChBP was active with alpha-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce beta-D-glucosyl-(1-->4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate . The enzyme allowed aryl-beta-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc . Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k(0)=5.5 s(-1), K(m)=2.0 mM; synthetic reaction, k(0)=10 s(-1), K(m)=14 mM, respectively . The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases . Substrate inhibition by GlcNAc was observed in the synthetic reaction . The enzyme was considered a unique biocatalyst for glycosidation. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1615 - 7 Reclassification of Vibrio hollisae as Grimontia hollisae gen . nov., comb . nov; Thompson FL et al.; The taxonomic positions of three representative strains of Vibrio hollisae (LMG 17719(T), LMG 21416 and LMG 21538) were investigated by means of 16S rDNA sequences and phenotypic data . V . hollisae strains (GenBank/EMBL accession nos AJ514909-AJ514911) shared 99.5 % 16S rDNA sequence similarity, but had only 94.6 % similarity to their closest phylogenetic neighbour, Enterovibrio norvegicus . 16S rDNA sequence similarity of V . hollisae and Vibrio cholerae was only 91 % . These results suggest that V . hollisae should be placed into a novel genus, for which the name Grimontia gen . nov . is proposed. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1569 - 73 Vibrio pacinii sp . nov., from cultured aquatic organisms; Gomez-Gil B et al.; Three strains were isolated from cultured aquatic organisms . They were Gram-negative, oxidase-positive, motile, fermentative, arginine dihydrolase-positive, lysine and ornithine decarboxylase-negative and sensitive to vibriostatic agent O/129 . These strains differ from other related Vibrio species by several phenotypic features, which include acetoin and indole production and utilization of amygdalin and D-mannitol . Comparison of 16S rDNA sequences showed a close relationship to the recently described species Vibrio kanaloae (96.6 %) and Vibrio pomeroyi (96.4 %) and to Vibrio furnissii (96.6 %), but DNA-DNA hybridization experiments showed that the three isolates form a tight novel species with </=30 % DNA-DNA similarity to its closest phylogenetic neighbours . Vibrio pacinii sp . nov . is proposed, with LMG 19999(T) (=CAIM 530(T)=STD3-1057(T); DNA G+C content, 44.9 mol%) as the type strain. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1495 - 501 Vibrio fortis sp . nov . and Vibrio hepatarius sp . nov., isolated from aquatic animals and the marine environment; Thompson FL et al.; In this study, the taxonomic positions of 19 Vibrio isolates disclosed in a previous study were evaluated . Phylogenetic analysis based on 16S rDNA sequences partitioned these isolates into groups that were closely related (98.8-99.1 % similarity) to Vibrio pelagius and Vibrio xuii, respectively . DNA-DNA hybridization experiments further showed that these groups had <70 % similarity to other Vibrio species . Two novel Vibrio species are proposed to accommodate these groups: Vibrio fortis sp . nov . (type strain, LMG 21557(T)=CAIM 629(T)) and Vibrio hepatarius sp . nov . (type strain, LMG 20362(T)=CAIM 693(T)) . The DNA G+C content of both novel species is 45.6 mol% . Useful phenotypic features for discriminating V . fortis and V . hepatarius from other Vibrio species include production of indole and acetoin, utilization of cellobiose, fermentation of amygdalin, melibiose and mannitol, beta-galactosidase and tryptophan deaminase activities and fatty acid composition. Int J Syst Evol Microbiol, 2003 Sep, 53(Pt 5), 1327 - 32 Desulfonatronum thiodismutans sp . nov., a novel alkaliphilic, sulfate-reducing bacterium capable of lithoautotrophic growth; Pikuta EV et al.; A novel alkaliphilic, sulfate-reducing bacterium, strain MLF1(T), was isolated from sediments of soda Mono Lake, California . Gram-negative vibrio-shaped cells were observed, which were 0.6-0.7x1.2-2.7 micro m in size, motile by a single polar flagellum and occurred singly, in pairs or as short spirilla . Growth was observed at 15-48 degrees C (optimum, 37 degrees C), >1-7 % NaCl, w/v (optimum, 3 %) and pH 8.0-10.0 (optimum, 9.5) . The novel isolate is strictly alkaliphilic, requires a high concentration of carbonate in the growth medium and is obligately anaerobic and catalase-negative . As electron donors, strain MLF1(T) uses hydrogen, formate and ethanol . Sulfate, sulfite and thiosulfate (but not sulfur or nitrate) can be used as electron acceptors . The novel isolate is a lithoheterotroph and a facultative lithoautotroph that is able to grow on hydrogen without an organic source of carbon . Strain MLF1(T) is resistant to kanamycin and gentamicin, but sensitive to chloramphenicol and tetracycline . The DNA G+C content is 63.0 mol% (HPLC) . DNA-DNA hybridization with the most closely related species, Desulfonatronum lacustre Z-7951(T), exhibited 51 % homology . Also, the genome size (1.6x10(9) Da) and T(m) value of the genomic DNA (71+/-2 degrees C) for strain MLF1(T) were significantly different from the genome size (2.1x10(9) Da) and T(m) value (63+/-2 degrees C) for Desulfonatronum lacustre Z-7951(T) . On the basis of physiological and molecular properties, the isolate was considered to be a novel species of the genus Desulfonatronum, for which the name Desulfonatronum thiodismutans sp . nov . is proposed (the type strain is MLF1(T)=ATCC BAA-395(T)=DSM 14708(T)). J Bacteriol, 2003 Oct, 185(19), 5891 - 6 Regulation of fur expression by RpoS and fur in Vibrio vulnificus; Lee HJ et al.; In a proteomic analysis of rpoS-deficient Vibrio vulnificus versus the wild type, one of the down-regulated proteins in the rpoS mutant strain was identified as a Fur protein, a ferric uptake regulator . The expression of a fur::luxAB fusion was significantly influenced by sigma factor S, the rpoS gene product, and positively regulated by Fur under iron-limited conditions. J Bacteriol, 2003 Oct, 185(19), 5822 - 30 Complete sequence of virulence plasmid pJM1 from the marine fish pathogen Vibrio anguillarum strain 775; Di Lorenzo M et al.; The virulence plasmid pJM1 enables the fish pathogen Vibrio anguillarum, a gram-negative polarly flagellated comma-shaped rod bacterium, to cause a highly fatal hemorrhagic septicemic disease in salmonids and other fishes, leading to epizootics throughout the world . The pJM1 plasmid 65,009-nucleotide sequence, with an overall G+C content of 42.6%, revealed genes and open reading frames (ORFs) encoding iron transporters, nonribosomal peptide enzymes, and other proteins essential for the biosynthesis of the siderophore anguibactin . Of the 59 ORFs, approximately 32% were related to iron metabolic functions . The plasmid pJM1 confers on V . anguillarum the ability to take up ferric iron as a complex with anguibactin from a medium in which iron is chelated by transferrin, ethylenediamine-di(o-hydroxyphenyl-acetic acid), or other iron-chelating compounds . The fatDCBA-angRT operon as well as other downstream biosynthetic genes is bracketed by the homologous ISV-A1 and ISV-A2 insertion sequences . Other clusters on the plasmid also show an insertion element-flanked organization, including ORFs homologous to genes involved in the biosynthesis of 2,3-dihydroxybenzoic acid . Homologues of replication and partition genes are also identified on pJM1 adjacent to this region . ORFs with no known function represent approximately 30% of the pJM1 sequence . The insertion sequence elements in the composite transposon-like structures, corroborated by the G+C content of the pJM1 sequence, suggest a modular composition of plasmid pJM1, biased towards acquisition of modules containing genes related to iron metabolic functions . We also show that there is considerable microheterogeneity in pJM1-like plasmids from virulent strains of V . anguillarum isolated from different geographical sources. J Bacteriol, 2003 Oct, 185(19), 5685 - 96 VGJ phi, a novel filamentous phage of Vibrio cholerae, integrates into the same chromosomal site as CTX phi; Campos J et al.; We describe a novel filamentous phage, designated VGJ phi, isolated from strain SG25-1 of Vibrio cholerae O139, which infects all O1 (classical and El Tor) and O139 strains tested . The sequence of the 7,542 nucleotides of the phage genome reveals that VGJ phi has a distinctive region of 775 nucleotides and a conserved region with an overall genomic organization similar to that of previously characterized filamentous phages, such as CTX phi of V . cholerae and Ff phages of Escherichia coli . The conserved region carries 10 open reading frames (ORFs) coding for products homologous to previously reported peptides of other filamentous phages, and the distinctive region carries one ORF whose product is not homologous to any known peptide . VGJ phi, like other filamentous phages, uses a type IV pilus to infect V . cholerae; in this case, the pilus is the mannose-sensitive hemagglutinin . VGJ phi-infected V . cholerae overexpresses the product of one ORF of the phage (ORF112), which is similar to single-stranded DNA binding proteins of other filamentous phages . Once inside a cell, VGJ phi is able to integrate its genome into the same chromosomal attB site as CTX phi, entering into a lysogenic state . Additionally, we found an attP structure in VGJ phi, which is also conserved in several lysogenic filamentous phages from different bacterial hosts . Finally, since different filamentous phages seem to integrate into the bacterial dif locus by a general mechanism, we propose a model in which repeated integration events with different phages might have contributed to the evolution of the CTX chromosomal region in V . cholerae El Tor. Lett Appl Microbiol, 2003, 37(4), 349 - 53 Misidentification of Aeromonas veronii biovar sobria as Vibrio alginolyticus by the Vitek system; Park TS et al.; AIMS: To find the cause of misidentification of aeromonads when using the Vitek system . METHODS AND RESULTS: Two Aeromonas veronii biovar sobria isolates were misidentified as Vibrio alginolyticus by the Vitek system . Both strains' identification was confirmed by biochemical testing, API 20E/20NE kits and/or 16S RFLP analysis . Thirty-one known Aeromonas species were tested by the Vitek system using 0.45 and 0.85% saline in the suspension medium . It was not clear whether low salinity causes misidentification of Aeromonas species more frequently . CONCLUSIONS: The specified reaction time may be inappropriately short for some critical biochemical tests of some strains . An ingenious reading strategy regarding incubation time is necessary to improve identification of Aeromonas species by the Vitek system . SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of misidentification of A . veronii biovar sobria as V . alginolyticus in the Vitek system. J Appl Microbiol, 2003, 95(4), 693 - 703 Characterization of Vibrio strains isolated from turbot (Scophthalmus maximus) culture by phenotypic analysis, ribotyping and 16S rRNA gene sequence comparison; Montes M et al.; AIM: The aim of the present study was to clarify the taxonomic status of Vibrio strains isolated from an aquaculture system and to compare the results of the identifications made by phenotypic and molecular methods . METHODS AND RESULTS: Fifty-one Vibrio strains isolated from a turbot (Scophthalmus maximus) aquaculture system were characterized by ribotyping and 16S rRNA gene sequencing . The strains had been identified phenotypically in a previous numerical taxonomy analysis as Vibrio anguillarum, V . mediterranei, V . splendidus, V . aestuarianus, V . ordalii, V . fischeri and V . scophthalmi . Cluster analysis of ribotype patterns showed that the strains were separated into two main groups: V . splendidus-V . lentus and V . scophthalmi groups . The use of 16S rRNA gene sequence allowed differentiation among V . splendidus biovar I and V . lentus strains . CONCLUSIONS: The molecular methods identified strains of V . splendidus biovar I, V . lentus and V . scophthalmi, showing discrepancies with phenotypic characterization . SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular methods, as 16S rRNA gene sequence analysis, are necessary for the identification of phenotypically close species to avoid mis-identifications . Interestingly, this is the first report of V . lentus strains associated to turbot culture. J Basic Microbiol, 2003, 43(5), 414 - 22 Characterization by restriction fragment length polymorphism and plasmid profiling of Vibrio tapetis strains; Le Chevalier P et al.; A total of nine Vibrio tapetis strains acquired from France, the United Kingdom and Norway were studied for their plasmid content . All the isolates contained from two to four large plasmids, ranging from approximately 60 to 100 kpb . in size . These plasmids were subsequently subjected to restriction fragment length polymorphism analysis (RFLP) . Using the EcoRI enzyme, three different restriction patterns were demonstrated, two of which were closely related . On the basis of RFLP patterns, the strain from Norway differed noticeably from the French and British strains. J Fish Dis, 2003 May, 26(5), 293 - 303 Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot, Scophthalmus maximus (L.), larvae; Villamil L et al.; A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain . Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected . The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection . The bacterial strain was recovered in pure culture from the internal organs of infected fish . Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney . Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C . Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli . In both cell lines, the number of adhered bacteria increased with incubation time. J Fish Dis, 2003 Feb, 26(2), 117 - 20 Oral administration of formalin-inactivated cells of Aeromonas hydrophila A3-51 controls infection by atypical A . salmonicida in goldfish, Carassius auratus (L.); Irianto A et al.; There has been increasing interest in the use of probiotics in aquaculture to control fish diseases (e.g . Douillet Langdon 1994; Gildberg, Mikkelsen, Sandaker & Ringo 1997; Kennedy, Tucker, Neidic, Vermeer, Cooper, Jarrell & Sennett 1998; DeSchrijver & Ollevier 2000; Robertson, O'Dowd, Burrells, Williams & Austin 2000), and a diverse range of Gram-positive (such as Carnobacterium inhibens) (Robertson et al . 2000) and Gram-negative bacteria, including Vibrio alginolyticus (Garriques & Arevalo 1995), have been evaluated at various times . However, the possible use of probiotics to control diseases of ornamental fish has been neglected . Consequently, following previous success with live (Irianto & Austin 2002) and inactivated probiotics in salmonids (Irianto & Austin, in press), research has focused on the usefulness of inactivated bacterial preparations for the control of atypical Aeromonas salmonicida infections in goldfish, Carassius auratus (L.) . Goldfish, of average 40-50 mm in length, were obtained from a commercial farm in England, and maintained in aerated re-circulating dechlorinated fresh water at 17 degrees C . Their health status was examined immediately upon arrival in the aquaria and at 1-2-week periods thereafter (after Austin & Austin 1989). J Fish Dis, 2003 Feb, 26(2), 103 - 8 Vibrio species isolated from diseased farmed sole, Solea senegalensis (Kaup), and evaluation of the potential virulence role of their extracellular products; Zorrilla I et al.; Bacteria isolated from an outbreak with moderate mortalities in farmed sole, Solea senegalensis (Kaup), in the south of Spain were identified as Vibrio harveyi and V . parahaemolyticus . Only bacterial strains showing swarming were virulent in sole and caused mortalities in experimentally inoculated fish . However, the signs of the disease were only reproduced with V . harveyi . The intramuscular inoculation of the extracellular products (ECPs) of both species produced mortalities in inoculated fish and the appearance of surface ulcers in the case of V . harveyi . However, the inoculation of sublethal doses of ECPs to fish showed a protective effect against V . harveyi. J Fish Dis, 2003 Jan, 26(1), 43 - 9 Hepatic cholangiocarcinoma and testicular mesothelioma in a wild-caught blue shark, Prionace glauca (L.); Borucinska JD et al.; An adult male blue shark, Prionace glauca (L.), caught in July 2000 by a recreational fisherman off Long Island, New York, USA, had a retained fishing hook from a previous capture . The hook penetrated the gastric wall and lacerated the right liver lobe . Macroscopic lesions consisted of transmural gastritis and peritonitis . Alteromonas sp . and Vibrio alginolyticus were isolated from the peritoneal fluid . In addition, a well delineated, sessile mass was found on the otherwise normal serosa of the right testis . Histopathological findings included mesothelial hyperplasia and hypertrophy involving diffusely the gastric, hepatic and parietal serosae, and forming a discrete testicular capsular mass compatible with mesothelioma . In the liver an intrahepatic cholangiocarcinoma, chronic hepatitis, biliary hyperplasia and increased numbers of melanomacrophages were found . In addition organisms compatible with histozoic and coelozoic myxosporeans were found within the skeletal muscle of the abdomen and intrahepatic bile ducts, respectively . This is the third literature report of a liver tumour and the first report of a coelomic mesothelioma from a shark. Appl Environ Microbiol, 2003 Sep, 69(9), 5711 - 5 Role for mannose-sensitive hemagglutinin in promoting interactions between Vibrio cholerae El Tor and mussel hemolymph; Zampini M et al.; The role of mannose-sensitive hemagglutinin (MSHA) in Vibrio cholerae O1 El Tor interactions with hemolymph of the mussel Mytilus galloprovincialis was studied . Bacterial adherence to and association with hemocytes were evaluated at 4 and 18 degrees C, respectively . In hemolymph serum, the wild-type strain N16961 adhered to and associated with hemocytes about twofold more efficiently than its mutant lacking MSHA . In artificial seawater (ASW), no significant differences between the two strains were observed . N16961 was also more sensitive to hemocyte bactericidal activity than its MSHA mutant; in fact, the percentages of killed bacteria after 120 min of incubation were 60 and 34%, respectively . The addition of D-mannose abolished the serum-mediated increase in adherence, association, and sensitivity to killing of the wild-type strain without affecting the interactions of the mutant . A similar increase in N16961 adherence to hemocytes was observed when serum was adsorbed with MSHA-deficient bacteria . In contrast, serum adsorbed with either wild-type V . cholerae El Tor or wild-type Escherichia coli carrying type 1 fimbriae was no longer able to increase adherence of N16961 to hemocytes . The results indicate that hemolymph-soluble factors are involved in interactions between hemocytes and mannose-sensitive adhesins. In Silico Biol, 2003, 3(3), 287 - 300 Identification of a unique IAHP (IcmF associated homologous proteins) cluster in Vibrio cholerae and other proteobacteria through in silico analysis; Das S et al.; Previously using global transcription profile approach, the icmF gene of V . cholerae was identified as an in vivo induced gene involved in the regulation of motility, adherence to intestinal epithelial cells . The present report shows that icmF has 27 homologues in the finished and unfinished microbial genome sequences available in the database and among them only Legionella pneumophila IcmF has been partially characterized . We first identified a gene cluster containing 15 genes surrounding IcmF and designated as IcmF associated homologous protein (IAHP), present in nine gram-negative proteo-bacteria that need an intimate contact with the eukaryotic host cells . Only in P . aeruginosa the cluster is present in three different regions of the genome . In Y . pestis CO92 the cluster is in the same orientation as that of V . cholerae . But in E . coli O157:H7 VT2 Sakai, E . coli O157:H7 EDL933 and Y . pestis KIM, the cluster is in the opposite orientation to that of V . cholerae though the relative position of respective ORFs are the same . In other proteobacteria like M . loti, A . tumefaciens, P . aeruginosa and S . typhimurum, the genes are reshuffled indicating the frequent rearrangement in microbial genomes . Annotation, motif and domain prediction suggested a role of the proteins of IAHP cluster in cell surface architecture or secretion . Phylogenetic analysis of each protein belonging to the cluster present in all the nine organisms showed that only E . coli O157:H7 VT2 Sakai, E . coli O157:H7 EDL933, Y . pestis KIM and Y . pestis CO92 are closer to the V . cholerae and this pattern was similar for all proteins in the cluster . Presence of low G + C regions before and after the gene cluster in V . cholerae and both of the E . coli O157: H7 strains provide possibility of transfer of the gene cluster from a foreign source . From our finding it can be speculated that both of the E . coli O157:H7 strains and V . cholerae has a common ancestor, but the other organism where the cluster is reshuffled has undergone a long term of evolution. Microbiology, 2003 Sep, 149(Pt 9), 2463 - 74 The variation of dTDP-L-rhamnose pathway genes in Vibrio cholerae; Li Q et al.; The genetic variation in the dTDP-L-rhamnose pathway genes (rmlA, rmlB, rmlC and rmlD) in Vibrio cholerae was investigated . The genes are part of the O antigen gene cluster and the aim was to study lateral gene transfer of O antigen gene clusters . The rml genes of an O6 strain were cloned using an Escherichia coli K-12 strain designed for selecting cloned rml genes . Thirty-three strains carrying the known rhamnose-containing O antigens were probed with O6-based rml gene probes, and 19 were positive with from one to all four of the gene probes . Nine rml gene sets from this group were sequenced and found to be in the order rmlBADC, at the 5' end of the gene clusters . A gradient in the level of variation was observed, with highly similar sequences at the 5' end rmlB gene, but very divergent and strain-specific sequences at the 3' end of the rml gene set . The change in level of similarity varied in position, but was always abrupt and coincided with a change in GC content, indicating that the 5' and 3' parts are of different origin, and that recombination within rml genes has occurred . The rml gene sets of two of the strains that did not hybridize with any O6 rml gene probes were also cloned and sequenced . Both gene sets were in the middle of the O antigen gene cluster and were very divergent from each other and all other rml gene sets . This supports the hypothesis that presence of rml genes at the end of the O antigen gene cluster facilitates lateral gene transfer of rml-containing O antigen gene clusters in V . cholerae . The sequence relationships make it possible to identify sites of recombination and to distinguish DNA that has long been in V . cholerae and DNA that probably came into the species with the O antigen gene cluster. Epidemiol Infect, 2003 Aug, 131(1), 621 - 6 Peak occurrences of ciguatera fish poisoning precede cholera outbreaks in Hong Kong; Kwan LC et al.; Occurrences of ciguatera fish poisoning (CFP) and Vibrio cholerae infected patients in Hong Kong were reviewed for the 13-year period 1989-2001 . Peak activity of CFP preceded peak activity of cholera in nine of the years except in 4 years (1990, 1991, 1992, 1996) where it was observed that the total number of cholera cases were all less than or equal to five per year (P < 0.05) . Average time interval was 2.4 months between peaks of CFP and Vibrio cholerae outbreaks . Findings suggested that the factors that affect cholera and ciguatera occurrences may not be operating in some years but when they are operating, they will affect both cholera and CFP . CFP peaks have consistently occurred before Vibrio cholerae peaks in our locality so much so that the occurrence of the latter can now be almost accurately predicted since 1998 . CFP peaks served as an early warning for public measures to be in place before occurrence of cholera outbreaks. Epidemiol Infect, 2003 Aug, 131(1), 613 - 9 Survey of Vibrio cholerae O1 and its survival over the winter in marine water of Port of Osaka; Miyagi K et al.; The survey of Vibrio cholerae O1 in marine area was carried out in the Port of Osaka, Japan in 1987-2001, and 51 V . cholerae O1 strains were isolated . All strains were identified to be of El Tor biotype, Ogawa serotype and classic Ubon Kappa-phage type, and were cholera toxin (CT)-negative and CT gene-negative . In order to clarify certain ecological aspects of V . cholerae O1 in the marine environment of the temperate zone, we performed molecular analysis of the isolated strains using pulsed-field gel electrophoresis (PFGE) with NotI and SfiI restriction enzymes . We found the indistinguishable strains by DNA analysis using PFGE with strains passed for 1 year, and also found the closely related strains with that passed for 3 and 12 years . Those results indicated that V . cholerae O1 can survive over one winter at least, and that it survives in marine water for a long time by undergoing continuous mutation. J Biol Chem, 2003 Nov 14, 278(46), 45072 - 81 Epub 2003 Aug 28. SmcR and cyclic AMP receptor protein coactivate Vibrio vulnificus vvpE encoding elastase through the RpoS-dependent promoter in a synergistic manner; Jeong HS et al.; The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE . We previously demonstrated that vvpE expression is differentially directed by two different promoters in a growth phase-dependent manner . The activity of the stationaryphase promoter (promoter S (PS)) is dependent on RpoS and is also under the positive control of cyclic AMP receptor protein (CRP) . In this study, primer extension analyses revealed that SmcR, the Vibrio harveyi LuxR homolog, is also involved in the regulation of vvpE transcription by activating PS . Although the influence of CRP on PS is mediated by SmcR, the level of PS activity observed when CRP and SmcR function together was found to be greater than the sum of the PS activities achieved by each activator alone . Western blot analyses demonstrated that the cellular levels of RpoS, CRP, and SmcR were not significantly affected by one other, indicating that CRP and SmcR function cooperatively to activate PS rather than sequentially in a regulatory cascade . The binding sites for CRP and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro DNase I protection assays . The binding sites for CRP and SmcR were juxtapositioned and centered 220 and 198 bp upstream of the transcription start site of PS, respectively . Accordingly, these results reveal that CRP and SmcR function synergistically to coactivate the expression of vvpE by the RpoS-dependent promoter (PS) and that the activators exert their effect by directly binding to the promoter in the stationary phase. Water Res, 2003 Oct, 37(17), 4091 - 8 Combined toxicity effects of MTBE and pesticides measured with Vibrio fischeri and Daphnia magna bioassays; Hernando MD et al.; Methyl-tert-butyl ether (MTBE), a fuel oxygenate that is added to gasoline, commonly contaminates aquatic systems, many of which are already contaminated with pesticides . The toxic effects (EC(50) value) of several pure pesticides (Diuron, Linuron, Dichlofluanid, Sea nine, Irgarol and tributyltin (TBT)) were measured and compared with the EC(50) value of the pesticide mixed with MTBE, using the Vibrio fischeri and Daphnia magna acute toxicity assays . The interaction between chemicals was evaluated in terms of the effects of mixing on the EC(50) value (i.e . the concentration (mg/L) of a compound or mixture that is required to produce a 50% change in a toxic response parameter) and the time required to generate the toxic response . Presence of MTBE enhanced the EC(50) value of several pesticides (Diuron, Dichlofluanid, TBT and Linuron) and/or the toxic response manifested more rapidly than with pure pesticides . Toxicity enhancements were quite substantial in many cases . For example, the presence of MTBE increased the toxicity of Diuron by more than 50% when tested with the V . fischeri assay (5, 15 and 30 min exposure) . Also, the toxic response manifested itself within 5 min whereas without the MTBE the same response arose in 30 min . Presence of MTBE increased the toxicity of Dichlofluanid by 30% when measured with the D . magna assay . Toxicities of only two pesticides (Sea nine and Irgarol) were not raised by the presence of MTBE. Biochem Biophys Res Commun, 2003 Sep 12, 309(1), 66 - 70 Bright stable luminescent yeast using bacterial luciferase as a sensor; Szittner R et al.; Bright luminescent yeast cells with light intensities similar to bacteria containing luciferase (LuxAB) were generated by providing saturating nontoxic levels of the substrates for the bioluminescence reaction (FMNH(2)+O(2) and fatty aldehyde-->light) . Z-9-Tetradecenal added to yeast (+luxAB) gave a luminescent signal close to that with decanal with the signal remaining strong for >24h while luminescence of yeast with decanal decayed to less than 0.01% of that with Z-9-tetradecenal after 2min . Moreover, yeast survived in 0.5% (v/v) Z-9-tetradecenal while 0.005% (v/v) decanal was lethal . Luminescence of yeast (+luxAB) was also stimulated 100-fold by transformation with the NADPH-specific FMN reductase (FRP) from Vibrio harveyi . The recognition of the nontoxicity and high luminescence generated by Z-9-tetradecenal and the generation of high levels of FMNH(2) in yeast by transformation with a flavin reductase provide evidence for the strong potential use of bacterial luciferase as the light-emitting sensor of choice in eukaryotic organisms. Mol Biol (Mosk), 2003 Jul-Aug, 37(4), 704 - 11 {The effects of the regulatory proteins RcsA and RcsB on the expression of the Vibrio fischeri lux operon in Escherichia coli}; Zavil'gel'skii GB et al.; A study was made of the effect of RcsA and RcsB on the Vibrio fischeri lux expression in Escherichia coli . RcsA suppressed the LuxR activity and thereby inhibited expression of the lux genes coding for luciferase and reductase . In osmotic shock, RcsA-RcsB activated lux expression and, consequently, bioluminescence of E . coli cells in the early log phase. Clin Infect Dis, 2003 Sep 1, 37(5), e63 - 7 Epub 2003 Aug 12. Vibrio vulnificus infection in a hemodialysis patient receiving intravenous iron therapy; Barton JC et al.; A 73-year-old man treated with long-term hemodialysis, erythropoietin, and intravenous iron sucrose infusions developed Vibrio vulnificus infection after eating raw oysters harvested from the Alabama coast . Five of the 31 persons with cases of V . vulnificus infection reported to the Alabama Department of Public Health (Montgomery) during 1996-2002 (including the patient described here) also had renal disease . Persons with renal disease, especially those treated with long-term hemodialysis and intravenous iron infusions, may have an increased risk of infection with V . vulnificus. Cell, 2003 Aug 22, 114(4), 521 - 30 Distinct replication requirements for the two Vibrio cholerae chromosomes; Egan ES et al.; Studies of prokaryotic chromosome replication have focused almost exclusively on organisms with one chromosome . We defined and characterized the origins of replication of the two Vibrio cholerae chromosomes, oriCI(vc) and oriCII(vc) . OriCII(vc) differs from the origin assigned by bioinformatic analysis and is unrelated to oriCI(vc) . OriCII(vc)-based replication requires an internal 12 base pair repeat and two hypothetical genes that flank oriCII(vc) . One of these genes is conserved among diverse genera of the family Vibrionaceae and encodes an origin binding protein . The other gene codes for an RNA and not a protein . OriCII(vc)- but not oriCI(vc)-based replication is negatively regulated by a DNA sequence adjacent to oriCII(vc) . There is an unprecedented requirement for DNA adenine methyltransferase in both oriCI(vc)- and oriCII(vc)-based replication . Our studies of replication in V . cholerae indicate that microorganisms having multiple chromosomes may utilize unique mechanisms for the control of replication. Org Biomol Chem, 2003 Mar 7, 1(5), 775 - 84 Neoglycoconjugates from synthetic tetra- and hexasaccharides that mimic the terminus of the O-PS of Vibrio cholerae O:1, serotype Inaba; Ma X et al.; A glycosyl acceptor and a glycosyl donor having the N-3-deoxy-L-glycero-tetronic acid side chain already attached have been prepared and used for the synthesis of the di-through to the hexasaccharide that mimic the upsteam terminus of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Inaba . The target tetra- and the hexasaccharide, which were obtained in the form of 5-methoxycarbonylpentyl glycosides, were linked to BSA using squaric acid diester chemistry . The conjugation reactions were monitored by surface enhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) . This allowed the progression of the conjugation of the synthetic oligosaccharides in a controlled way and termination of the reaction when the desired molar hapten/BSA ratio had been reached, yielding neoglycoconjugates with predetermined carbohydrate/carrier ratios . The ability to monitor the conjugation by the SELDI-TOF MS technique made it possible to prepare, from one hapten in a one-pot reaction, several neoglycoconjugates having different, predetermined carbohydrate/carrier ratios. J Bacteriol, 2003 Sep, 185(17), 5220 - 33 Complete genome sequence of the broad-host-range vibriophage KVP40: comparative genomics of a T4-related bacteriophage; Miller ES et al.; The complete genome sequence of the T4-like, broad-host-range vibriophage KVP40 has been determined . The genome sequence is 244,835 bp, with an overall G+C content of 42.6% . It encodes 386 putative protein-encoding open reading frames (CDSs), 30 tRNAs, 33 T4-like late promoters, and 57 potential rho-independent terminators . Overall, 92.1% of the KVP40 genome is coding, with an average CDS size of 587 bp . While 65% of the CDSs were unique to KVP40 and had no known function, the genome sequence and organization show specific regions of extensive conservation with phage T4 . At least 99 KVP40 CDSs have homologs in the T4 genome (Blast alignments of 45 to 68% amino acid similarity) . The shared CDSs represent 36% of all T4 CDSs but only 26% of those from KVP40 . There is extensive representation of the DNA replication, recombination, and repair enzymes as well as the viral capsid and tail structural genes . KVP40 lacks several T4 enzymes involved in host DNA degradation, appears not to synthesize the modified cytosine (hydroxymethyl glucose) present in T-even phages, and lacks group I introns . KVP40 likely utilizes the T4-type sigma-55 late transcription apparatus, but features of early- or middle-mode transcription were not identified . There are 26 CDSs that have no viral homolog, and many did not necessarily originate from Vibrio spp., suggesting an even broader host range for KVP40 . From these latter CDSs, an NAD salvage pathway was inferred that appears to be unique among bacteriophages . Features of the KVP40 genome that distinguish it from T4 are presented, as well as those, such as the replication and virion gene clusters, that are substantially conserved. J Bacteriol, 2003 Sep, 185(17), 5045 - 54 Control of SXT integration and excision; Burrus V et al.; The Vibrio cholerae SXT element is a conjugative self-transmissible chromosomally integrating element that encodes resistance to multiple antibiotics . SXT integrates in a site-specific fashion at prfC and excises from the chromosome to form a circular but nonreplicative extrachromosomal form . Both chromosomal integration and excision depend on an SXT-encoded recombinase, Int . Here we found that Int is necessary and sufficient for SXT integration and that int expression in recipient cells requires the SXT activators SetC and SetD . Although no xis-like gene was annotated in the SXT genome, Int was not sufficient to mediate efficient SXT chromosomal excision . We identified a novel SXT Xis that seems to function as a recombination directionality factor (RDF), facilitating SXT excision and inhibiting SXT integration . Although unrelated to any previously characterized RDF, Xis is similar to five hypothetical proteins that together may constitute a new family of RDFs . Using real-time quantitative PCR assays to study SXT excision from the chromosome, we determined that while SXT excision is required for SXT transfer, the percentage of cells containing an excised circular SXT does not appear to be a major factor limiting SXT transfer; i.e., we found that most cells harboring an excised circular SXT molecule do not act as SXT donors . In the absence of prfC, SXT integrated into several secondary attachment sites but preferentially into the 5' end of pntB . SXT excision and transfer from a donor containing pntB::SXT were reduced, suggesting that the SXT integration site may also influence the element's transmissibility. Vaccine, 2003 Sep 8, 21(25-26), 3663 - 74 Evaluation of the protective efficacy of Vibrio cholerae ghost (VCG) candidate vaccines in rabbits; Eko FO et al.; An effective Vibrio cholerae vaccine is needed to reduce the morbidity and mortality caused by this pathogen . Despite the availability of current oral vaccines with measurable efficacy, there is need for more effective vaccines with broad-spectrum efficacy in target populations . Recent studies have shown that bacterial ghosts, produced by the expression of cloned lysis gene E, possess adjuvant properties and are immunogenic . In this study, ghosts were prepared from V . cholerae O1 or O139 and evaluated as vaccines in the reversible intestinal tie adult rabbit diarrhea (RITARD) model . Rabbits were orally immunized with different doses of V . cholerae ghost (VCG) formulations . The vaccine formulations elicited high levels of serum vibriocidal titers against indicator strains . The magnitude of the response was measured as the geometric mean titer (GMT) increase for all rabbits in relation to prevaccination titers . The induction of cross protection was evidenced by the ability of serum from VCG-immunized rabbits to mediate complement-dependent killing of both the homologous and the heterologous strains . Immunized rabbits were protected against intraduodenal challenge 30 days after primary immunization . Protective immunity against challenge appeared to be dose dependent and was associated with marked inhibition of colonization . These results indicate that VCGs represent a novel approach to cholera vaccine development and constitute an effective vaccine delivery vehicle. J Appl Microbiol, 2003, 95(3), 602 - 11 Isolation of partial toxR gene of Vibrio harveyi and design of toxR-targeted PCR primers for species detection; Conejero MJ et al.; AIMS: To differentiate Vibrio harveyi from closely related Vibrio species by toxR sequence analysis and design primers for the specific detection of the shellfish pathogen . METHODS AND RESULTS: The partial toxR homologue from the shellfish pathogen V . harveyi was isolated by PCR using degenerate primers . The 578-bp toxR fragment from V . harveyi, that exhibited highest homology with partial toxR of V . parahaemolyticus (68%), is predicted to encode for a polypeptide with 192 amino acid residues . Alignment of the V . harveyi toxR nucleotide and deduced amino acid sequence with those from other Vibrio species revealed the presence of the fairly characteristic conserved transcription activation and transmembrane domain as well as the divergent membrane tether region that may be targeted for the development of species-specific oligonucleotide primers . Consequently, PCR primers that could amplify a 390-bp gene fragment in V . harveyi were designed by targeting portions of the V . harveyi toxR that display variability with toxR sequences from other Vibrio species . The 390-bp-amplicon was detected in all V . harveyi strains examined except in the nontarget bacteria and unexpectedly, in two shrimp-derived strains (VIB 391 and STD 3-101) from Thailand and Ecuador . Results show that strains exhibiting the 390-bp amplicon mostly belong to the same cluster based on previous amplified fragment length polymorphism data while strains which were previously unclustered or unclassified did not display the 390-bp PCR product . CONCLUSIONS: The toxR sequence variation could differentiate V . harveyi from closely related Vibrio species . A PCR protocol amplifying a 390-bp fragment of the V . harveyi toxR was established and could be useful in the specific and rapid detection of the species . SIGNIFICANCE AND IMPACT OF THE STUDY: The molecular approaches reported in this study could facilitate the early diagnosis and surveillance of luminous vibriosis in hatchery-reared fish and shellfish species through rapid identification and specific detection of causal agent. J Appl Microbiol, 2003, 95(3), 479 - 83 Acclimation and repair of DNA damage in recombinant bioluminescent Escherichia coli; Min J et al.; AIMS: The aim of this study is to understand different adaptive responses in bacteria caused by three different mutagens, namely, an intercalating agent, an alkylating agent and a hydroxylating agent, and the repair systems according to the type of DNA damage, that is, DNA cross-linking and delayed DNA synthesis, alkylation and hydroxylation of DNA . A recombinant bioluminescent Escherichia coli, DPD2794 with the recA promoter fused to luxCDABE originating from Vibrio fischeri, was used in this study . METHODS AND RESULTS: The recombinant bioluminescent E . coli strain DPD2794, containing a recA promoter fused to luxCDABE from V . fischeri, was used to detect adaptive and repair responses to DNA damage caused by mitomycin C (MMC), and these responses were compared with those when the cells were induced with N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and hydrogen peroxide (H2O2) . The response ratio between the induced samples and that of the controls decreased suddenly when the induced culture was used in further inductions, indicating a possible adaptive response to DNA damage . DNA damage, or the proteins produced, because of MMC addition does not appear to be completely resolved until the seventh sub-culture after the initial induction, whereas simple damage, such as the base modification caused by MNNG and H2O2, appears to be repaired rapidly as evidenced by the quick recovery of sensitivity . CONCLUSIONS: These results suggest that it takes more time to completely repair DNA damage caused by MMC, as compared with a simple repair such as that required for the damage caused by MNNG and H2O2 . Therefore, repair of the damage caused by these three mutagens is controlled by different regulons, even though they all induced the recA promoter . SIGNIFICANCE AND IMPACT OF THE STUDY: Using a bioluminescent E . coli harbouring a recA promoter-lux fusion, it was found that different adaptive responses and repair systems for DNA damage caused by several mutagens exists in E . coli. Dis Aquat Organ, 2003 Jul 8, 55(2), 169 - 73 Enhanced growth and resistance to Vibrio challenge in pond-reared black tiger shrimp Penaeus monodon fed a Bacillus probiotic; Rengpipat S et al.; The bacterial probiont Bacillus S11 (BS11) was used as a supplement in feed (PF) for black tiger shrimp Penaeus monodon in 2 earthen pond field-trials carried out for 100 d during 2 different seasons in Thailand . Growth and survival were compared with those of shrimp receiving an unsupplemented feed (UF) . In the hot and cool seasons, respectively, shrimp fed PF grew significantly larger and had significantly higher survival than shrimp fed UF (p < 0.05) . Projected yields on an annual basis (two 100 d crops) were 49% greater with PF-fed shrimp . In 8 d challenge tests using the luminescent bacterium Vibrio harveyi 1526, shrimp fed UF all died within 6 d while survival for shrimp fed PF was 5 and 9%. Gene, 2003 Jul 17, 312, 181 - 8 Cloning and characterization of a novel lipase from Vibrio harveyi strain AP6; Teo JW et al.; A lipolytic enzyme designated Vst, was cloned from Vibrio harveyi strain AP6 . The vst gene was 1650 bp and was predicted to encode a 549 amino acid precursor with a molecular mass of about 61 kDa . The predicted polypeptide shared about 30% sequence identity with Bacillus esterases . Sequence alignment of Vst and related esterases revealed the presence of an active site serine located in the middle of the GESAG motif, at positions 212-216 . This motif resembled the GXSXG consensus motif characteristic of lipolytic enzymes . Vst was expressed as a carboxy-terminal 6 x His tagged recombinant enzyme from E . coli TOP10 cells . SDS-PAGE and Western blot analysis using anti-His antibodies confirmed that the size of the mature protein was about 61 kDa . Substrate specificity of Vst was investigated using p -nitrophenyl (p NP) esters with varying carbon chain lengths, from C2 to C18 . Vst showed the highest activity with the long chain p -nitrophenyl ester p NPC12 but was able to hydrolyze longer chain esters (p NPC14-p NPC16) as well as short and medium chain esters (p NPC4 and p NPC8). Toxicon, 2003 Aug, 42(2), 183 - 9 Role of mast cells and pro-inflammatory mediators on the intestinal secretion induced by cholera toxin; Rocha MF et al.; Recent data suggest that diarrhea caused by Vibrio cholerae involves a pro-inflammatory mediators release, such as cytokines, prostaglandin and nitric oxide . The aim of this study was to investigate the role of mast cells and their mediators in the intestinal secretion induced by cholera toxin . We examined the dose responses, time course and role of mast cells and pro-inflammatory mediators in cholera toxin intestinal secretory response, in vivo . Cholera toxin caused a dose-dependent secretion, in ligated small intestine loops, at 18 h . Rats treated with 48/80 compound or ketotifen had a significant decrease in the intestinal secretory response . Cholera toxin secretion was significantly reduced by an unspecific histamine/serotonin receptor antagonist, histamine receptor antagonist, phospholipase A2 and cyclooxygenase inhibitors, platelet-activating factor (PAF) receptor antagonists and TNF-alpha synthesis blockers . On the other hand, pretreatment with a specific serotonin receptor antagonist and lipoxygenase inhibitors failed to block this effect . Analysis of the intestinal fluid from rats injected with cholera toxin, revealed that cholera toxin induces the release of IL-1beta and TNF-alpha into fluid . The data suggest that, at least in part, mast cells are involved in cholera toxin-induced secretion, as well as point to the importance of histamine, prostaglandins, PAF, IL-1beta and TNF-alpha in this process. Microbiol Immunol, 2003, 47(6), 419 - 27 Gene cloning and characterization of VcrM, a Na+-coupled multidrug efflux pump, from Vibrio cholerae non-O1; Huda MN et al.; We cloned a DNA fragment responsible for drug resistance from chromosome of Vibrio cholerae non-O1 . Nucleotide sequence analysis of this fragment revealed the presence of a single open reading frame encoding a protein consisting of 445 amino acid residues . We designated the gene as vcrM . Hydropathy analysis of the deduced amino acid sequence of VcrM suggests the presence of 12 trans-membrane segments . A dendrogram showed that VcrM is a member of the DinF-subfamily within the MATE family of multidrug efflux pumps . Expression of the cloned vcrM gene in drug-hypersensitive Escherichia coli KAM32 cells made them resistant to acriflavine, 4', 6-diamidino-2-phenylindole, Hoechst 33342, rhodamine 6G, tetraphenylphosphonium chloride (TPPCl) and ethidium bromide . Efflux of acriflavine due to VcrM was dependent on Na+ or Li+ . Moreover, Na+ efflux was observed with VcrM when TPPCl was added to Na+-loaded cells . Therefore, we conclude that VcrM is a Na+/drug antiporter-type multidrug efflux pump. J Clin Microbiol, 2003 Aug, 41(8), 3939 - 41 Use of dipsticks for rapid diagnosis of cholera caused by Vibrio cholerae O1 and O139 from rectal swabs; Bhuiyan NA et al.; We evaluated the recently developed dipsticks for the rapid detection of Vibrio cholerae serotypes O1 and O139 from rectal swabs of hospitalized diarrheal patients after enrichment for 4 h in alkaline peptone water . The sensitivity and specificity of the dipsticks were above 92 and 91%, respectively . The dipsticks represent the first rapid test which has been successfully used to diagnose cholera from rectal swabs, and this would immensely improve surveillance for cholera, especially in remote settings. Appl Environ Microbiol, 2003 Aug, 69(8), 4706 - 13 Photobactin: a catechol siderophore produced by Photorhabdus luminescens, an entomopathogen mutually associated with Heterorhabditis bacteriophora NC1 nematodes; Ciche TA et al.; The nematode Heterorhabditis bacteriophora transmits a monoculture of Photorhabdus luminescens bacteria to insect hosts, where it requires the bacteria for efficient insect pathogenicity and as a substrate for growth and reproduction . Siderophore production was implicated as being involved in the symbiosis because an ngrA mutant inadequate for supporting nematode growth and reproduction was also deficient in producing siderophore activity and ngrA is homologous to a siderophore biosynthetic gene, entD . The role of the siderophore in the symbiosis with the nematode was determined by isolating and characterizing a mini-Tn5-induced mutant, NS414, producing no detectable siderophore activity . This mutant, being defective for growth in iron-depleted medium, was normal in supporting nematode growth and reproduction, in transmission by the dauer juvenile nematode, and in insect pathogenicity . The mini-Tn5 transposon was inserted into phbH; whose protein product is a putative peptidyl carrier protein homologous to the nonribosomal peptide synthetase VibF of Vibrio cholerae . Other putative siderophore biosynthetic and transport genes flanking phbH were characterized . The catecholate siderophore was purified, its structure was determined to be 2-(2,3-dihydroxyphenyl)-5-methyl-4,5-dihydro-oxazole-4-carboxylic acid {4-(2,3-dihydroxybenzoylamino)-butyl}-amide, and it was given the generic name photobactin . Antibiotic activity was detected with purified photobactin, indicating that the siderophore may contribute to antibiosis of the insect cadaver . These results eliminate the lack of siderophore activity as the cause for the inadequacy of the ngrA mutant in supporting nematode growth and reproduction. Appl Environ Microbiol, 2003 Aug, 69(8), 4390 - 5 Vibrio harveyi nitroreductase is also a chromate reductase; Kwak YH et al.; The chromate reductase purified from Pseudomonas ambigua was found to be homologous with several nitroreductases . Escherichia coli DH5alpha and Vibrio harveyi KCTC 2720 nitroreductases were chosen for the present study, and their chromate-reducing activities were determined . A fusion between glutathione S-transferase (GST) and E . coli DH5alpha NfsA (GST-EcNfsA), a fusion between GST and E . coli DH5alpha NfsB (GST-EcNfsB), and a fusion between GST and V . harveyi KCTC 2720 NfsA (GST-VhNfsA) were prepared for their overproduction and easy purification . GST-EcNfsA, GST-EcNFsB, and GST-VhNFsA efficiently reduced nitrofurazone and 2,4,6-trinitrotoluene (TNT) as their nitro substrates . The K(m) values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 11.8, 23.5, and 5.4 micro M, respectively . The V(max) values for GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA were 3.8, 3.9, and 10.7 nmol/min/mg of protein, respectively . GST-VhNfsA was the most effective of the three chromate reductases, as determined by each V(max)/K(m) value . The optimal temperatures of GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA for chromate reduction were 55, 30, and 30 degrees C, respectively . Thus, it is confirmed that nitroreductase can also act as a chromate reductase . Nitroreductases may be used in chromate remediation . GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA have a molecular mass of 50 kDa and exist as a monomer in solution . Thin-layer chromatography showed that GST-EcNfsA, GST-EcNfsB, and GST-VhNfsA contain FMN as a cofactor . GST-VhNfsA reduced Cr(VI) to Cr(III) . Cr(III) was much less toxic to E . coli than Cr(VI). J Fish Dis, 2003 Jun, 26(6), 339 - 47 A single-dose pharmacokinetic study of oxolinic acid and vetoquinol, an oxolinic acid ester, in cod, Gadus morhua L., held in sea water at 8 degrees C and in vitro antibacterial activity of oxolinic acid against Vibrio anguillarum strains isolated from diseased cod; Samuelsen OB et al.; The pharmacokinetic properties of the antibacterial agent oxolinic acid and vetoquinol, the carbitol ester of oxolinic acid, were studied after intravenous (i.v.) and oral (p.o.) administration to 100-150 g cod, Gadus morhua L., held in sea water at 8 degrees C . Following i.v . injection, the plasma drug concentration-time profile showed two distinct phases . The distribution half-life (t1/2alpha) was estimated at 1.3 h, the elimination half-life (t1/2beta) as 84 h and the total body clearance (Cl(T)) as 0.047 L kg(-1) h(-1) . The volume of distribution at steady state, Vd(ss) was calculated to be 5.5 L kg(-1), indicating good tissue penetration of oxolinic acid in cod . Following p.o . administration of oxolinic acid or vetoquinol, the peak plasma concentrations (C(max)) of oxolinic acid and the time to peak plasma concentrations (T(max) were estimated to be 1.2 and 2.5 microg mL(-1) and 24 and 12 h, respectively . The bioavailabilities of oxolinic acid following p.o . administration of oxolinic acid and vetoquinol were calculated to be 55 and 72%, respectively . The in vitro minimum inhibitory concentration (MIC) values of oxolinic acid against three strains of Vibrio anguillarum isolated from diseased cod were 0.016 microg mL(-1) (HI-610), 0.250 microg mL(-1) (HI-618) and 0.250 microg mL(-1) (HI-A21) . Based on a MIC value of 0.016 microg mmL(-1) a single p.o . administration of 25 mg kg(-1) of oxolinic acid maintains plasma levels in excess of 0.064 microg mL(-1), corresponding to four times the MIC-value, for approximately 12 days . The analogous value for a single p.o . dose of 25 mg kg(-1) of oxolinic acid administered as vetoquinol was 13 days. J Fish Dis, 2003 Jun, 26(6), 321 - 9 Non-specific immune response of turbot, Scophthalmus maximus (L.), experimentally infected with a pathogenic Vibrio pelagius; Villamil L et al.; The effect of a pathogenic Vibrio pelagius, isolated during a mass mortality of turbot larvae, on the non-specific immune response of turbot, Scophthalmus maximus (L.), macrophages was studied both in vitro and in vivo . The in vitro treatment of head kidney (HK) macrophages with viable V . pelagius caused a significant inhibition of the chemiluminescence (CL) response in comparison with untreated macrophages, while incubation with heat-killed bacteria did not affect this response . In vivo, the intraperitoneal injection of V . pelagius resulted in a significant inhibition of the CL response in infected fish at days 1 and 4 post-infection compared with the control fish response . The HK macrophage nitric oxide (NO) production was enhanced by in vitro incubation with intermediate doses of viable V . pelagius (5 x 10(3) and 5 x 10(4) bacteria mL(-1)) and higher doses of the heat-killed bacteria (5 x 10(4)-5 x 10(6) bacteria mL(-1)) . In both cases, the NO inhibitor N-omega -nitro-L-arginine was capable of down-regulating the specific NO induction caused by incubation with the bacterial treatments . In contrast, incubation with ECPs at higher doses caused a reduction in NO production . In vivo, a significant enhancement in NO production was also observed in macrophage supernatants at day 10 post-infection . Lysozyme concentration in the serum was also significantly increased in the experimentally infected fish at days 4 and 10 post-injection . In addition, viable V . pelagius and its ECPs significantly reduced HK macrophage viability in vitro, whereas no significant differences in viability were observed during the incubation with heat-killed bacteria . As NO production was enhanced in the experimentally infected fish, the inhibitory effect of the NO donor, S-nitroso-acetyl-penicillamine (SNAP), was tested in vitro in a cell-free assay . The results showed that growth of V . pelagius was significantly inhibited using SNAP at a high concentration (1 mM). J Bacteriol, 2003 Aug, 185(16), 4825 - 36 The virulence activator AphA links quorum sensing to pathogenesis and physiology in Vibrio cholerae by repressing the expression of a penicillin amidase gene on the small chromosome; Kovacikova G et al.; Activation of the tcpPH promoter on the Vibrio pathogenicity island by AphA and AphB initiates the Vibrio cholerae virulence cascade and is regulated by quorum sensing through the repressive action of HapR on aphA expression . To further understand how the chromosomally encoded AphA protein activates tcpPH expression, site-directed mutagenesis was used to identify the base pairs critical for AphA binding and transcriptional activation . This analysis revealed a region of partial dyad symmetry, TATGCA-N6-TNCNNA, that is important for both of these activities . Searching the V . cholerae genome for this binding site permitted the identification of a second one upstream of a penicillin V amidase (PVA) gene on the small chromosome . AphA binds to and footprints this site, which overlaps the pva transcriptional start, consistent with its role as a repressor at this promoter . Since aphA expression is under quorum-sensing control, the response regulators LuxO and HapR also influence pva expression . Thus, pva is repressed at low cell density when AphA levels are high, and it is derepressed at high cell density when AphA levels are reduced . Penicillin amidases are thought to function as scavengers for phenylacetylated compounds in the nonparasitic environment . That AphA oppositely regulates the expression of pva from that of virulence, together with the observation that PVA does not play a role in virulence, suggests that these activities are coordinated to serve V . cholerae in different biological niches. J Bacteriol, 2003 Aug, 185(16), 4672 - 82 Mutation in the relA gene of Vibrio cholerae affects in vitro and in vivo expression of virulence factors; Haralalka S et al.; The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing . In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA(VCH)) was cloned and sequenced . The relA(VCH) gene encodes a 738-amino-acid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli . A deltarelA::kan allele was generated by replacing approximately 31% of the open reading frame of wild-type relA of V . cholerae El Tor strain C6709 with a kanamycin resistance gene . The V . cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation . Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions . In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results . The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V . cholerae . Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17 . Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V . cholerae . In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT. Comp Biochem Physiol B Biochem Mol Biol, 2003 Aug, 135(4), 611 - 25 Replacement of fish oil with sunflower oil in feeds for Atlantic salmon (Salmo salar L.): effect on growth performance, tissue fatty acid composition and disease resistance; Bransden MP et al.; Dietary sunflower oil (SFO) was used to gradually replace fish oil (FO) in six diets (which also contained fish meal) for Atlantic salmon parr (initial mass: 21.7 g) . The effect on growth performance, tissue fatty acid profiles and disease resistance was monitored after 63 days . At the conclusion of the trial, no significant differences were detected in growth between any of the feeds . Fatty acid composition of whole carcass, dorsal muscle and liver generally reflected that of the diets . Forty percent of the FO could be replaced by SFO before tissue 22:6n-3 was significantly reduced, although other essential and non-essential fatty acids were more susceptible to change . Significant differences were detected in cumulative mortality of Atlantic salmon challenged with Vibrio anguillarum at the trials conclusion, although this was not correlated to the inclusion level of SFO . Despite the changes observed to the tissue fatty acid profile, there was no significant effect on growth suggesting that SFO is a suitable alternative to FO in diets for Atlantic salmon parr when fish meal is also included. Fish Shellfish Immunol, 2003 Sep, 15(3), 225 - 40 Measurement of Crassostrea gigas hemocyte oxidative metabolism by flow cytometry and the inhibiting capacity of pathogenic vibrios; Lambert C et al.; A flow cytometric method to measure the production of oxidative metabolism products was adapted for use with Crassostrea gigas hemocytes . The method is based upon the oxidation, by hydrogen peroxide (H2O2), of intracellular 2',7'-dichlorofluorescin (DCFH) to green-fluorescent dichlorofluorescein . Activation of the respiratory burst (RB) was tested using phorbol myristate acetate with no success . By contrast, activation by zymosan particles increased oxidation of DCFH in C . gigas hemocytes, mainly granulocytes, and optimization tests showed a good response with 20 zymosan particles per hemocyte . Anti-aggregant solution, used to prevent hemocytes from clumping during bleeding, inhibited the RB activity measured by DCFH oxidation . The flow cytometric method developed during this work was used to evaluate the DCFH oxidation-inhibiting capacity of four strains of vibrio bacteria, known or suspected to be pathogenic for bivalves. Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1111 - 4 Rhodocista pekingensis sp . nov., a cyst-forming phototrophic bacterium from a municipal wastewater treatment plant; Zhang D et al.; A novel bacterial species, Rhodocista pekingensis sp . nov., was isolated from a municipal wastewater treatment plant and characterized by polyphasic taxonomy . Cells of R . pekingensis were gram-negative, motile by a single polar flagellum, vibrioid to spiral, 0.6-0.8 microm in width and 0.8-1.5 microm in length . R-bodies were not observed . Phototrophically grown cells contained lamellar photosynthetic membranes and bacteriochlorophyll a . Cell growth was anaerobically phototrophic or aerobically chemoheterotrophic . Anaerobically grown cultures were pink-reddish . Thiamin and vitamin B12, but not biotin, were required for growth and 0.05% yeast extract stimulated growth . Acetate, lactate, pyruvate and succinate supported growth . Cysts were formed when butyrate was used as the sole carbon source . Molecular hydrogen (H2), but not sulfide or thiosulfate, was used as an electron donor . The major cellular quinone was Q-9 . The DNA G + C content of cells was 68.8 mol% . The type strain of Rhodocista pekingensis is 3-pT (=AS 1.2194T=JCM 11689T). Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 941 - 51 Phylogenetic taxonomy of the family Chlorobiaceae on the basis of 16S rRNA and fmo (Fenna-Matthews-Olson protein) gene sequences; Imhoff JF; A new taxonomy of the green sulfur bacteria is proposed, based on phylogenetic relationships determined using the sequences of the independent 16S rRNA and fmo (Fenna-Matthews-Olson protein) genes, and supported by the DNA G + C content and sequence signatures . Comparison of the traditional classification system for these bacteria with their phylogenetic relationship yielded a confusing picture, because properties used for classification (such as cell morphology, photosynthetic pigments and substrate utilization) do not concur with their phylogeny . Using the genetic information available, strains and species assigned to the genera Chlorobium, Pelodictyon and Prosthecochloris are considered, and the following changes are proposed . Pelodictyon luteolum is transferred to the genus Chlorobium as Chlorobium luteolum comb . nov . Pelodictyon clathratiforme and Pelodictyon phaeoclathratiforme are transferred to the genus Chlorobium and combined into one species, Chlorobium clathratiforme comb . nov . The name Pelodictyon will become a synonym of Chlorobium . Strains known as Chlorobium limicola subsp . thiosulfatophilum that have a low DNA G + C content (52-52.5 mol%) are treated as strains of Chlorobium limicola; those with a high DNA G + C content (58.1 mol%) are transferred to Chlorobaculum gen . nov., as Chlorobaculum thiosulfatiphilum sp . nov . Chlorobium tepidum is transferred to Chlorobaculum tepidum comb . nov., and defined as the type species of the genus Chlorobaculum . Strains assigned to Chlorobium phaeobacteroides, but phylogenetically distant from the type strain of this species, are assigned to Chlorobium limicola and to Chlorobaculum limnaeum sp . nov . Strains known as Chlorobium vibrioforme subsp . thiosulfatophilum are transferred to Chlorobaculum parvum sp . nov . Chlorobium chlorovibrioides is transferred to 'Chlorobaculum chlorovibrioides' comb . nov . The type strain of Chlorobium vibrioforme is phylogenetically related to Prosthecochloris, and is therefore transferred to this genus as Prosthecochloris vibrioformis comb . nov . Consequently, the name Chlorobium vibrioforme will become a synonym of Prosthecochloris vibrioformis, and other strains that were assigned to this species are now considered to belong to Chlorobium luteolum, Chlorobium phaeovibrioides and 'Chlorobaculum chlorovibrioides', according to their phylogenetic relatedness. Chin Med J (Engl), 2003 Jul, 116(7), 1115 - 7 Highly efficient expression, purification of recombinant LTB protein and its activity against mucosal immunoadjuvant by nasal immunization; Wang J et al.; OBJECTIVE: To develop an efficient expression, purification system of recombinant Escherichia coli heat-labile enterotoxin B subunit (rLTB) and study its activity against mucosal immunoadjuvant by nasal immunization . METHODS: A recombinant, pMMB68-LTB was generated by cloning the LTB cDNA fragment into an expression vector (pMMB68) and transformed it into the host strain marine vibrio VSP60 . The relevant target protein was identified using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot . Sephacryl S-100 gel filtration chromatography was carried out for purification of rLTB in engineering bacteria VSP60 . BALB/c mice received hen egg lysozyme (HEL) alone or combined with rLTB by nasal administration . After three times immunization, IgG and IgA antibody levels in serum or small intestine wash samples were determined using ELISA . RESULTS: rLTB protein was highly expressed in VSP60 . After gel filtration with Sephacryl S-100, the purity of rLTB reached 98.1%, the yield rate was about 52% . After immunization, IgG and IgA antibody responses specific to HEL in system and mucosa of HEL + rLTB groups were significantly increased, compared with the HEL alone group (P < 0.001) . CONCLUSIONS: A set of protocols for large-scale rLTB preparation has been established, which is simple, efficient and applicable . The rLTB protein we prepared was proved to be a powerful mucocal adjuvant, which could greatly enhance systemic and mucosal immune responses to nasally co-administered antigen. Emerg Infect Dis, 2003 Jul, 9(7), 810 - 4 Molecular epidemiology of O139 Vibrio cholerae: mutation, lateral gene transfer, and founder flush; Garg P et al.; Vibrio cholerae in O-group 139 was first isolated in 1992 and by 1993 had been found throughout the Indian subcontinent . This epidemic expansion probably resulted from a single source after a lateral gene transfer (LGT) event that changed the serotype of an epidemic V . cholerae O1 El Tor strain to O139 . However, some studies found substantial genetic diversity, perhaps caused by multiple origins . To further explore the relatedness of O139 strains, we analyzed nine sequenced loci from 96 isolates from patients at the Infectious Diseases Hospital, Calcutta, from 1992 to 2000 . We found 64 novel alleles distributed among 51 sequence types . LGT events produced three times the number of nucleotide changes compared to mutation . In contrast to the traditional concept of epidemic spread of a homogeneous clone, the establishment of variant alleles generated by LGT during the rapid expansion of a clonal bacterial population may be a paradigm in infections and epidemics. Int J Oncol, 2003 Sep, 23(3), 769 - 74 Sialic acids linked to glycoconjugates of Fas regulate the caspase-9-dependent and mitochondria-mediated pathway of Fas-induced apoptosis in Jurkat T cell lymphoma; Suzuki O et al.; To clarify the functions of sialic acids linked to glycoconjugates of Fas in Fas-induced apoptosis, Jurkat T cells, untreated and treated with neuraminidase, were incubated with anti-Fas monoclonal antibody, CH11 . Apoptosis of Jurkat T cells induced by incubation with CH11 was enhanced by the pre-treatment with neuraminidase . By flow cytometry sialylated glycoconjugates were detected on the cell surface of Jurkat T cells using LFA lectin, which specifically reacts with sialic acid, and pre-treatment with Vibrio Cholerae neuraminidase resulted in desialylation of Jurkat cell surface glycoconjugates . The enhancement of Fas-induced apoptosis by pre-treatment with neuraminidase was inhibited by z-VAD-fmk, a broad caspase inhibitor, and Ac-LEHD-CHO, an inhibitor of caspase-9, but not by Ac-IETD-CHO an inhibitor of caspase-8 or 6, imipramine, an inhibitor of acidic sphingomyelinase, glutathione, an inhibitor of neutral sphingomyelinase and Fumonisin B1, an inhibitor of ceramide synthase . Mitochondrial membrane potentials (Deltapsim) measured with a Mitocapture assay kit demonstrated that the loss of Deltapsim involved in Fas-induced apoptosis was enhanced by pre-treatment with neuraminidase . Furthermore, Western blot analysis using polyclonal antibody (C-20) against Fas detected Fas at about 45 kDa, and pre-treatment with neuraminidase resulted in a reduction of the molecular weight of Fas of about 8 kDa . These data suggest that the enhancement of Fas-induced apoptosis by pre-treatment with neuraminidase was mediated by a caspase-9 dependent pathway closely associated with the loss of Deltapsim, not by activation of caspase-8, -6 or acidic and neutral sphingomyelinases, and that sialic acid linked to glycoconjugates of Fas may regulate Fas-induced apoptosis in human T cell lymphoma. Mol Cell, 2003 Jul, 12(1), 157 - 65 DNA binding and ToxR responsiveness by the wing domain of TcpP, an activator of virulence gene expression in Vibrio cholerae; Krukonis ES et al.; Virulence in Vibrio cholerae requires activation of toxT by two membrane-localized activators, TcpP and ToxR . We isolated 12 tcpP activation mutants that fell into two classes: class I mutants were inactive irrespective of the presence of ToxR, and class II mutants exhibited near wild-type activity when coexpressed with ToxR . Most class I mutants had lesions in the wing domain predicted by homology with the winged helix-turn-helix family of activators . Class I mutants bound promoter DNA poorly and were largely unable to interact with ToxR in a crosslinking assay, whereas class II mutants retained physical interaction with ToxR . One mutant constructed in vitro bound DNA poorly but nevertheless responded to ToxR by activating toxT and also maintained ToxR interaction . We propose that ToxR interaction, but not DNA binding, is essential for TcpP function and that the wing domain of TcpP enables contact with ToxR required for productive TcpP-RNA polymerase association. J Microbiol Immunol Infect, 2003 Jun, 36(2), 117 - 22 Independent prognostic factors for fatality in patients with invasive vibrio cholerae non-O1 infections; Ou TY et al.; To identify independent prognostic factors for fatality, 73 patients with a total of 75 episodes of invasive Vibrio . cholerae non-O1 infections treated from July 1998 through October 2001 at 2 medical centers were retrospectively studied . The demographic, laboratory, and clinical information of these patients were collected and analyzed . The overall mortality rate was 36% . Multivariate analysis revealed that severe liver cirrhosis (p=0.003; odds ratio {OR}, 14.12, with 95% confidence interval {CI} 2.49-79.91), malignancy (p=0.034; OR, 3.9, with 95% CI 1.11-13 . 7), and steroid use (p=0.011; OR, 12.37, with 95% CI 1.79-85.49) were independent risk factors for fatality . These findings suggest that patients at high risk of fatality should be hospitalized and aggressively treated when V . cholerae non-O1 infections develop, and that public education on how to avoid exposure to V . cholerae non-O1 is important for the high-risk population. J Microbiol Immunol Infect, 2003 Jun, 36(2), 81 - 8 Vibrio vulnificus infection: clinical manifestations, pathogenesis, and antimicrobial therapy; Chiang SR et al.; There has been a dramatic increase in the number of reported cases of infection due to Vibrio vulnificus in Taiwan . Although the organism has been etiologically implicated in a variety of clinical syndromes, most cases of V . vulnificus infection are categorized as primary bacteremia, skin and soft tissue infection . The mortality was up to 50% in septic patients, most of them dying within 48 h of admission . In most of the cases involving V . vulnificus infection have underlying disease, particularly liver cirrhosis . The pathogenesis may attribute to several virulent factors, such as lipopolysaccharide, capsular lipopolysaccharide, cytolysin, metalloprotease and siderophore . Tetracycline was suggested as the drug of choice based on an animal study . Our previous in vitro data showed that cefotaxime and minocycline acted synergistically in inhibiting V . vulnificus . Furthermore, another in vivo animal study indicated that therapy using combined with cefotaxime and minocycline was distinctly more advantageous than therapy with the single antibiotic regimen for the treatment of severe experimental murine V . vulnificus infection . Recently, we also demonstrated that the newer fluoroquinolones, as single agents were as effective as the combination therapy both in vitro and in vivo. Pediatr Infect Dis J, 2003 Jul, 22(7), 666 - 8 Vibrio vulnificus necrotizing fasciitis of the calf presenting with compartment syndrome; Miron D et al.; We describe a 17-year-old boy with congenital spherocytosis and iron overload who presented with compartment syndrome of the calf as the initial manifestation of Vibrio vulnificus infection after minor trauma in a contaminated fish pond . The disease was complicated by necrotizing fasciitis requiring above the knee amputation . Childhood diseases associated with iron overload pose an increased risk for complicated V . vulnificus infections. Zh Mikrobiol Epidemiol Immunobiol, 2003 May-Jun, (3), 81 - 2 {Antigenic specificity of Vibrio cholerae O139 nitrosoguanidine mutants}; Lobanov VV et al.; The action of nitrosoguanidine (NG) on the culture of V . cholerae O139 P-16064 resulted in the appearance of mutant 16064 NG6, not agglutinating with commercial diagnostic serum O139 . Its incapacity of agglutination was due to the sorption of the specific serum with strains V . cholerae O22 and R-variant RCA-385, which caused the loss of antibodies to common determinants . Experiments with the sorption of immune sera made it possible to suggest that one of the determinants of LPS O139, phosphate-galactose, was absent in NG mutant. Zh Mikrobiol Epidemiol Immunobiol, 2003 May-Jun, (3), 77 - 9 {Dependence of the expression of the biological features of Vibrio cholerae on the conditions of their cultivation}; Mironova AV et al.; The biological activity of toxigenic and non-toxigenic V . cholerae supernatants was found to depend on the cultivation medium . The use of iron-free tryptone medium made it possible to obtain supernatants of toxigenic V . cholerae with haemolytic activity and destructive action on passaged cell cultures . In the experimental infection of suckling rabbits the influence of the cultivation conditions of V . cholerae on the character and expression of their pathogenic properties was determined . The dissemination of V . cholerae into the internal organs of rabbits after their infection with both toxigenic and non-toxigenic strains correlated neither with the cultivation conditions of these strains, nor with the character of changes in the intestine of the infected animals. Zh Mikrobiol Epidemiol Immunobiol, 2003 May-Jun, (3), 16 - 21 {Effect of various amino acids and ammonium salts in a synthetic culture media on the cholera enterotoxin production}; Ovsova LM et al.; The influence of amino acids and ammonium salts on the production of cholera enterotoxin (CT) by 3 Vibrio cholerae strains of different biovars and serogroups was evaluated . As revealed in this study, toxin formation in each of the strains was quantitatively and qualitatively determined by their individual sets of amino acids . The amino acid compositions ensuring the maximum production of CT by the V . cholerae strains under study were formed . The use of ammonium salts as the only source of nitrogen in the composition of a synthetic nutrient medium for the accumulation of CT was shown to be inexpedient. Zh Mikrobiol Epidemiol Immunobiol, 2003 Mar-Apr, (2), 12 - 6 {Dynamics of the isolation and biological properties of Vibrio cholerae cultures, serogroups 01 and 0139, isolated from water reservoirs and sewage water on the territory of the city of Rostov-on-Don}; Mazrukho BL et al.; The dynamics of the isolation of V . cholerae cultures from various water objects on the territory of Rostov-on-Don during the period of 1994-2001 was analyzed and biological properties of 14 such cultures were studied . In the absence of epidemic complications during the above-mentioned period, a growth in the amount of V . cholerae isolates, serogroups 01 and 0139, including toxigenic V . cholerae 01, was registered . The microbiological and epidemiological aspects of the monitoring of surface reservoirs and sewage were considered and the expediency of the profound and systematic study of its results for epidemiological surveillance on cholera was emphasized. An Sist Sanit Navar, 2000 Jan-Apr, 23(1), 85 - 107 {Dr . D . Nicasio Landa, official epidemic doctor during the cholera epidemic of 1854-1855}; Vines JJ; In this unpublished work from 1861, which we have transcribed, Dr . Nicasio Landa (1830-1891) is revealed to have been at the forefront in carrying out epidemiological studies in Spain . He traced the medical topography, or geographical pathology, of cholera by provinces, by means of calculating the incidence rate ("millesimal proportion of those infected") and lethality rates ("millesimal proportion of the dead") using the official data on the sick and deceased in the cholera epidemic of 1854-1855, published by the General Directorate of Health and Charity, as well as the data of the population census of 1857 . He worked with an infectionist conception and, besides tracing the epidemiological map, he proposed an association of cholera with the geographical constitution of the terrain in order to explain the anomalous distribution of the disease . That same "anomalous" distribution was repeated in the cholera epidemic in Spain in 1885, and in that of 1971, which is in accordance with the environmental characteristics of certain terrains that make possible the maintenance and persistence of the vibrio cholerae, according to recent research explaining the varied endemo-epidemic distribution of cholera. J Chromatogr A, 2003 Jun 20, 1002(1-2), 79 - 92 On-line high-performance liquid chromatography-mass spectrometric detection and quantification of N-acylhomoserine lactones, quorum sensing signal molecules, in the presence of biological matrices; Morin D et al.; A protocol using reversed-phase liquid chromatography coupled with positive-ion electrospray ionization and ion trap mass spectrometry is described for the identification and quantification of N-acylhomoserine lactones (HSLs) in crude cell-free supernatants of bacterial cultures . The HSLs are produced by gram-negative bacteria and act as intercellular signals inducing density-dependent gene expression . Compared with the multi-step procedures previously reported, which included chemical extraction, purification and the use of Escherichia coli HSL biosensors, this on-line LC-MS-MS method is fast and detects 11 HSLs . Its speed and robustness allow the analysis of a large number of samples without loss of performance (no signal variation for a control sample after 90 chromatographic injections) . The selectivity is based on the MS-MS fragment ions of the molecular {M+H}- ions and on their relative intensities . For quantification, the m/z 102 ion, specific for the lactone ring and detected with a good signal-to-noise ratio, allows low detection limits even in complex matrix samples (0.28 up to 9.3 pmol) . Moreover, this method allows the quantification of 11 HSLs whatever their chemical structure, substituted or not . The protocol was applied to Vibrio vulnificus, a marine bacterium . Six HSLs were detected and quantified with relative standard deviations for repeatability of < 10%. J Ind Microbiol Biotechnol, 2003 Jul, 30(7), 433 - 9 Epub 2003 Jul 23. Antibiotic activity of lectins from marine algae against marine vibrios; Liao WR et al.; Saline and aqueous ethanol extracts of marine algae and the lectins from two red algal species were assayed for their antibiotic activity against marine vibrios . Experimental studies were also carried out on the influence of environmental factors on such activity, using batch cultures . The results indicated that many of the saline extracts of the algal species were active and that the activity was selective against those vibrios assayed . The algal extracts were active against Vibrio pelagius and the fish pathogen V . vulnificus, but inactive against V . neresis . Algal lectins from Eucheuma serra (ESA) and Galaxaura marginata (GMA) strongly inhibited V . vulnificus but were inactive against the other two vibrios . The antibacterial activity of algal extracts was inhibited by pretreatment with various sugars and glycoprotein . Extracts of the two red algae, E . serra and Pterocladia capillacea, in saline and aqueous ethanol, inhibited markedly the growth rate of V . vulnificus at very low concentrations . Culture results indicated that metabolites active against V . vulnificus were invariably produced in P . capillacea over a wide range of temperature, light intensity, and nutritional conditions . Enhanced antibacterial activity occurred when P . capillacea was grown under higher irradiance, severe nutrient stress and moderate temperature (20 degrees C), reflecting the specific antibiotic characteristics of this alga . The strong antibiotic activity of lectins towards fish pathogenic bacteria reveals one of the important roles played by algal lectins, as well as the potential high economic value of those marine algae assayed for aquaculture and for biomedical purposes. EMBO J, 2003 Aug 1, 22(15), 4014 - 25 DNA binding and cleavage by the periplasmic nuclease Vvn: a novel structure with a known active site; Li CL et al.; The Vibrio vulnificus nuclease, Vvn, is a non-specific periplasmic nuclease capable of digesting DNA and RNA . The crystal structure of Vvn and that of Vvn mutant H80A in complex with DNA were resolved at 2.3 A resolution . Vvn has a novel mixed alpha/beta topology containing four disulfide bridges, suggesting that Vvn is not active under reducing conditions in the cytoplasm . The overall structure of Vvn shows no similarity to other endonucleases; however, a known 'betabetaalpha-metal' motif is identified in the central cleft region . The crystal structure of the mutant Vvn-DNA complex demonstrates that Vvn binds mainly at the minor groove of DNA, resulting in duplex bending towards the major groove by approximately 20 degrees . Only the DNA phosphate backbones make hydrogen bonds with Vvn, suggesting a structural basis for its sequence-independent recognition of DNA and RNA . Based on the enzyme-substrate and enzyme-product structures observed in the mutant Vvn-DNA crystals, a catalytic mechanism is proposed . This structural study suggests that Vvn hydrolyzes DNA by a general single-metal ion mechanism, and indicates how non-specific DNA-binding proteins may recognize DNA. J Biol Chem, 2003 Oct 3, 278(40), 39143 - 54 Epub 2003 Jul 22. Purification and characterization of three members of the photolyase/cryptochrome family glue-light photoreceptors from Vibrio cholerae; Worthington EN et al.; The sequence of Vibrio cholerae genome revealed three genes belonging to the photolyase/cryptochrome blue-light photoreceptor family . The proteins encoded by the three genes were purified and characterized . All three proteins contain folate and flavin cofactors and have absorption peaks in the range of 350-500 nm . Only one of the three, VcPhr, is a photolyase specific for cyclobutane pyrimidine dimers . The other two are cryptochromes and were designated VcCry1 and VcCry2, respectively . Mutation of phr abolishes photoreactivation of UV-induced killing, whereas mutations in cry1 and cry2 do not affect photorepair activity . VcCry1 exhibits some unique features . Of all cryptochromes characterized to date, it is the only one that contains stoichiometric amounts of both chromophores and retains its flavin cofactor in the two-electron reduced FADH2 form . In addition, VcCry1 exhibits RNA binding activity and co-purifies with an RNA of 60-70 nucleotides in length. J Biol Chem, 2003 Oct 3, 278(40), 38470 - 5 Epub 2003 Jul 23. Unfolding of Vibrio cholerae hemolysin induces oligomerization of the toxin monomer; Chattopadhyay K et al.; Vibrio cholerae hemolysin (HlyA) is a pore-forming toxin that exists in two stable forms: a hemolytically active water-soluble monomer with a native molecular weight of 65,000 and a hemolytically inactive SDS-stable heptamer with the configuration of a transmembrane diffusion channel . Transformation of the monomer into the oligomer is spontaneous but very slow in the absence of interaction with specific membrane components like cholesterol and sphingolipids . In this report, we show that mild disruption of the native tertiary structure of HlyA by 1.75 M urea triggered rapid and quantitative conversion of the monomer to an oligomer . Furthermore, the HlyA monomer when unfolded in 8 M urea refolded and reconstituted on renaturation into the oligomer biochemically and functionally similar to the heptamer formed in target lipid bilayer, suggesting that the HlyA polypeptide had a strong propensity to adopt the oligomer as the stable native state in preference to the monomer . On the basis of our results, we propose that (a) the hemolytically active HlyA monomer represents a quasi-stable conformation corresponding to a local free energy minimum and the transmembrane heptameric pore represents a stable conformation corresponding to an absolute free energy minimum and (b) any perturbation of the native tertiary structure of the HlyA monomer causing re