Infect Immun, 2001 Oct, 69(10), 6091 - 101 The toxR gene of Vibrio (Listonella) anguillarum controls expression of the major outer membrane proteins but not virulence in a natural host model; Okuda J et al.; To examine the hypothesis that the ancestral role of the toxR gene in the family Vibrionaceae is control of the expression of outer membrane protein (OMP)-encoding genes for adaptation to environmental change, we investigated the role of the toxR gene in Vibrio anguillarum, an important fish pathogen . The toxR gene of V . angullarum (Va-toxR) was cloned from strain PT-87050 isolated from diseased ayu (Plecoglossus altivelis), and the sequence was analyzed . The toxR sequence was 63 to 51% identical to those reported for other species of the family Vibrionaceae . Distribution of the Va-toxR gene sequence in V . anguillarum strains of various serotypes was confirmed by using DNA probe and PCR methods . An isogenic toxR mutant of V . anguillarum PT-24, isolated from diseased ayu, was constructed by using an allelic exchange method . The wild-type strain and the toxR mutant did not differ in the ability to produce a protease(s) and a hemolysin(s) or in pathogenicity for ayu when examined by the intramuscular injection and immersion methods . A 35-kDa major OMP was not produced by the toxR mutant . However, a 46-kDa OMP was hardly detected in the wild-type strain but was produced as the major OMP by the toxR mutant . For the toxR mutant, the MICs of two beta-lactam antibiotics were higher and the minimum bactericidal concentration of sodium dodecyl sulfate was lower than for the wild-type strain . Analysis of the N-terminal amino acid sequences of the 35- and 46-kDa OMPs indicated that these proteins are the porin-like OMPs and are related to the toxR-regulated major OMPs of the family Vibrionaceae . The results indicate that the toxR gene is not involved in virulence expression in V . anguillarum PT-24 and that toxR regulation of major OMPs is universal in the family Vibrionaceae . These results support the hypothesis that the ancestral role of the toxR gene is regulation of OMP gene expression and that only in some Vibrio species has ToxR been appropriated for the regulation of a virulence gene(s). Infect Immun, 2001 Oct, 69(10), 6084 - 90 Diminished diarrheal response to Vibrio cholerae strains carrying the replicative form of the CTX(Phi) genome instead of CTX(Phi) lysogens in adult rabbits; Faruque SM et al.; Toxigenic Vibrio cholerae strains are lysogens of CTX(Phi), a filamentous bacteriophage which encodes cholera toxin (CT) . Following infection of recipient V . cholerae cells by CTX(Phi), the phage genome either integrates into the host chromosome at a specific attachment site (attRS) or exists as a replicative-form (RF) plasmid . We infected naturally occurring attRS-negative nontoxigenic V . cholerae or attenuated (CTX(-) attRS negative) derivatives of wild-type toxigenic strains with CTX(Phi) and examined the diarrheagenic potential of the strains carrying the RF of the CTX(Phi) genome using the adult rabbit diarrhea model . Under laboratory conditions, strains carrying the RF of CTX(Phi) produced more CT than corresponding lysogens as assayed by a G(M1)-based enzyme-linked immunosorbent assay and by fluid accumulation in ligated ileal loops of rabbits . However, when tested for diarrhea in rabbits, the attRS-negative strains (which carried the CTX(Phi) genome as the RF) were either negative or produced mild diarrhea, whereas the attRS-positive strains with integrated CTX(Phi) produced severe fatal diarrhea . Analysis of the strains after intestinal passage showed that the attRS-negative strains lost the phage genome at approximately a fivefold higher frequency than under in vitro conditions, and 75 to 90% of cells recovered from challenged rabbits after 24 h were CT negative . These results suggested that strains carrying the RF of CTX(Phi) are unable to cause severe disease due to rapid loss of the phage in vivo, and the gastrointestinal environment thus provides selection of toxigenic strains with an integrated CTX(Phi) genome . These results may have implications for the development of live V . cholerae vaccine candidates impaired in chromosomal integration of CTX(Phi) . These findings may also contribute to understanding of the etiology of diarrhea occasionally associated with nontoxigenic V . cholerae strains. Zh Mikrobiol Epidemiol Immunobiol, 2001 May-Jun, (3), 69 - 72 {Comparative study of the diagnostic value of new cholera eltor bacteriophages ctx+, ctx-, and KhDF-3, 4, and 5}; Saiapina LV et al.; The comparative evaluation of the diagnostic value of new cholera eltor bacteriophages ctx+ and ctx-, as well as monophages X{symbol: see text}-3, 4, 5, demonstrated their high activity and specificity . Using of these bacteriophages epidemic potential of 95% Vibrio cholerae eltor strains ctx+ and 84.5% of V . cholerae eltor stains ctx- was determined . Commercial monophages X{symbol: see text}-3, 4, 5 were inferior to bacteriophages ctx+ and ctx- in their diagnostic value: only 55% of strains having gene ctxAB were found to be epidemically dangerous, i.e . 45% of strains capable of causing the disease were not detected . On the basis of the results obtained in this investigation cholera eltor bacteriophages ctx+ and ctx- were recommended for introduction into practical use, while further production of cholera diagnostic monophages X{symbol: see text}-3, 4, 5 was recommended to be stopped. Appl Microbiol Biotechnol, 2001 Aug, 56(3-4), 448 - 52 Isolation and characterization of a bacterium that produces hydrocarbons extracellularly which are equivalent to light oil; Park MO et al.; A halotorelant bacterial strain that produces a significant amount of lipids from short-chain fatty acids was isolated from the sludge of a sewage disposal plant . This strain displayed a significant extracellular accumulation of lipids . The yield of lipids including hydrocarbons was highest (120% of cell dry weight) at the end of the linear growth phase . Fractionation of the lipids using thin-layer chromatography and subsequent gas chromatography showed that hydrocarbons were also obtained following an increase in total lipids . Their yield was the highest (50% of cell dry weight) in the linear growth phase . Additional analysis using infrared absorption spectrum and gas chromatography-mass spectrometry showed that the hydrocarbon fraction was composed of alkanes, such as C15H32, C18H38, C21H44, C22H46 and C24H50 . Homology analysis of the 16s rDNA sequence as well as studies of the morphological and physiological characteristics indicated that the bacterium is a strain of Vibrio furnissii. Zh Mikrobiol Epidemiol Immunobiol, 2001 Mar-Apr, (2), 6 - 9 {Comparative pathomorphological assessment of the action of Vibrio cholerae serogroups 01 and 0139}; Isupov IV et al.; The pathomorphological picture of experimental infection caused by the infective agent of cholera was shown to have some specific features observed in infections caused by vibrios belonging to the serogroups under study . Infection caused by V . cholerae of serogroup O139 induced some morphological changes in the gastrointestinal tract which were quite characteristic of this disease, but inflammatory changes with the prevalence of proliferative infiltrative processes came to the foreground simultaneously with less developed processes of edema and dystrophic lesions of enterocytes . These specific morphological features in animals infected with V . cholerae of serogroup O139 appeared to be probably due to the production of new surface structures by these strains. Zh Mikrobiol Epidemiol Immunobiol, 2001 Mar-Apr, (2), 3 - 6 {Isolation and phenotyping of Vibrio cholerae strains with the enhanced production of main protective antigens}; Zadnova SP et al.; Two V . cholerae strains of classical biovar, 2414 (serovar Ogawa) and 2415 (serovar Inaba), with of increased production of main protective antigens--cholera toxin, toxin-coregulated pili of adhesion (TCP), outer membrane protein OmpU, as well as phospholipases and proteases, have been detected among natural and recombinant strains under study . A simultaneous increase in the production of the above-mentioned main immunogenicity factors in strains 2414 and 2415 is seemingly linked with the presence of recombinant plasmid pCT105 with cloned genes of cholera toxin in these microbial cells . As the result of plasmid-chromosomal relationships, this plasmid probably ensures the effective expression of global regulating gene toxR . The strains capable of the hyperproduction of cholera toxin, TCP and protein OmpU may be used for the manufacture of more effective chemical vaccine (choleragen-toxoid). Zh Mikrobiol Epidemiol Immunobiol, 2001 Mar-Apr, (2), 25 - 9 {Analysis of toxin genetic determinants of Vibrio cholerae virulence cassette and neuraminidase with DNA probes}; Monakhova EV et al.; V . cholerae strains of different origin have been studied for the presence of cholera toxin genes (vct), the proximal part of the virulence cassette including genes zot, ace and orfU, as well as neuraminidase genes (neu), in their genomes with the use of molecular DNA probes . The possibility, in principle, for some strains to lose only a part of their virulence cassette (gene vct), while retaining its proximal part has been shown . In most cases such strains are isolated from patients with diarrhea of different severity and may probably play some etiological role, provided that the expression of the genes of additional toxins of the virulence cassette occurs . The gene expressing neuraminidase which facilitates the penetration of cholera toxin into the epithelial cells of the intestine is always present in vct+ strains and may be absent in vct- strains . The absence of genetic relationship between neuraminidases in V . cholerae O139 and V . cholerae O1 and non-O1 (non-O139) has been confirmed . The problems in connection with the integration and deletion of genetic determinants of V . cholerae virulence factors are discussed. J Bacteriol, 2001 Oct, 183(19), 5762 - 7 Residue aspartate-147 from the third transmembrane region of Na(+)/H(+) antiporter NhaB of Vibrio alginolyticus plays a role in its activity; Nakamura T et al.; NhaB is a bacterial Na(+)/H(+) antiporter with unique topology . The pH dependence of NhaB from Vibrio alginolyticus differs from that of the Escherichia coli NhaB homolog . Replacement of Asp-147 with Glu made high H(+) concentrations a requirement for the NhaB activity . Replacement of Asp-147 with neutral amino acids inactivated NhaB. J Bacteriol, 2001 Oct, 183(19), 5751 - 5 Identification, characterization, and functional analysis of a gene encoding the ferric uptake regulation protein in Bartonella species; Park SY et al.; Environmental iron concentrations coordinately regulate transcription of genes involved in iron acquisition and virulence via the ferric uptake regulation (fur) system . We identified and sequenced the fur gene and flanking regions of three Bartonella species . The most notable difference between Bartonella Fur and other Fur proteins was a substantially higher predicted isoelectric point . No promoter activity or Fur autoregulation was detected using a gfp reporter gene fused to the 204 nucleotides immediately upstream of the Bartonella fur gene . Bartonella henselae fur gene expression complemented a Vibrio cholerae fur mutant. Syst Appl Microbiol, 1997 Jan, 20(1), 133 - 6 A low molecular weight artificial RNA of unique size with multiple probe target regions; Pitulle C et al.; Artificial RNAs (aRNAs) containing novel sequence segments embedded in a deletion mutant of Vibrio proteolyticus 5S rRNA have previously been shown to be expressed from a plasmid borne growth rate regulated promoter in E . coli . These aRNAs accumulate to high levels and their detection is a promising tool for studies in molecular microbial ecology and in environmental monitoring . Herein a new construct is described which illustrates the versatility of detection that is possible with aRNAs . This 3xPen aRNA construct carries a 72 nucleotide insert with three copies of a unique 17 base probe target sequence . This aRNA is 160 nucleotides in length and again accumulates to high levels in the E . coli cytoplasm without incorporating into ribosomes . The 3xPen aRNA illustrates two improvements in detection . First, by appropriate selection of insert size, we obtained an aRNA which provides a unique and hence, easily quantifiable peak, on a high resolution gel profile of low molecular weight RNAs . Second, the existence of multiple probe targets results in a nearly commensurate increase in signal when detection is by hybridization . These aRNAs are naturally amplified and carry sequence segments that are not found in known rRNA sequences . It thus may be possible to detect them directly . An experimental step involving RT-PCR or PCR amplification of the gene could therefore be avoided. Syst Appl Microbiol, 1993, 16, 280 - 6 A prototype stable RNA identification cassette for monitoring plasmids of genetically engineered microorganisms; Hedenstierna KO et al.; A prototype stable RNA identification cassette for monitoring genetically engineered plasmids carried by strains of Escherichia coli has been developed . The cassette consists of a Vibrio proteolyticus 5S ribosomal RNA (rRNA) gene surrounded by promoters and terminators from the rrnB operon of Escherischia coli . The identifier RNA is expressed and successfully processed so that approximately 30% of the 5S rRNA isolated from either whole cells or 70S ribosomes is of the V . proteolyticus type . Cells carrying the identifier are readily detectable by hybridization . Accurate measurements show that the identification cassette has little effect on fitness compared to a strain containing an analogous plasmid carrying wild type E . coli 5S rRNA, and the V . proteolyticus 5S rRNA gene is not inactivated after prolonged growth . These results demonstrate the feasibility of developing small standardized identification cassettes that can utilize already existing highly sensitive rRNA detection methods . Cassettes of this type could in principle be incorporated into either the engineered regions of recombinant plasmids or their hosts. Appl Environ Microbiol, 1988 Jan, 54(1), 250 - 6 34S/32S fractionation in sulfur cycles catalyzed by anaerobic bacteria; Fry B et al.; Stable isotopic distributions in the sulfur cycle were studied with pure and mixed cultures of the anaerobic bacteria, Chlorobium vibrioforme and Desulfovibrio vulgaris . D . vulgaris and C . vibrioforme can catalyze three reactions constituting a complete anaerobic sulfur cycle: reduction of sulfate to sulfide (D . vulgaris), oxidation of sulfide to elemental sulfur (C . vibrioforme), and oxidation of sulfur to sulfate (C . vibrioforme) . In all experiments, the first and last reactions favored concentration of the light 32S isotope in products (isotopic fractionation factor epsilon = -7.2 and -1.7%, respectively), whereas oxidation of sulfide favored concentration of the heavy 34S isotope in products (epsilon = +1.7%) . Experimental results and model calculations suggest that elemental sulfur enriched in 34S versus sulfide may be a biogeochemical marker for the presence of sulfide-oxidizing bacteria in modern and ancient environments. J Photochem Photobiol B, 1992 Aug 14, 15(1-2), 171 - 9 Förster energy transfer in chlorosomes of green photosynthetic bacteria; Causgrove TP et al.; Energy transfer properties of whole cells and chlorosome antenna complexes isolated from the green sulfur bacteria Chlorobium limicola (containing bacteriochlorophyll c), Chlorobium vibrioforme (containing bacteriochlorophyll d) and Pelodictyon phaeoclathratiforme (containing bacteriochlorophyll e) were measured . The spectral overlap of the major chlorosome pigment (bacteriochlorophyll c, d or, e) with the bacteriochlorophyll a B795 chlorosome baseplate pigment is greatest for bacteriochlorophyll c and smallest for bacteriochlorophyll e . The absorbance and fluorescence spectra of isolated chlorosomes were measured, fitted to gaussian curves and the overlap factors with B795 calculated . Energy transfer times from the bacteriochlorophyll c, d or e to B795 were measured in whole cells and the results interpreted in terms of the Forster theory of energy transfer. Biochim Biophys Acta, 1990 Feb 22, 1015(3), 457 - 63 Effects of oxidants and reductants on the efficiency of excitation transfer in green photosynthetic bacteria; Wang J et al.; The efficiency of energy transfer in chlorosome antennas in the green sulfur bacteria Chlorobium vibrioforme and Chlorobium limicola was found to be highly sensitive to the redox potential of the suspension . Energy transfer efficiencies were measured by comparing the absorption spectrum of the bacteriochlorophyll c or d pigments in the chlorosome to the excitation spectrum for fluorescence arising from the chlorosome baseplate and membrane-bound antenna complexes . The efficiency of energy transfer approaches 100% at low redox potentials induced by addition of sodium dithionite or other strong reductants, and is lowered to 10-20% under aerobic conditions or after addition of a variety of membrane-permeable oxidizing agents . The redox effect on energy transfer is observed in whole cells, isolated membranes and purified chlorosomes, indicating that the modulation of energy transfer efficiency arises within the antenna complexes and is not directly mediated by the redox state of the reaction center . It is proposed that chlorosomes contain a component that acts as a highly quenching center in its oxidized state, but is an inefficient quencher when reduced by endogenous or exogenous reductants . This effect may be a control mechanism that prevents cellular damage resulting from reaction of oxygen with reduced low-potential electron acceptors found in the green sulfur bacteria . The redox modulation effect is not observed in the green gliding bacterium Chloroflexus aurantiacus, which contains chlorosomes but does not contain low-potential electron acceptors. Mol Gen Mikrobiol Virusol, 2001, (3), 23 - 8 {Prevalence of major virulence genes among various Vibrio cholerae El-TOR strains for evaluating their epidemiological significance}; Smirnova NI et al.; Specific oligonucleotide primers were chosen for identifying the fragments of the four major virulence genes of V . cholerae eltor (ctxA, tcpA, toxR, and hap) using the polymerase chain reaction (PCR) . In order to estimate the efficiency of complex PCR testing of V . cholerae for evaluation of their epidemiological significance, a collection of 80 V . cholerae eltor strains with known virulence was selected, whose most important specific features had been studied previously . The hap was appropriate species-specific gene making it possible to detect V . cholerae strains regardless of their virulence . The most complete and objective data for evaluating the epidemic significance can be obtained by detecting the presence of three virulence genes (ctxA, tcpA, and toxR) in their chromosome . The prevalence of the above four genes in various V . cholerae strains isolated from the environment during epidemic and non-epidemic periods was studied. J Biol Chem, 2001 Nov 23, 276(47), 43645 - 52 Epub 2001 Aug 31. A secreted aminopeptidase of Pseudomonas aeruginosa . Identification, primary structure, and relationship to other aminopeptidases; Cahan R et al.; Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains . The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable . The apparent molecular mass of the enzyme was approximately 56 kDa; hence, it was designated AP(56) . Heating (70 degrees C) of the partially purified aminopeptidase preparations led to the conversion of AP(56) to a approximately 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB) . The pH optimum for Leu-pNA hydrolysis by AP(28) was 8.5 . This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme . Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate . The sequences of the first 20 residues of AP(56) and AP(28) were determined . A search of the P . aeruginosa genomic data base revealed a perfect match of these sequences with positions 39-58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase . A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, approximately 35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases . The residues potentially involved in zinc coordination were conserved in all these proteins . Thus, P . aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases. J Mol Biol, 2001 Aug 31, 311(5), 1001 - 9 Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts; Daniell H et al.; The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens . Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome) . However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen . PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome . Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB . Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts . In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts . Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses . Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible . Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants . Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts . Microbiol Immunol, 2001, 45(7), 543 - 7 Identification of a H+/glucose and galactose symporter gene glt from Xanthomonas oryzae pv . oryzae; Tsuge S et al.; We identified a glucose and galactose transporter gene from the plant-pathogenic bacterium Xanthomonas oryzae pv . oryzae . Sequence analysis indicated that the gene, named glt, encoded a polypeptide of 592 amino acid residues and the product was significantly homologous with members of the Na+/glucose cotransporter (SGLT) family from mammalian and bacterial origin, especially with vSGLT from Vibrio parahaemolyticus (50% identity) . GLT functioned as a glucose and galactose transporter in an Escherichia coli mutant deficient in glucose and galactose transport activity . A protonophore inhibited the transport activity, suggesting that GLT is a H+-coupled glucose/galactose symporter. Microbiol Mol Biol Rev, 2001 Sep, 65(3), 445 - 62, table of contents Polar flagellar motility of the Vibrionaceae; McCarter LL; Polar flagella of Vibrio species can rotate at speeds as high as 100,000 rpm and effectively propel the bacteria in liquid as fast as 60 microm/s . The sodium motive force powers rotation of the filament, which acts as a propeller . The filament is complex, composed of multiple subunits, and sheathed by an extension of the cell outer membrane . The regulatory circuitry controlling expression of the polar flagellar genes of members of the Vibrionaceae is different from the peritrichous system of enteric bacteria or the polar system of Caulobacter crescentus . The scheme of gene control is also pertinent to other members of the gamma purple bacteria, in particular to Pseudomonas species . This review uses the framework of the polar flagellar system of Vibrio parahaemolyticus to provide a synthesis of what is known about polar motility systems of the Vibrionaceae . In addition to its propulsive role, the single polar flagellum of V . parahaemolyticus is believed to act as a tactile sensor controlling surface-induced gene expression . Under conditions that impede rotation of the polar flagellum, an alternate, lateral flagellar motility system is induced that enables movement through viscous environments and over surfaces . Although the dual flagellar systems possess no shared structural components and although distinct type III secretion systems direct the simultaneous placement and assembly of polar and lateral organelles, movement is coordinated by shared chemotaxis machinery. J Clin Microbiol, 2001 Sep, 39(9), 3241 - 6 Concomitant infection of enterotoxigenic Escherichia coli in an outbreak of cholera caused by Vibrio cholerae O1 and O139 in Ahmedabad, India; Chakraborty S et al.; In Ahmedabad, a major city in the state of Gujarat, India, an outbreak of acute secretory diarrhea caused by Vibrio cholerae O1 Ogawa El Tor, V . cholerae O139, and multiple serotypes of enterotoxigenic Escherichia coli (ETEC) occurred in January 2000 . All of the representative V . cholerae O1 and O139 isolates examined harbored the ctxA gene (encoding the A subunit of cholera toxin) and the El Tor variant of the tcpA gene (encoding toxin-coregulated pilus) . ETEC isolates of different serotypes were positive for the elt gene, encoding heat-labile enterotoxin . To further understand the molecular characteristics of the pathogens, representative isolates were examined by ribotyping and pulsed-field gel electrophoresis (PFGE) . Ribotyping showed that the isolates of V . cholerae O1 Ogawa exhibited a pattern identical to that of the prevailing clone of O1 in areas where cholera is endemic in India, and all of the O139 isolates were identical to the BII clone of V . cholerae O139 . PFGE of the representative O1 Ogawa isolates exhibited an identical pattern, comparable to the H pattern of the new clone of O1 reported in Calcutta, India . PFGE analysis of the V . cholerae O139 isolates showed identical patterns, but these differed from the PFGE patterns of O139 isolates reported during 1992 to 1997 in Calcutta . ETEC isolates showed genetic heterogeneity among isolates belonging to the same serotype, although the identical PFGE pattern was also observed among ETEC isolates of different serotypes . Antibiograms of the isolates were unusual, because all of the O139 isolates were resistant to nalidixic acid . Likewise, all of the E . coli isolates showed resistance to ciprofloxacin, norfloxacin, and nalidixic acid . This is a unique outbreak, and we believe that it is the first in which V . cholerae and ETEC were concomitantly involved. Biochemistry, 2001 Sep 4, 40(35), 10655 - 63 Heterocycle formation in vibriobactin biosynthesis: alternative substrate utilization and identification of a condensed intermediate; Marshall CG et al.; The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit nonribosomal peptide synthetase complex, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold . We have tested the ability of the six-domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analogues and found the beta-functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihydrobenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclization to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N(5) and N(9) positions (condensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) analogues of vibriobactin . When L-threonyl-S-pantetheine or L-threonyl-S-(N-acetyl)cysteamine was used as a small-molecule thioester analogue of the threonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step . This clean separation of condensation from cyclization validates a two-stage mechanism for threonyl, seryl, and cysteinyl heterocyclization domains in siderophore and antibiotic synthetases . Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl carrier protein (PCP)-C2, suggesting an important role for the protein scaffold component of the heterocyclization acceptor substrate . We also examined heterocyclization donor substrate specificity at the level of acyl group and protein scaffold and observed intolerance for substitution at either position. Eur J Pharm Biopharm, 2001 Sep, 52(2), 145 - 50 Enhancing the permeation of marker compounds and enaminone anticonvulsants across Caco-2 monolayers by modulating tight junctions using zonula occludens toxin; Cox DS et al.; Zonula occludens toxin (Zot), a protein elaborated from Vibrio cholerae, has been shown to be capable of reversibly opening tight junctions between intestinal cells The objective of this study was to examine the effect of Zot on the flux of various molecules across Caco-2 cell monolayers . In addition, the transport of a series of anticonvulsants, the enaminones was also evaluated in the presence of Zot . The flux of {(14)C}mannitol, {(14)C}inulin and various enaminones across Caco-2 cell monolayers (n=6) was examined after pre-incubation for 1h with Zot (0 or 4000ng/ml) or phosphate-buffered saline (PBS) . At the end of the incubation period, the flux of radiolabeled compounds or enaminones (1x10(-4)M) was assessed over a 2-h period . In addition, dose-response studies with Zot (0, 1000, 2000 or 4000ng/ml) were performed using mannitol . The flux of both mannitol and inulin significantly increased (P<0.05) in the presence of Zot . The transport of the enaminones with Zot ranged from 9.42 to 26.83x10(-5)cm/s vs . 4.68 to 13.83x10(-5)cm/s without Zot . Zot significantly increased the transport of all agents tested . This suggests that the co-administration of drugs with Zot may be a useful delivery strategy to increase the intestinal permeability and hence oral absorption of poorly bioavailable agents. J Infect Dis, 2001 Sep 15, 184(6), 799 - 802 Epub 2001 Aug 07. Cholera in the United States, 1995-2000: trends at the end of the twentieth century; Steinberg EB et al.; To evaluate recent trends in cholera in the United States, surveillance data from all cases of laboratory-confirmed toxigenic Vibrio cholerae O1 and O139 infection reported to the Centers for Disease Control and Prevention between 1995 and 2000 were reviewed . Sixty-one cases of cholera, all caused by V . cholerae O1, were reported . There was 1 death, and 35 (57%) of the patients were hospitalized . Thirty-seven (61%) infections were acquired outside the United States; 14 (23%) were acquired through undercooked seafood consumed in the United States, 2 (3%) were acquired through sliced cantaloupe contaminated by an asymptomatically infected food handler, and no source was identified for 8 (13%) infections . The proportion of travel-associated infections resistant to trimethoprim-sulfamethoxazole, sulfisoxazole, streptomycin, and furazolidone increased from 7 (8%) of 88 in 1990-1994 to 11 (31%) of 35 in 1995-2000 . Foreign travel and undercooked seafood continue to account for most US cholera cases . Antimicrobial resistance has increased among V . cholerae O1 strains isolated from ill travelers. Carbohydr Res, 2001 Aug 30, 334(3), 195 - 205 Synthetic explorations towards 3-deoxy-3-fluoro derivatives of D-perosamine; Poirot E et al.; Based on a literature precedent, preparation of methyl 4-azido-3,4,6-trideoxy-3-fluoro-alpha-D-mannopyranoside (18) was attempted via fluorination of methyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-altropyranoside with diethylaminosulfur trifluoride (DAST) . Contrary to expectations, the reaction took place with retention of configuration at the site of the fluorination yielding methyl 4-azido-2-O-benzyl-3,4,6-trideoxy-3-fluoro-alpha-D-altropyranoside . Treatment with DAST of methyl 4-azido-2-O-benzyl-4,6-dideoxy-alpha-D-allopyranoside (8), or its 2-(p-methoxybenzyl) analog 9 resulted in fluorination with inversion of configuration at position 3, to give the corresponding 3-deoxy-3-fluoro glucopyranosides 10 and 11, respectively . Accordingly, compound 18 was prepared from 11, by de-p-methoxybenzylation at O-2, followed by inversion of configuration at C-2 in the resulting methyl 4-azido-3,4,6-trideoxy-3-fluoro-alpha-D-glucopyranoside . The 2-O-methyl analog of 18 (19) was prepared by methylation of 18 . Compounds 18 and 19 were converted, conventionally, into the 3-fluoro analogs of the terminal determinants of the O-PS of Vibrio cholerae O:1, serotype Inaba and Ogawa, respectively. J Food Prot, 2001 Aug, 64(8), 1172 - 7 Heterogeneity of environmental, retail, and clinical isolates of Vibrio vulnificus as determined by lipopolysaccharide-specific monoclonal antibodies; Zuppardo AB et al.; The opportunistic pathogen Vibrio vulnificus expresses lipopolysaccharide (LPS) antigens on its outer membrane surface . A serological typing system was developed for these antigens, utilizing the discriminatory recognition of monoclonal antibodies (MAb) by ELISA . MAb were used to recognize five unique types of LPS-associated antigens for examination of clinical . environmental, and retail isolates of V . vulnificus . The overall serotype profile of the clinical isolates was significantly different (P < 0.05) from that of the environmental and retail isolates . A higher percentage of clinical isolates were typable (61%) compared to the environmental isolates (41%) and retail isolates (44%) . In particular, the percentage of serotype 1/5 among clinical isolates (33%), compared to that of environmental (9%) and retail (4%), was highly significant (P < 0.0001) . Among the environmental Gulf Coast isolates, there were differences in the prevalence of serotypes 2 and 3 (P < 0.05), depending on whether isolates were obtained from Louisiana or Alabama harvest sites . There were no statistically significant differences between the serotype profiles of Gulf and Atlantic Coast retail isolates despite the absence of serotype 1/5 from the Atlantic Coast . While some serotype diversity was detected in V . vulnificus isolated during different seasons, from different geographic locations, and at retail versus at harvest, there was no apparent concordance between any of the serotype distributions obtained from oysters versus that isolated clinically . The heterogeneity of environmental isolates and relative homogeneity among clinical isolates suggest that human risk may not be predicted on quantitative exposure alone. FEMS Microbiol Lett, 2001 Aug 7, 202(1), 79 - 83 Presence of high-affinity iron uptake systems in fish-isolated and environmental strains of Vibrio anguillarum serotype O3; Muino L et al.; This work describes the presence of high-affinity iron uptake systems in Vibrio anguillarum serotype O3 strains (subgroups A and B), isolated from diseased fish and environmental samples, as well as the presumptive effect of iron on their virulence . All strains demonstrated an ability to grow under iron-limiting conditions, production of catechol-type siderophores and synthesis of iron-regulated outer membrane proteins . However, clear differences were found depending on the isolation source, suggesting a more efficient iron uptake system in fish-isolated strains . Comparing the iron-regulated outer membrane protein profiles with those described for O1 and O2 serotypes, we found a protein which is specific for serotype O3, and another one that allows the differentiation of serotype O3 fish isolates from environmental strains . Moreover, only the strains showing this protein increased their virulence when iron was added to the inoculum in pathogenicity assays. Med J Malaysia, 2001 Mar, 56(1), 4 - 9 Detection of Vibrio cholerae 01 from aquatic environment in Sarawak; Norazah A et al.; The detection of Vibrio cholerae 01 from the aquatic environment of Daro and Bintulu in Sarawak was carried out following an outbreak of cholera . Conventional culture methods and detection of ctx gene by polymerase chain reaction technique were carried out on 80 water samples . Only one sample was positive by culture methods while 8 were positive by PCR . DNA finger printing by pulsed-field gel electrophoresis showed that the clinical isolates in Daro and Bintulu were genetically identical while the environmental isolate was closely related . Recovery of Vibrio cholerae by culture method is poor and newer methods of detection should be developed. Infect Immun, 2001 Sep, 69(9), 5943 - 8 Isolation and characterization of a Vibrio vulnificus mutant deficient in both extracellular metalloprotease and cytolysin; Fan JJ et al.; We isolated a Vibrio vulnificus mutant that was deficient in both metalloprotease and cytolysin by allelic exchange . The virulence of this mutant in mice and its cytotoxicity for HEp-2 cells were comparable to those of the wild-type strain, indicating that neither factor was essential for these properties . The cytolysin, but not the protease, seemed to be important for causing damage in the alimentary tract of the mice. Infect Immun, 2001 Sep, 69(9), 5864 - 73 Gene cluster for assembly of pilus colonization factor antigen III of enterotoxigenic Escherichia coli; Taniguchi T et al.; The assembly of pilus colonization factor antigen III (CFA/III) of enterotoxigenic Escherichia coli (ETEC) requires the processing of CFA/III major pilin (CofA) by a prepilin peptidase (CofP), similar to other type IV pilus formation systems . CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to a 20.5-kDa mature pilin by CofP which is predicted to be localized in the inner membrane . In the present experiment, we determined the nucleotide sequence of the whole region for CFA/III formation and identified a cluster of 14 genes, including cofA and cofP . Several proteins encoded by cof genes were similar to previously described proteins, such as the toxin-coregulated pili of Vibrio cholerae and the bundle-forming pili of enteropathogenic E . coli . The G+C content of the cof gene cluster was 37%, which was significantly lower than the average for the E . coli genome (50%) . The introduction of a recombinant plasmid containing the cof gene cluster into the E . coli K-12 strain conferred CFA/III biogenesis and the ability of adhesion to the human colon carcinoma cell line Caco-2 . This is the first report of a complete nucleotide sequence of the type IV pili found in human ETEC, and our results provide a useful model for studying the molecular mechanism of CFA/III biogenesis and the role of CFA/III in ETEC infection. Microbiol Immunol, 2001, 45(6), 417 - 24 Gene analysis of Vibrio cholerae NAGV14 pilus and its distribution; Kuroki H et al.; Adhesive pilus of Vibrio cholerae 034, strain NAGV14, was genetically analyzed . The deduced amino acid (aa) sequence of the major pilin structural gene (VcfA) was 67% homologous to the MshA pilin in the N-terminal region, but no homology was found in the C-terminal region which contained the antigenic epitopes . Upstream and downstream flanking regions examined were highly homologous to mshB and mshC of the MSHA (mannose-sensitive hemagglutinin) gene locus . A short leader sequence and a pair of cysteines near the C-terminus which are the characteristics of type 4a pilus family were found . The major pilin structural gene of NAGV14 was compared to that of a strain V10 producing non-adhesive pili . The deduced aa sequences showed 60% homology, and the distance between two cysteines in the C-terminal region was different . A total of 177 V . cholerae strains were investigated for the presence of a type 4 pilus gene locus by PCR, and 95% were positive . The major pilin gene of NAGV14 was detected in 4 of 93 V . cholerae non-O1, non-0139 strains tested, but none of the V . cholerae O1 and O139 (72 and 12 strains, respectively) . Our result suggested that a type 4 pilus gene locus similar to the MSHA gene locus is widely distributed among V . cholerae strains . We proposed naming this type 4 pilus gene locus the VCF (for V . cholerae flexible pili) gene locus. New Microbiol, 2001 Jul, 24(3), 273 - 80 Virulence factors in Vibrios and Aeromonads isolated from seafood; Scoglio ME et al.; Thirty-one isolates from seafood, identified as Aeromonas hydrophila (7), Aeromonas caviae (11), Vibrio parahaemolyticus (3), Vibrio fluvialis (5), Vibrio alginolytictus (3), Vibrio metschnikovii (1) and Vibrio damsela (1), were tested for possible virulence factors including extracellular hydrolytic enzymes, haemolysins, cytotoxins (VERO and HEp-2 cells) and adherence ability (HEp-2 cells) . All the A . hydrophila strains were beta-haemolytic and produced cytotoxins as well as one strain of V . fluvialis . A . hydrophila and A . caviae strains, frequently adhesive, showed both aggregative and diffusive patterns, while five Vibrio strains only (three V . fluvialis, one V . parahaemolyticus and one V . alginolyticus) were adhesive with an aggregative pattern. Microbiology, 2001 Aug, 147(Pt 8), 2089 - 101 Endogenous isolation of replicon probes for assessing plasmid ecology of marine sediment microbial communities; Cook MA et al.; Six functional replication origins (repGA14, repGA33, repGA70, repSD41, repSD164 and repSD172), obtained from endogenously isolated, broad-host-range (BHR) marine plasmids ranging in size from 5 to 60 kb, were used to determine plasmid occurrence in three coastal marine sediment sites (in California, Georgia and South Carolina, USA) . The plasmid-specific replicons were isolated from plasmid-bearing marine sediment bacteria belonging to the alpha and gamma subclasses of the Proteobacteria . The plasmid sources of the endogenous replicons were considered to be cryptic due to a lack of identifiable phenotypic traits . The putative Rep proteins from a number of these replicons showed similarity to replicons of two recognized families: RCR group III (repSD164) and the FIA family of theta group A (repSD41, repSD121, repGA33 and repGA14) . Plasmids isolated from marine bacteria belonging to the genera Pseudoalteromonas, Shewanella and Vibrio cultivated from geographically different coastal sites exhibited homology to two of the marine plasmid replicons, repSD41 and repGA70, obtained from a Vibrio sp . The repGA33 plasmid origin, obtained from a Shewanella sp . isolated from coastal Georgia, was detected in 7% of the Georgia marine sediment Shewanella sp . isolates . Microbial community DNA extracted from marine sediments was also screened for the presence of the plasmid replication sequences . Community DNA samples amplified by PCR yielded a positive signal for the repSD172 and repGA14 replication sequences . The replication origin of BHR plasmid RK2 (IncP) was also detected in marine Vibrio sp . and microbial community DNA extracted from the three coastal sites . These findings provide molecular evidence that marine sediment bacteria harbour an untapped population of BHR plasmids. Kansenshogaku Zasshi, 2001 Jun, 75(6), 485 - 9 {The trends of Vibrio parahaemolyticus foodborne outbreaks in Tokyo: 1989-2000}; Obata H et al.; The total number of foodborne outbreaks due to Vibrio parahaemolyticus in Tokyo during the last 12 years between 1989 and 2000 were 710 . The number of outbreaks in a year was 55 in 1989, 75 in 1990, and there was a gradual decrease to 24 outbreaks in 1993 which was the smallest number during those 12 years . After 1994, the number of outbreaks increased dramatically year by year until 1998 (107 outbreaks) . Then they had decreased slightly to 74 in 1999, 65 in 2000 . The monthly incidence of V . parahaemolyticus foodborne outbreaks showed a peak in August (44.2%) each year . In the last 12 years, 88.7% of V . parahaemolyticus foodborne outbreaks occurred during the 3 months between July and September, while 99.9% occurred between June and October . The most prevalent serotype of V . parahaemolyticus also changed, the most prevalent was O4:K4 in 1989, O4:K8 in both 1990 and 1991, O1:K56 in 1992, and O4:K8 from 1993 through 1995 . Serotype O3:K6 became the most prevalent in 1996 and has remained so to date . In addition, the new serotype O4:K68 had also appeared in 1998 . The number of outbreaks due to serotype O4:K68 followed that of O3:K6 . Thus, the trends of V . parahaemolyticus foodborne outbreaks during the last 12 years in Tokyo showed various characteristics and dramatic changes in causal organisms. J Infect Dis, 2001 Sep 1, 184(5), 643 - 7 Epub 2001 Aug 09. Local production of anti-vibrio cholerae mucosal antibody in reproductive tract tissues after cholera; Ryan ET et al.; To investigate whether intestinal presentation of an antigen by Vibrio cholerae, a noninvasive organism, could induce an anatomically distant mucosal immune response in reproductive tract tissues, the endocervical immune responses of women in Bangladesh were evaluated after cholera . Endocervical secretions were analyzed for secretory IgA (sIgA) antibody against the B subunit of cholera toxin (CtxB) in 9 women with cholera and 8 women with diarrhea caused by neither V . cholerae nor heat labile enterotoxin-producing Escherichia coli . Women infected with V . cholerae developed significant sIgA anti-CtxB responses in endocervical samples (P< or =.02) . Antibody subtype analysis of endocervical IgA was consistent with local mucosal production (P< or =.001) . Women with cholera did not develop sIgA anti-CtxB responses in serum . The ability to generate specific mucosal immune responses in reproductive tract tissues after intestinal presentation of antigen could facilitate development of vaccines effective against reproductive tract pathogens. Environ Toxicol Chem, 2001 Aug, 20(8), 1733 - 9 Freeze concentration of ambient waters for toxicity testing; deBruyn AM et al.; We have developed a method to concentrate aqueous samples for toxicity testing . This method relies on the phenomenon of freezing exclusion, whereby solutes are rejected from the interstices of a growing ice crystal . Tenfold freeze concentration gave excellent recoveries of inorganic and organic analytes, phenol and ZnSO4 toxicity from spiked natural waters, and toxicity of both pre- and postdischarge municipal wastewater . Simultaneous 10-fold concentration of strong mineral or humic ambient matrices did not substantially modify the expressed toxicity of phenol or ZnSO4, and it did not seem to generate spurious toxicity to the marine bioassay organism used (Vibrio fischeri) . Hundredfold freeze concentration permitted the quantification of low levels of ambient toxicity in a wide variety of natural waters using a rapid, inexpensive microbioassay . Precipitation of matrix elements may limit the degree of concentration that can be achieved with highly mineralized or strongly humic waters . This approach is well suited to ambient toxicity testing, because it is nonspecific and has low potential for solvent contamination . Furthermore, the low temperatures involved minimize volatilization and degradation of organic contaminants. Int J Syst Evol Microbiol, 2001 Jul, 51(Pt 4), 1383 - 8 Vibrio shiloi sp . nov., the causative agent of bleaching of the coral Oculina patagonica; Kushmaro A et al.; The aetiological agent of bleaching of the coral Oculina patagonica was characterized as a new Vibrio species on the basis of 16S rDNA sequence, DNA-DNA hybridization data and phenotypic properties, including the cellular fatty acid profile . Based on its 16S rDNA and DNA-DNA hybridization, the new Vibrio species is closely related to Vibrio mediterranei . The name Vibrio shiloi sp . nov . is proposed for the new coral-bleaching species, the type strain being AK1T (= ATCC BAA-91T = DSM 13774T). Mol Microbiol, 2001 Jul, 41(2), 393 - 407 Overlapping binding sites for the virulence gene regulators AphA, AphB and cAMP-CRP at the Vibrio cholerae tcpPH promoter; Kovacikova G et al.; The expression of the Vibrio cholerae virulence factors, toxin-co-regulated pilus (TCP) and cholera toxin (CT), are dependent on the ability of the LysR regulator AphB to co-operate with a second protein, AphA, to activate the expression of the membrane-bound transcription factors TcpP and TcpH . To gain insights into the mechanism by which AphA and AphB co-operate to activate the expression of tcpPH, we have purified these two proteins to near homogeneity and show that they are each capable of interacting with the classical tcpPH promoter at distinct binding sites . As shown by tcpP-lacZ promoter deletion experiments, gel shift and DNase I footprinting, AphA binds to and activates from a region of the promoter between -101 and -71 from the start of transcription . AphB binds to and activates from a partially overlapping downstream site between -78 and -43, and these functions are dependent upon a region of partial dyad symmetry that resembles the well-characterized LysR-binding motif . A single basepair difference in this region of dyad symmetry has been shown previously to play a critical role in the expression of virulence genes between the two disease-causing biotypes of V . cholerae, classical and El Tor . We also show here that the tcpPH promoter is negatively influenced by the global regulator cAMP-CRP . Purified CRP binds to a near-consensus sequence in the tcpPH promoter in a cAMP-dependent manner and protects from DNase I digestion a region that is completely within the region protected by AphA and AphB . These findings raise the possibility that the negative effect of cAMP-CRP on virulence gene expression is the result of its ability to influence AphA- and AphB-dependent transcriptional activation of tcpPH under various conditions. Mol Microbiol, 2001 Jul, 41(2), 311 - 23 Evidence for a rolling-circle mechanism of phage DNA synthesis from both replicative and integrated forms of CTXphi; Moyer KE et al.; The genes encoding cholera toxin, the principal virulence factor of Vibrio cholerae, are part of the circular single-stranded DNA genome of CTXphi . In toxigenic V . cholerae strains, the CTXphi genome is typically found in integrated arrays of tandemly arranged CTX prophages . Infected cells that lack a chromosomal integration site harbour the CTXphi genome as a plasmid (pCTX) . We studied the replication of pCTX and found several indications that this plasmid replicates via a rolling-circle (RC) mechanism . The initiation and termination sites for pCTX plus-strand DNA synthesis were mapped to a 22 bp sequence that contains inverted repeats and a nonanucleotide motif found in the plus-strand origins of several RC replicons . Furthermore, similar to other RC replicons, replication of plasmids containing duplicated pCTX origins resulted in the deletion of sequences between the two origins and the formation of a single chimeric origin . Our previous work revealed that CTX prophage arrays give rise to hybrid CTX virions that contain sequences derived from two adjacent prophages . We now report that the boundaries between the sequences contributed to virions by the upstream and the downstream prophages in an array correspond to the site at which synthesis of plus-strand pCTX DNA is initiated and terminated . These data support the model that plus-strand CTXphi DNA is generated from chromosomal prophages via a novel process analogous to RC replication. Eur J Biochem, 2001 Aug, 268(15), 4261 - 8 Identification of protein-coding genes in the genome of Vibrio cholerae with more than 98% accuracy using occurrence frequencies of single nucleotides; Wang J et al.; The published sequence of the Vibrio cholerae genome indicates that, in addition to the genes that encode proteins of known and unknown function, there are 1577 ORFs identified as conserved hypothetical or hypothetical gene candidates . Because the annotation is not 100% accurate, it is not known which of the 1577 ORFs are true protein-coding genes . In this paper, an algorithm based on the Z curve method, with sensitivity, specificity and accuracy greater than 98%, is used to solve this problem . Twenty-fold cross-validation tests show that the accuracy of the algorithm is 98.8% . A detailed discussion of the mechanism of the algorithm is also presented . It was found that 172 of the 1577 ORFs are unlikely to be protein-coding genes . The number of protein-coding genes in the V . cholerae genome was re-estimated and found to be approximately 3716 . This result should be of use in microarray analysis of gene expression in the genome, because the cost of preparing chips may be somewhat decreased . A computer program was written to calculate a coding score called VCZ for gene identification in the genome . Coding/noncoding is simply determined by VCZ > 0/VCZ < 0 . The program is freely available on request for academic use. Emerg Infect Dis, 2001, 7(3 Suppl), 583 - 7 Cholera outbreak in southern Tanzania: risk factors and patterns of transmission; Acosta CJ et al.; To identify risk factors and describe the pattern of spread of the 1997 cholera epidemic in a rural area (Ifakara) in southern Tanzania, we conducted a prospective hospital-based, matched case- control study, with analysis based on the first 180 cases and 360 matched controls . Bathing in the river, long distance to water source, and eating dried fish were significantly associated with risk for cholera . Toxigenic Vibrio cholerae O1, biotype El Tor, serotype Ogawa, was isolated in samples from Ifakara's main water source and patients' stools . DNA molecular analyses showed identical patterns for all isolates. Southeast Asian J Trop Med Public Health, 2001 Mar, 32(1), 95 - 9 Transition of drug susceptibilities of Vibrio cholerae O1 in Lao People's Democratic Republic; Phantouamath B et al.; The changes of drug susceptibilities of Vibrio cholerae O1 isolated during the past 7 years (1993-1999) in Lao PDR were investigated . The most noteworthy finding was the appearance of polymyxin B sensitive El Tor vibrios . Until 1996, the susceptibilities were almost as expected and cholera disappeared in 1997 . When a cholera outbreak resurfaced in 1998, the susceptibilities of isolated V . cholerae O1 against tetracycline, sulfamethoxazol-trimethoprim, chloramphenicol and polymyxin B were quite different from those of previously isolated organisms . Minimum inhibitory concentrations (MICs) of tetracycline and chloramphenicol against the isolates in 1998 were about 16 times higher than those against the previous isolates, and the MICs of sulfamethoxazol-trimethoprim were about 256 times higher than those against the previous isolates, (trimethoprim 32 microg/ml: sulfamethoxazol 608 microg/ml) . Eleven percent of the isolates (11/99) were as sensitive to polymyxin B as the classic cholera vibrios (MIC < 2 microg/ml) . In 1999, the susceptibility pattern was almost the same as that in 1998 except for polymyxin B to which 58% of the isolates (21/36) became sensitive. Southeast Asian J Trop Med Public Health, 2001 Mar, 32(1), 100 - 4 Detection and molecular characterization of the zot gene in Vibrio cholerae and V . alginolyticus isolates; Raja N et al.; A total of 11 Vibrio cholerae isolates from 1996-1998 outbreaks in Malaysia and 4 V . alginolyticus were analyzed . Isolates were characterized by polymerase chain reaction (PCR) and Southern hybridization for the presence of the gene encoding zonula occludens toxin (zot) . Screening of zot gene by PCR revealed the presence of this gene in V . cholerae and V . alginolyticus . The zot gene from one V . cholerae Ogawa isolate that was cloned in a pCR 2.1 TOPO vector was sequenced . The sequences obtained were 99% homologous to the zot gene sequence from the Gene Bank. Glycobiology, 2001 Aug, 11(8), 655 - 61 Expression and identification of the RfbE protein from Vibrio cholerae O1 and its use for the enzymatic synthesis of GDP-D-perosamine; Albermann C et al.; The 4-amino-6-deoxy-monosaccharide D-perosamine is an important element in the glycosylation of interesting cell products, such as antibiotics and lipopolysaccharides (LPS) of Gram-positive and Gram-negative bacteria . The biosynthetic pathway of the precursor molecule, GDP-D-perosamine, in Vibrio cholerae O1 starts with an isomerisation of fructose-6-phosphate catalyzed by the bifunctional enzyme phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase (RfbA; E.C . 2.7.7.22) creating the intermediate mannose-6-phosphate, which is subsequently converted by the phosphomanno-mutase (RfbB; E.C . 5.4.2.8) and further by RfbA to GDP-D-mannose, to GDP-4 keto-6-deoxymannose by a 4,6-dehydratase (RfbD; E.C . 4.2.1.47) and finally to GDP-D-perosamine by an aminotransferase (RfbE; E.C . not yet classified) . We cloned the rfbD and the rfbE genes of V . cholerae O1 in Escherichia coli expression vectors . Both biosynthetic enzymes were overproduced in E . coli BL21 (DE3) and their activities were analyzed . The enzymatic conversion from GDP-D-mannose to GDP-D-perosamine was optimized and the final product, GDP-D-perosamine, was purified and identified by nuclear magnetic resonance, mass spectrometry, and chromatography . The catalytically active form of the GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase seems to be a tetramer of 170 kDa . The His-tag RfbE fusion protein has a Km of 0.06 mM and a Vmax value of 38 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose . The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively . The intention of this work is to establish a basis for both the in vitro production of GDP-D-perosamine and for an in vivo perosaminylation system in a suitable bacterial host, preferably E . coli. Fish Shellfish Immunol, 2001 Jul, 11(5), 415 - 31 In vitro interactions between rainbow trout (Oncorhynchus mykiss) macrophages and Vibrio anguillarum serogroup O2a; Boesen HT et al.; The sensitivity of Vibrio anguillarum serogroup O2a to killing by rainbow trout macrophages in the presence or absence of specific antibodies and complement components was evaluated using an in vitro assay . Fluorescence microscopy revealed that V . anguillarum serogroup O2a was phagocytosed by rainbow trout macrophages . In the absence of specific antibodies and complement components the bacteria were killed to a limited extent by the macrophages and there was no increased killing if the bacteria were opsonised with either antibodies or antibodies and complement . Furthermore, activated macrophages did not show enhanced ability to kill the bacteria . Vibrio anguillarum serogroup O2a were susceptible to both cell-free superoxide anion (O2-) and hydrogen peroxide (H2O2), which might be generated during the macrophage respiratory burst and the bacteria did not quench cell-free O2- . However, the production of O2- by macrophages was undetectable during the first 30 min following infection and no respiratory burst was inducible by phorbol myristate acetate (PMA) 4 h after infection with V . anguillarum . This suggests that the bacteria were able to inhibit the production of O2- by the infected macrophages . Naive fish were protected when passively immunised with anti-V . anguillarum serogroup O2a antiserum . However, previous results suggest that antibodies are unlikely to provide the fish with protective immunity directly through activation of the complement system and lysis of the bacterial cells . The present in vitro findings suggest that the protective mechanisms of antibody against V . anguillarum serogroup O2a may not involve the opsonising effect of antibodies for enhanced killing by macrophages . However, the possibility exists that such antibodies may prevent the attachment of the pathogen to the host's tissues. Fish Shellfish Immunol, 2001 Jul, 11(5), 399 - 413 Immunoglobulin genes and antibody responses in the spotted wolffish (Anarhichas minor Olafsen); Espelid S et al.; The spotted wolffish Anarhichas minor Olafsen is a promising new species in aquaculture in the cold waters of northern Norway . In this paper, some basic immunological studies of this marine species are reported . Of comparative interest are the cDNA sequences of the immunoglobulin transcript and the antibody responses to model antigens . Of more practical importance are the humoral immune responses and antibody specificities to potentially pathogenic bacteria . Full length cDNA clones encoding the immunoglobulin heavy and light chains in the spotted wolffish were sequenced demonstrating variable degrees of similarity to other teleost fish species . Also in the spotted wolffish the CH4 domain was deleted in the transmembrane form of the immunoglobulin heavy chain (IgH) as a receptor on B cells, with the transmembrane exon spliced directly to the CH3 domain . The antibody responses to various antigens like hapten-carrier molecules, protein antigens and bacterial pathogens were relatively high, but with some interesting exceptions . Anti-hapten responses to NIP and FITC were high while anti-DNS responses were low, but more surprisingly, there was hardly any B-cell response to the carrier molecule LPH . On the other hand, protein antigens like CGG and BSA were highly immunogenic in the spotted wolffish as were the bacterial antigens Vibrio anguillarum, V . salmonicida and Aeromonas salmonicida. J Appl Microbiol, 2001 Aug, 91(2), 322 - 7 Comparison of selective media for the detection of Vibrio vulnificus in environmental samples; Cerda-Cuellar M et al.; AIMS: To compare two selective agars, cellobiose-colistin (CC) agar and a modification of the Vibrio vulnificus medium (VVMc agar), for the isolation of Vibrio vulnificus from environmental samples . METHODS AND RESULTS: The efficiencies of recovery of V . vulnificus collection strains on CC, VVM, VVMc and on thiosulphate-citrate-bile salts-sucrose (TCBS) agar were compared and similar efficiencies were obtained . A slightly higher recovery was observed on VVMc agar . The detection of V . vulnificus in environmental samples (eels and water) was performed by combining culture-based methods (CC and VVMc agars) with DNA-based methods using species-specific probes based on the cytolysin-haemolysin and the 16S rDNA genes . A lower accompanying microbiota was found on CC agar than on VVMc agar . CONCLUSION: The comparison between CC and VVMc agars confirms that both are useful for the detection of V . vulnificus in environmental samples . However, the use of any of these media should be combined with a species-specific probe . SIGNIFICANCE AND IMPACT OF THE STUDY: The combined use of a selective medium and a specific probe provides a feasible method for the detection of V . vulnificus for epidemiological and ecological studies. Arch Pathol Lab Med, 2001 Aug, 125(8), 1107 - 9 Vibrio vulnificus septicemia in a patient with the hemochromatosis HFE C282Y mutation; Gerhard GS et al.; Vibrio vulnificus is an extremely invasive gram-negative bacillus found in marine waters that causes overwhelming bacteremia and shock that is associated with high mortality . Impaired iron metabolism has been implicated in the susceptibility to V vulnificus bacterial infections . We report a case of fatal V vulnificus sepsis in a 56-year-old man who died within 1 to 3 days after consuming raw seafood . At autopsy, he was found to have micronodular cirrhosis and iron overload . Postmortem genetic analysis revealed the presence of the hemochromatosis gene (HFE) C282Y mutation . To our knowledge, this is this first documented fatal case of V vulnificus infection in a patient proven to carry the HFE C282Y mutation . Because this patient was heterozygous for the major hereditary hemochromatosis mutation and was not previously diagnosed with clinical iron overload, the spectrum of clinical susceptibilities to V vulnificus infection may include carriers of the C282Y mutation. Appl Environ Microbiol, 2001 Aug, 67(8), 3707 - 11 Purification and characterization of a vulnificolysin-like cytolysin produced by Vibrio tubiashii; Kothary MH et al.; An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200 . This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3 . In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture . Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH . Twelve of the first 17 N-terminal amino acid residues (Asp-Asp-Tyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin. Lett Appl Microbiol, 2001 Aug, 33(2), 137 - 43 Diversity of Vibrio spp . populations in several exhibition aquaria with a shared water supply; Blanch AR et al.; AIMS: Abiotic factors may influence the settlement of bacterial populations in similar marine environments . Exhibition aquaria are a model for the study of the settlement of bacteria in different environment . Vibrio populations in the seawater reservoir, the Mediterranean tank and the Tropical tank from an exhibition aquarium on the western coast of the Mediterranean were compared and the effect of abiotic factors on the structure of these populations was considered . METHODS AND RESULTS: High diversity indexes and similar Vibrio populations were found in the water of the reservoir and of the Mediterranean tank, whereas a lower diversity and different main populations were found in the water of the Tropical tank . The antibiotic resistance profiles of the most representative strains, presented a number of differences depending on the origin of the sample . CONCLUSION: Abiotic conditions, mainly temperature, may determine the structure and composition of Vibrio populations in exhibition aquaria . SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial monitoring of water could be useful for health management of aquatic environments. Microbiol Immunol, 2001, 45(5), 393 - 7 Analysis of seawaters for the recovery of culturable Vibrio parahaemolyticus and some other vibrios; Alam MJ et al.; We investigated the recovery of dormant and injured cells along with the normally culturable cells of Vibrio species with special emphasis on V . parahaemolyticus using both selective and non-selective media at moderate (20 C) and standard (37 C) culture temperatures from a bay water environment . Culture temperatures (20 or 37 C) did not affect the recovery of V . parahaemolyticus but did for other vibrios . We observed similar seasonality of V parahaemolyticus as in most other environmental studies . V . parahaemolyticus and other Vibrio species were recovered in higher numbers by a replica plating method compared to most probable number (MPN) and direct TCBS (thiosulfate citrate bile-salt sucrose) agar counts . Even with the replica plating method, however, vibrios number goes down to a minimum level and V . parahaemolyticus was undetectable during the cool temperature period of the year, although total bacterial cells and CFU on nutrient agar (with 2% NaCl) did not vary so much during the study period. Arch Biochem Biophys, 2001 Aug 1, 392(1), 110 - 6 Flavin specificity and subunit interaction of Vibrio fischeri general NAD(P)H-flavin oxidoreductase FRG/FRase I; Tang CK et al.; Apoenzyme of the major NAD(P)H-utilizing flavin reductase FRG/FRase I from Vibrio fischeri was prepared . The apoenzyme bound one FMN cofactor per enzyme monomer to yield fully active holoenzyme . The FMN cofactor binding resulted in substantial quenching of both the flavin and the protein fluorescence intensities without any significant shifts in the emission peaks . In addition to FMN binding (K(d) 0.5 microM at 23 degrees C), the apoenzyme also bound 2-thioFMN, FAD and riboflavin as a cofactor with K(d) values of 1, 12, and 37 microM, respectively, at 23 degrees C . The 2-thioFMN containing holoenzyme was about 40% active in specific activity as compared to the FMN-containing holoenzyme . The FAD- and riboflavin-reconstituted holoenzymes were also catalytically active but their specific activities were not determined . FRG/FRase I followed a ping-pong kinetic mechanism . It is proposed that the enzyme-bound FMN cofactor shuttles between the oxidized and the reduced form during catalysis . For both the FMN- and 2-thioFMN-containing holoenzymes, 2-thioFMN was about 30% active as compared to FMN as a substrate . FAD and riboflavin were also active substrates . FRG/FRase I was shown by ultracentrifugation at 4 degrees C to undergo a monomer-dimer equilibrium, with K(d) values of 18.0 and 13.4 microM for the apo- and holoenzymes, respectively . All the spectral, ligand equilibrium binding, and kinetic properties described above are most likely associated with the monomeric species of FRG/FRase I . Many aspects of these properties are compared with a structurally and functionally related Vibrio harveyi NADPH-specific flavin reductase FRP . Protein Sci, 2001 Aug, 10(8), 1563 - 71 Modeling of the bacterial luciferase-flavin mononucleotide complex combining flexible docking with structure-activity data; Lin LY et al.; Although the crystal structure of Vibrio harveyi luciferase has been elucidated, the binding sites for the flavin mononucleotide and fatty aldehyde substrates are still unknown . The determined location of the phosphate-binding site close to Arg 107 on the alpha subunit of luciferase is supported here by point mutagenesis . This information, together with previous structure-activity data for the length of the linker connecting the phosphate group to the isoalloxazine ring represent important characteristics of the luciferase-bound conformation of the flavin mononucleotide . A model of the luciferase-flavin complex is developed here using flexible docking supplemented by these structural constraints . The location of the phosphate moiety was used as the anchor in a flexible docking procedure performed by conformation search by using the Monte Carlo minimization approach . The resulting databases of energy-ranked feasible conformations of the luciferase complexes with flavin mononucleotide, omega-phosphopentylflavin, omega-phosphobutylflavin, and omega-phosphopropylflavin were filtered according to the structure-activity profile of these analogs . A unique model was sought not only on energetic criteria but also on the geometric requirement that the isoalloxazine ring of the active flavin analogs must assume a common orientation in the luciferase-binding site, an orientation that is also inaccessible to the inactive flavin analog . The resulting model of the bacterial luciferase-flavin mononucleotide complex is consistent with the experimental data available in the literature . Specifically, the isoalloxazine ring of the flavin mononucleotide interacts with the Ala 74-Ala 75 cis-peptide bond as well as with the Cys 106 side chain in the alpha subunit of luciferase . The model of the binary complex reveals a distinct cavity suitable for aldehyde binding adjacent to the isoalloxazine ring and flanked by other key residues (His 44 and Trp 250) implicated in the active site. J Bacteriol, 2001 Aug, 183(16), 4737 - 46 Characterization of VPI pathogenicity island and CTXphi prophage in environmental strains of Vibrio cholerae; Mukhopadhyay AK et al.; Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V . cholerae evolution . We found that each isolate contained a variant form of the VPI pathogenicity island . Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5' half and nearly identical in its 3' half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT . Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI . Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTXphi-like prophages . Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types . New possible prophage repressor and replication genes were also identified . Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V . cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species. Mar Environ Res, 2000 Jul-Dec, 50(1-5), 163 - 8 Immune function, hepatic CYP1A, and reproductive biomarker responses in the gulf killifish, Fundulus grandis, during dietary exposures to endocrine disrupters; Rice CD et al.; The gulf killifish, Fundulus grandis, was used to determine the influence of biological rhythms on three biomarker responses . We first developed monoclonal antibodies against the model's immunoglobulins and vitellogenin in order to measure antibody responses and vitellogenesis, respectively . We then treated adults with 10, 1, 1, and 10 ppm of Aroclor 1254, tribuyltin, 3-methylcholanthrene, and nonyl-phenol, respectively, in mixtures over a 16-week period . The study followed Vibrio anguillarum-specific antibody responses, hepatic CYP1A, and plasma vitellogenin levels in the morning and again in the evening at 2-week intervals . The contaminated diet suppressed secondary antibody responses, but only in the morning . The contaminated diet also altered CYP1A, but not vitellogenesis . In addition, fish in the control group exhibited daily and seasonal differences in specific antibody levels and CYP1A induction . Moreover, circulating vitellogenin levels in control males sampled in the morning increased throughout the exposure, but remained below those of females . This study underscores the need to consider normal physiological rhythms when employing biomarkers in toxicology. Carbohydr Res, 2001 Jul 19, 333(4), 263 - 9 Depolymerization of the capsular polysaccharide from Vibrio cholerae O139 by a lyase associated with the bacteriophage JA1; Linnerborg M et al.; We have studied the interaction between the Vibrio cholerae O139 specific phage JA1, belonging to the Podoviridae family, and the capsular polysaccharide (CPS) of the parent strain from which the phage was isolated . Upon incubation of the JA1 phage with the CPS, oligosaccharides were isolated and purified . The oligosaccharides derived from one (shown below) and two repeating units of the CPS were characterized using NMR spectroscopy, mass spectrometry and sugar analysis (structure: see text) . The cleavage was found to occur by beta-elimination at the 4-substituted alpha-linked galacturonic acid, which results in a 4-deoxy-beta-L-threo-hex-4-enopyranosyl uronic acid group (Sug) . The enzyme associated with the JA1 phage responsible for the depolymerization of the V . cholerae O139 CPS is thus a lyase. Environ Sci Technol, 2001 Jul 1, 35(13), 2773 - 7 Rhizosphere effects on microbial community structure and dissipation and toxicity of polycyclic aromatic hydrocarbons (PAHs) in spiked soil; Joner EJ et al.; Phytoremediation of soils polluted with polycyclic aromatic hydrocarbons (PAHs) has so far neglected the possible role of the ubiquitous symbiotic associations between plant roots and fungi known as arbuscular mycorrhizas . A time course laboratory experiment with clover and ryegrass grown on spiked {500 + 500 + 50 mg kg-1 of anthracene, chrysene and dibenz(a,h)anthracene} soil demonstrated for the first time that dissipation of condensed PAHs may be enhanced in the presence of arbuscular mycorrhiza {66 and 42% reductions in chrysene and dibenz(a,h)anthracene, respectively, versus 56 and 20% reductions in nonmycorrhizal controls} . Addition of a surfactant accelerated initial PAH dissipation but did not attain final PAH concentrations below those obtained with nonmycorrhizal plants . Toxicity tests (earthworm survival and bioluminescence inhibition in Vibrio fischeri) indicated that mycorrhiza reduced the toxicity of PAHs and/or their metabolites and counteracted a temporally enhanced toxicity mediated by surfactant addition . Phospholipid fatty acid profiles demonstrated that the imposed treatments altered the microbial community structure and indicated that the mycorrhiza-associated microflora was responsible for the observed reductions in PAH concentrations in the presence of mycorrhiza. Mol Gen Mikrobiol Virusol, 2001, (2), 24 - 6 {PCR-detection of markers for epidemiologically-significant strains of Vibrio cholerae 0139, isolated in Siberia}; Balakhonov SV et al.; Fourteen V . cholerae 0139 strains were isolated in 1996-1999 in Siberia from the Ob river (Novosibirsk) and bogs and lakes (Irkutsk) . The strains were tested in PCR for the key virulence determinants (ctx AB, tcp, acf) . The genomes lacked these elements, and therefore the strains were acknowledged avirulent . The results correlate completely with the data of phenotypical analysis, characterizing the pathogenic characteristics of isolated strains. J Biomol Struct Dyn, 2001 Jun, 18(6), 872 - 80 Analysis of the codon usage pattern in the Vibrio cholerae genome; Wang J et al.; The codon usage in the Vibrio cholerae genome is analyzed in this paper . Although there are much more genes on the chromosome 1 than on chromosome 2, the codon usage patterns of genes on the two chromosomes are quite similar, indicating that the two chromosomes may have coexisted in the same cell for a very long history . Unlike the base frequency pattern observed in other genomes, the G+C content at the third codon position of the V . cholerae genome varies in a rather small interval . The most notable feature of codon usage of V . cholerae genome is that there is a fraction of genes show significant bias in base choice at the second codon position . The 2,006 known genes can be classified into two clusters according to the base frequencies at this position . The smaller cluster contains 227 genes, most of which code for proteins involved in transport and binding functions . The encoding products of these genes have significant bias in amino acids composition as compared with other genes . The codon usage patterns for the 1,836 function unknown ORFs are also analyzed, which is useful to study their functions. Prikl Biokhim Mikrobiol, 2001 May-Jun, 37(3), 359 - 63 {Lipids of microorganisms from the family Vibrionaceae causing diseases in fishes}; Bogdan VV et al.; Lipid fractional and fatty acid compositions of microorganisms from the genera Aeromonas, Pseudomonas, and Vibrio (the family Vibrionaceae), causing diseases of different fish species, were studied . Motile aeromonads and vibrios displayed higher relative contents of membrane lipids and oleic acid and lower relative contents of storage lipids compared with immotile aeromonads and pseudomonads, which is connected with the activities of their movements . Immotile aeromonads and vibrios exhibited higher levels of polyunsaturated fatty acids and higher absolute phospholipid contents compared to motile aeromonads and pseudomonads . This is likely to be related to host specificity of these bacteria and reflects the specific patterns of fatty acid compositions of the infected fish (salmonid and cyprinid) tissues. Arch Environ Contam Toxicol, 2001 Apr, 40(3), 386 - 91 The effects of tributyltin (TBT) and 3,3',4,4',5-pentachlorobiphenyl (PCB-126) mixtures on antibody responses and phagocyte oxidative burst activity in channel catfish, Ictalurus punctatus; Regala RP et al.; The organotin tributyltin (TBT) is an antifouling biocide used in marine paints and is a common pollutant in harbor estuaries . We previously demonstrated that the immune system of channel catfish, Ictalurus punctatus, is a sensitive target organ of TBT . Exposure strongly suppresses humoral immune responses . Harbor estuaries often contain polychlorinated biphenyls (PCBs) due to their ubuquitous distribution . The coplanar congener 3,3',4,4'5'-polychlorinated biphenyl (PCB-126) is also immunotoxic to channel catfish, but it suppresses only the innate immune responses and only at high doses . In this study we exposed channel catfish to TBT, PCB-126, or both in mixtures, with canola oil (CO) serving as the carrier control . Antibody responses to Vibrio anguillarum and phagocyte oxidative burst activity were measured after (1) a single dose of 0.01 or 1 mg/kg of each or both in combination, and (2) six injections of 1.7 or 170 microg/kg of each (or in combination) given every 3 days over a 16-day period to yield a cumulative dose of 0.01 or 1 mg/kg, respectively . We measured antibody responses to V . anguillarum 21 days after immunization and oxidative burst activities 14 and 21 days after the final treatment . The highest dose of TBT suppressed antibody responses after a single exposure . The high dose of PCB-126 also suppressed antibody responses . The addition of PCB-126 to TBT doses did not alter the antibody responses beyond the effects of TBT alone . In the repeated exposure group, only the high dose of TBT suppressed antibody responses . In animals exposed to mixtures, high levels of PCB-126 enhanced suppression associated with low levels of TBT, whereas PCB-126 protected against suppression associated with high levels of TBT . Single exposures to TBT or PCB-126 suppressed phagocyte oxidative burst activity . In animals exposed to mixtures, as a single exposure, the addition of a low dose PCB-126 protected against low dose TBT-related oxidative burst activity suppression . In the repeated exposure groups TBT suppressed oxidative burst activity, but only at the highest dose on day 21, while high doses of PCB-126 suppressed activity on day 14 . Furthermore, low levels of PCB-126 reversed the suppressed oxidative burst activity associated with high levels of TBT on day 21 . Overall, this study demonstrates moderate additivity in terms of the immunotoxicity of TBT and PCB-126 mixtures using these two endpoints of immune function in the channel catfish model. J Biol Chem, 2001 Sep 21, 276(38), 35934 - 9 Epub 2001 Jul 06. Site-directed mutagenesis of acyl carrier protein (ACP) reveals amino acid residues involved in ACP structure and acyl-ACP synthetase activity; Flaman AS et al.; Acyl carrier protein (ACP) interacts with many different enzymes during the synthesis of fatty acids, phospholipids, and other specialized products in bacteria . To examine the structural and functional roles of amino acids previously implicated in interactions between the ACP polypeptide and fatty acids attached to the phosphopantetheine prosthetic group, recombinant Vibrio harveyi ACP and mutant derivatives of conserved residues Phe-50, Ile-54, Ala-59, and Tyr-71 were prepared from glutathione S-transferase fusion proteins . Circular dichroism revealed that, unlike Escherichia coli ACP, V . harveyi-derived ACPs are unfolded at neutral pH in the absence of divalent cations; all except F50A and I54A recovered native conformation upon addition of MgCl(2) . Mutant I54A was not processed to the holo form by ACP synthase . Some mutations significantly decreased catalytic efficiency of ACP fatty acylation by V . harveyi acyl-ACP synthetase relative to recombinant ACP, e.g . F50A (4%), I54L (20%), and I54V (31%), whereas others (V12G, Y71A, and A59G) had less effect . By contrast, all myristoylated ACPs examined were effective substrates for the luminescence-specific V . harveyi myristoyl-ACP thioesterase . Conformationally sensitive gel electrophoresis at pH 9 indicated that fatty acid attachment stabilizes mutant ACPs in a chain length-dependent manner, although stabilization was decreased for mutants F50A and A59G . Our results indicate that (i) residues Ile-54 and Phe-50 are important in maintaining native ACP conformation, (ii) residue Ala-59 may be directly involved in stabilization of ACP structure by acyl chain binding, and (iii) acyl-ACP synthetase requires native ACP conformation and involves interaction with fatty acid binding pocket residues, whereas myristoyl-ACP thioesterase is insensitive to acyl donor structure. Ecotoxicol Environ Saf, 2001 Jul, 49(3), 293 - 301 Use of the ciliated protozoan Tetrahymena pyriformis for the assessment of toxicity and quantitative structure--activity relationships of xenobiotics: comparison with the Microtox test; Bogaerts P et al.; Cytotoxicity and quantitative structure-activity relationships of 13 inorganic and 21 organic substances were determined using three bioassays performed on the ciliated protozoan Tetrahymena pyriformis and the luminescent bacterium Vibrio fischeri . The best concordance of toxicity results was observed between the T . pyriformis FDA--esterase activity and population growth inhibition tests for the organic compounds . The sensitivity of these two assays is compared with that of the Microtox test . The T . pyriformis FDA test showed a high sensitivity is most cases . The aim of the current research was to determine whether the relative toxicity of metal ions and organic molecules, with these three bioassays, was predictable using three ion characteristics and hydrophobicity, respectively . For metal ions, the variable that best modeled the toxicity data obtained with the two T . pyriformis tests was the softness index {sigma(p), i.e., (coordinate bond energy of the metal fluoride--coordinate bond energy of the metal iodide)/(coordinate bond energy of the metal fluoride)} . No correlation was found with the Microtox test . For organic compounds, a significant correlation was observed between the hydrophobicity coefficient and the toxicity data . This correlation is closer with the two tests using Tetrahymena . J Microbiol Methods, 2001 Sep, 46(3), 217 - 25 Isolation and characterization of a temperature-sensitive generalized transducing bacteriophage for Vibrio cholerae; Hava DL et al.; CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown . Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool . To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation . Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size . Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci . Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V . cholerae. Int J Med Microbiol, 2001 May, 291(2), 81 - 8 Regulation of virulence in Vibrio cholerae; Klose KE; Vibrio cholerae causes the diarrheal disease cholera primarily because it expresses a colonization factor (toxin-coregulated pilus; TCP) and a potent toxin (cholera toxin; CT) within the human intestine . While the true environmental signals that induce CT and TCP expression within the intestine remain unknown, much progress has been made identifying the regulatory factors that modulate their expression . Transcriptional regulation of the genes encoding TCP and CT involves a cascade consisting of a number of regulatory factors located on recently acquired mobile genetic elements as well as others residing within the ancestral Vibrio genome . In vivo studies have revealed interesting differences between the regulation of TCP and CT expression in the laboratory and within the intestine. Environ Toxicol Chem, 2001 Jul, 20(7), 1438 - 49 The use of acute and chronic bioassays to determine the ecological risk and bioremediation efficiency of oil-polluted soils; van Gestel CA et al.; To compare the effectiveness of acute and chronic bioassays for the ecological risk assessment of polluted soils, soil samples from a site with an historical mineral oil contamination (< 50-3,300 mg oil/kg dry soil) at the Petroleum Harbour in Amsterdam, The Netherlands, were screened for ecological effects using acute and chronic bioassays . A two-step 0.001 M Ca(NO3)2 extraction at a final solution-to-soil ratio of 1:1 was used to prepare extracts for the acute bioassays . Acute bioassays (< or = 5 d) applied to the 0.001 M Ca(NO3)2 extracts from the polluted and reference soils included growth tests with bacteria (Bacillus sp.), algae (Raphidocelis subcapitata), and plants (Lactuca sativa), immobility tests with nematodes (Plectus acuminatus), springtails (Folsomia candida), and cladocerans (Daphnia magna), and the Microtox test (Vibrio fischeri) . Chronic bioassays (four weeks) performed on the same soil samples included tests with L . sativa, F . candida, and earthworms (Eisenia fetida) and the bait-lamina test (substrate consumption) . The acute bioassays on Microtox showed a response that corresponded with the level of oil pollution . All other acute bioassays did not show such a consistent response, probably because pollutant levels were too low to cause acute effects . All chronic bioassays showed sublethal responses according to the contaminant levels (oil and in some soils also metals) . This shows that chronic bioassays on soil samples are more sensitive in assessing the toxicity of mineral oil contamination in soil than acute bioassays on soil extracts . A pilot scale bioremediation study on soils taken from the two most polluted sites and a control site showed a rapid decline of oil concentrations to reach a stable level within eight weeks . Acute bioassays applied to the soils, using Microtox, algae, and D . magna, and chronic bioassays, using plants, Collembola, earthworms, and bait-lamina consumption, in all cases showed a rapid reduction of toxicity, which could be attributed to the degradation of light oil fractions. FEMS Microbiol Lett, 2001 May 1, 198(2), 141 - 6 Structures of ribonuclease P RNAs of Vibrio core species; Maeda T et al.; The structures of an RNA component of ribonuclease P (RNase P RNA) were examined for Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio carchariae, Vibrio natriegens, Vibrio campbellii, Vibrio proteolyticus, Vibrio pelagius and Vibrio harveyi to clearly determine their genetic differences . The RNase P RNAs ranged from 382 to 454 nucleotides (nt) in size, and were remarkably different from each other in the structure of two helices, P3 and P12 . The P3 helices were comprised of tandem repeats of a palindromic sequence (24 nt), resulting in the longitudinal repetition of a stem structure . The number of repetitions ranged from four in V . harveyi, to one in both V . alginolyticus and V . proteolyticus . The genes for the RNase P RNAs of all species were located between two open reading frames, the amino acid sequences of which were similar to the hypothetical proteins located at 70.92 and 1.94 min in the Escherichia coli chromosome. FEMS Microbiol Lett, 2001 May 1, 198(2), 123 - 4 Helical growth and the curved shape of Vibrio cholerae; Cooper S; The curved, comma, or bent shape of Vibrio cholerae is attributed to, and explained by, the normal helical growth of the cell . The comma-like shape of V . cholerae is not due to an asymmetrical positioning of peptidoglycan such that some chains of peptidoglycan are placed so they are more spread out on one side of the cell and squeezed together on the other side. J Zoo Wildl Med, 2000 Dec, 31(4), 523 - 31 Common cuttlefish (Sepia officinalis) mortality at the National Zoological Park: implications for clinical management; Sherrill J et al.; Six out of seven cuttlefish acquired by the Smithsonian National Zoological Park in July 1998 died before 1 November 1998 . Postmortem examinations showed mantle ulcers, secondary bacterial infections, inanition, and cuttlebone fractures . The surviving cuttlefish developed a progressive focal mantle ulcer, was treated with oral chloramphenicol intermittently for 9 wk, and maintained a normal appetite and growth rate until death at 7 mo of age . The National Zoological Park pathology database showed signalments, histories, and causes of mortality of 186 common cuttlefish, each 1-14 mo old, that received gross and histologic examinations; for example, the largest group of cuttlefish of known sex, age, and body weight at postmortem were 7-9 mo old and weighed an average of 376.2 g (males, n = 18) and 299.0 g (females, n = 15) . Many cuttlefish had multiple pathologic diagnoses . Significant diseases included inflammation and secondary bacterial infections, especially gastrointestinal, cardiovascular, respiratory, reproductive, and ophthalmic, and septicemia due to Vibrio spp . or other gram-negative bacteria . Mantle lesions, including ulceration/dermatitis, abscess/granuloma, necrosis/fibrosis/cellulitis, and laceration/abrasion/erosion, were also identified, along with inanition, cuttlebone lesions, and trauma . Mantle lesions were associated with secondary bacterial infections and death . On the basis of this information, if captive cuttlefish behavior creates risk for development of mantle lesions, administration of antibiotics effective against gram-negative bacteria may delay or halt disease progression . Cuttlefish exhibits require proper design, husbandry, economic resources, and staffing to minimize disease syndromes and mortality. J Clin Microbiol, 2001 Jul, 39(7), 2594 - 7 Detection of RTX toxin gene in Vibrio cholerae by PCR; Chow KH et al.; A PCR that amplifies a recently discovered Vibrio cholerae RTX (repeat in toxin) toxin gene was developed . Among 166 clinical and environmental isolates of V . cholerae causing epidemics and sporadic cases of cholera in various parts of the world, all were found to be toxigenic by both PCR and HEp-2 cell cytotoxicity assay . Standard strains of the classical biotype containing a deletion within the gene cluster exhibited negative results by both assays . This is the first rapid genotyping method for differentiation of V . cholerae O1 classical biotype strains from El Tor biotype strains as well as strains of other non-O1 serogroups including serogroup O139 . The PCR assay that was developed also specifically detects RTX toxin genes in V . cholerae, as clinical isolates of Vibrio parahaemolyticus, diarrheagenic Escherichia coli, Aeromonas species, and Plesiomonas species were all negative by the RTX toxin-specific PCR as well as the HEp-2 cytotoxicity assay . These findings highlight the characteristics of the RTX toxins in V . cholerae . Their role in the pathogenicity of the bacterium requires further investigation. Appl Environ Microbiol, 2001 Jul, 67(7), 3220 - 5 The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton; Chiavelli DA et al.; The bacterium Vibrio cholerae, the etiological agent of cholera, is often found attached to plankton, a property that is thought to contribute to its environmental persistence in aquatic habitats . The V . cholerae O1 El Tor biotype and V . cholerae O139 strains produce a surface pilus termed the mannose-sensitive hemagglutinin (MSHA), whereas V . cholerae O1 classical biotype strains do not . Although V . cholerae O1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the other strains . Since V . cholerae O1 El Tor and V . cholerae O139 have displaced V . cholerae O1 classical as the major epidemic strains over the last fifteen years, we investigated the potential role of MSHA in mediating adherence to plankton . We found that mutation of mshA in V . cholerae O1 El Tor significantly diminished, but did not eliminate, adherence to exoskeletons of the planktonic crustacean Daphnia pulex . The effect of the mutation was more pronounced for V . cholerae O139, essentially eliminating adherence . Adherence of the V . cholerae O1 classical mshA mutant was unaffected . The results suggest that MSHA is a factor contributing to the ability of V . cholerae to adhere to plankton . The results also showed that both biotypes of V . cholerae O1 utilize factors in addition to MSHA for zooplankton adherence . The expression of MSHA and these additional, yet to be defined, adherence factors differ in a serogroup- and biotype-specific manner. Appl Environ Microbiol, 2001 Jul, 67(7), 3161 - 7 Duplication of hemolysin genes in a virulent isolate of Vibrio harveyi; Zhang XH et al.; Vibrio harveyi VIB 645, which is very pathogenic towards salmonids and produces extracellular product with a high titer of hemolytic activity towards fish erythrocytes, was found to contain two closely related hemolysin genes (designated vhhA and vhhB), whereas the majority of strains examined (11 of 13) carried only a single hemolysin gene . Both genes from VIB 645 were cloned and sequenced . The open reading frames (ORFs) of vhhA and vhhB shared a high level of identity (98.8%) and were predicted to encode identical polypeptides comprising 418 amino acid residues . The VHH protein shows homology to the lecithinase of V . mimicus and V . cholerae . Transformants of Escherichia coli containing the ORF of either vhhA or vhhB displayed weak hemolytic activity in rainbow trout blood agar . The hemolytic activity was very high when the ORF of vhhB was cloned in E . coli together with the native promoter . Surprisingly, the level of vhh-specific RNA transcript produced by VIB 645 was found to be very low . We conclude that the hemolytic phenotype of VIB 645 is not due to increased expression of one or both copies of the vhh gene. Appl Environ Microbiol, 2001 Jul, 67(7), 2895 - 902 Antacid increases survival of Vibrio vulnificus and Vibrio vulnificus phage in a gastrointestinal model; Koo J et al.; Viable counts of three strains of Vibrio vulnificus and its phage were determined during exposure to a mechanical gastrointestinal model with or without antacid for 9 h at 37 degrees C . V . vulnificus was eliminated (>4-log reduction) within 30 min in the gastric compartment (pH decline from 5.0 to 3.5) . Viable V . vulnificus cells delivered from the gastric compartment during the first 30 min of exposure reached 10(6) to 10(8) CFU/ml in the intestinal compartment after 9 h (pH 7.0) . Phages were eliminated within 45 min in the gastric compartment (pH decline from 5.1 to 2.5) . Less than a 2-log reduction of phage was observed in the intestinal compartment after 9 h (pH 7.0) . When the gastric compartment contained antacid V . vulnificus counts decreased slightly (<2 log) during 2 h of exposure (pH decline from 7.7 to 6.0), while counts in the intestinal compartment (pH 7.5) reached 10(7) to 10(9) CFU/ml . Phage numbers decreased 1 log after 2 h in the gastric compartment (pH decline from 7.7 to 5.7) containing antacid and decreased 1 log in the intestinal compartment (pH 7.6) after 9 h . Presence of antacid in the gastric compartment of the model greatly increased the ability of both V . vulnificus and its phage to survive simulated gastrointestinal transit and may be a factor involved with oyster-associated illness. Biophys J, 2001 Jul, 81(1), 382 - 93 Photoinduced transient absorbance spectra of P840/P840(+) and the FMO protein in reaction centers of Chlorobium vibrioforme; Vassiliev IR et al.; The kinetics of photoinduced absorbance changes in the 400-ns to 100-ms time range were studied between 770 and 1025 nm in reaction center core (RCC) complexes isolated from the green sulfur bacterium Chlorobium vibrioforme . A global, multiple stretched-exponential analysis shows the presence of two distinct but strongly overlapping spectra . The spectrum of the 70-micros component consists of a broad bleaching with two minima at 810 and 825 nm and a broad positive band at wavelengths greater than 865 nm and is assigned to the decay of (3)Bchl a of the Fenna-Matthews-Olson (FMO) protein . The contribution of the 70-micros component correlates with the amount of FMO protein in the isolated RCC complex . The spectrum of the 1.6-micros component has a sharp bleaching at 835 nm, a maximum at 805 nm, a broad positive band at wavelengths higher than 865 nm, and a broad negative band at wavelengths higher than 960 nm . When the RCC is incubated with inorganic iron and sulfur, the 1.6-micros component is replaced by a component with a lifetime of approximately 40 micros, consistent with the reconstruction of the F(X) cluster . We propose that the 1.6-micros component results from charge recombination between P840(+) and an intermediate electron acceptor operating between A(0) and F(X) . Our studies in Chlorobium RCCs show that approaches that employ a single wavelength in the measurement of absorption changes have inherent limitations and that a global kinetic analysis at multiple wavelengths in the near-infrared is required to reliably separate absorption changes due to P840/P840(+) from the decay of (3)Bchl a in the FMO protein. Environ Toxicol, 2001 Jun, 16(3), 277 - 86 Effects of a pre-incubation period on the photoinduced toxicity of polycyclic aromatic hydrocarbons to the luminescent bacterium Vibrio fischeri; el-Alawi YS et al.; Irradiation of polycyclic aromatic hydrocarbons (PAHs) in aqueous solution with simulated solar radiation (SSR; a light source with a visible light: UV-A:UV-B ratio similar to that of sunlight) can greatly enhance their toxicity . Two microbial toxicity tests with Vibrio fischeri were used to investigate the effect of composition of the growth medium and pre-incubation on the photoinduced toxicity of PAHs . The assays were a short-term test (15 min) and long-term test (18 h) . Both assays were carried out in SSR and darkness to examine for photoinduced toxicity of PAHs . For the short-term toxicity assay, inhibition of bacterial luminescence was measured . For the long-term toxicity assay, both inhibition of bacterial luminescence and inhibition of growth were recorded . To broaden this test, V . fischeri cells were pre-incubated with PAHs in medium without a carbon source (minimal medium) for 8 h to facilitate assimilation and photooxidation of the contaminants, and to prevent bacterial growth at the outset of the assay . V . fischeri was more sensitive in minimal medium than in complex medium in both the short- and long-term toxicity assays . Moreover, in the long-term assay, SSR greatly increased toxicity, especially if there was a pre-incubation period in minimal medium . This indicates that both assimilation and photooxidation of PAHs are important to their toxicity to V . fischeri. J Appl Microbiol, 2001 Jun, 90(6), 919 - 27 Characterization of Aeromonas and Vibrio species isolated from a drinking water reservoir; Ivanova EP et al.; AIMS: To study the phenotypic and chemotaxonomic (i.e . phospholipid and cellular fatty acid composition) characteristics of environmental Aeromonas spp . and Vibrio spp . isolated from a drinking water reservoir near Vladivostok City, and the application of some chemotaxonomic markers for discrimination of the two genera and species . METHODS AND RESULTS: Presumptive Aeromonas species were dominant in surface water samples (up to 25% of the total number of bacteria recovered) . These strains were consistent with respect to the cultural and biochemical properties used to define the species Aeromonas sobria (seven strains) and Aer . popoffii (three strains) . Vibrio mimicus (two strains) and Vibrio metschnikovii (one strain) were identified according to phenotypic features and cellular fatty acid composition . CONCLUSION: Environmental Aer . sobria isolates were atypical in their ability to grow at 42 degrees C, and were haemolytic, proteolytic and cytotoxic . Although it was present in a high proportion in the water samples, atypical Aer . sobria is not an indicator of polluted water . SIGNIFICANCE AND IMPACT OF THE STUDY: The incidence of Aeromonas in the drinking water reservoirs in the Far East of Russia is reported for the first time. Int J Syst Evol Microbiol, 2001 May, 51(Pt 3), 1035 - 44 Isolation of Desulfomicrobium orale sp . nov . and Desulfovibrio strain NY682, oral sulfate-reducing bacteria involved in human periodontal disease; Langendijk PS et al.; The species of sulfate-reducing bacteria that prevail in sites affected by periodontal disease may be different from those commonly occurring in the digestive tracts of healthy individuals . Ten strains of mesophilic sulfate-reducing bacteria (SRB) were isolated from subgingival plaque in periodontal lesions of ten patients with periodontitis . Characterization on the basis of morphological, physiological and phylogenetic properties demonstrated two distinct types of oral SRB . One strain was a curved rod with high motility . For dissimilatory sulfate reduction, lactate or pyruvate was oxidized incompletely to equimolar amounts of acetate . Desulfoviridin and cytochrome c3 were present in this mesophilic vibrio and the cellular lipid profile was similar to that from members of the genus Desulfovibrio . The 16S rDNA sequence was similar to that of the proposed 'Desulfovibrio fairfieldensis' . Cells of the nine other strains were straight, rod-shaped, exhibited a low growth rate and oxidized substrates incompletely to acetate . These SRB, like members of the genus Desulfomicrobium, lacked desulfoviridin . Analysis of the 16S rDNA sequences of seven of the nine isolates showed a high degree of similarity among these oral strains, forming a distinct lineage within the genus Desulfomicrobium . The cellular lipid profile of a representative oral strain, NY678T, was in accordance with that of other Desulfomicrobium species, but also showed dissimilar features . The phenotypic and phylogenetic analyses indicate that these rod-shaped SRB from the oral cavity could be regarded as a new species, for which the designation Desulfomicrobium orale sp . nov . is proposed. FEMS Microbiol Lett, 2001 Jun 12, 200(1), 111 - 6 Unusual adaptive, cross protection responses and growth phase resistance against peroxide killing in a bacterial shrimp pathogen, Vibrio harveyi; Vattanaviboon P et al.; Oxidant induced protection against peroxide killing was investigated in a prawn bacterial pathogen, Vibrio harveyi . Exposure to 250 microM H(2)O(2) induced adaptive protection against subsequent exposure to killing concentrations of H(2)O(2) . In addition, 200 microM t-butyl hydroperoxide (tBOOH) induced cross protection to H(2)O(2) killing . On the other hand, peroxide pretreatment did not induce protection against tBOOH killing . Peroxide induced adaptive and cross protection responses required new protein synthesis and were abolished by addition of a protein synthesis inhibitor . Pretreatments of V . harveyi with 250 microM H(2)O(2) and 200 microM tBOOH induced an increase in peroxide scavenging enzymes, catalase and alkyl hydroperoxide reductase subunit C . In addition, stationary phase cells of V . harveyi were more resistant to H(2)O(2) and iodoacetamide killing but highly susceptible to tBOOH killing compared to exponential phase cells . Many aspects of the oxidative stress response of V . harveyi are different from those of other bacteria and these factors may be important for bacterial survival in the environment and during interactions with host shrimp. J Commun Dis, 2000 Sep, 32(3), 207 - 11 An outbreak of Eltor cholera in Aizwal town of Mizoram, India; Sengupta PG et al.; During the months of May, June and through early part of July 1994, an unusual occurrence of severe dehydrating watery diarrhoea cases and deaths were reported from Aizwal town, the capital of Mizoram, a North-Eastern state of India . Vibrio cholerae 01 biotype Eltor, the causative agent responsible for this outbreak, was isolated from 50.0% of hospitalised cases . The disease affected older children and adults more (52.9%) than younger children below five years of age . Vibrio cholerae 01 strains isolated were uniformly resistant to furazolidone and co-trimoxazole, which are commonly advocated in the treatment of cholera specially in children of developing countries . Emergence of such resistant strain is alarming and is of great public health importance. Infect Immun, 2001 Jul, 69(7), 4681 - 5 Vibrio cholerae tolC is required for bile resistance and colonization; Bina JE et al.; TolC and its homologues are outer membrane proteins that are essential for the transport of many molecules across the cell envelope . In this study we characterized the gene encoding Vibrio cholerae TolC . V . cholerae tolC mutants failed to secrete the RTX cytotoxin, were hypersensitive to antimicrobial agents, and were deficient in intestinal colonization. Biochemistry, 2001 Jun 19, 40(24), 7318 - 23 Sodium-dependent steps in