A tetrodotoxin-producing Vibrio strain, LM-1, from the puffer fish Fugu vermicularis radiatus

Identification of tetrodotoxin (TTX) and its derivatives produced from a Vibrio strain in the intestine of the puffer fish Fugu vermicularis radiatus was performed by thin-layer chromatography, electrophoresis, high-performance liquid chromatography, and gas chromatography-mass spectrometry, together with a mouse bioassay for toxicity. It was demonstrated that the isolated bacterium produced TTX, 4-epi-TTX, and anhTTX during cultivation, suggesting that Vibrio strains are responsible for the toxification of the puffer fish.     

Reexamination of tetrodotoxin production by bacteria.

Vibrio alginolyticus has been reported as a good producer of tetrodotoxin (TTX), but the toxin extracted from this bacterium did not react to the monoclonal antibody against TTX. Surprisingly, chromatographic analyses detected high TTX peaks for polypeptone and yeast extracts used as medium materials, which were, as expected, all negative by the mouse bioassay. These results may require us to revise the bacterial production of TTX.     

Effects of different dietary levels of fish protein hydrolysates on growth, digestive enzymes, gut microbiota, and resistance to Vibrio anguillarum in European sea bass (Dicentrarchus labrax) larvae

Two fish protein hydrolysates (FPH) were incorporated into four diets prepared for start-feeding sea bass larvae, at two different levels (10% and 19% of total ingredients): a commercial FPH, CPSP, in which the molecular mass of the main fraction of soluble peptides (51%) was between 500-2500 Da, and an experimental FPH obtained by acidic silage of sardine offal, SH, with a main portion of soluble peptides (54%) ranging from 200 to 500 Da. The diet with 10% of the commercial FPH gave the best results in terms of growth, survival and intestinal development, as evaluated by the early activity of digestive enzymes in the brush border membrane (alkaline phosphatase and aminopeptidase N). This was related to the low level of Vibrio spp. counted in the larvae of group C10. The high dose of FPH, especially in the experimental preparation rich in short peptides, seemed to favour the dominance of Vibrio sp. TYH3, which behaved opportunistically. The effect of the experimental FPH was ambiguous, since early larvae challenged with Vibrio anguillarum were more resistant to the pathogen, especially at high FPH dose (group S19). This might be due either to direct antagonism between V. anguillarum and Vibrio sp. TYH3, or to the stimulation of the immune response in the larvae. These results indicate that different molecular weight fractions and concentrations of feed-soluble peptides may affect the growth performance and immunological status of sea bass larvae. Consequently, a low dose of commercial FPH seems advisable, both for larval development and for the bacterial environment, although further research is required to determine and characterize peptide fractions that may have a beneficial effect on growth and immune response, and to determine their optimal inclusion levels in diets for sea bass larvae.     

Vibrios of some deep-water invertebrates

Abstract The gut microbiota of 7 species of deep-water (300–400 m) invertebrates from the Gulf of Mexico was examined. High populations of Vibrio spp. were observed in crustaceans (ranging from 10⁵ to 7 × 10⁶ cells/g gut content) while relatively low populations of Vibrio spp. were found in annelids, the water column, and sediment. Although saprophytic Vibrio species were isolated, Vibrio fluvialis and Vibrio hollisae , potential human pathogens, were isolated from the crustaceans, Pleoticus robustus, Nematocarcinas sp., Plesionika sp., and Munida sp., and Vibrio vulnificus was isolated from Nereis sp. These observations confirm the finding of Ohwada et al. [18] that the gut of deep-water invertebrates has a bacterial flora abundant in Vibrio spp. These results also suggest that some marine invertebrates may serve as reservoirs for certain potential pathogenic Vibrio species.     

The induction of stress proteins in three marine Vibrio during carbon starvation

Abstract The induction of DnaK and GroEL homologous proteins by heat-shock and long-term carbon starvation was studied in Vibrio vulnificus, Vibrio sp. strain S14, and Vibrio sp. strain DW1. In each Vibrio strain one protein (60 kDa) reacted with antibodies against Escherichia coli -GroEL and two proteins, DnaK (69 kDa) and Sis1 (62-60 kDa), reacted with antibodies against E. coli -Dnak. The carbon starvation elicited induction of the stress proteins was strain-specific, suggesting that the induction of stress proteins like DnaK and GroEL in marine Vibrios might not be a uniform starvation response. It appears as of these proteins, only DnaK in Vibrio sp. strain S14 remains induced after long-term carbon starvation in the three marine bacterial strains that were tested.     

Expression of the B subunit of Escherichia coli heat-labile enterotoxin in a marine Vibrio and in a mutant that is pleiotropically defective in the secretion of extracellular proteins.

A marine Vibrio (designated Vibrio sp. 60) that is related to Vibrio anguillarum was used as a host for a plasmid that encodes the non-toxic B subunit (EtxB) of Escherichia coli heat-labile enterotoxin. Expression of EtxB in Vibrio sp. 60 resulted in the efficient and selective secretion of the B subunit into the extracellular growth medium. This indicated that Vibrio sp. 60, which does not normally produce cholera-like enterotoxins, nonetheless possesses a secretory machinery that permits these toxins to be translocated across its cytoplasmic and outer membranes. Expression of EtxB in a sec mutant of Vibrio sp. 60 (MVT1192), which had previously been shown to be defective in the secretion of several extracellular proteins, resulted in approximately 95% of the B subunit remaining entrapped within the periplasm of the bacterial cell envelope. This implies that the mutation in MVT1192 defines a locus that determines a common step in the secretion of extracellular proteins, including oligomeric toxins.     

罗尼氏弧菌Vibrio shilonii BY新琼胶酶基因的克隆、序列分析及表达

【目的】从海洋来源的罗尼氏弧菌菌株BY中克隆得到一个具有琼胶酶活性的新基因,并对其进行重组表达。【方法】对实验室保藏的产琼胶酶菌株BY进行16S rRNA基因序列分析,并构建系统发育树。根据已报道的琼胶酶基因序列的同源性,设计简并引物,利用降落PCR (Touch-down PCR)及染色体步移技术扩增琼胶酶基因序列全长,对基因序列进行生物信息学分析。将目的基因插入pET22a(+)载体,转化大肠杆菌BL21(DE3),对重组酶进行表达,利用DNS法测定了重组酶的酶活,对该重组琼胶酶酶学性质进行研究。【结果】克隆得到一条新的琼胶酶基因,命名为Vibrio sp. BY (GenBank登录号：AIW39921.1),Vibrio sp. BY基因序列全长2 232 bp,编码744个氨基酸,理论分子量为85 kD,Vibrio sp. BY的氨基酸序列基因库中与已知的琼胶酶氨基酸序列Vibrio sp. EJY3的相似度为86%。发酵液琼胶酶酶活力为71.73 U/mL,证明表达的蛋白为琼胶酶。酶学性质研究表明重组琼胶酶的最适温度及pH分别为50 °C和7.0,并且具有较好的稳定性。【结论】利用染色体步移技术克隆得到一条新的琼胶酶基因,并在大肠杆菌BL21(DE3)中实现了重组表达,为琼胶酶的应用奠定了基础。     

三种蜘蛛粗毒对NG108—15细胞电压门控钠通道的抑制作用

利用小鼠神经细胞瘤×大鼠神经胶质细胞的杂交细胞NG108-15,通过全细胞记录(whole-cellrecording)模式的膜片钳技术,检验了虎纹捕鸟蛛(Selenocosmia huwena)、海南捕鸟蛛(Selenocosmia hainana)和广西大疣蛛(Macrothele guangxiasp)的粗毒对NG108-15细胞膜上电压门控TTX敏感型钠电流和延迟整流钾电流的作用.结果表明,三种蜘蛛粗毒对外向延迟整流钾电流没有明显作用,但对TTX敏感型的快钠电流表现出较强的抑制效应.抑制效应呈量效关系.三种粗毒抑制钠电流的EC     

Bacterial production of histamine in some tropical fish

Quantitative and qualitative distribution of histamine-forming bacteria associated with the fish Rastrelliger kanagurta, Sardinella longiceps, Sillago sihama and Liza subviridis, were investigated. These bacteria constituted a significant portion of the total bacterial population of fish and the values obtained in the present study were higher than those previously reported. The order of quantitative abundance of histamine-forming bacteria in the fish examined was: S. longiceps greater than R. kanagurta greater than S. sihama greater than L. subviridis. The bacterial genera isolated were Vibrio sp., Bacillus sp., Pseudomonas sp., Aeromonas sp. and Micrococcus sp., and among them Vibrio was dominant. Growth of the isolates (Vibrio sp., V. fischeri and Bacillus sp.) at different temperatures, pH and sodium chloride concentrations indicated them to be mesophilic, euryhaline and tolerant to acidic and alkaline pH. Bacillus sp. produced more histamine in R. kanagurta, while V. fisheri produced more histamine in S. longiceps.     

Correlation of taxonomic criteria for a collection of marine bacteria

Numerical taxonomy was done on 208 strains of marine bacteria. The collection was segregated into eight groups, seven of which contained Vibrio sp. Nucleic acid base ratio studies on a typical Vibrio sp. from each group and other genera were done. The phenotypically different Vibrio sp. had a narrow range of base ratios. The other genera had base ratios more similar to the base ratios reported for their genus than to each other as marine bacteria. The taxonomic groups are compared with generic classification and the strains' sources of isolations.     

Evidence for cis-trans isomerization of a double bond in the fatty acids of the psychrophilic bacterium Vibrio sp. strain ABE-1.

Vibrio sp. strain ABE-1 was grown in a medium that contained as its stable isotope tracer either [2,2-2H2]cis-9-hexadecenoic or [2,2-2H2]trans-9-hexadecenoic acid. Gas chromatographic-mass spectrometric analysis of the cis-9-hexadecenoic and trans-9-hexadecenoic acid fractions from the cells revealed the formation of an intracellularly isomerized 2,2-2H2-fatty acid which differed from the tracer only in the geometrical configuration of the double bond. This observation shows that cis-trans isomerization without a shift in double-bond position between these two geometric hexadecenoic acid isomers can occur in the cells.     

The production of tetrodotoxin-like substances by nemertean worms in conjunction with bacteria

Evidence is presented which strongly indicates a relationship between the presence of Vibrio bacteria, probably Vibrio alginolyticus, and the synthesis of tetrodotoxin (TTX)-like chemicals in seven species of British nemerteans. The occurrence of these substances and associated Vibrio bacteria in these species was investigated by bacteriological, chromatographic, spectroscopic and ultraviolet spectrometric techniques. It is suggested that these toxins are utilised by the nemerteans as a chemical defence against potential predators.     

Vibrio sp. as a potentially important member of the Black Band Disease (BBD) consortium in Favia sp. corals

Black Band Disease (BBD) is a well-described disease plaguing corals worldwide. It has been established that ecological and environmental stress factors contribute to the appearance and progression of the disease, believed to be caused by a diverse microbial consortium. We have identified and characterized Vibrio sp. associated with BBD in Eilat reef corals using both culture-dependent and -independent methods. Direct sampling using 16S rRNA gene clone libraries showed seasonal dynamics in the diversity of BBD-associated Vibrios . In the two sampling periods, BBD-associated Vibrio clones showed similarities to different groups: October samples were similar to known pathogens, while December samples were similar to general aquatic Vibrio sp. Cultured bacterial isolates of Vibrio sp. were highly homologous (≥99%) to previously documented BBD-associated bacteria from the Caribbean, Bahamas and Red Seas, and were similar to several known coral pathogens, such as Vibrio coralliilyticus . The proteolytic activity of Vibrio sp., as measured using casein- and azocasein-based assays, directly correlated with temperature elevation and peaked at 26–28 °C, with the microorganisms producing more proteases per bacterial cell or increasing the rate of proteolytic activity of the same proteases (potentially metalloproteases). This activity may promote coral tissue necrosis and aid in ensuing progression of the coral BBD.     

Cloned aerolysin of Aeromonas hydrophila is exported by a wild-type marine Vibrio strain but remains periplasmic in pleiotropic export mutants.

With a wide host range vector, the structural gene aerA for the hole-forming extracellular protein aerolysin of Aeromonas hydrophila was cloned into the marine Vibrio sp. strain 60 and into three pleiotropic export mutants (epr mutants). The parent strain and all of the mutants were able to express the protein with the aerA promoter in the plasmid. The parent strain exported proaerolysin into the medium, while all of the mutants accumulated the protoxin in their periplasms. Two of the mutants also accumulated protease; however, as we have found earlier with A. hydrophila, the periplasmic form of proaerolysin in the Vibrio sp. must somehow be protected from proteolysis because it was not converted to active toxin until the cells were shocked. Conversion could be prevented by adding o-phenanthroline to the solutions used in shocking. These results show that the export pathway in the marine Vibrio sp. is very similar to the pathway in A. hydrophila.     

Effect of Bacteria Associated with the Green Alga Ulva reticulata on Marine Micro- and Macrofouling

The green alga Ulva reticulata (Forsskal) is often free from biofouling in Hong Kong waters. An early study indicated that bioactive substances from this alga inhibit settlement of the polychaete Hydroides elegans (Haswell). It is also predicted that epibiotic bacteria protect this alga from micro- and macrofouling. In this study, bacterial strains from the surface of U. reticulata were isolated and their inhibitive activities on micro- and macrofouling assayed. The strains were identified by 16S rRNA analysis as belonging to the genera Alteromonas , Pseudoalteromonas and Vibrio . There was no significant effect of these strains or their extracts (aqueous and ethanol) on the growth of five Vibrio strains isolated from natural biofilm. Two bacterial strains ( Alteromonas sp. and Vibrio sp. 3) were non-toxic to the benthic diatom Nitzschia paleacea (Grunow) while the other five strains caused a low level of mortality. No one bacterial strain was toxic to the larvae of H. elegans . Aqueous extract of one of the isolated bacterial species, i.e. Vibrio sp. 2, significantly ( p <0.00001) inhibited the settlement and metamorphosis of H. elegans larvae. The putative antifouling compounds have a molecular weight of >100 kD. On the other hand, biofilm of Pseudoalteromonas sp. 2 and aqueous extract of Vibrio sp. 2 suppressed the settlement of larvae induced by 3-isobutyl-1-methylxanthine (IBMX). Other epibiotic bacteria and their extracts had neither inhibitive nor inductive effects on larval settlement of H. elegans . The results indicate that the antifouling mechanism of U. reticulata may be dependent not only on materials from the macroalga itself but also on the epibiotic bacteria on the algal surface.     

Global analysis of the carbon starvation response of a marine Vibrio species with disruptions in genes homologous to relA and spoT.

The stringent control response, which involves a rapid accumulation of ppGpp, is triggered if the marine Vibrio sp. strain S14 is subjected to carbon and energy starvation. By means of high-resolution two-dimensional gel electrophoresis analysis, we addressed the role of the major ppGpp-synthesizing enzyme (RelA) in the regulation of the carbon starvation response of Vibrio sp. strain S14. The finding that a large number of the carbon starvation-induced proteins were underexpressed in the Vibrio sp. S14 relA mutant strain after the onset of glucose starvation suggests that a rapid accumulation of ppGpp is required for induction of many of the carbon starvation-induced proteins. However, it was also found that a majority of the carbon starvation-induced proteins were significantly less induced if the stringent control response was provoked by amino acid starvation. We therefore also addressed the notion that a carbon starvation-specific signal transduction pathway, complementary to the stringent control, may exist in Vibrio sp. strain S14. It was found that a majority of the proteins that were underexpressed in the relA mutant strain were also underexpressed in the Vibrio sp. S14 spoT mutant strain (csrS1). Interestingly, a large proportion of these underexpressed proteins were found to belong to a group of proteins that are not, or significantly less, induced by starvation conditions that do not promote starvation survival. On the basis of these observations and the finding that the csrS1 strain survives poorly but accumulates ppGpp in a fashion similar to the wild type during carbon and energy source starvation, the gene product of the csrS gene is suggested to be responsible for the mediation of a signal which is complementary to ppGpp and essential for the successful development of the starvation- and stress-resistant cell. This conclusion was also supported by experiments in which changes in phenotypic characteristics known to be induced during carbon starvation were studied. The starvation induction of the high-affinity glucose uptake system was found to be dependent on the csrS gene but not relA, and the synthesis of carbon starvation-specific periplasmic space proteins was dependent, at different times of starvation, on both the relA and the csrS gene products.     

Purification and characterization of an extracellular beta-1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138.

A beta-mannanase (EC 3.2.1.78) from Vibrio sp. strain MA-138 was purified by ammonium sulfate precipitation and several chromatographic procedures including gel filtration, adsorption, and ion-exchange chromatographies. The final ion-exchange chromatography Mono Q yielded one major active fraction and three minor active fractions. The major active fraction was purified to homogeneity on the basis of native polyacrylamide gel electrophoresis (PAGE). This purified enzyme was identified as a glycoprotein by periodic acid-Schiff staining and a monomeric protein with a molecular mass of 49 kDa by sodium dodecyl sulfate-PAGE. The pI of the enzyme was 3.8. The purified enzyme exhibited maximal activity at pH 6.5 and 40 degrees C and hydrolyzed at random the internal beta-1,4-mannosidic linkages in beta-mannan to give various sizes of oligosaccharides. The first 20 N-terminal amino acid sequence of the purified enzyme showed high homology with the N-terminal region of beta-mannanase from Streptomyces lividans 66.     

Isolation and characterization of mutants of a marine Vibrio strain that are defective in production of extracellular proteins

A marine Vibrio strain, Vibrio sp. strain 60, produces several extracellular proteins, including protease, amylase, DNase, and hemagglutinin. Mutants of Vibrio sp. strain 60 (epr mutants) pleiotropically defective in production of these extracellular proteins were isolated. They fell into two classes, A and B. In class A, no protease activity was detected in the cells either, whereas in class B, considerable protease activity was detected in the cells. Gel electrophoretic analysis revealed that the protease detected in class B mutant cells was similar to the protease excreted by the parent strain. In addition, the protease in class B mutant cells was found to be localized in the periplasmic space. These results suggest that the passage of the protease through the outer membrane is blocked in class B mutants. Comparison of membrane protein profiles by polyacrylamide gel electrophoresis revealed that all the epr mutants contained an increased amount of a 94,000-Mr protein that may be an outer membrane protein. Four epr mutations were mapped in two different regions of the Vibrio chromosome by transduction; two class A mutations and one class B mutation were located close to each other, whereas another class B mutation was located in a different region of the chromosome.     

Microbial diversity within early-stage cultured Panulirus ornatus phyllosomas

A thorough understanding of the microorganisms and pathogens associated with the larval stage of the tropical ornate rock lobster, Panulirus ornatus, is required to overcome disease outbreaks that currently block aquaculture attempts. This study used microscopy in addition to culture and molecularly based microbiological techniques to characterize the bacterial community associated with cultured, developmental stage PI to PII P. ornatus phyllosomas. Scanning electron microscopy demonstrated colonization of phyllosomas by filamentous, rod-shaped, and coccus-shaped bacteria. A clone library constructed from dead phyllosomas sampled from the larval rearing tank on day 10 was dominated by Thiothrix-affiliated sequences (56% of clones). A comparable library from live phyllosomas also contained Thiothrix-affiliated sequences, though these only represented 19% of clones within the library. Fluorescent in situ hybridization (FISH) confirmed identification of the filamentous bacteria as Thiothrix sp., being present on dead phyllosomas. FISH also identified Leucothrix sp. and Vibrio sp., as well as a range of other rod- and coccus-shaped bacteria, colonizing both live and dead phyllosomas. The development of the microbial community associated with phyllosomas was monitored through a standard larval rearing run using denaturing gradient gel electrophoresis (DGGE). Vibrio sp.-affiliated bands dominated the profiles of live animals through the rearing period and dead phyllosomas sampled on selected days. The population of Vibrio sp. associated with phyllosomas was monitored with culture-based analysis on selective media and demonstrated to increase significantly on day 7, coinciding with the beginning of the larval molt. An isolated Vibrio harveyi strain demonstrated an identical 16S rRNA sequence with retrieved DGGE and clone library sequences. Colonization of phyllosomas with filamentous bacterial species potentially hinders the ability of the animals to molt and, combined with the added stress of the molt process, likely results in reduced immune function, allowing opportunistic pathogenic Vibrio sp. to cause larval mortalities.     

Effects of bacteria on the growth of an amoeba infecting the gills of turbot.

We analysed the influence of various bacteria on the in vitro growth of trophozoites of a Platyamoeba strain isolated from diseased gill tissues of cultured turbot. Little or no growth was shown by amoebae cultured in the presence of (1) the turbot-pathogenic bacteria Vibrio anguillarum, Aeromonas salmonicida or Streptococcus sp., (2) Pasteurella piscicida or Vibrio vulnificus (pathogenic for some fishes but not turbot), or (3) the non-pathogenic 'environmental' bacteria Vibrio campbelli, Vibrio fluvialis or Pseudomonas dondorofii. The only bacteria which were successfully utilized as food sources were Aeromonas hydrophila (pathogenic for some fishes but not turbot) and the non-pathogens Vibrio natriegens, Pseudomonas nautica and Escherichia coli. These results suggest that the colonization of the gills of cultured turbot by the epizoic amoeba Platyamoeba may be an indicator of faecal contamination.