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Lipopolysaccharides (LPS) from an opaque and a translucent colony variant of Vibrio vulnificus were isolated by several methods and the electrophoretic profiles were analysed. The phenol-water extraction method provided a better yield compared to the phenol-chloroform-petroleum ether method. In addition, two rapid micro-assays were used to isolate LPS for electrophoretic analysis. The electrophoretic pattern was the same for all LPS extracts and was similar in both variants. No high molecular weight bands, characteristic of smooth LPS, were detected in the LPS of this organism. This was in contrast to the smooth nature of the LPS of another member of the Vibrionaceae examined, V. cholerae. The result of this study showed no correlation between LPS and colony morphology in V. vulnificus.

A high level of a trans-unsaturated fatty acid was found in the phospholipids of a psychrophilic bacterium, Vibrio sp. strain ABE-1. This fatty acid was identified as 9-trans-hexadecenoic acid (C16:19t) by gas-liquid chromatography and infrared absorption spectrometry. C16:1(9)t accounted for less than 1% of the total fatty acids in cells grown at 5 degrees C and reached 12% of the total at 20 degrees C. We suggest that the increase in the level of the trans-unsaturated fatty acid is related to the high growth rate of this bacterium at elevated temperatures. Possible biological roles of the trans-unsaturated fatty acid in the adaptation of the microorganism to the ambient temperature are discussed.

A rapid particle agglutination assay (PAA) utilizing latex beads coated with connective tissue and serum proteins was evaluated for its ability to identify fibronectin, collagen (types I and IV), fibrinogen, and transferrin cell surface receptors on Vibrio and Aeromonas strains isolated from diseased fish, human infections, and the environment. Similar tests were performed to screen for cell surface lectins. Vibrio as well as Aeromonas strains were found to bind connective tissue proteins (collagen types I, II, and IV and fibronectin), serum proteins (i.e., fibrinogen), and glycoproteins (bovine submaxillary mucin, hog gastric mucin, orosomucoid, and fetuin) immobilized on the latex particles. The specificity of the agglutination reaction was studied by particle agglutination inhibition assays performed by preincubating bacterial suspensions in solutions containing either gelatin (for the various connective tissue protein PAA reagents) or sialic acid-rich glycoproteins (for the various glycoprotein PAA reagents). Expression of cell surface receptors for connective tissue proteins was found to depend on culture methods.

Vibrio vulnificus is a marine bacterium associated with both primary septicemias and wound infections in humans. The lipopolysaccharides of a virulent and an avirulent strain of Vibrio vulnificus were compared with respect to their chemical constituents and electrophoretic characteristics. 2-Keto-3-deoxyoctonic acid, a normal constituent of the lipopolysaccharide of typical Enterobacteriaceae, was not found in the lipopolysaccharide of either strain. Hexadecenoate (C16:1) was the predominant fatty acid of the lipid A moiety of the lipopolysaccharides and of the membrane phospholipids of both strains. Hydroxy fatty acids composed 44% of the total fatty acids of the lipid A of the avirulent and 40% of those in the virulent strain. In addition, odd-numbered fatty acids were detected in both lipopolysaccharides. The electrophoretic profile was similar for both strains, but demonstrated no "ladder-like" pattern characteristic of "smooth" lipopolysaccharides. The result of this study showed no significant differences between the lipopolysaccharides of the virulent and avirulent strains of Vibrio vulnificus. The possible role for lipopolysaccharide in pathogenesis of Vibrio vulnificus infections is discussed.

Thermograms obtained by differential scanning calorimetry of a range of bacteria of different heat resistances were compared. Equations were derived to calculate the rate at which the numbers of viable organisms in a calorimeter decline as the temperature is raised at a constant rate. Vegetative bacteria scanned at 10 degrees C min-1 showed multi-peaked thermograms with four major peaks (denoted m, n, p and q) occurring in the regions 68-73, 77-84, 89-99 and 105-110 degrees C respectively. Exceptions were that peak m (the largest peak) occurred at 79-82 degrees C in Bacillus stearothermophilus and an additional peak, r, was detected in Escherichia coli at 119 degrees C. At temperatures below the main peak m there were major differences in thermograms between species. There was a direct relationship between the onset of thermal denaturation and the thermoresistance of different organisms. Heat-sensitive organisms displayed thermogram features which were absent in the more heat-resistant types. When samples were cooled to 5 degrees C and re-heated, a small endothermic peak, pr, was observed at the same temperature as p. Peaks p and pr were identified as the melting endotherms of DNA. In all vegetative organisms examined, maximum death rates, computed from published D and z values, occurred at temperatures above the onset of thermal denaturation, i.e. cell death and irreversible denaturation of cell components occurred within the same temperature range.

The systematic position of some Vibrio species was ascertained by the method of molecular DNA -- DNA hybridization. The DNA of the brine vibrios V. costicola and V. fischeri were shown to have about 10% of sequences homologous with DNA of a typical cholera vibrio (V. cholerae eltor No. 334). Similarity between the genomes of other representatives of the Vibrionaceae family, as well as in DNA hybridization of V. costicola and V. fischeri, was found to be approximately on the same level. All species included into the genus, Vibrio on account of their phenotypic characteristics may be considered to have essential differences in the structures of their genomes.

Large amounts of an extremely potent extracellular cytolysin produced by the halophilic bacterium Vibrio damsela were obtained free of detectable contamination with medium constituents and other bacterial products by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-100, and hydrophobic interaction chromatography with phenyl-Sepharose CL-4B. The cytolysin is heat labile and protease sensitive and has a molecular weight (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of ca. 69,000 and an isoelectric point of ca. 5.6. The first 10 amino-terminal amino acid residues of the cytolysin are Phe-Thr-Gln-Trp-Gly-Gly-Ser-Gly-Leu-Thr. The cytolysin was very active against erythrocytes from 4 of the 18 animal species examined (mice, rats, rabbits, damselfish) and against Chinese hamster ovary cells and was lethal for mice (ca. 1 microgram/kg, intraperitoneal median lethal dose). Lysis of mouse erythrocytes by the cytolysin is a multi-hit, at least two-step process consisting of a temperature-independent, toxin-binding step followed by a temperature-dependent, membrane-perturbation step(s).

Proteinograms of 112 strains of vibrios and closely affiliated microorganisms were studied by disc electrophoresis in polyacrylamide gel. Up to 25 protein peaks with definite mobility coefficients were revealed. The influence of the culture medium on the protein spectrum of the microbes was found. The frequency of peak formation was of great significance for the differentiation of the microbes under study. The quantitative characteristics of the peak area could not be used for differentiation.

Several proteins and polypeptides of reptilian, amphibian, insect, and microbial origin share a common cytolytic property. However, these cytolysins fulfill different objectives. They provide offensive armament in the case of toxins, but defensive systems in the case of antibacterial peptides. The sequences of several nonenzymatic cytolysins and their analogues were compared to identify the structural requirements for cytolytic activity. These cytolysins, although isolated from phylogenetically unrelated organisms, possess the common sequence features of a cationic site flanked by a hydrophobic surface. The presence of such a region apparently confers the cytolytic activity of various cytolysins. The concept of a cytolytic region is strongly supported by the existence of several natural and synthetic analogues of cytolysins and by chemical modification studies of these cytolysins. This prediction provides a new focus for cytolysin research. The understanding of this structure-function relationship should facilitate the design, synthesis, and development of better antibacterial and anticancer peptides.

Strains representative of species of the marine genera Beneckea and Photobacterium were used as reference standards in in vitro DNA/DNA competition experiments. Within a given species, strains were found to be related by over 80% competition. (Competition was defined as the amount of radioactive DNA displaced by heterologous DNA relative to the amount displaced by homologous DNA.) On the basis of interspecies competition values (expressed as averages), the following groupings could be made: 1. "Photobacterium" fischeri was related to strain ATCC 15382 by a competition of 38% and was distinct from all the other strains tested (competition less than or equal to 11%). 2. The genus Photobacterium consisted of 3 species, P.phosphoreum, P.leiognathi, and a newly designated species, P.angustum (composed of non-luminous strains). The latter species was found to be related to P.leiognathi and P.phosphoreum by 56 and 28% competition, respectively, while P.phosphoreum was related to P.leiognathi by 29%. 3. In the genus Beneckea, 65% competition was detected between B.harveyi and B.campbellii as well as between B.parahaemolytica and B.alginolytica. These pairs of species were related to each other by 51-58% and to B.natriegens by 34-56% competition. A newly designated pathogenic species, B.vulnifica, appeared to have a low but significant relationship to all the above mentioned species of Beneckea. 4. Two biotypes, related by 68% competition, were recognized in the species B.splendida. Similarly, B.pelagia was found to consist of 2 biotypes related by a competition of 67%. The competition values between these species were 38-40%. 5. B.nereida, B.nigrapulchrituda, and "Vibrio" anguillarum had competition values less than or equal to 30% to each other as well as to other species of Beneckea. 6. With Vibrio cholerae as the reference standard, V.albensis was found to be related by a competition of 82%, while V.proteus and V.metschnikovii had competition values of 22 and 12%, respectively. These results suggested that V.albensis should be synonymized with V.cholerae, while the latter two organisms should remain distinct from this species. V.cholerae as well as the other terrestrial organisms tested did not appear to be significantly related to any of the marine strains (competition values less than or equal to 27%). The speciation derived from the results of the DNA/DNA competition experiments was compared to previous speciation based on phenotypic similarities.

It was revealed that in fractionation of nucleic acids by Schmidt-Tannhauser's method up to 90% of the DNA of bacteria was destroyed in the course of alkaline hydrolysis changing into the RNA fraction (the alkali-labile fraction of the DNA). The authors suggest a modification of the method based on the precipitation of the alkali-labile DNA fraction from the RNA fraction with ethanol. This modified method permits to record over 96% of all the cell DNA of bacteria.

The coenzyme Q system was examined on 55 strains of Gram negative aerobic or facultatively anaerobic rods. No bacteria contain Co-Q7 nor Co-Q10. Ubiquinone Q8 predominates in Flavobacterium and in Enterobacteriaceae; Q9 was the only homolog found in the Pseudomonas, and predominates in the Acinetobacter.

A low molecular weight protein (approximately 25,000 D) exhibiting a yellow fluorescence emission peaking at approximately 540 nm was isolated from Vibrio fischeri (strain Y-1) and purified to apparent homogeneity. FMN is the chromophore, but it exhibits marked red shifts in both the absorption (lambda max = 380, 460 nm) and the fluorescence emission. When added to purified luciferase from the same strain, which itself catalyzes an emission of blue-green light (lambda max approximately 495 nm), this protein induces a bright yellow luminescence (lambda max approximately 540 nm); this corresponds to the emission of the Y-1 strain in vivo. This yellow bioluminescence emission is thus ascribed to the interaction of these two proteins, and to the excitation of the singlet FMN bound to this fluorescent protein.

The characteristics of Vh-rTDH, a hemolysin similar to Vp-TDH of Vibrio parahaemolyticus produced by clinical and environmental isolates of Vibrio hollisae, were comparatively studied. All 7 strains of V. hollisae tested were found to produce indistinguishable Vh-rTDH when they were examined by heat-stability test, Western blotting analysis and conventional polyacrylamide slab gel electrophoresis.

Fifteen stations, in two estuaries, along the Northumberland Strait of Nova Scotia were examined between June and September 1981 for a relationship between the concentrations of commonly monitored fecal indicator bacteria and the potential pathogens Candida albicans, Pseudomonas aeruginosa, and Vibrio parahaemolyticus. Increased densities of these three organisms were usually associated with high densities of indicator bacteria. Whereas C. albicans and P. aeruginosa occur in human fecal wastes, V. parahaemolyticus is indigenous to the marine environment and positively responds to elevated nutrient levels in sewage. There is also some evidence that these bacteria survive as long or longer in marine waters than the common indicator bacteria. While membrane-filtration techniques for the enumeration of C. albicans and P. aeruginosa proved satisfactory, a V. parahaemolyticus membrane-filtration method lacked specificity and was supplemented by a most-probable-number method. In marine recreational and shellfish waters, these three organisms could complement fecal coliforms and fecal streptococci as indicators of human fecal contamination.

The 16S ribosomal RNAs from two species of methanogenic bacteria, the mesophile Methanobacterium ruminantium and the thermophile Methanobacterium thermoautotrophicum, have been characterized in terms of the oligonucleotides produced by digestion with T1 ribonuclease. These two organisms are found to be sufficiently related that they can be considered members of the same genus or family. However, they bear only slight resemblance to "typical" Procaryotic genera; such as Escherichia, Bacillus and Anacystis. The divergence of the methanogenic bacteria from other bacteria may be the most ancient phylogenetic event yet detected--antedating considerably the divergence of the blue green algal line for example, from the main bacterial line.

Hemolytic activity of 3 pathogenic strains, of fish Vibrio commonly associated with vibriosis (V anguillarum NCMB6 and NCMB571 strains, and Vibrio sp N7802 strain) was examined, using chicken and mammalian erythrocytes. Vibrio strains NCMB6 and NCMB571 and their culture filtrates had hemolytic activity against 8 kinds of erythrocytes tested, whereas Vibrio strain N7802 produced only a little amount of hemolysin. Strain NCMB571 culture filtrate and its material partially purified by column chromatography were lethal in mice. From 2 peaks of protein, hemolysin was recovered from the 1st peak, which coincided with toxicity in mice. Heat-inactivation of culture filtrate indicated that hemolytic materials may be thermolabile proteins, but that toxic material may be comparatively thermostable.

In the course of the El Tor cholera epidemic in Togo (West Africa), Vibrio parahaemolyticus was identified as the causative agent of a cholera-like gastroenteritis. From September 1971 to March 1973 81 cases were confirmed bacteriologically. Seventy patients developed cholera-like symptoms and at first were wrongly diagnosed as cholera cases; 6 other patients were simultaneoulsy infected with El Tor vibrios and V. parachaemolyticus. There was a markedly higher incidence in males than in females. Only sporadic cases occurred, and outbreaks and secondary cases have not been observed. It is suggested that V. parahaemolyticus might be an important agent of acute gastroenteritis on the coast of West Africa.

The structure of the O-specific polysaccharide of the phenol-soluble cellular lipopolysaccharide of Vibrio anguillarum has been investigated. The studies involved the use of methylation analysis, partial hydrolysis with 48% hydrogen fluoride, Smith degradation, oxidation with chromium trioxide, and comprehensive proton and carbon-13 nuclear magnetic resonance studies, in which one- and two-dimensional experiments were carried out. As a result of these studies it is proposed that the O-specific polysaccharide of Vibrio anguillarum is composed of a regular heteropolymer, i.e., a main chain of (1----4)-linked 3-acetamido-3,6-dideoxy-beta-L-glucose residues alternately substituted through O-2 with side chain residues of 2-acetamido-2,6-dideoxy-alpha-D-glucose, which seem to be substituted through either O-3 or O-4 with propionyl groups (R), as in the following structure. (Formula: see text)

Enzyme-linked immunosorbent assays using polyclonal and monoclonal antibodies specific for V. vulnificus cytolysin detected the toxin in an extract of skin lesions and in serum from mice showing local and systemic V. vulnificus disease after subcutaneous injection of the bacterium. The cytolysin also was detected in skin lesions by an indirect immunofluorescence procedure using polyclonal and monoclonal antibodies. Our findings provide direct evidence that the cytolysin is produced in vivo during the development of the disease process, and this observation is consistent with the hypothesis that the toxin is involved in the pathogenesis of V. vulnificus disease.

Polyacrylamide gel electrophoresis and infrared spectroscopy were used to study the relationship between Vibrio cholerae (classical), Vibrio cholerae (El Tor), and nonagglutinable (NAG) vibrios. Acid extracts of the different strains produced type-specific electrophoretic patterns, and the infrared spectra revealed broad absorption maxima which largely correspond to those found in other organisms. With the exception of the NAG vibrios, the infrared spectra of cholera El Tor vibrios were identical. Strain-specific differences were found in the exoprotein spectra of these organisms by the sodium dodecyl sulphate - polyacrylamide gel electrophoretic technique.

The dot blot assay, modified and adapted for detection of antigens from Vibrio anguillarum in fish tissues, was specific for V. anguillarum and did not react with antigens of V. ordalii, Pseudomonas sp., or Yersinia ruckeri. The blot assay enabled detection of as little as 2.3 ng of a mixture of protein antigens obtained from cell-free extracts of V. anguillarum; it was about 100 times more sensitive than either the indirect fluorescent antibody technique or bacterial isolation for detecting V. anguillarum in fish tissues.

Over most of the range of salt concentrations in which the moderately halophilic bacterium Vibrio costicola could grow, the sum of the cell-associated Na+ + K+ ions was at least as high as in the external medium. This is in contrast to other moderate halophiles, which have substantially lower internal than external salt concentrations for most of their growth range. The relative amounts of Na+ and K+ in V. costicola varied with environmental conditions. The K+/Na+ ratio fell during anaerobic incubation or when cells were poisoned. As Na+ ions left the cells, K+ ions entered. However, movement of these ions was not tightly coupled, since K+ content of cells could increase without a corresponding decrease in Na+ content. The Mg2+ contents of cells varied little with environmental conditions.

Conditions are described for the production of an extracellular cytotoxin or CHO cell-killing factor by Vibrio fluvialis, a recently recognized enteric pathogen. The cell-killing factor was ammonium sulfate precipitable, heat labile, and inactivated by proteases, and had an isoelectric point (estimated by sucrose density gradient electrofocusing) and an apparent molecular weight (estimated by gel filtration) of ca. 4.8 and 12,200, respectively. The culture supernatant fluids also possessed hemolytic and phospholipase A2 activities; however, they were separable from cell-killing factor activity by gel filtration. The substance(s) possessing the hemolytic and phospholipase activities coeluted when fractionated by gel filtration with Sephacryl S-200 (apparent molecular weight of ca. 34,500) and had isoelectric points of ca. 4.4 and 4.5, respectively.

Lipopolysaccharide (LPS) prepared from Vibrio cholerae and a non-agglutinable (NAG; not agglutinable with O-group I serum according to Gardner and Venkatraman [13]) vibrio strain, isolated from a patient with cholera-like clinical symptoms, have been compared with respect to their chemical composition and immunological behavior. In addition to a significant difference in the chemical composition between the two lipopolysaccharides, the LPS from V. cholerae, unlike that from the NAG vibrio, requires prior treatment with alkali for it to be an effective antigen in the indirect hemagglutination test with sheep cells. It has been suggested that the alkali acts by removing excess O-acetyl group from LPS of agglutinable vibrios. LPS from the NAG vibrio consistently showed a lower antibody response in rabbits in terms of agglutinin and vibriocidal titer. Also, the class of agglutinin antibody elicited by LPS of the NAG vibrio was predominantly immunoglobin M, and that from V. cholerae was immunoglobulin G under comparable conditions.

Nitrofurantoin resistance brought about decreased level of membrane bound ATPase activity in Vibrio el tor as compared to the normal strain while phospholipid as well as total lipid levels in the membrane fraction was increased. Mg++ was found to be the mose effective co-factor for ATPase. At higher concentrations of sodium and potassium ions, ATPase of normal and nitrofurantoin resistant Vibrio el tor responded quite differently. A soluble ATPase extract having low phospholipid content was found to be activated by the phospholipid extracts of Vibrio el tor, phosphatidyl ethanolamine, and also by phosphatidyl choline which is not a constituent of the membrane preparation of Vibrio el tor strains.

Anguibactin, a siderophore produced by cells of Vibrio anguillarum 775 harboring the pJM1 plasmid, has now been isolated from the supernatants of iron-deficient cultures. This iron-reactive material was purified by adsorption onto an XAD-7 resin and subsequent gel filtration on a Sephadex LH-20 column. The resulting neutral compound produced an ion at m/z 348 in mass spectrometry and contained one sulfur, four oxygen, and four nitrogen atoms as determined by elemental analysis. Its strong UV absorbance and blue fluorescence were suggestive of a phenolic moiety. In colorimetric reactions anguibactin behaved like a catechol. The catechol assignment was supported by the appearance of a new absorption band at 510 nm in the ferric complex and by the appearance of peaks at 1,367, 1,447, 1,469, and 1,538 cm-1 in the resonance Raman spectrum. In addition, the infrared spectrum gave evidence of a secondary amide function, but no free carboxylic acid or hydroxamic acid groups were observed. A third iron-ligating group was suggested by the liberation of three protons during iron binding; mass spectrometry of the resulting material yielded a molecular ion characteristic of a 1:1 complex of ferric anguibactin. The purified anguibactin exhibited specific growth-promoting activity under iron-limiting conditions for a siderophore-deficient mutant of V. anguillarum 775(pJM1). A novel structure for anguibactin was indicated by the failure of a large number of known siderophores and synthetic chelators to yield a similar type of specific cross-feeding in the V. anguillarum bioassay.

The nucleotide composition of DNA was correlated with the peculiarities of the antigenic structure of vibrios and aeromonades. The authors propose a supplementary test for the differentiation between these microorganisms by the detection of thermolabile "indicator" antigens, most characteristic of cholera vibrios and pathogenic NAG vibrios, in immunoelectrophoresis with diagnostic serum to alcoholic preparation of NAG-vibrio flagellin.

DNA content in bacterium Bacillus subtilis, Pseudomonas aeruginosa. Proteus vulgaris, Vibrio proteus at different growth condition has been studied. It has been shown that during the cultures growth DNA content is various independently of culture age and as a rule exceeds one chromosome weight and depends on growth condition.

Cell-free culture filtrate boiled for 15 minutes has been found to retain its biological activity in various experimental models used for the determination of the toxicogenicity of cholera vibrio filtrates. During gel filtration of the concentrated filrate o. NAG vibrio, strain NO. 9852, through Sephadex G-75 toxic activity could be detected in the free volume of the column, which was indicative of the fact that the molecular weight of the thermostable enterotoxin was about 70,000 daltons and greater. The methods of gel diffusion and aggregated hemagglutination have been used to show that the thermostable enterotoxin of NAG vibrio No. 9852 is immunologically unrelated to cholerogen. Some data obtained in experimental models suggest that the thermostable enterotoxin probably differs from cholera enterotoxin in the mechanism of its action.

Brief treatment, with 10 M hydrochloric acid, of the lipopolysaccharides of Vibrio cholerae and related organisms led to the release of 4-amino-4, 6-dideoxy-D-mannose (D-perosamine) and 4-amino-4-deoxyl-L-arabinose. These sugar are highly unstable and undergo complex changes in neutral and basic media. Of the strains examined, the lipopolysaccharides from representatives of the Inaba group contain only D-perosamine, while those of the Ogawa biotype have 4-amino-4-deoxyl-L-arabinose as an additional component. The other Vibrios produce lipopolysaccharides which lack these sugars.

A comparative study was made of the quantitative sugar composition of lipopolysaccharides (LPS) isolated from Vibrio cholerae (O1 and non O1 groups), 'V. albensis', 'V. proteus' and V. metschnikovii. The amino sugars 4-amino-4,6-dideoxy-D-mannose (perosamine) and 2-amino-2,6-dideoxy-D-glucose (quinovosamine) were present exclusively in LPS isolated from S-form O1 group of V. cholerae regardless of serotype (i.e. Ogawa or Inaba) and biotype (i.e. classical or eltor). Classical O1 group V. cholerae was distinguishable from eltor O1 group V. cholerae on the basis of the fructose content of the LPS: greater than 3% and less than or equal to 1%, respectively. Distinct differences in the sugar composition of LPS were observed between V. cholerae and 'V. albensis', 'V. proteus' and V. metschnikovii.

The 'A' protein, equivalent to ribosomal protein EL7/L12 from Escherichia coli, has been isolated and purified from two moderate halophiles Vibrio costicola and NRCC 11227. The 'A' protein from V. costicola contained an N-terminal serine and separated into two forms on DEAE-cellulose and two-dimensional electrophoresis while the equivalent protein in NRCC 11227 contained an N-terminal alanine residue and was present in only one form. The amino acid composition and mobility on two-dimensional gels indicated these proteins were very similar to EL7/L12. The first 38 residues of the 'A' proteins were sequenced and compared to the equivalent protein from E. coli and the extreme halophile Halobacterium cutirubrum. The N-terminal region of the 'A' protein from both moderate halophiles showed substantial homology to EL 12 (75--80%) but no evidence of any homology to the equivalent protein from the extreme halophile. The ribosomal proteins equivalent to ES1A in E. coli were also isolated and their amino acid compositions determined.

Deep-sea bacteria were isolated from the digestive tract of animals inhabiting depths of 5900 m in the Puerto Rico Trench and 4300 m near the Walvis Ridge. Growth of two bacterial strains was measured in marine broth and in solid media under a range of pressures and temperatures. Both strains were barophilic at 2 degrees C (+/- 1 degrees C) with an optimal growth rate of 0.22 h-1 at a pressure 30% lower than that encountered in situ. At 1 atm they grew at temperatures ranging from 1.2 to 18.2 degrees C (+/- 0.3 degrees C), while in situ pressures increased the upper temperature limit to 23.3 degrees C. Both strains were identified as members of the genus Vibrio, based on standard taxonomic tests and mol% G + C values (47.0 and 47.1). Ribonucleotide sequences determined for 5S ribosomal RNA from each strain confirmed relationship to the Vibrio-Photobacterium group, as represented by V. harveyi and P. phosphoreum, but the barophiles were clearly distinct from these species. Secondary structure conformed to the established model for eubacterial 5S rRNA.