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A cAMP receptor protein (CRP) species was purified from the luminous Vibrio harveyi cells to apparent homogeneity. This protein had a dimeric structure with a molecular weight of 23,000 per subunit. Among all eight nucleotides tested, only cAMP (Kd = 3 to 4 microM at 0 degrees C and 52 microM at 23 degrees C) and cGMP (Kd = 6 to 10 microM at 0 degrees C and 67 microM at 23 degrees C) bound to this protein. Its binding to poly(dI-dC), poly(dA-dT), and DNA fragments isolated from V. harveyi cells were all significantly enhanced by the addition of cAMP. Based on patterns of limited proteolysis by trypsin, this CRP assumes different conformations in the absence and presence of cAMP. Also consistent with this conclusion is the finding that the binding of cAMP to CRP induced about 50% quenching of the CRP fluorescence with a concomitant 3-nm blue shift from the original 336-nm emission peak. The binding of cGMP resulted in similar fluorescence changes but had no apparent effect on the pattern of proteolysis by trypsin. Using an in vitro transcription system known to be dependent on cAMP and Escherichia coli CRP, the synthesis of a run-off transcript product was also significantly enhanced by cAMP and this V. harveyi CRP.

Colonies which varied in opacity were isolated from the four strains of Vibrio vulnificus. Opaque and translucent colonial types of the strains were distinguished from the corresponding parent strains. Variation in the opacity of colonies formed by each strain was accompanied by variation of capsular material formation, which was clarified by electron microscopy of the organisms stained with ruthenium red. The opaque-type colonies of the strains had capsular materials. On the other hand, three translucent-type colonies had no observable capsular materials, and one had incomplete capsular materials, in contrast to the corresponding opaque type. The corresponding opaque and translucent types of the strains were compared for points of virulence in mice and guinea pigs. By having capsular materials, the bacterial strains acquired resistance to serum bactericidal action, antiphagocytic activity, high lethality for mice, and strong invasiveness in the subcutaneous tissue of guinea pigs. Capsular materials of V. vulnificus were considered to be important for the expression of virulence.

An extracellular toxic substance was separated from the cell-free culture filtrate of Vibrio anguillarum (strain NCMB571). Two fractions (GI and GII + III) obtained by Sephadex G-200 chromatography following DEAE-cellulose chromatography were lethal to rainbow trout and mice. Material separated from the GI fraction by Sepharose 4B affinity chromatography (GI-A fraction) was lethal to these animals. By sodium dodecylsulfate polyacrylamide gel electrophoresis, the GI and GI-A fractions were found to be composed of components with molecular weights of 44K and 34K, and 44K, respectively. The 44K protein band was associated with carbohydrate. Peripheral vascular disorder was observed in fish and mice that died after inoculation with GI or GI-A fraction. The toxic substance was sensitive to potassium periodate but was resistant to trypsin and acetone. Heat inactivation of the toxic substance was almost complete at 100 C for 20 min and complete at 121 C for 20 min. The toxic activity was not associated with hemolytic or proteolytic activity. Homologous antitoxin completely neutralized the toxic activity.

Vibrio hollisae, a halophilic bacterium isolated from patients with diarrhea, was examined for virulence factor production. Intragastric administration of 2 X 10(7) CFU per mouse elicited fluid accumulation which peaked at ca. 6 h postchallenge in infant mice. An enterotoxin which elongated Chinese hamster ovary (CHO) cells was detected in extracts of infected-mouse intestines and in culture fluids from various growth media. The yield of the enterotoxin was maximal beginning at the onset of the stationary phase of growth in heart infusion broth supplemented with 0.5% NaCl. A concentrated preparation obtained by ammonium sulfate precipitation of culture supernatant fluids induced intestinal fluid accumulation which peaked at 2 h postchallenge in infant mice. The abilities of the enterotoxin preparation to elongate CHO cells and to elicit fluid accumulation in infant mice were inseparable by gel filtration, isoelectric focusing, and hydrophobic interaction chromatography. The enterotoxin has a molecular weight of ca. 33,000 by gel filtration and an isoelectric point of ca. 4 and is sensitive to heat.

The technique of immunoblotting was used to identify the surface antigens of the marine vibrios pathogenic for fish, Vibrio anguillarum and Vibrio ordalii. Polyclonal antisera raised in rabbits to strains representing the two most common serotypes causing Vibriosis in fish in North America were used. The results demonstrated that antigenic specificity was conferred by the lipopolysaccharides, with three serotypes being displayed among the strains examined. The lipopolysaccharides of strains chosen as type species for V. anguillarum and V. ordalii displayed antigenic cross-reactivity. The morphological heterogeneity of the Vibrio lipopolysaccharides was also analyzed in silver-stained polyacrylamide gels and by intrinsic 32P-radiolabelling. Two distinct lipopolysaccharide morphologies were exhibited, one with 0 polysaccharide chains of heterogeneous chain length, the other having 0 polysaccharide chains of more uniform chain length but displaying microheterogeneity. These lipopolysaccharide morphologies corresponded to different serogroups. Two minor proteins of apparent molecular weights 49000-51000 present in outer membrane preparations isolated by the sarcosinate extraction procedure were also strong antigens, and common to all strains of V. anguillarum tested and to several strains of V. ordalii. The major outer membrane protein was a weak antigen common to both species.

Cholera toxin-like (CT-like) enterotoxins produced by two strains of Vibrio mimicus, 61892 and 63616, isolated from diarrhea patients in Bangladesh, were purified, and their physicochemical, biological, and immunological properties were compared with those of CT produced by classical Vibrio cholerae 569B. The CT-like toxins were produced by lincomycin-resistant mutants grown in the presence of lincomycin at 200 micrograms/ml for strain 63616 and 250 micrograms/ml for strain 61892 and were purified by coprecipitation with hexametaphosphate followed by chromatography on phosphocellulose. The pure CT-like toxins were indistinguishable from 569B CT in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, rabbit intestinal loop and Y-1 adrenal cell assays, antiserum neutralization and binding inhibition assays, and Ouchterlony immunodiffusion, except that the CT-like toxins appeared to consist almost entirely of A subunit which was proteolytically unnicked. Trypsin nicking, however, resulted in fragments that appeared to be identical to those of 569B CT. These results indicate that at least one species of Vibrio other than V. cholerae can produce enterotoxins which are virtually identical to CT.

The occurrence of uronic acid as a sugar constituent of lipopolysaccharides (LPS) in Vibrionaceae was demonstrated for the first time. More than 100 strains were examined. Of five genera constituting Vibrionaceae, i.e., Vibrio, Aeromonas, Plesiomonas, Photobacterium, and Lucibacterium, the latter three contained uronic acid in LPS of all of their constituting members examined, while it was totally lacking in Aeromonas LPS so far tested. Only the members of genus Vibrio were found to be divided into uronic acid-containing and -lacking groups; V. parahaemolyticus, V. alginolyticus, V. fisheri, V. costicola, Beneckea ('Vibrio'), and V. fluvialis belonged to the former, while all four biotypes of V. cholerae regardless of their serotypes, V. vulnificus and V. anguillarum, belonged to the latter group. The uronic acid content of V. parahaemolyticus O1 to O12 LPS ranged from 1.6 to 4.2%. The uronic acid residue released from V. parahaemolyticus O1, O4, O10, and O12 LPS by heating in 5% acetic acid at 100 C for 2 hr was identified as galacturonic acid; in particular, that from O12 LPS was characterized as D-galacturonic acid.

Electron microscopic analysis of basal bodies of the flagella Vibrio alginolyticus revealed a structure composed of four discs. The diameters of two discs localized in the cytoplasmic membrane appeared to be twice as little as those of the other two discs. In this respect the basal body of V. alginolyticus resembles that of V. cholerae. The 5S sequence of ribosomal RNA from V. alginolyticus appeared to be similar to those of V. cholerae, V. harveyi and some other vibrios. Comparison of 5S-RNA sequence culminated in a dendrogram of evolutionary relationships of various bacterial species, suggesting that V. alginolyticus is a typical representative of the Vibrionacea family. The data obtained are discussed in terms of the role of Na+ energy metabolism in living cells.

The authors present the results of a comparative study of the protein spectra of 80 strains of various vibrios. Proteinograms were elaborated by the method of disc-electrophoresis in polyacrylamide gel for each strain and species. There were revealed 25 protein peaks with definite mobility coefficients. The number of peaks and their areas varied in different species. The qualitative and the quantitative differences between the vibrio species were established. Results of investigations demonstrated a possibliity of using the protein spectra for the differentiation of microorganisms.

Vibrio anguillarum, one of the causative agents of fish vibriosis, is serologically and biochemically divided into three groups (A, B and C). The chemical composition and molecular architecture of lipopolysaccharide (LPS) isolated from V. anguillarum PT 514, which belongs to serogroup B, were investigated. The LPS contained glucose (Glc), fructose (Fru), L-glycero-D-mannoheptose (L-D Hep), glucosamine (GlcN) and 4-amino-4,6-dideoxyglucose as sugar constituents in molar ratios of 8.9:0.7:3.0:1.1:1.6. Sephadex G-50 gel-chromatography of a degraded polysaccharide fraction separated from the LPS by 5% acetic acid hydrolysis suggested that the O-specific polysaccharide region consists of, in average, as much as 29 moles of Glc per 3 moles of L-D Hep, while the core polysaccharide contains at least Glc, L-D Hep and GlcN in molar ratios of 3.2 : 3.0 : 0.2. Fru and 4-amino-4,6-dideoxyglucose components were released from LPS on weak-acid hydrolysis, indicating that PT 514 LPS is distinguishable from those of Vibrio anguillarum belonging to the other serogroups. 2-Keto-3-deoxyoctonate (KDO), a common sugar constituent of gram-negative bacterial LPS, was not detected by Weissbach's color reaction under the conventional hydrolysis condition, but O-phosphoryl KDO was found in the strong-acid hydrolysate (4 M HCl, 100 C, 45 min). This substance was identical, at least in high-voltage paper electrophoresis, to 5-O-phosphoryl KDO.(ABSTRACT TRUNCATED AT 250 WORDS)

The red pigment of Vibrio psychroerythrus (formerly marine psychrophile NRC 1004) was identified as prodigiosin by comparison of its mass spectrum, absorption spectrum in the visible range, and chromatographic behavior with prodigiosin isolated from Serratia marcescens. The properties of the V. psychroerythrus pigment were clearly distinguishable from five other prodigiosin-like compounds isolated from three different microorganisms.

An extracellular cytolysin produced by Vibrio metschnikovii was purified by acid precipitation, phenyl-Sepharose CL-4B chromatography, and rechromatography on a phenyl-Sepharose CL-4B column and high-performance liquid chromatography on a Mono Q (anion-exchange) column. The purified cytolysin had a molecular weight of 50,000 and an isoelectric point of 5.1. It was inactivated by heating at 60 degrees C for 5 min and was inhibited by Zn2+, Cu2+, and high concentrations of cholesterol. Lysis of calf erythrocytes by cytolysin was temperature dependent and occurred only above 18 degrees C. Moreover, no lysis was observed at high concentrations of erythrocytes, suggesting that the cytolysin lyses erythrocytes by a multihit mechanism. This cytolysin had no immunological cross-reactivities with hemolysins from other Vibrio species tested, indicating that it is a new cytolysin. V. metschnikovii cytolysin lysed erythrocytes from several animal species (calf, rabbit, guinea pig, mouse, human, sheep, chicken, and horse) and cultured cells (Vero and Chinese hamster ovary), caused fluid accumulation in the intestines of infant mice, and increased vascular permeability in rabbit skin.

Cholera and El Tor lipopolysaccharide (LPS) were identical in respect of chemical composition except that the hexosamine content was significantly lower and galactosamine was absent in El Tor LPS. Mukerjee's group IV cholera phage phi149 was inactivated by cholera LPS but was resistant to El Tor LPS.

Polynucleotide relationships among selected Vibrio species were examined by means of deoxyribonucleic acid (DNA) reassociation reactions and chromatography on hydroxyapatite. Relative levels of intraspecific DNA duplex formation (V. cholerae-V. cholerae and V. parahaemolyticus-V. parahaemolyticus) were found to be high at 60 C (>80%), and only minimally reduced at 75 C. Interspecific DNA duplexes between V. cholerae DNA and that of the non-cholera vibrios also exhibited high relative levels of formation at 60 C (>80%) and, with one exception, were only slightly reduced at 75 C. The thermal stability of these duplexes formed at 60 or 75 C was virtually identical to that of homologous V. cholerae DNA duplexes. The degree of reassociation and the thermal stability of V. cholerae-non-cholera vibrio DNA duplexes suggests relatively little evolutionary divergence in these organisms. In all other interspecific DNA reassociation reactions, only low levels of DNA duplex formation were noted at 60 C (<25%), and these were drastically reduced (>50%) at 75 C. The degree of nucleotide sequence divergence indicated by these reactions suggests that these Vibrio species are not significantly related to V. cholerae or V. parahaemolyticus. Reassociation reactions between V. cholerae DNA and the DNA of V. parahaemolyticus indicated these species were not significantly related to each other.

Phospholipid compositions of 20 strains of marine and estuarine bacteria were determined. Results showed that phospholipids of marine bacteria differed very little from those of nonmarine organisms with phosphatidylethanolamine, phosphatidylglycerol, and diphosphatidylglycerol being the predominant phospholipids in all strains examined. Lyso-phosphatidylethanolamine occurred in significant quantities among a number of the marine bacteria, and two of the isolates contained significant quantities of poly-beta-hydroxybutyrate. Effects of age and growth temperature on the phospholipid composition were also investigated. It is suggested that phylogenetic relationships among bacteria may be correlated with phospholipid composition.

Streptomycin-dependent cholera vibrio strains were derived from Inaba, Ogawa, and NAG vibrios by the method of Mel. These phenotypes grew more slowly and attacked fermentable substances after a longer period of time than the streptomycin-sensitive parent strains. Rabbits injected with streptomycin-sensitive strains and their streptomycin-dependent forms showed homologous agglutinin production. Patas monkeys fed with 10(9) streptomycin-dependent strains shed them for 1 to 2 days without ill effect, whereas the same number of streptomycin-independent organisms caused disease. The possibility of the application of multiple doses of streptomycin-dependent organisms in oral immunization against cholera was considered.

The deoxyribonucleic acid (DNA) relationships of 80 strains identified either as Vibrio parahaemolyticus, V. alginolyticus, and V. anguillarum, or as allied marine vibrios were delineated by DNA-DNA competition experiments as well as by measuring the thermal stabilities of the DNA-DNA duplexes formed in direct binding studies. The tested strains included isolates from Japan, Europe, and the United States. The V. parahaemolyticus and V. alginolyticus groups showed an average of 67% homology to one another and 30% to strains of V. anguillarum. Significantly, a number of the isolates from the Pacific Northwest which had been previously identified as V. parahaemolyticus based on morphological, biochemical, and serological evidence were shown either to be strains of V. anguillarum or to belong to as yet unnamed groups. Most strains isolated from diseased salmon in the Pacific Northwest proved to be virtually identical with V. anguillarum type C by DNA homology experiments, thereby differentiating them from similar strains isolated from diseased herring and occasionally from salmon. The latter Pacific Northwest isolates fell into two distinct genotypic groups. A plot of the per cent homology by competition versus the difference in the thermal stabilities of heterologous and homologous duplexes (DeltaT(m,e)) between the same DNA species shows a linear decline in homology of 4.25% per degree of DeltaT(m,e). The use of this relationship for estimating the percentage of the mispaired bases distinguishing DNA preparations directly from competition experiments is discussed.

Spectral changes accompanying the thermal denaturation of phage deoxyribonucleic acid suggested that lambda is not unique in possessing large-scale intramolecular heterogeneity and nucleotide clustering; instead, lambda seems to share this property with other enteric phages.

The particulate hydrogenase of Vibrio succinogenes is solubilized during treatment of cell envelopes at pH 11.0. Alkali-solubilized enzyme requires sulfhydryl compounds for activity. At neutral pH, soluble enzyme is reincorporated into alkalitreated cell envelopes and no longer requires an additional activator. In the present study, cell envelopes prepared by lysing cells with ethylenediaminetetraacetic acid plus lysozyme (EDTA-lysozyme) were used to determine the chemical composition of cell envelopes and derived pH 11.0 soluble and insoluble fractions and to investigate some properties of the binding and activation of alkali-solubilized hydrogenase. Lysis with EDTA-lysozyme resulted in the formation of spheroplast ghosts. The derived cell envelopes contained 61% protein, 3% ash, 23% lipid, and 1% phosphorus. The alkali-treated cell envelopes contained 50% protein, 2% ash, 24% lipid, and 1% phosphorus. The ash from cell envelopes and alkali-treated cell envelopes was rich in iron and phosphorus and also contained calcium, copper, magnesium, sodium, and zinc. Virtually all of the weight of the ashed samples was accounted for by the oxides of these metals. Since the reconstitution of particulate hydrogenase was achieved with pH 11.0 supernatant solution and precipitate, intact mucopeptide is not essential for hydrogenase binding. Release of hydrogenase during EDTA-lysozyme lysis was found to depend upon an apparent structural change which occurs in the membranes during extended storage at -20 C.

The exo-enterotoxin of Vibrio cholerae has been obtained in crystalline form. A solution of the crystalline protein was equal in potency to the parent pure toxin in both choleragenicity and skin reactivity. Crystals of the natural toxoid, choleragenoid, resemble those of the toxin in appearance. A solution of crystalline choleragenoid was equivalent to the parent preparation in the flocculation test.

In response to intraluminal challenge with crude cholera exotoxin, canine Thiry-Vella duodenal loops consistently produced isotonic fluid for a 24-36 hr period. Isotonic fluid production generally began within 15 min after challenge. Mean bicarbonate concentration of fluid produced by duodenal loops was 24+/-6 (SD) mEq/liter. Perfusion of exotoxin-treated duodenal loops with an isotonic electrolyte solution containing glucose 60 mOsm/liter caused a significant decrease in exotoxin-induced isotonic fluid output. The net effects of glucose on isotonic fluid absorption by perfused duodenal loops were not significantly different before and after administration of crude cholera exotoxin. The response of canine duodenal loops to challenge by cholera exotoxin differs from responses of jejunal and ileal loops in a) absence of a detectable "lag period" between administration of exotoxin and initiation of net fluid output; b) a longer period of fluid production following exotoxin administration; and c) a significantly greater net fluid output per unit length of gut. The mean bicarbonate concentration of the fluid produced by duodenum is less than that produced by ileum, but is not significantly different from that produced by jejunum. The duodenal response is similar to that of the more distal small bowel segments in that an effect on isotonic fluid movement is observed shortly after exotoxin administration and the maximum rate of exotoxin-induced isotonic fluid production is not reached until 4-5 hr after exotoxin administration. The basis for the consistent delay of 4-5 hr between intraluminal exotoxin administration and maximum gut fluid production has not yet been determined. Current data are consistent with the hypothesis that the rate of secretion of isotonic fluid induced by cholera exotoxin is not significantly different per unit length, in duodenum and ileum and that the lesser net fluid output in the ileum is due to the greater capacity for isotonic fluid absorption by the more distal small bowel segment.

Actively growing strains of Vibrio fetus venerealis and V. fetus intestinalis, none of which produced penicillinase, were treated with inhibitory levels of penicillin or glycine, primarily to gain insight into the differential sensitivities of the two varieties to both of these compounds. Treatments induced the accumulation of uridine nucleotide glycopeptide precursors which contained amino sugars and amino acids in various molar ratios. Penicillin-induced nucleotides all contained muramic acid and sometimes glucosamine; they generally contained alanine, glutamic acid, diaminopimelic acid, and glycine. Approximately equimolar ratios of these components were observed in some compounds, but ratios varied considerably in others. Glycine-induced nucleotides contained muramic acid and, in some instances, glucosamine. Amino acids were detected only infrequently and usually in low molar ratios. The data suggest that penicillinase production, differences in the chemical composition of glycopeptide, and variations in modes of action of penicillin and glycine cannot individually account for the differential sensitivities of venereal and intestinal strains of V. fetus to these substances.

Four gram-negative bacterial species, including Escherichia coli strain B, Serratia marcescens, Pseudomonas fluorescens, and Vibrio cholerae (comma) strain NIH 41, were investigated for fatty acid content by gas-liquid chromatography involving a preparatory technique which facilitated detection of cyclopropane fatty acids. Methyl esters of fatty acids were subjected to mild catalytic hydrogenation to eliminate unsaturates. Hydrogenation was followed by bromination which removed cyclopropane acids from chromatographic profile patterns. Lactobacillic acid (cis-11,12-methyleneoctanoate) and cis-9,10-methylenehexadecanoate, previously reported in lipids of E. coli and S. marcescens, were found in small amounts in P. fluorescens but were not detected in V. cholerae.