Vibrio cholerae strains with the transmissible fertility factor P contained a supercoiled circular deoxyribonucleic acid (DNA) component amounting to between 2 and 6% of the total DNA obtained from the cells. Such a component was not observed in V. cholerae strains lacking the fertility factor. This supercoiled circular DNA was isolated from P+ cells, and the molecular weight was determined by sedimentation velocity experiments and electron microscopy to be approximately 80 million daltons. These supercoiled circular DNA molecules, which have a guanine plus cytosine (G + C) composition of 42%, were concluded to be the extrachromosomal P factor. It was calculated that there is approximately one copy of the P factor per chromosome. A small amount of,supercoiled circular DNA was occasionally isolated from the Pstrains of V. cholerae. The function of this component, which has a molecular weight of 40 million daltons, is not known. The molecules found in the Pstrains were readily distinguished from the P+ circular molecules by their smaller molecular weight and different G + C composition. Recombination of chromosomal genes inVibrio cholerae depends on the presence of a fertility factor designated P (3, 4). V. cholerae strains containing this factor are referred to as P+ and behave as donor strains. V. cholerae cultures lacking the P factor, which can act as recipients, are termed P-. Although the recombination frequency for chromosomal characters in matings between P+ and Pstrains is low, the P factor itself is transmitted at high frequency from P+ to P-cells. Apparently, the P factor only occasionally causes transfer of chromosomal genes. High-frequency donors, similar to the Hfr types of Escherichia coli in which the fertility factor is chromosomally fixed, have not been found in the V. cholerae mating system.A number of recent studies have established that fertility agents as well as other plasmids exist as autonomously replicating deoxyribonucleic acid (DNA) molecules, functionally and physically distinct from the bacterial chromosome. In this extrachromosomal state, such elements can be isolated as supercoiled circular DNA molecules (13). In the present . 90024. report, we describe the isolation of the P factor from the P+ V. cholerae strains and its characterization as a supercoiled circular DNA molecule.MATERIALS AND METHODS Media. Meat extract agar (MEA) and Difco brain heart infusion (BHI) broth were used for routine cultivation of the V. cholerae strains. The nutrient gelatin agar (NGA) used for the lacunae assay was described by Enomoto (10). Tritium-labeled DNA was isolated from cells grown in the minimal medium for V. cholerae described by Bhaskaran and Rowley (5) to which 1% casein hydrolysate and 20 gCi of 3Hthymidine/ml (specific activity 6.7 Ci/mmole) was added.Bacterial strains. All bacterial strains used are listed in Table 1. The P+ strains were lyophilized to ensure active donor cultures, and a fresh vial was opened for each experiment. Infection of nondonor strains with the P factor. Amounts ...
An N‐acetyl‐d‐glucosamine‐specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P‐150. A single stained protein band of 47 kDa in sodium dodecyl sulfate‐polyacryl‐amide gel electrophoresis (SDS‐PAGE) was observed with the purified HA. HA‐antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid‐phase antigen in an enzyme‐linked immunosorbent assay (ELISA), reacted strongly with HA‐antisera but cross‐reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N‐acetyl‐d‐glucosamine. The immunogold‐labelling method using HA‐antisera confirmed the location of the HA on the surface of the bacterial cells. The HA‐antisera reacted with
Previously a N-acetyl-D-glucosamine specific cell-associated haemagglutinin (HA) had been purified from a Vibrio cholerae O1 strain. This study documents the role of this purified HA as an adhesin of V. cholerae O1. A significant inhibition in the adhesion of V. cholerae O1 bacterial cells to isolated rabbit intestinal brush borders (RIBB) was observed when the latter were pretreated with purified HA in ELISA. Antibody raised against purified HA and Fab (IgG) fragment of this serum inhibited adhesion of the bacteria to isolated rabbit intestinal epithelial cells (RIEC). V. cholerae O1 (both Ogawa and Inaba serovars) showed less adherence to isolated RIEC of animals immunised with the purified HA. Patients convalescing from V. cholerae O1 infection showed high ELISA titres against the purified HA indicating that it is expressed in the host during the disease process.
Summary. Adhesion of bacteria to guinea-pig colonic epithelial cells in vitro was inhibited by fucose with all the four strains tested (two of Shigella dysenteriae type 1 and two of S. Jexneri). N-acetyl neuraminic acid and N-acetyl mannosamine also caused inhibition, suggesting a multiplicity of receptors on the epithelial cell. Congo-red binding of the strains correlated with their adhesive ability, whereas haemagglutination of rabbit erythrocytes by the bacteria did not.
Two hybrid clones producing monoclonal antibodies (MAbs) raised against the purified enterotoxic hemolysin-phospholipase C (HlyPC) bifunctional molecule of a Vibrio cholerae O139 strain were used to study its enterotoxicity in relation to its hemolytic and enzymatic activities. Fab fragments of MAbs from ascites produced by the two hybrids neutralized the hemolytic activity of HlyPC, leaving the enzymatic activity unaffected. In ligated rabbit ileal loop and infant mouse intestine, the Fab fragments of the MAbs were not able to neutralize the enterotoxicity of HlyPC, suggesting that PC rather than Hly is the enterotoxic moiety of the molecule. The enterotoxicity of the purified PC molecule isolated from an Hly− spontaneous mutant of the HlyPC-producing parent strain further confirms this contention. The Hly molecule isolated from a PC− mutant was not diarrheagenic.
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