The diarrheal response of orally inoculated infant mice to viable Vibrio cholerae and purified cholera toxin was quantitated by means of a fluid accumulation (FA) ratio. The FA ratio is defined as the gut weight/remaining body weight. FA ratios were determined in relation to time of exposure and dose. Onset of fluid accumulation with viable cells of strains CA401 and 569B occurred 8 h postinoculation and reached a near maximum at 16 h. A dose of 4 x 106 colony-forming units of strain CA401 was required for a positive response 16 to 18 h postinoculation. Several other classical cholera strains demonstrated a similar dose-related response. Strain 569B, however, required a 100-fold higher dose to give a positive response. Several mutant cholera strains with decreased virulence in other model systems elicited FA ratios decreased from wild-type values. Onset of fluid accumulation with cholera toxin occurred 6 to 8 h postinoculation and reached a maximum by 10 h. A dose of 0.5 Ag was required for a positive response 10 to 12 h postinoculation. The positive response to toxin could be inhibited by preincubation with specific antitoxin.

Outer membrane proteins of Vibrio cholerae were purified by sucrose density cenatifugation and Triton X-100 extraction at 10 mM Mg2e. The proteins were separated by polyacrylamide gel electrophoresis in the presence ofsodium dodecyl sulfate. V. cholerae outer membrane proteins presented a unique pattern when compared with the patterns of other gram-negative rods. There were 8 to 10 major bands (Mr 94,000 to 27,000), with most of the protein located in band 5 000), which thus appears to be the major structural protein of the outer membrane. Lipid and carbohydrate were associated with band 6.Outer membranes of gram-negative bacilli are composed primarily of proteins, lipids, and lipopolysaccharide. The outer membranes of members of the Enterobacteriaceae and Pseudomonadaceae (8,10,18,19,26,28,34,36) have been characterized, but little has been reported on the outer membrane of Vibrio cholerae (23, 37). V. cholerae differs from members of the Enterobacteriaceae and Pseudomonadaceae by having a lipopolysaccharide which lacks 2-keto-3-deoxyoctonate (29-31).Rough cholera strains are avirulent (16), and recent mutant studies suggest that other cell surface changes may play a role in the virulence of V. cholerae (6). We studied the outer membrane proteins of V. cholerae strain CA401, a virulent wild-type strain.Slightly modified methods, similar to those used for isolation of outer membranes of Enterobacteriaceae, were successful in obtaining outer membrane proteins of V. cholerae. Characterization of these proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis revealed 8 to 10 major proteins, with more than 90% of the outer envelope protein appearing in a band having an Mr of -45,000.In this paper we present a useful method for isolating outer membrane proteins of V. cholerae, assign numbers to outer membrane proteins, and discuss some of the unique features of the V. cholerae outer envelope. MATERIALS AND MErHODS Bacterial strain. V. cholerae CA401 is a classical Inaba strain isolated in 1953 in Calcutta, India (22), which has been extensively characterized (3-6, 35). t Present address: MO 65212.Stock cultures were kept lyophilized or frozen at -70°C in brain heart infusion broth plus 15% glycerol. Working stocks were kept on meat extract agar slants (2) at 49C for no longer than 3 weeks.Cell culture and harvesting. V. cholerae was routinely grown in the semisynthetic medium (Syncase) of R. A. Finkelstein and C. E. Lankford (Bacteriol. Proc., p. 43, 1955) to mid-logarithmic growth phase and was occasionally grown in other media for comparison: complex medium, meat extract broth (2), and defined minimal A medium (27). Cultures were grown aerobically by inoculating approximately 20 ml of medium with 2 ml of an overnight culture in the same medium and incubating it at 35 to 370C with shaking (180 to 200 rpm) in a New Brunswick shaking waterbath until mid-logarithmic phase. Ten milliliters of this culture was used to inoculate 400 ml of the same medium in a 2-liter Erlenmeyer flask and incubated at 37°...

Several conditions of growth of Bordetella pertussis cause a reversible phenotypic alteration in properties termed modulation. Growth in medium containing nicotinic acid induces normal (X-mode) cells to change to modulated (C-mode) cells. We examined several pyridines and compounds resembling pyridines for their ability to affect modulation, using envelope protein patterns and serological reactivity as indicators of modulation. We found that 6-chloronicotinic acid and quinaldic acid were more effective modulating stimuli than was nicotinic acid on a molar basis. Both 2-chloronicotinamide and isoniazid interfered with nicotinic acid-induced modulation, and can be called antimodulators. Picolinic acid inhibited growth.

Mucinase from Vibrio cholerae strain CA401 was purified by precipitation with ammonium sulfate and chromatography on a column of BioGel P-100 (Bio-Rad Laboratories, Richmond, California). Ovomucinase, intestinal mucinase, and protease appeared as two peaks of slightly different molecular weights, comigrated in nondenaturing polyacrylamide gel electrophoresis, demonstrated similar patterns of inhibition by heavy metals, and were inhibited by antiserum to purified mucinase. Both molecular weight forms exhibited similar properties, which were identical to those of fresh culture supernatants. Specific activity was only slightly increased by purification. Antiserum to mucinase passively protected infant mice from diarrhea due to V. cholerae.

Mutants of Vibrio cholerae that were deficient in protease production were isolated by picking clones form gelatin or casein plates which showed reduced zones of proteolysis. All mutants showed reduced ability to degrade complex proteins (casein and gelatin), and those tested were deficient in ability to degrade chicken egg ovomucin. Some of the mutants demonstrated a decrease in neuraminidase activity. Almost all mutants showed a dramatic loss of virulence in the infant mouse, although toxin was still produced. A partial revertant, to protease-proficient, demonstrated a simultaneous increase in neuraminidase activity and also an increase in mouse virulence. The strains described had a variety of phenotypes.