We have devised a novel plate assay method for detecting mutants of Vibrio cholerae altered in the production of cholera toxin (tox mutants). Colonies replicated from a master plate are grown on the surface of a cellulose filter disc to which ganglioside-albumin conjugates have been attached. Toxin secreted by the colonies is tightly bound to the ganglioside filters. After removal of the cells by washing, the bound toxin may be detected by treating the filters with radioactively labeled antibodies against either whole toxin or one of its constituent polypeptide chains, followed by autoradiography.Colonies producing significantly greater or lesser amounts of toxin than the parental type are easily recognized and can be shown in liquid culture to have the corresponding hypertoxinogenic or hypotoxinogenic phenotype. This method, termed "the ganglioside filter assay," is applicable to screening large numbers of colonies and should facilitate isolation of various specific classes of mutants in cholera toxin production. In modified form the method will be applicable to various systems in which mutants of secreted proteins are sought.Isolation of bacterial mutants defective in the production of protein toxins (tox mutants) has proven to be laborious, primarily because toxin production usually confers no known selective advantage for bacterial growth or survival in vitro. Generally, large numbers of clones from mutagenized populations of bacteria must be tested for production of toxic material or specific antigen. Screening for these properties has been greatly facilitated in some instances by assay methods performed either directly on colonies growing in agar [e.g., immunoprecipitin assays (1)] or in microtiter plates [e.g., toxicity or immunological assays (2)] but there is a great need for improved methods in this area.Cholera toxin (choleragen), a multimeric protein of molecular weight about 84,000, consists of a B protomer containing five noncovalently linked chains (molecular weight, 11,590) perhaps in the form of a ring, and an A protomer containing two peptides, A1 (molecular weight, 24,000) and A2 (molecular weight, 9700) linked by a single disulfide bridge (3, 4). The B protomer has been shown to have a high affinity for the oligosaccharide moiety of ganglioside GM1, and there is evidence to indicate that this ganglioside may serve as the cell surface receptor for cholera toxin (5). Free B protomer, which has been termed "choleragenoid," is often found in culture fluids in addition to whole toxin and has been shown to block competitively the action of cholera toxin on intact cells (5). The A1 chain promotes the stimulation of adenylate cyclase activity in erythrocyte ghosts and certain other systems by a NAD-dependent reaction (6). A similar reaction in intestinal epithelium is believed to be responsible for the massive loss of fluid into the intestinal lumen, which gives rise to the major symptoms of clinical cholera (7). The A2 subunit is required for reassembly of choleragen from its subunits and ...

Cholera toxin was obtained in pure form by fractionation on two phosphocellulose columns successively. Cholera toxin and choleragenoid were quantitatively and selectively adsorbed to the first column in 10 mM phosphate buffer, pH 7.0, and were subsequently eluted with buffer of high ionic strength. The toxin was then separated from choleragenoid on the second column by chromatography at pH 8.3. The toxin obtained was highly active and pure as judged by electrophoresis, isoelectric focusing, and various immunological and chemical tests. Pure choleragenoid was a by-product of the procedure. The A1 chain of the toxin was obtained in pure form by treating phosphocellulose-bound toxin with urea and a reducing agent. The anionic Al peptide was thereby released, leaving a complex of the B and A2 chains (A25B) bound to the resin. The latter was then eluted and further purified to obtain nontoxic antigen. The overall yields of cholera toxin and choleragenoid were increased twoto threefold by the use of hypertoxinogenic mutants of Vibrio cholerae.

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Angeles), AND A. M. BRODETSKY. Isolation and preliminary characterization of bacteriophages for Bacillus subtilis. J. Bacteriol. 82:135-141. 1961.-A simplified procedure for direct isolation of phages for Bacillus subtilis from soil was developed. Phage enrichment was accomplished by growing streptomycin-resistant B. subtilis in medium previously inoculated with the soil sample. Contaminating soil bacteria were eliminated by adding bactericidal quantities of streptomycin and the phages were isolated by conventional agar layer techniques. By this method 1 or more subtilis phages were isolated from 15 of 18 soil samples tested. Several of these phages were unusually sensitive to chloroform and all of them were relatively unstable when stored at refrigerator temperatures. Of 6 phages retained for study, 1 was temperate for B. subtilis, but attempts to obtain stably lysogenic bacteria following infection with this phage were unsuccessful. All 6 phages had identical host ranges and were able to lyse all strains of B. subtilis tested, as well as several related species of Bacillus.