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C G Leonard

Publications and source records attributed to C G Leonard.

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Inhibition of transformation in group H streptococci by lysogeny.

Group H streptococcal strains Challis and WE4 were lysogenized with a bacteriophage isolated from strain Channon, after which their capacity for transformation to streptomycin and rifampin resistance was reduced by three orders of magnitude. The probable reason is the inability of the lysogenized strains to bind deoxyribonucleic acid irreversibly, even though they exhibit earlier stages of competence development during a competence regimen.

Autolysis↗

Early events in development of streptococcal competence.

Appropriately timed use of trypsin, which inactivates competence factor (CF), and chloramphenicol made feasible a separation and characterization of early events in the development of competence in group H streptococci. Step 1 is production of CF, which is inseparable in time from the concomitant release of free CF into the medium. The producing cells, which are noncompetent at the time, also accumulate cell-bound CF (CB-CF) from the onset of CF synthesis. In step 2, the released CF is adsorbed or taken up in a trypsin-insensitive state by the producing cells and is not destroyed as previously suggested. This occurs rapidly in a transformation-supporting (complete) medium. The rapid decline in free CF is concomitant with the rise in CB-CF, and a maximal increase in the latter does not occur in cultures exposed to trypsin, which inactivates any trypsin-accessible CF. The rapid increase in CB-CF (above trypsin-treated levels) leads to step 3, the induction of competence. All of these steps probably require protein synthesis, because each is inhibited by chloramphenicol. The data also indicate that only free CF that is subsequently adsorbed, and which thus leads to maximal levels of trypsin-insensitive CB-CF, is the effective inducer of competence in either CF-producing (Challis) or CF-nonproducing (Wicky) cultures. The processes induced by the newly bound CF are not fully understood, but certain new properties, previously described by others as indicating competence, were measured during the several steps of competence development. Cell aggregation at pH 2 appears to be related to CB-CF and can be shown before this bound CF has induced competence. The ability of cultures to autolyze maximally can be diminished by trypsin treatment of precompetent cells without affecting subsequent competence development as measured by transformation.

Autolysis↗

Purification and properties of Streptococcal competence factor isolated from chemically defined medium.

A procedure for the isolation and purification of competence factor produced in a defined medium by group H streptococci, strain Challis-6, is presented. Partial characterization and chemical analysis of the product are described. The procedure yields competence factor of high purity, as shown by homogeneity in electrofocusing, by electrophoresis in sodium dodecyl sulfate polyacrylamide gels, and by chemical analysis. The data indicate that competence factor is a small, dialyzable, highly basic compound. It is free from lipids, phosphorus, and carbohydrates, and is colorless and thermoresistant. Its biological activity is destroyed by trypsin but not by deoxyribonuclease, ribonuclease, lipase, or lysozyme. Its high isoelectric point of above pH 11.0 suggests that competence factor may be a protamine or a polymer of basic amino acids. The possibility that a polyamine may be an integral part of the polypeptide molecule has not been excluded.

Amino Acids↗

Intergroup lysis and transduction by streptococcal bacteriophages.

Bacteriophages from streptococci of groups A, E, and G lysed streptococci distributed among serogroups A, C, G, H, and L; propagated in some, but not all, of these; and transduced streptomycin resistance within group A or (by A phages only) from group A to group G streptococci.

Bacteriolysis↗

Autolytic activity associated with competent group H streptococci.

Competent cells of group H streptococci strains Wicky and Challis autolyzed markedly when placed at 37 C in 0.05 m tris(hydroxymethyl)methyl-amino-propane sulfonic acid buffer (pH 9.0 to 9.1) containing 0.02 m 2-mercaptoethanol, whereas noncompetent cells autolyzed slightly. Autolysis of competent Wicky cells did not occur at 0 C or after the cells were heated at 100 C for 5 min. Culture fluids derived from strain Challis that contained competence factor (CF) activity did not contain lytic activity. Addition of native deoxyribonucleic acid (DNA) to competent Wicky cells caused a retardation in the rate of autolysis; ribonucleic acid and alkali-denatured DNA had less of an effect. Supernantant fluids derived from competent cell lysates lysed noncompetent Wicky cells but were inactive against cells of Hydrogenomonas eutropha, a group A Streptococcus, and against a commercial lysozyme substrate (Micrococcus lysodeikticus). This lytic activity was inactivated by heat (5 min at 100 C). Electron microscopic observations of autolyzed cells showed that autolysis occurs only at the site of cross-wall formation. A close relationship between the development of competence and autolysis is suggested by the fact that certain conditions that prevent the establishment of the competent state in Wicky populations (such as no CF, addition of CF simultaneously with chloramphenicol, and addition of trypsin-inactivated CF) also prevent autolysis. This observation emphasizes the indirect or inductive nature of CF on these processes.

Autolysis↗

Lysis and lysogenization of groups A, C, and G streptococci by a transducing bacteriophage induced from a group G Streptococcus.

A temperate bacteriophage, designated GT-234, was isolated from a group G Streptococcus after ultraviolet irradiation. After several single-plaque passages in a group G indicator strain, this phage formed plaques in 3 of 14 group A strains, in 3 of 15 group C strains, and in 4 of 13 group G strains-but not in some representatives of several other serogroups. After propagation in each of the sensitive strains, the progeny from each was shown to be the same phage by (i) adsorption and plaque formation in each of the other groups, (ii) lysogenization in each of the other groups, (iii) high titers on infection of each serogroup, regardless of the group of propagating strain, and (iv) neutralization of infection in each of the other groups by antiserum against the phage propagated in group G. Phage GT-234 is serologically related to virulent group A phage A25, from which it is morphologically indistinguishable. Like A25, it is a transducing phage. Other studies showed that A25, as well as a group A temperate transducing phage (AT-298), could also infect strains of group C and G. These results indicate a need for reassessment of group specificity and phage receptors among streptococci of groups A, C, and G and raise possibilities for intergroup transduction.

Adsorption↗

Competence factor production in chemically defined media by noncompetent cells of group H Streptococcus strain Challis.

Chemically defined media for competence factor (CF) production by group H Streptococcus strain Challis-6 are described. CF is produced by noncompetent cells in a glutamate-free medium in which the cells cannot attain competence and by cells prior to their competence development in a glutamate-containing medium. Glutamate was required for competence development, but was not necessary for growth or CF production. Exacting cultural conditions required for the consistent production of relatively high amounts of CF in defined medium and for its recovery are detailed. The most important requirements include the selection of isolates (like Challis-6) which grew well in another defined medium, early harvest of CF because of its demonstrated instability on continued incubation in defined medium, incubation at 37 C, and the addition of glucose. The CF production was more rapid with increasing inocula and with reduced aeration. Aspartate, cystine, and NaCl were not required. Under the conditions described, large amounts of CF were consistently obtained in the culture filtrates of Challis-6 as measured by the induction of competence in strain Wicky cells and their subsequent transformation at frequencies of 6% or greater.

Amino Acids↗

Transformation of auxotrophic mutants of group H streptococci.

Stable auxotrophic mutants of a group H streptococcus (strain Challis) were isolated on a modified Mickelson defined medium after exposure to N-methyl- N-nitro-N-nitrosoguanidine. Such mutants were transformed to both prototrophy and streptomycin-resistance and hence may be used as nutritional markers in the study of streptococcal genetics.

Culture Media↗