The mechanism of competence in the transformation of streptococci of serological group H

Summary The production of a special competence factor (cpf) by the recipient cells is one of the key-elements of the complex mechanims of competence in transformation. The role of cpf and its interrelation with the other factors involved in competence remain obscure. The evidence regarding the genetic background of competence is also very scarce.The cpf production ability was demonstrated to be a genetic unit which could be transferred in transformation. The cpf marker was transformed in a heterospecific reaction, in which the S. challis (cpf ⁺) strain was the DNA donor, and S. wicky (cpf ^–) its recipient.As a result of the incorporation of this marker, S. wicky cells acquired the stable cpf production ability and, consequently, the transformation ability in a yield equal to that shown by the donor cells. DNA isolated from the S. wicky (cpf ⁺) transformants could be applied, in turn, as a donor of the cpf marker. The experiments were performed by a semiquantitative technique and the yield of the transfer of the cpf marker amounted to about 1%.     

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.     

Factors regulating competence in transformation of streptococci

Pakula, Roman (University of Toronto, Toronto, Ontario, Canada). Factors regulating competence in transformation of streptococci. J. Bacteriol. 90:1320-1324. 1965.-The highly transformable group H Streptococcus strain Challis produced an exocellular competence-provoking enzyme capable of converting to the state of competency incompetent cells of the homologous strain, and of the very poorly transformable strain Wicky. The competence-provoking activities of culture filtrates of strain Challis prepared at various periods of growth were tested on cells of strain Wicky. In the first 3 hr of growth, a strict correlation was found between the degree of competence and the competence-provoking activity. The period of maximal competency was followed by a rapid decline, although the competence-provoking activity of the filtrates remained at a maximum. The decay of competence was caused by a change in the structure of the cells which rendered them nonreceptive to the action of the competence-provoking enzyme.     

A system for insertion of heterologous DNA into chromosome of group H streptococci

A system for insertion of genes into the chromosome of group H streptococci has been elaborated. It consists of a recipient strain (Gallis GS10/1), having the fragments of lambda L-47-1 bacteriophage DNA inserted into the chromosome, and lambda 202 vector. The constructed system suggests the preliminary cloning of genes in E. coli cells with their subsequent insertion into the chromosome of group H streptococci.     

The mechanism of competence in the transformation of Streptococci of serological group H. Purification and some properties of the competence factor

Summary InStreptococcus challis, similarly as in the other transforming systems, the appearance of competence (i.e. the ability to take up DNA and undergo transformation) is strictly related to the production by the cells of a special competence factor (CF). As we reported previously,S. challis CF or an essential fragment of its seems to be a polypeptide.In the present work, the method of the purification ofS. challis CF on CM-cellulose is described. Highly purified (O.D. at 280 m-0,05–0.1) freeze-dried preparations of this factor were obtained. The results of chromatography on Sephadex and Bio-Gels columns suggest, that the mol. w. of theS. challis CF is about 5,000. Highly purified preparations of the CF, besides the competence inducing activity, contain a factor (s) which inactivates transforming DNAin vitro and appears to include some DNase-like activity.     

Relationship of macromolecular synthesis to competence induction in a group H streptococcus.

Group H streptococcus strain Wicky, which was induced to competence for genetic transformation with competence factor (CF) derived from a related strain, displayed reduced rates of ribonucleic acid (RNA) and peptidoglycan synthesis. Pulse-labeling studies revealed that the inhibition of both RNA and peptidoglycan synthesis was maximal at the peak of competence and decreased as competence declined. These studies indicated that competence induction had only a slight effect on the rate of protein synthesis. Trypsin inactivation of CF prevented the reductions in synthesis normally elicited by CF preparations. If the addition of trypsin was delayed until 5 min after the addition of CF, competence induction and decreased synthesis of RNA and peptidoglycan were again apparent. Thus, the alterations in the synthesis of these macromolecules appeared to be related to the induction of competence. Further studies indicated that the apparent reductions in biosynthesis were not caused by decreased uptake of the labeled precursors by intact Wicky cells. In addition, these effects were probably not the result of turnover of macromolecules induced by CF. The lack of turnover of labeled peptidoglycan suggested that competence induction may not involve an autolysin.