Cellular Sites for the Competence-provoking Factor of Streptococci

Immune globulins against competent cells of group H streptococci, strains Challis and Wicky, inhibited genetic transformation to streptomycin resistance when added to competent cultures. Antibodies against noncompetent cells did not inhibit transformation of competent cells. Strain Challis is spontaneously highly transformable. Strain Wicky is very poorly transformable but can be converted to high transformability with the exocellular competence-provoking factor (CPF) produced by strain Challis. Globulins against noncompetent cells of strain Challis and Wicky also inhibited transformation when added to noncompetent cultures prior to conversion to competence. Antibodies against cells of the related strain Blackburn, however, did not inhibit transformation under any circumstances. It is concluded that, although globulins prepared against competent cells block the deoxyribonucleic acid receptor sites present in these cells, the globulins prepared against noncompetent cells prevent conversion to competence by blocking the access of CPF to specific cellular sites for this factor. Strain Blackburn seems not to contain CPF-receptive sites and is, therefore, nontransformable.     

Transformability of Group H Streptococcus Challis

From a wild type strain Challis of the group H streptococcus, greening (Challis α) and β-hemolytic (Challis β) colonies were isolated on horse blood agar. Both colonies formed greening on sheep blood agar, and no significant differences were found in their biological, serological and chemical analyses. They, however, showed clear differences on the transformability. Transformability, the producibility of competence-provoking factor (CPF) and competency which have been reported on the Challis strain were all found in Challis β strain. On the other hand, Challis α strain did not produce antibiotic-resistant transformants with the addition of CPF, and could not produce CPF even when the cells were cultured under various conditions of incubation or treated with lysozyme or detergents. The transformabilities of antibiotic-resistant mutants obtained from the Challis β strain were lower than those of the original Challis β strain, as pointed out by other investigators, while the Challis α strain became transformable on antibiotic resistance only when it acquired streptomycin resistance. In the Challis β strain and the antibiotic-resistant mutants of Challis α strain, the separate markers of streptomycin, penicillin, tetracycline, mitomycin C, as well as the combinations of these markers were found to be transformed at the highest rate in the strains having transformation of streptomycin resistance. The findings are discussed with respect to incorporation of deoxyribonucleic acid into recipient cells and to the reports of other workers.     

Nuclease content of group H Streptococcus strain Challis cell surface extracts inactivating transforming deoxyribonucleic acid.

Besides the competence factor (cpf), the activity of nuclease present in cell surface extracts (iF) is most likely necessary for the occurrence of competence in transformation of Challis strain and other group H streptococci. Very small amounts of iF activity were available. For this reason there were no data on nucleases occurring in iF preparations. In our previous studies, three deoxyribonucleases (endonucleases) were isolated and partly purified from stationary Challis strain cells. In the present work, due to the application of stationary-phase cell endonucleases, gel electrophoresis, and the immunological method, we found that two of these nucleases occur in Challis strain iF preparations. These two nucleases are present in larger amounts at the cell surface only in cells originating from the early-logarithmic-phase culture. Only in this phase does competence occur in the Challis strain. Antibodies against the three endonucleases of the Challis strain do not block the occurrence of competence. We suggest that an increase in the permeability of the cell membrane for intracellular nucleases independent of cpf activity is a stage in the maturation of competence.     

Autolytic Activity and Its Association with the Development of Competence in Group H Streptococci

Seventeen strains of group H streptococci were tested for their ability to develop competence for genetic transformation, either spontaneously or with the addition of competence factor derived from strain Challis supernatant fluids, and for their ability to autolyze. Autolysis was measured as a decline in optical density after washed cells were placed in a buffer at pH 9. Kinetic experiments showed that, in strains Challis, SBE I/II, WE4, SR 30, and a strain (FW 227) cured of its bacteriophage, competence and the ability to autolyze occurred simultaneously. Since autolysis was observed only in (i) competent cells, (ii) cells that passed their peak of competence, and (iii) those cells that exhibit a potential for developing competence but never go on to transform (i.e., lysogenized Challis cells), it is concluded that, in the group H streptococci, autolytic events are associated with the competent state. Strains that transformed but did not autolyze were not found.     

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.     

Competitive inhibition of transformation in group H Streptococcus strain Challis by heterologous deoxyribonucleic acid.

Glucosylated deoxyribonucleic acid (DNA) from phages T4 and T6 competes poorly with homologous DNA causing only a slight decrease of transformation in Group H Streptococcus strain Challis. Other types of heterologous DNAs (Micrococcus luteus, Clostridium perfringens, Escherichia coli, calf thymus and non-glucosylated phage T6 DNA), in contrast to glucosylated T4 and T6 DNAs, compete with transforming DNA to the normal, high extent. These results indicate that as in transformation of Bacillus subtilis, the presence of glucose attached to 5-hydroxymethylcytosine in phage T6 DNA considerably decreases the interaction of such DNA with competent cells of the Challis strain. It also indicates that the guanine plus cytosine content of DNA is not decisive in determining its interaction with competent cells.     

Binding of Deoxyribonucleic Acid by Cell Walls of Transformable and Nontransformable Streptococci

Cell walls isolated from competent streptococci (group H strain Challis) were shown to bind more homologous and heterologous deoxyribonucleic acid (DNA) than noncompetent walls. Heat- and alkali-denatured DNA was not bound by either wall preparation. Pretreatment of cell walls with cetyltrimethylammonium bromide sharply increased the binding of DNA but did not increase transformation of whole cells. Pretreatment of the walls with either sodium dodecylsulfate, deoxyribonuclease and ribonuclease, or with crude competence-provoking factor did not affect the binding of DNA. Antiserum prepared against whole competent cells completely blocked transformation and also inhibited DNA binding to competent cell walls. Adsorption of this antiserum with competent Challis cells removed its blocking action for both binding and transformation. Pretreatment of walls with trypsin and Pronase destroyed their ability to bind DNA. Trypsin treatment also blocked transformation in whole cells. The transforming activity of DNA bound to cell walls was found to be protected from deoxyribonuclease action. Significant differences were observed in the arginine, proline, and phenylalanine content of competent and noncompetent walls. With few exceptions, the amino acids released from competent cell walls by trypsin were several-fold greater than from noncompetent walls. The results indicate that (i) two binding sites exist, one in competent cells only and essential for subsequent transformation, and a second, present in all cells, which is not involved in transformation; (ii) both sites are protein in nature; (iii) the transformation site is blocked by antibody; and (iv) the competent cell wall possesses tryptic-sensitive protein not present in the noncompetent wall.     

Accumulation of 14C-Streptomycin by Streptomycin-sensitive and Streptomycin-resistant Group H Streptococci

Three streptomycin-resistant mutants of group H streptococcus, strain Challis, were examined for ability to accumulate ¹⁴C-streptomycin. Although the mutants exhibited different levels of transformation, only the streptomycin-sensitive parent Challis strain accumulated significant amounts of ¹⁴C-streptomycin. It appears that impermeability to streptomycin does not necessarily result in reduction or loss of transformability. The amount of label accumulated by strain Challis was correlated with a loss of viability. In addition, accumulation of label was influenced by the concentration of ¹⁴C-streptomycin, the time of exposure, and the type of medium employed.     

Further studies on the deoxyribonucleic acid helping effect during transformation

Summary Transformation studies of the Challis strain of the H group of streptococcus were performed to further investigate the molecular basis of the deoxyribonucleic acid helping effect. Studies in the efficiency of transformation in the presence of non-transforming DNA support the notion that bacterial cells are indiscriminate in their uptake of donor DNA and that the helping effect occurs at a time when both transforming (T) and helping (H) DNAs have jointly entered the recipients. Furthermore, the ability of H DNA to promote transformation by T DNA is not directly altered by exposure of the former DNA to either UV-irradiation or nitrous acid. Nor does 5-bromouracil incorporation affect the capacity of H DNA to assist T DNA to transform a Challis cell.Increasing the concentration of denatured H DNA to a level that saturates the Challis bacteria in reaction mixtures produces a significant increase in the efficiency of genetic transformation. This increase in transformation frequency is greater with single DNA strands than with the corresponding amount of double strands.The extent of the helping effect with the Challis H DNA remains constant within an average molecular weight range of 3.5–7.0x10⁶ daltons. In the case of heterologous E. coli DNA, however, the helping function in this range is more pronounced as a result of decreasing molecular weight, even though the net incorporation of T DNA remains unaffected. When the average M. W. is reduced below 2×10⁶ a significant decline in the helping effect occurs in both cases.The effect of H DNA on the genetic transfer of two nonallelic antibiotic markers demonstrates that a saturating amount of non-transforming H DNA present in cells does not enhance the likelihood of co-integration of nonallelic factors. Evidence concerning the physiology of the competent cells and their ability to be helped reveals that the physiological basis of transformabilities and the helping capabilities of a culture are not identical.     

Bacteriocin Production by Transformable Group H Streptococci

Group H streptococci (strain Challis) which are competent for transformation release a bacteriocin into liquid medium which is bacteriocidal for another group H streptococcus (strain Wicky). The streptocin (STH(1)) is resistant to treatment with deoxyribonuclease and ribonuclease but is sensitive to trypsin, phospholipase C, and alkaline phosphatase. Such enzyme sensitivity experiments indicate that the bacteriocin may be a complex molecule (protein and lipid) containing phosphate groups essential for activity. STH(1), which is readily distinguishable from competence factor and bacteriophage activity, appears to have no role in the initiation of the competent state in strain Wicky. The presence of this factor in Challis culture supernatant fluids indicates that a reevaluation of earlier studies performed with the Challis-Wicky transformation system may be necessary.     

Growth and Development of Competence in the Group H Streptococci

The growth and development of competence by group H streptococci, strain Challis, were compared in synthetic, semisynthetic, and complex media with respect to the cultural conditions required, time of onset and persistence of competence and transformation efficiency. Provided that cultural conditions were strictly controlled in the synthetic system, transformation frequencies of 1% or above were routinely observed. The initial pH must be between 7.3 and 7.6, and the addition of freshly prepared bicarbonate ion was required. Furthermore, competence was sensitive to the degree of initial agitation of the culture. There was no evidence that "step-down" or "unbalanced" growth conditions were required. Competence could be provoked in the incompetent strain Wicky, growing in complex or semisynthetic media, by the addition of heat-killed or filtered cultures of strain Challis prepared during the competent period of growth in synthetic medium.     

Transformation of Streptococcus sanguis Challis with a plasmid of Streptococcus pneumoniae

Abstract The present work is concerned with plasmid transformation of Streptococcus sanguis strain Challis with derivatives of pDP1/pSMB1, the only plasmid found to occur naturally in Streptococcus pneumoniae . Two recombinant plasmids derived from the cryptic pSMB1 were used: pDP27 (4.5 kb) conferring resistance to chloramphenicol (Cm), and pDP28 (7.8 kb), a shuttle plasmid, conferring resistance to Cm in Escherichia coli , and resistance to erythromycin (Em) in pneumococcus. It could be shown that pSMB1 can replicate in S. sanguis ; in fact, Challis strain V288 was transformed to Cm-resistance and to Em-resistance by pDP27 and pDP28 respectively.
Shuttle plasmid pDP28 can transform S. sanguis both when isolated from pneumococcus and from E. coli , albeit with a different efficiency. The low frequency of transformation observed when pDP28 was isolated from E. coli DH1 ( recA ) was shown to be due to lack of multimeric forms of the plasmid in the DNA preparations obtained from this strain. When pDP28 was isolated from E. coli C600 (RecA⁺), multimeric forms were present, and transformations of S. sanguis was more efficiency Using pDP28 as vector in cloning experiments, where S. sanguis was the host of the recombinant DNA molecules, treatment of the vector with alkaline phosphatase inhibited the recovery of recombinant clones.     

Protein Difference Between Competent and Noncompetent Cultures of a Group H Streptococcus

Acidified phenol extracts prepared from competent cultures of a group H Streptococcus strain Wicky made competent with competence factor derived from cultures of another group H Streptococcus, strain Challis, showed a difference in polyacrylamide-gel protein patterns when compared to extracts prepared from noncompetent cultures of strain Wicky. The prominent single protein band difference did not appear when Wicky cells were simultaneously treated with competence factor and chloramphenicol, an inhibitor of the development of competence. Chloramphenicol had no effect on transformation nor the appearance of the "new" protein band when added to fully competent cells. This new protein, which is associated with the appearance of competence, seems to be synthesized as a result of induction by competence factor; its exact role, however, is as yet unknown.     

Group H Streptococcal Bacteriocins Having No Relation to Bacterial Transformation

The culture filtrate of group H streptococcus strain Challis produced a competence factor (CF) for bacterial transformation as well as a bactericidal factor(s) against Wicky cells. Strain 36658, in the same streptococcal group, also produced the bactericidal factor(s) but not CF. The effect of the Challis bacteriocin was limited to strains Wicky and 58, whereas the 36658 bacteriocin affected 67% of 49 strains tested. Strain 58, one of the indicator strains, was affected by the bactericidal activity of these bacteriocins but not by CF activity, and failed to transform. No relationship between the bacteriocin-producing strains and indicator strains was observed. Both Challis and 36658 bacteriocin activities decreased markedly either when the bacteriocins were heated at 50 C for 30 min or with the addition of a protein synthesis inhibitor, but showed different sensitivities to trypsin, papain and lipase. The bacteriocins were of at least protein nature and their molecular weight was roughly estimated as 100,000 daltons by membrane filtration experiments. The 36658 bacteriocin is a new type of streptocin previously not reported. The possible absence of bacteriophage or phage-like particles in the preparations is discussed.     

Deoxyribonuclease-sensitive transfer of an R plasmid in Streptococcus pyogenes (group A)

Abstract The 11.6-megadalton (MDa) plasmid pIP955, encoding resistance to macrolides and related drugs and chloramphenicol, harbored by Streptococcus pyogenes strain A449, transferred only by filter mating and only into Streptococcus sanguis strain Challis recipient. This transfer was completely inhibited by low concentrations of deoxyribonuclease I and occurred even if the donor strain had been killed.     

Novobiocin-resistant mutants of Streptococcus sanguis with reduced cell hydrophobicity and defective in coaggregation

Mutants of Streptococcus sanguis resistant to novobiocin (NovR-mutants) were isolated after mutagenesis of strain Challis with ethyl methanesulphonate. The resistance phenotype was transferred by DNA-mediated transformation back into the parent strain at high frequency suggesting resistance was due to mutation(s) in a single gene or in closely-linked genes. Cells of NovR-mutants had normal morphology and secreted similar proteins to the wild-type strain. However, mutant cultures had slower growth rates, the mutant cells had reduced hydrophobicity, and they showed a reduced degree of coaggregation with Actinomyces viscosus and Actinomyces naeslundii. Cell envelopes prepared from NovR-mutants differed from wild-type cell envelopes in that they (a) were impaired in ability to coaggregate with A. viscosus cells, and (b) had altered protein composition as detected by SDS-PAGE. The results suggest that hydrophobic proteins in the cell envelope of S. sanguis may be necessary for coaggregation of this bacterium with actinomycetes.     

Transformation of Streptococcus sanguis Challis by plasmid deoxyribonucleic acid from Streptococcus faecalis.

Plasmid deoxyribonucleic acid (DNA) from Streptococcus faecalis, strain DS5, was transferred to the Challis strain of Streptococcus sanguis by transformation. Two antibiotic resistance markers carried by the beta plasmid from strain DS5, erythromycin and lincomycin, were transferred to S. sanguis at a maximum frequency of 1.8 x 10-5/colony-forming unit. Approximately 70% of the covalently closed circular DNA isolated from transformant cultures by dye buoyant density gradients was shown to be hybridizable to beta plasmid DNA. Two major differences were observed between the beta plasmid from S. faecalis and the plasmid isolated from transformed S. sanguis: (i) the beta plasmid from strain DS5 sedimented in velocity gradients at 43S, whereas the covalently closed circular DNA from transformed Challis sedimented at 41S, suggesting a 1.5-Mdal deletion from the beta plasmid occurred; (ii) although the 43S beta plasmid remained in the supercoiled configuration for several weeks after isolation, the 41S plasmid was rapidly converted to a linear double-stranded molecule. Attempts to transform S. sanguis with the alpha plasmid from S. faecalis, strain DS5, were unsuccessful.     

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.     

Role of Streptococcus sanguis (strain Wicky) cell surface-located deoxyribonucleic acid-binding factor in transformation of a homologous strain

In a previous report we demonstrated the presence of a factor binding deoxyribonucleic acid (DNA) in vitro (BF) in cell leakage fluids from transformable Streptococcus sanguis strains Wicky, Challis, and Blackburn. BF originating from strain Wicky was purified to homogeneity, and its properties are described. In this work, it was found that BF occurs at the surface of Wicky cells in two forms, loosely bound (LB-BF) and strongly bound to the cell envelope. It was demonstrated that LB-BF formed fast-sedimenting complexes with exogenous DNA at the surface of Wicky cells. About 10-fold-more DNA became associated as a fast-sedimenting complex in competent than in incompetent cells. Thus, LB-BF is a cell receptor for exogenous DNA. However, the comparison of the effects of some agents on the transformation yield and the formation of LB-BF-DNA complexes, showed that the influence of these agents on both observed phenomena is not parallel and may be even opposite. These results are interpreted to mean that the LB-BF-DNA complexes do not take part in transformation. The problem of participation of BF strongly bound with the cell membrane fraction remains to be elucidated.     

Natural genetic transformation in Streptococcus gordonii: comX imparts spontaneous competence on strain wicky.

Streptococcus gordonii Wicky becomes competent only after stimulation with conditioned medium from strain Challis as a source of competence factor (CF). A 3.2-kbp genomic fragment from Challis was found to impart spontaneous competence on Wicky by a complementation assay. Wicky clones containing the fragment secreted a heat-sensitive activity that induced competence in Wicky and in a comA insertion mutant of Challis. Activity was localized to a putative open reading frame, comX, with the potential to encode a 52-amino-acid peptide. comX had no similarity to known sequences, and a comX::ermAM insertion mutant of Challis transformed normally and secreted CF. These data suggest that a CF-independent pathway for competence induction exists in S. gordonii.