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.     

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.     

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.     

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.     

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.     

Purification and Properties of Deoxyribonucleic Acid Binding Factor Isolated from the Surface of Streptococcus sanguis Cells

Deoxyribonucleic acid (DNA) binding factor (BF) was found in surface fluids from competent and noncompetent cells of Streptococcus sanguis strains Challis, Wicky, and Blackburn. Fluids from noncompetent cells exhibited about 10% BF activity compared with extracts from competent cells. BF from competent Wicky cells was purified to homogeneity by electrophoresis and immunodiffusion. Purified BF preparations exhibited slight endonucleolytic activity, directed mainly against single-stranded DNA. Nucleolytic and DNA binding activities present in purified BF could be separated by polyacrylamide gel electrophoresis. Purified BF was sensitive to proteolytic enzymes and to phospholipase D, and its activity was stimulated in the presence of low Triton X-100 concentrations. The protein component of BF is a single, monomeric polypeptide with a molecular weight of 56,000 and an isoelectric point of pH 5.8. Binding of purified BF to DNA was a very rapid process at the optimum temperature, pH, and ionic strength and led to the formation of fast-sedimenting complexes. Purified BF was tested for several properties. It exhibited higher affinity to single- than to double-stranded DNA. It bound poorly to glucosylated phage T4 and single-stranded, synthetic polydeoxyribonucleotides and did not bind to RNA. It protected single-stranded DNA against nuclease S₁ action but did not protect native DNA against deoxyribonuclease I action. No evidence was found for unwinding activity, using double-stranded DNA as a substrate.     

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.     

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.     

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.     

Effect of Competence Induction on Macromolecular Synthesis in a Group H Streptococcus

The effects of competence induction by competence factor (CF) on macromolecular synthesis in group H streptococcus strain Wicky were investigated. CF preparations (culture filtrates from competent group H streptococcus strain Challis) were either heated or partially purified to remove a bacteriocin. These preparations did not inhibit growth, although they induced high levels of competence in strain Wicky. The action of the CF preparations did not affect the overall rates of deoxyribonucleic acid and protein synthesis, but caused a reduction in the rates of ribonucleic acid (RNA) and peptidoglycan synthesis. When competence induction by CF was prevented, no alterations in RNA or peptidoglycan synthesis were observed, indicating that these changes are in fact related to the development of competence.     

Competence-related increased enzyme release from Streptococcus sanguis (Wicky) cells.

The ablity of competent and noncompetent Streptococcus sanguis (strain Wicky) cells to release enzymes to the environment was studied. Both competent and noncompetent cells leaked the enzymes tested (aldolase, phosphatase and deoxyribonuclease), but the activities liberated from the competent cells were always roughly 2-fold higher than those released from noncompetent cells. This increased enzyme leakage from competent cells occured in all kinds of media and procedures employed. The leakage of enzymes followed a time-dependent kinetics (different for aldolase and phosphatase), was temperature sensitive and had a pH optimum. The increased enzyme release was most likely not due to cell disruption, but seemed to be rather a consequence of alteration in cell barrier permeability. These results strongly support the "unmasking" model proposed for explanation of competence development in bacteria.     

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.     

Effect of D-cycloserine on transformation in group H streptococcus, strain Challis

d-Cycloserine (d-CS), a selective inhibitor of bacterial cell wall biosynthesis, inhibited transformation in group H streptococcus, strain Challis, by preventing the development of the competent state. The incubation of strain Challis cells with d-CS resulted in the production of a substance which inhibited the action of competence factor on these cells. d-CS had an enhancing effect on transformation when deoxyribonucleic acid uptake or phenotypic expression was allowed to occur in its presence.     

Properties of a Streptococcus sanguis (Group H) Bacteriocin and Its Separation from the Competence Factor of Transformation

STH(1), a streptocin elaborated by group H streptococcus strain Challis, is lethal for group H streptococcus strain Wicky and is produced maximally during the exponential growth phase of liquid medium cultures. Crude streptocin preparations are resistant to oxidation and display a biphasic pH stability (stability being maximal at pH 5.0 and 10.0). Survivor studies indicate that streptocin-mediated killing is a "one-hit" phenomenon and proceeds rapidly. The streptocin has been purified 50-fold with (NH(4))(2)SO(4) fractionation and Sephadex G100 chromatography and appears to exist in equilibrium between two molecular weight forms. Low ionic strength and neutral pH buffers favor the isolation of the 110,000 molecular weight form, whereas high ionic strength and alkaline pH conditions facilitate isolation of the 28,000 to 30,000 molecular weight form. These findings suggest an association-dissociation relationship between macromolecules of 28,000 to 30,000 molecular weight. Purified STH(1) has no "competence factor" (CF) activity. In addition, CF has no STH(1) activity and displays no inhibitory effect on exponential-phase Wicky cultures as determined by absorbancy measurements. It appears, therefore, that initiation of the competent state for transformation in strain Wicky is not necessarily accompanied by gross alterations in cell growth.     

Intercellular Effects on Development of Competence in Bacillus subtilis

The intercellular transfer of competence during growth under the conditions specified by the transformation procedure of Spizizen was investigated with Bacillus subtilis 168. The rate of competence development as assayed uniformly in medium B was not affected by variations in the cell concentration, although the first appearance of transformants occurred earlier with high cell densities in medium A, approximately in proportion to the onset of the stationary phase in the culture. Growth in the presence of Pronase enhanced the frequency of transformation, but did not detectably alter the kinetics of competence development. The rate of competence increase in physiologically noncompetent cultures was not changed by mixing with competent cultures either in medium A or in medium B; however, an early appearance of transformants was noted in mixed cultures in which the proportion of competent to noncompetent cells prevented exponential growth of the noncompetent strain. These experiments indicate that the normal development of competence in B. subtilis is not mediated by a soluble or loosely bound protein factor capable of transmitting competence directly via cell contact. The onset of competence is thus a function of internal physiological changes which are induced by the overall metabolic state of the culture.     

Inhibition of the Development of Competence in Streptococcus sanguis (Wicky) by Reagents that Interact with Sulfhydryl Groups: Discernment of the Competence Process

Reagents that interact with sulfhydryl groups are shown to inhibit competence factor (CF)-induced competence development in Streptococcus sanguis (Wicky) strain WE4 (Wicky 4 Ery(R)). Inhibition is correlated with specific inhibition of either the function or biosynthesis of three competent cell-related proteins and is reversed by either 2-mercaptoethanol or dithiothreitol. Mercuric chloride (5 muM) or N-ethylmaleimide (NEM; 50 muM) inhibited (i) the function but not the biosynthesis or activation of the competent cell-associated autolysin; (ii) the biosynthesis of a competent cell-associated protein of unknown function, demonstrated by polyacrylamide gel electrophoresis of acidified phenol extracts; and (iii) the biosynthesis or activation of distinct deoxyribonucleic acid (DNA)-binding sites. Neither reagent at the indicated concentration interfered with the uptake of CF by cells or with the uptake and expression of DNA by competent cells. Neither reagent inactivated CF or genetic markers coded by the transforming DNA, nor did they inhibit cell growth or viability appreciably. The data reveal that either mercuric chloride or NEM can differentially inhibit induced protein synthesis and, in addition, conclusively show that some autolytic activity is essential for the onset of the competent state.     

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.     

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.     

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.