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.     

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.     

Effect of pH on competence development and deoxyribonucleic acid uptake in Streptococcus sanguis (Wicky).

Streptococcus sanguis (Wicky) cells, strain WE4, developed little or no competence and failed to autolyze in permissive conditions when treated with competence factor (CF) below PH 7.0. This lack of activity was directly correlated with the inability of the cells to bind or take up CF at pH values of 5.5, 6.0, and 6.5. On the other hand, competent cells bound deoxyribonucleic acid molecules maximally below pH 7.0 and transformed maximally at pH 6.5. Deoxyribonucleic acid was optimally bound to cells in a deoxyribonuclease-resistant form at pH values between 7.0 and 8.5. Concomitant with this binding, undefined acid-soluble DNA fragments appeared in the culture menstrua. CF binding and uptake by cells was not only influenced by low pH but also by low temperature. At 0 C, WE4 cells bound only 4% of the input CF and took up less than 1% into a trypsin-insensitive state compared to cells treated at 37 C. Cells treated with CF at 0 C did not autolyze when transferred to permissive conditions. The results presented in this report extend earlier findings that showed that competence development and autolysis are related to the uptake of CF.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Plasmid-Encoded ComI Inhibits Competence in the Ancestral 3610 Strain of Bacillus subtilis

Natural competence is a process by which bacteria construct a membrane-associated machine for the uptake and integration of exogenous DNA. Many bacteria harbor genes for the DNA uptake machinery and yet are recalcitrant to DNA uptake for unknown reasons. For example, domesticated laboratory strains of Bacillus subtilis are renowned for high-frequency natural transformation, but the ancestral B. subtilis strain NCIB3610 is poorly competent. Here we find that endogenous plasmid pBS32 encodes a small protein, ComI, that inhibits transformation in the 3610 strain. ComI is a single-pass trans-membrane protein that appears to functionally inhibit the competence DNA uptake machinery. Functional inhibition of transformation may be common, and abolishing such inhibitors could be the key to permitting convenient genetic manipulation of a variety of industrially and medically relevant bacteria.     

Tracing the Evolution of Competence in Haemophilus influenzae

Natural competence is the genetically encoded ability of some bacteria to take up DNA from the environment. Although most of the incoming DNA is degraded, occasionally intact homologous fragments can recombine with the chromosome, displacing one resident strand. This potential to use DNA as a source of both nutrients and genetic novelty has important implications for the ecology and evolution of competent bacteria. However, it is not known how frequently competence changes during evolution, or whether non-competent strains can persist for long periods of time. We have previously studied competence in H. influenzae and found that both the amount of DNA taken up and the amount recombined varies extensively between different strains. In addition, several strains are unable to become competent, suggesting that competence has been lost at least once. To investigate how many times competence has increased or decreased during the divergence of these strains, we inferred the evolutionary relationships of strains using the largest datasets currently available. However, despite the use of three datasets and multiple inference methods, few nodes were resolved with high support, perhaps due to extensive mixing by recombination. Tracing the evolution of competence in those clades that were well supported identified changes in DNA uptake and/or transformation in most strains. The recency of these events suggests that competence has changed frequently during evolution but the poor support of basal relationships precludes the determination of whether non-competent strains can persist for long periods of time. In some strains, changes in transformation have occurred that cannot be due to changes in DNA uptake, suggesting that selection can act on transformation independent of DNA uptake.     

Competence for Deoxyribonucleic Acid Uptake and Deoxyribonuclease Action External to Cells in the Genetic Transformation of Diplococcus pneumoniae

A mutant of Diplococcus pneumoniae that apparently does not require activator can become competent for uptake of deoxyribonucleic acid (DNA) when grown in dilute cultures or in the presence of trypsin. Development of competence in both mutant and wild strains is temperature dependent, being 10-fold greater at 30 C than at 37 C. Induction of competence on a shift from 37 to 30 C requires protein synthesis and the presence of Mg(2+) and Ca(2+); uptake of DNA does not require protein synthesis. Competence decays exponentially at higher temperatures. As well as taking up DNA, competent cells release oligonucleotide fragments of donor DNA in the medium external to the cells. Normal strains release fragments comparable in amount to the DNA taken up; but, in a mutant selected for inability to degrade DNA in agar, the amount of fragments formed external to the cells is only 40% of DNA uptake. Requirements for external deoxyribonuclease action are identical to those for DNA uptake: prior development of competence and the presence during treatment with DNA of Mg(2+) ions and a source of energy.     

Natural Competence and the Evolution of DNA Uptake Specificity

Many bacteria are naturally competent, able to actively transport environmental DNA fragments across their cell envelope and into their cytoplasm. Because incoming DNA fragments can recombine with and replace homologous segments of the chromosome, competence provides cells with a potent mechanism of horizontal gene transfer as well as access to the nutrients in extracellular DNA. This review starts with an introductory overview of competence and continues with a detailed consideration of the DNA uptake specificity of competent proteobacteria in the Pasteurellaceae and Neisseriaceae. Species in these distantly related families exhibit strong preferences for genomic DNA from close relatives, a self-specificity arising from the combined effects of biases in the uptake machinery and genomic overrepresentation of the sequences this machinery prefers. Other competent species tested lack obvious uptake bias or uptake sequences, suggesting that strong convergent evolutionary forces have acted on these two families. Recent results show that uptake sequences have multiple “dialects,” with clades within each family preferring distinct sequence variants and having corresponding variants enriched in their genomes. Although the genomic consensus uptake sequences are 12 and 29 to 34 bp, uptake assays have found that only central cores of 3 to 4 bp, conserved across dialects, are crucial for uptake. The other bases, which differ between dialects, make weaker individual contributions but have important cooperative interactions. Together, these results make predictions about the mechanism of DNA uptake across the outer membrane, supporting a model for the evolutionary accumulation and stability of uptake sequences and suggesting that uptake biases may be more widespread than currently thought.     

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.     

Relationship Between Competence for Transfection and for Transformation

Deoxyribonucleic acid (DNA) extracted from phage SPP1 is highly infectious on Bacillus subtilis competent cells; the efficiency of infection is 5 x 10(3) to 6 x 10(3) phage equivalents per plaque-forming unit. This DNA was used to study the relationship between competence for transfection and for transformation. The experiments were concerned with the frequency of infection and transformation in mutants exhibiting different levels of competence, the effect of periodate on competence for infection and for transformation, the competition between phage and bacterial DNA, the transformation of cells preinfected with phage DNA, and the infection of cells pretreated with bacterial DNA. The data show that B. subtilis cells competent for transformation are also competent for transfection and vice versa; transfection with phage DNA represents, therefore, a simple way to measure the total number of competent cells in a culture. The fraction of competent cells, determined by SPP1 DNA infection, varied from 10(-2) to 7 x 10(-2).     

Inhibition of the light-induced H+ release from uncoupled thylakoid membranes by N-ethylmaleimide.

The light-induced H+ release from thylakoids, which can be observed under completely uncoupled conditions, was inhibited by the SH reagent N-ethylmaleimide (NEM) and its analogs, while the conventional H+ uptake and electron transfer were not affected. The half-inhibiting concentration of NEM for the H+ release was 10 mM and 4 mM in thylakoids in the presence of nigericin and in CF1-depleted thylakoids, respectively. The inhibitory effect increased with the increase in hydrophobicity of the NEM analogs: N-methylmaleimide less than N-ethylmaleimide less than N-phenylmaleimide. It is suggested that SH groups in hydrophobic interior within the membrane are essential to the release of protons.