Competence Factor Production in Chemically Defined Media by Noncompetent Cells of Group H

Transcription

1 JOURNAL of BACTRIoLoGY, Nov. 1970, p Copydght American Society for Microbiology Vol. 104, No. 2 Printed In U.S.A. Competence Factor Production in Chemically Defined Media by Noncompetent Cells of Group H Streptococcus Strain Challis C. GOMEZ LEONARD, JON M. RANHAND, AND ROGER M. COLE National Institute ofallergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland Received for publication 9 July 1970 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. Pakula et al. (11, 12) showed that competent cultures of group H Streptococcus strain Challis produced, in complex media, exocellular factors which, on addition to cells of the nontransformable strain Wicky made them competent as detected by their subsequent transformation. Competence factors (CF) have also been demonstrated in culture fluids of competent cells of Diplococcus pneumoniae (16) and Bacillus subtilis (1). The nature and modes of action of these factors are not defined (17). It is clear that studies on the chemical composition of CF, as well as -of the mechanism and nature of competence, would be greatly enhanced by the use of chemically defined media. We previously described such media for both growth and transformation of strain Challis (8), and similar defined media have been reported by others for transformation of Challis (7) D. pneumoniae (Tomasz, Bacteriol. Proc., p. 29,1964; 15) and Haemophilus influenzae (4, 5). However, although our initial studies showed reproducibly good frequencies of transformation in defined medium MS6-C (8), the recovery of CF from this medium was erratic. We have now further modified the defined medium and established exacting cultural conditions for the consistent production of CF therein. This report presents the media and conditions developed, and discusses factors influencing the production and detection of CF. It also presents evidence for the production of CF by cells which do not become competent (in the same medium), or by cells prior to attainment of competence in another defined medium. MATERIALS AND METHODS Organisms. Strains Challis, SBE: 12, and Wicky of group H streptococci were used. These strains were originally used in transformation studies by Pakula et al. (11, 12). Strain Challis is a spontaneously transformable strain which releases CF into the medium (11). The culture used in this work, designated Challis-6, was isolated in this laboratory by repeated transfers for its rapid and luxuriant growth in chemically defined medium MS6 (Table 1). Wicky is a nontransformable strain which requires the addition of CF for transformation and, therefore, it was used to measure CF activity (12). Strain SBE: 12 is resistant to 2 mg of dihydrostreptomycin/ml and was used only as a.source of deoxyribonucleic acid (DNA). 674

2 VOL. 104, 1970 STREPTOCOCCAL COMPETENCE FACTOR 675 Growth and storage of cultures. Unless otherwise stated, all cultures were initiated with a 5% (v/v) inoculum of an overnight culture and incubated at 37C in stationary, tightly stoppered test tubes (16 by 150 mm) or Erlenmeyer screw-cap flasks (Bellco Glass Co., Vineland, N.J.). Stock cultures of Challis-6 were prepared as follows. Cells were grown overnight in MS6, and 0.5-ml amounts were placed in test tubes, quickly frozen to -60 C, and stored at this temperature for up to 4 months. For studies of transformation or CF production, the frozen inocula were thawed at room temperature and diluted with 5 ml of MS6 per tube. The cultures were incubated overnight and transferred at least once in MS6 before use in CF production or transformation studies. The same culture may be transferred daily up to 7 consecutive days before it is discarded. These procedures assured reproducibility in the optimal incubation time required by cells of Challis-6 for CF production in medium MS6-F (Table 1). Stock cultures of Wicky were grown and stored as described for Challis-6, except that Brain Heart Infusion broth (Difco) containing 2.5% heat-inactivated horse serum (BHI-HS) was used. For studies on induction to competence by added CF, the frozen inocula were thawed at room temperature, diluted with 5 ml of BHI-HS per tube, and incubated overnight. Media. The compositions of the three chemically defined media are given in Table 1. The ingredients were obtained from several sources. Challis-6 growth medium, MS6. Medium MS6 was prepared by dissolving, in 850 ml of double glass-distilled water, the ingredients in the amounts listed in Table 1, avoiding temperatures in excess of 80 C. We prepared the following dry mixtures and used the amounts indicated per liter of medium: amino acids (except aspartic acid, glutamic acid, and cystine), 3.60 g; the first four vitamins (niacinamide, thiamine, riboflavine, and calcium-pantothenate), 0.04 g; the next four vitamins, (p-aminobenzoic acid, pyridoxalhydrochloride, pyridoxamine, and folic acid) g; and a mixture of adenine, guanine, and uracil, 0.03 g. All the other ingredients except NaHCO3 were then added individually. The medium was sterilized by filtration with 0.2-,um Nalgene filters and was dispensed in 170-ml amounts into 500-ml screw-cap flasks. The medium was stable at 4 C for several weeks. Prior to its use, 20 ml of a 5% solution of NaHCO3 (filter-sterilized) and 10 ml of inoculum were added to give a final volume of 200 ml per flask. CF production medium, MS6-F. Medium MS6-F was prepared as MS6 except that all of the ingredients minus the last four salts and glucose were dissolved and autoclaved at 121 C for 20 min. MS6-F was stored at 4 C. Before use, glucose, FeSO4, MnSO4, MgSO4, and NaHCO3, each filter-sterilized separately, were added aseptically. Challis-6-transformation medium, MS6-T. Medium MS6-T was prepared similarly to MS6-F except that 2.5 mg of L-glutamate (sodium salt) per ml was added aseptically. Isolation of transforming DNA. DNA was isolated from cultures of strain SBE: 12 resistant to 2 mg of dihvdrostreptomycin per ml. Cultures were grown for 18 hr in a 1:1 mixture of Todd-Hewitt Broth (Difco) and Trypticase Soy Broth (BBL), and the cells were harvested by centrifugation. The cells were lysed with a crude C-phage enzyme preparation obtained from group C Streptococcus, strain 26RP66 (6, 10). DNA was extracted from the cell lysate by the method of Marmur (9) with omission of the ribonuclease step, and was stored in 0.85% NaCl at 4 C. The DNA concentration was determined by the method of Burton (2). Procedure for transformation of strain Challis-6. Transformation of strain Challis-6 was done in four phases. Phase 1: inoculum growth. Challis-6 inoculum was grown overnight in MS6 as described above. As a rule, 1010 colony-forming units (CFU)/ml were obtained. The inoculum was centrifuged cold at 16,300 X g (Sorvall GSA rotor) for 20 min. The cell pellet was suspended in MS6-T to its original volume. Phase 2: competence development. Test tubes containing 4.75 ml of MS6-T or 1-liter flasks containing 1 liter of MS6-T were inoculated with an initial cell density of 2 X 108 CFU/ml and incubated for 60 to 90 min for competence development. Phase 3: DNA uptake. Transformation experiments were performed by mixing 0.5 ml of cells from phase 2, 0.4 ml of MS6-T medium, and 5 pg of DNA in a final volume of 1 ml. The tubes were incubated for 20 min, and then 5 pug of deoxyribonuclease (Worthington), in 0.02 M MgSO4 was added and incubated for an additional 10 min. Phase 4: scoring of transformants. Transformants were scored as follows. Samples were serially diluted in 0.9 ml of 0.85% NaCl, and 0.1 ml of appropriate dilutions were spread on the surface of Brain Heart Infusion Agar (BHIA) plates. After 2 to 3 hr of incubation to allow expression of the dihydrostreptomycin marker, the plates were overlaid with BHIA containing 600 pug of dihydrostreptomycin (Sigma Co.) per ml. Viable counts of recipient cells were done as described above, but no dihydrostreptomycin was added. Transformants and recipient cells were counted after 40 hr of incubation. The percentage of transformation was obtained by dividing the number of transformants per milliliter by the number of recipient cells per milliliter times 100. Appropriate transformation controls with cells alone and DNA alone were included, and no colonies were found. Procedure for CF production in MS6-F. Production of CF in MS6-F was accomplished in two phases. Phase 1: inoculum growth. The inoculum was grown overnight in MS6 and centrifuged as described for Challis-6 transformation, except that the cells were resuspended in MS6-F. Phase 2: production of CF. A 5% inoculum was added to any desired volume of MS6-F. We used either 5 ml of medium per test tube or 1 liter of medium per 1-liter Erlenmeyer flask. The tubes or flasks were submerged almost to the cap in a water bath and warmed to 37 C before inoculation. The time required for the optimal release of CF into the culture medium must be predetermined for each batch of stock cells and for the desired medium volume. Under our conditions, 40 to 60 min of incubation usually

3 676 LEONARD, RANHAND, AND COLE J. BACTERIOL. gave the best results when using 1 liter of MS6-F per 1-liter flask. After maximal release of CF, the cultures were immediately chilled in an ice bath and centrifuged (20 min) in a refrigerated centrifuge (Sorvall GSA rotor) at a speed not exceeding 7,000 rev/min (8,000 X g). The chilled supemnatants were filtered as soon as possible through 47-mm Gelman filter membranes (0.45 jm pore size) held in 500-ml filter holders. Since some CF is lost on the membranes, only two membranes were used per liter of culture supernatant. Small volumes of CF were used without filtration. Initially, small volumes of supernatants were sterilized by exposure to ultraviolet light, but this procedure was found to be unnecessary. Filtered CF preparations could be stored at -20 C for over 6 months without significant loss in activity. Determination of CF activity by inductio of corpeane In Wicky cels. Induction of competence in Wicky cells, for the determination of CF activity, was done in five phases. Phase 1: lnoclum growth. Five milliliters of BHI- HS was added to 0.5 ml of a thawed stock suspension of Wicky cells which had been stored at -70 C, and the cells were incubated overnight. With this inoculum, 5 X 108 CFU/ml were obtained after overniglht growth. Phase 2: prenduction growth. Test tubes containing 4.75 ml of BHI-HS were inoculated with 0.25 ml of ceflls from phase 1 and incubated for 60 min. Phase 3: Iduction of compet. Routinely, 0.5 ml of Wicky cells from phase 2 was mixed with 0.1 ml (larger volumes were also tested) of the desired CF, and the tubes were incubated for 30 min. Phs 4: DNA uptake. DNA (5 pg per tube) and fresh BHI-HS, to a final l-ml volume, were added to the tubes from phase 3. Incubation was continued for an additional 30 min, at which time 5 4g of deoxyribonuclease was added, and incubation was continued for another 10 min. Pae 5: scoring for ta. The transfonnants to streptomycin resistance were scored as described for Challis-6 transformants. Appropriate transformation controls of Wicky cells without CF and CF alone gave no colonies. CF prol don or activity. CF activity is expressed as the number of Wicky transformants obtained by miig0.1 ml of CF (supernatants or filtrates), 108 CFIJ of Wicky, and 5 ;g of DNA in a 1-ml volume of BHI-HS. RESULTS Selection and maintenance of strain Challis isolates. Isolates which grew well in MS6 were selected by repeated daily transfers of Challis cells in MS6. Initially Challis grew poorly, but after many daily transfers (over 50), a culture was isolated which grew from a 5% inoculum to over 1010 CFU/ml in 8 hr of incubation (Fig. 1). This isolate was designated Challis-6. It is most important first to select such isolates and to maintain and store them in MS6 as described above, if one is to detect production of ii, 101 V~ * * 7 * 20 FIG. 1. Effiect ofcomposition ofmedia on growth of Challis-6 cells. A series of tubes containing 5 ml of MS6, MS6-F, or MS6-T were tested for viable counts at the times indicated. competence factor and transformation consistently in defined media. Effect of medium composition. The compositions of the three media used in this study are shown in Table 1. Each had different effects on growth, CF production, and competence development. The only change A iade in the composition of the gmowth medium MS6, as shown in Table 1, from that originally described (8) was a reduction in tyrosine from 0.4 to 0.2 g/liter. In previous efforts with another isolate of Challis (8), no transformation was detected in this growth medium, but we have now shown transformation (10% frequency) in tahiis-6 cells grown for 60 to 125 min in MS6, when they were exposed to go25 g of DNA/ml in MS6. Rapidand growndoptimal transformation occurred in MS6, but either the CF production or its detection was poor. As previously shown (8), growth was reduced, transformation was optimal (10%), but detection of large amounts of CF (at the incubation ti(es tested) was inconsistent in medium MSf6C. This medium contained NaClw aspartate, less phosphate, and less glucose, and the concentrations of most amino acids were higher than in MS6 (8). Further efforts to detect CF production consistently in defined media led to the finding that CF detection and recovery wera e consistent as

4 VOL. 104, 1970 STREPTOCOCCAL COMPETENCE FACTOR 677 Component TABLE 1. Chemically defined media Amt (g/liter) Amt (g/liter) Component MS6 MS6-Fa MS6 MS6-Fa L-Amino acids Vitamins-continued Alanine Calcium-pantothenate Arginine p-aminobenzoic acid Asparticacid Pyridoxal. HCI Cysteine Pyridoxamine Cystine Folic acid Glutamic acid Biotin Glycine Histidine Purines and pyrimidines Isoleucine Adenine Leucine Guanine Lysine Uanine raci....0 Methionme Uai Phenylalanine Salts Proline Serine K2HPO Threonme KH2PO Tryptophan (NH4)2SO Tyrosine MgSO47H Valine MnSO4*H FeS47H2O Vitamins NaHCO, Glucose Niacinamide ph Thiamine Water (double-distilled to Riboflavine make 1 liter) MS6-T is similar to MS6-F except that 2.5 g of L-glutamate/liter is added. the lag phase of growth was prolonged. The most important overall changes from the growth medium MS6 which resulted in an increased lag phase and in a consistent detection of CF were alterations in the content of glutamate, cystine, aspartate, glucose, and phosphate (8). These new combinations resulted in media MS6-F and MS6-T (Table 1). The effects of these and other changes on growth, CF production, and competence development are presented. Effect of medium composition on growth. Chalis-6 cells in MS6, with or without glutamate, grew as shown in Fig. 1. The lag phase was less than 1 hr, and a level greater than 1010 CFU/ml was achieved by 8 hr. Reduction of glucose concentration from 1.0 to 0.3%, as in media MS6-F and MS6-T, caused lower growth (109 CFU/ml) in these media (Fig. 1). Aspartate or glutamate were not essential for growth (or CF production), but, when aspartate was added and glutamate eliminated (MS6-F), the lag phase lasted 4 hr as compared with 2 hr in the presence of glutamate (MS6-T). Although there was a difference in the duration of the lag phases, the level of growth of Challis-6 reached in 20 hr was similar in MS6-F and MS6-T (Fig. 1). Effect of glutamate on CF production and competence development. Glutamate was not essential for CF production in MS6-F, but its addition (resulting in MS6-T; 2.5 g/liter was optimal) was required for competence development. These findings, which are shown in Fig. 2 and 3, were obtained with 1-liter amounts of each medium in 1-liter flasks. The flasks were inoculated with Challis-6 and incubated for CF production. At times indicated in Fig. 2 and 3, 5-ml samples were removed from each flask and tested for viable counts, transformation, and CF production. In addition, 0.1-ml amounts of the supernatant fluids (not filtrates) of the samples incubated in MS6-F and MS6-T were tested for CF activity by transformation of Wicky cells. It is clear that, when cultures of Challis-6 incubated in MS6-F were tested for transformation in fresh MS6-F, no transformation was detected at any cell age tested (Fig. 2, curve B). Nevertheless, the same culture in MS6-F produced CF (Fig. 2, curve A). As shown, there was good production of CF

5 678 LEONARD, RANHAND, AND COLE J. BACTERIOL. I I aa U I CI rowth Challis)./ ~St ChaWaB, F Wicky) A I I " " 240 Minutes FIG. 2. Growth and production of CF by noncompetent cells of Challis-6 in MS6-F. One liter of MS6-F in a 1-liter flask was inoculated with Challis-6 cells and incubatedfor CF production. Five-milliliter samples were removed from the flask at the times indicated and were testedfor CF production (curve A), transformation in MS6-F (curve B), and for viable counts (curve C). Transformation tests were performed as follows: 0.5 ml of culture, 0.4 ml of MS6-F, and 5,ug of DNA were mixed in test tubes in I ml final volume. The tubes were incubated for 20 min before deoxyribonuclease was added. Part of the sample was centrifuged immediately, and the supernatant fluids were stored at -20 C until tested for CF production. CF production or activity was measured by transformation of Wicky cells using 0.1 ml of culture supernatants, and is expressed as the number of Wicky transformants. Procedures are detailed in Materials and Methods. for production of CF in this medium MS6-F was thus shown. In addition, Challis-6 cells incubated in the presence of glutamate (MS6-T) became competent as shown (Fig. 3, curve B). Transformation frequencies of 3 to 5% were obtained with cells incubated for 60 to 90 min before exposure to DNA for 20 min. However, in MS6-T, maximal production of CF (6% frequencies of transformation in Wicky cells) occurred between 30 and 40 min of incubation and declined rapidly therafter (Fig. 3, curve A). No CF was detected after 60 min with 0.1 ml of CF, but some low amounts were detectable up to 150 min when 0.5 ml of CF was tested. The rapid decline in CF detectability coincided with the development of competence. Therefore, even in a medium (MS6-T) which supports both competence development and transformation, maximal production of CF occurred prior to competence development. Similar findings were obtained in MS6-C(8) by early sampling for CF activity. Although equivalent amounts of CF were detected in MS6-F and MS6-T, maximal CF aci &t 4 12 S. Ito 0 from 30 to 120 min. Maximal levels were found between 40 and 80 min (6% frequencies of transformation of Wicky cells). After 180 min, no CF was detected, even when 0.5-ml samples of supernatant fluids instead of the routine 0.1 ml-samples were tested. It is evident (Fig. 2) that cells that never attained competence produced maximal amounts of CF. Although not shown in Fig. 2, cultures incubated in MS6-F for 50 to 120 min (but not for shorter nor longer times) were shown to be transformable when removed to glutamate medium (MS6-T) and further incubated for 45 min in the presence of DNA. A requirement for glutamate for competence development but not Minutes FiG. 3. Growth, CF production, and transformation of Challis-6 cells in MS6-T. The experiment was performed essentially as described in Fig. 2, except that I liter of MS6-T was used. The samples were testedfor CF production (curve A) by transformation of Wicky cells as described in Fig. 2, for transformation in MS6-T (curve B), and for viable counts (curve C).

6 VOL. 104, 1970 STREPTOCOCCAL COMPETENCE FACTOR 679 tivity was detectable in MS6-F for a longer period of incubation and was therefore the medium of choice for CF production and recovery. It seemed that, by extending the lag phase, CF recovery, but not production, was facilitated and therefore was consistently detected. MS6-T was the medium of choice for competence development and transformation. It is equivalent to MS6-C (8); both media supported good transformation of Challis-6 cells. Furthermore, by reference to the growth curves (curves C) in Fig. 2 and 3 and comparison with the extended scale curves in Fig. 1, it is clear that CF production occurred only during the lag phase in either MS6-F or MS6-T, and became undetectable by the time exponential growth was initiated. Effect of glucose on CF production. Figure 4 shows that glucose was essential for CF production when using 1 liter of MS6-F. Concentrations of 0.1 to 0.5% glucose were found optimal; higher concentrations resulted in more rapid loss of CF activity. In the studies reported here, 0.3% glucose was used routinely. Effect of NaCI on CF production. In previous studies, we found that the addition of 0.5% NaCl to MS6-C stimulated CF production (8). However, in 1 liter of MS6-F, this concentration did not markedly alter the onset or maximal levels of CF found, as compared to no NaCl (Fig. 5). As shown, 1.5% NaCl was partially inhibitory and 3% was completely inhibitory. All the results presented in this paper were obtained in MS6-F or MS6-T containing 0.5% NaCl. Effect of inoculum size on CF production. Increasing initial cell densities resulted in an earlier production of CF in 1 liter of MS6-F. Maximal CF release was obtained with initial concentrations of 6 X 107 to 6 X 108 CFU/ml in the inoculated medium. Initial concentrations of over 5 X 109 CFU/ml were inhibitory. Routinely, a starting cell concentration of about 2 X 108 CFU/ml was used. Effect of temperature on CF production. Flasks with 1 liter of MS6-F each were warmed to 25, 30, 34, 40, and 45 C (Fig. 6). Samples were taken at the times indicated in Fig. 6 and tested for CF activity using Wicky cells as described for Fig. 2. No CF was detected at the times tested in the cultures incubated at 25, and 45 C. At other temperatures, as shown, the times of onset, levels detected, and duration of CF activity were variable. Early and maximal CF production occurred at 37 C, which was therefore the temperature routinely used. 7 Ai. 03 0~~~~~~~~ S TIME In MINUTES FIG. 4. Effect of glucose concentrations on CF production. To 1-liter flasks, each containing I liter of MS6-F, was added 0, 0.1, 0.3, 1.0, or 5% glucose, respectively. Five-milliliter samples were removed from each flask at the incubation times indicated, and a 0.1-ml amount of each supernatant fluid was tested for CF activity by transformation of Wicky cells as described in Fig s0 120 I" TIME In MINUTES FIG. 5. Effect of NaCI concentration on CF production. To 1-literflasks, each containing I liter ofms6-f, was added 0, 0.5, 1.5, or 3.0% NaCI, respectively. Fivemilliliter samples were removed from each flask at the incubation times indicated, and a 0.1-ml amount of each supernatant fluid was tested for CF activity by transformation of Wicky cells as described in Fig. 2.

7 680 LEONARD, RANHAND, AND COLE J. BACTERIOL. 7 indicated in Fig. 7, 5-ml samples were tested for CF activity by transformation of Wicky cells as previously described (seelegend, Fig. 2). As seen in Fig. 7, CF production in 5 ml/tube 6 37C was first detected at 40 min of incubation and reached good levels which persisted for a long 2 *T> N time (curve A). In 500-ml volume in a 1-liter l\ts N flask, CF appeared at the same time and was 4o0 detectable for a similar period, but the levels were le.\never as high and decreased more rapidly (curve _ B). When a similar flask was aerated by shaking, X10XX\ \ \ \CF did not appear until 60 min, the maximum 34C level was less, and the duration of detectability o 3\ was less (curve D). In a i-liter flask with 0 1,000 Z Z \ \ \ \ ml of MS6-F, CF appeared at a high level in 20 min and maintained a high level for a relatively o l t \ \ \. \ short time, after which it declined rapidly (curve 2 soc C). These results suggest a relationship between CF production and the ratio of air-exposed medium surface to medium volume, but a more de- s f I \ \ \ tailed study is required for precise definition. The o21 25C e 45C; 0. O * P0 160 IS0VO70 TIMIE i MINUTES FIG. 6. Effect of temperature of incubation on CF * b / production. One-liter flasks, each containing I liter of MS6-F, were incubated at 25, 30, 34, 37, 40, and * 45 C, respectively. Five-milliliter samples were removed from each flask at the indicated times, and a 0.1-ml amount of each supernatant fluid was tested for CF 4 activity using Wicky cells as described in Fig. 2. data are presented here only to emphasize the r '* Effect of ph on CF production. The effect of., MOWis initial ph of the medium was studied by using 5 ml of MS6-F/tube. MS6-F was buffered with M phosphate adjusted to a ph of 7.0, 7.5, \ 8.0, or 8.5. No siificant differences were found J. in the amounts of CF produced at these ph a values. Routinely, an initial ph of 8.0 was used. 3 Effect of medium volume, container size, and agitation on CF production. The onset, level, and * o *m e a duration of CF production were influenced not I only by medium components, temperature, and FIG. 7. Effect of the ratio of MS6-F volume to inoculum size, but also apparently by available available air in a given container on CF production was air in relation to medium volume. This was shown studied as follows. A series of test tubes containing S by using different volumes of media in different ml/tube (curve A), a 1-liter flask containing 500 ml containers which allowed exposure of different (curve B), and another containing 1,000 ml of MS6-F surface areas of medium to the air. We used a (curve C) were incubated statically as previously deseries of test tubes (16 by 150, 20-ml capacity) scribed. Another 1-liter flask, containing 500 ml (curve containing S MS6-F per as well. D) was covered with a cotton plug and incubated with 5'ml.oflMs6 p shaking in tube,na ellhas a New Brunswick G-25 rotary incubator -liter screw-capped nfasks containing either shaker set at 200 rev/min. At the times indicated, S-ml 500 or I,000 ml of MS6F per flask. All containers samples were taken, and the supernatant fluids were were incubated statically, except one (500 ml/liter testedfor CF activity by transformation of Wicky cells flask) which was rotated at 220 rev/min. At times as described in Fig. 2.

8 VOL. 104, 1970 STREPTOCOCCAL COMPETENCE FACTOR 681 importance of titrating CF detectability over a complete sampling range whenever medium volumes, containers, or conditions of incubation are changed. Unless otherwise stated, we use 1 liter of MS6-F per 1-liter flask for CF production. Biological properties of CF produced in defined and complex media. Since we are the first to show adequate and consistent production of CF in a defined medium, we felt it important to compare this CF with that produced in a complex medium. For this purpose, CF was produced by incubation of Challis-6 in the complex medium and by methods described by Pakula et al. (13). The results in Table 2 show a similarity of the two preparations, the properties of which conform to those described by Pakula and co-workers (11-14) and Dobrzanski and Osowiecki (3). The preparations were similar in the following ways. (i) Each induced competence in otherwise non- TABLE 2. Comparison of competence factor (CF) produced in defined medium with CF produced in complex mediuma Residual activity ty (%)b Pretreatment of CF ti CF CF-HS Incubation of cultures for 2 hr in production media Storage of culture filtrate at 4 C for 20 hr Storage of culture filtrates at -20 C for 6 months Single filtration of 5 ml of cultures supernatant through Gelman membranes (0.4,pm) Trypsin (2 pg/ml); pretreatment terminated by the addition of 20 pg of soybean inhibitorc Inactivated trypsin (2 pug of trypsin with 20 pg of soybean inhibitor) Subtilisin (1 pg/ml) Antiserum to noncompetent Challis cells Antiserum to competent Challis cells a CF was produced in defined medium MS6-F and CF-HS in complex medium ET-3 with 2.5% horse serum added (13). b CF activity was measured by transformation of Wicky cells. A 100% CF activity represents 6 X 106 Wicky transformants/ml (using 0.1 ml of appropriate dilutions of culture filtrates initially containing comparable CF activity). c In the CF pretreatments, 0.1 ml of CF was incubated at 37 C with each enzyme for 15 min or with antisera for 30 min; samples were tested for residual CF activity by the described technique. transformable Wicky cells as shown by 6% or higher frequencies of transformation; (ii) each resulted in identical rates of DNA uptake by strain Wicky cells; (iii) the competence-inducing activity of each was destroyed by trypsin, and subtilisin (3); and (iv) the activity of either CF was inhibited by antiserum to competent Challis cells, but not by antiserum to noncompetent Challis cells (8, 14). Although not shown, the addition of chloramphenicol within 20 min of the addition of either factor prevented the acquisition of competence by Wicky cells (13). The outstanding difference between the two preparations was in stability. In defined medium, CF activity was lost rapidly (after maximal production) on continued incubation of the cultures, whereas the activity of CF made in the complex medium was not lost on continued incubation of the cultures for as long as 8 hr. Even after harvesting, the CF-containing filtrates from defined medium showed the greater and more rapid loss of CF activity upon storage at 4 C or upon filtration through membranes when preparations of initially comparable CF activity were compared. The CF activity of both preparations with or without dilution, was stable to storage at -20 C for at least 6 months (Table 2). DISCUSSION The development of chemically defined media for growth, production of CF, and transformation in group H streptococci was a prerequisite to any precise studies on the mechanism and nature of competence as well as the characterization of CF. We previously reported defined media for growth (MS6) and transformation (MS6-C), but CF detection was inconsistent (8). This study was directed toward the identification and control of conditions influencing CF production and recovery in defined media. The results emphasize the importance of first selecting isolates from Challis cultures which grow well in defined media and still exhibit a high degree of CF production and transformability. Lawson and Gooder (7) also showed recently the importance of culture selection and variability in the transformation of different Challis isolates. In addition, Challis isolates must be maintained and stored in defined medium (MS6) if one is to obtain consistent production of CF in MS6-F. The results also point out the exacting cultural conditions required by Challis-6 cells for the consistent production and for the recovery of high levels (106 Wicky transformants per 0.1 ml of filtrate) of CF in defined media. Since this is the first report on the consistent production and recovery of CF in defined medium, it was impor-

9 682 LEONARD, RANHAND, AND COLE J. BACTERIOL. tant to compare this CF with that produced in to transformation samples in MS6-T inhibited complex media. Evidence is presented for the competence development in cells which had similarity of the two preparations, the properties already released CF maximally in either MS6-F of which conform to those described by others or MS&T, and which would otherwise transform for CF produced in complex media (3, 11-14). in MS6-T in the absence of chloramphenicol. This study also revealed important relationships among medium components, growth, CF strated requirement for protein synthesis for These results reaffirm the previously demon- production and detectability, and the development of the competent state. Some of these relatake and subsequent transformation (4, 13, 15). competence development, but not for DNA uptionships are discussed. In defined medium, CF The findings also suggest that the normal course was produced early, its activity declined rapidly, of events is release of CF by Challis-6 cells prior and by the time exponential growth was initiated to competence development, and that these cells only low levels of CF activity were detectable in are then induced to competence by interaction 0.5 ml or higher volumes of culture filtrates. The with the extracellular CF in a manner analogous instability of CF produced in defined medium, to the induction in Wicky cells by added exogenous CF. in contrast to the more stable CF produced in complex medium (11, 12), accounts in part for The availability of the defined media described, the exacting conditions required for its recovery and the separability of events produced therein, and emphasizes the need for early harvest from now permits independent study of the various MS6-F or MS6-T culture medium. Continued stages and mechanisms of CF production and detectability of CF, but not its initial production competence development which terminate in nor transformability of Challis-6 cells, improved genetic transformation of group H streptococci. as the lag phase of growth was prolonged. The relationship between CF detectability and onset ACKNOWLEDGMENTS of growth is not known. Under our conditions, We thank David Brand and Catherine Dixon for their excellent the important events in the production of CF technical assistance. We also thank Benjamin Prescott, Roy Repaske, and Arthur Schade for helpful discussions. and development of genetic competence in Challis-6 occurred during the prolonged lag LITERATURE CITED phase of cells incubated in MS6-F or MS6-T. But these events may also occur during the early 1. Akrigg, A., S. Ayad, and G. Barker The nature of a logarithmic phase of cells incubated in MS6 and competence-inducing factor in Bacillus subtills. Biochem. Biophys. Res. Commun. 28: SM (7), in which transformation was adequate 2. Burton, K A study of the condition and mechanism of but CF detection was poor-due to a greater diphenylamine reaction for the colorimetric estimation of instability of CF produced in these latter media deoxyribonucleic acid. Biochem. J. 62: Dobrzanski, W. T., and H. Osowiecki Isolation and which support rapid onset of growth. Furthermore, CF production and transformation have Streptococcus strain Challis. J. Gen. Microbiol. 48: some properties of the competence factor from group H been shown in early log-phase cells grown in 4. Herriott, R. M., E. M. Meyer, and M. Vogt Defined nongrowth media for stage II development ofcompetence in complex media (11-13), in which the more stable Haemophilus influenzae. J. Bacteriol. 101: CF remained detectable several hours after the 5. Herriott, R. M., E. Y. Meyer, M. Vogt, and M. Modan decline in competence. Defined medium for growth of Haemophilus lnfluenzae. J. Bacteriol. 101: Glutamate, although not required for growth 6. Krause, R. M Studies on bacteriophages of hemolytic or production of CF, was required for competence streptococci. II. Antigens released from the streptococcal development. Furthermore, the two events of cell wall by a phage-associated lysin. J. Exp. Med. 108: CF production and competence development are 7. Lawson, J. W., and H. Gooder Growth and development of competence in group H streptococci. J. Bacterol. separable, depending on the presence or absence of glutamate, since noncompetent cells of 102: Challis-6 produced CF in a medium without 8. Leonard, C. G., D. Corley, and R. M. Cole Transformation of streptococci in chemically defined media. glutamate (MS6-F), in which the cells cannot Biochem. Biophys. Res. Commun. 26: attain competence. In addition, kinetic studies 9. Marmur, J A Arocedure for the isolation of deoxyribonucleic acid from microorganisms. J. Mol. Biol. 3: showed that cells release CF prior to competence 10. Maxted, W. R The active agent in nascent phage lysis development in a medium with glutamate, such of streptococci. J. Gen. Microbiol. 16: as MS6-T or MS6-C (8), in which competence 11. Pakula, R., M. Piechowska, E. Bankawska, and W. Walczak. development and transformation occur A characteristic of DNA mediated transformation systems of two streptococcal strains. Acta Microbiol. Polon. We also found (to be published) that addition 11: of sublethal concentrations of chloramphenicol 12. Pakula, R., and W. Walczak On the nature of compe-

10 VOL. 104, 1970 STREPTOCOCCAL COMPETENCE FACTOR 683 tence of transformable streptococci. J. Gen. Microbiol. 15. Tomasz, A Cellular metabolism in genetic transforma- 31: tion of pneumococci: requirement for protein synthesis 13. Pakula, R., J. Cybuiska, and W. Walczak The effect of during induction of competence. J. Bacteriol. 101: environmental factors on transformability of a strepto- 16. Tomasz, A., and R. Hotchkiss Regulation of the transcoccus. Acta Microbiol. Polon. 12: formability of pneumococcal cultures by macromolecular 14. Pakula, R., P. Ray, and L. R. Spencer Some charac- cell products. Proc. Natl. Acad. Sci. U.S.A. 51: teristics of streptococci competent for uptake of deoxyribo- 17. Tomasz, A Some aspects of the competent state in nucleic acid. Can. J. Microbiol. 16: genetic transformation. Annu. Rev. Genet. 3: