Vol. 130, No. 3 Printed in U.S.A.
JOURNAL OF BACTzRIOLOGY, June 1977, p. 1064-1071 Copyright C) 1977 American Society for Microbiology
Genetic Loci of Hemolysin Production in Streptococcus faecalis subsp. zymogenes' MARSHA L. FRAZIER AND LEONARD N. ZIMMERMAN*
Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 18 November 1976
Two plasmids corresponding to molecular weights of 38.5 x 106 and 3.6 x 106 have been identified in Streptococcus faecalis subsp. zymogenes strain X-14. The larger plasmid is required for hemolysin-bacteriolysin production. Strain L2, a nonlytic nitrosoguanidine mutant of strain X-14, still harbors the hemolytic plasmid and produces the lysin component, but not the activator component, of the lytic system. Conjugal transfer of this plasmid from strain L2 to plasmid-free strains and strains cured of the 38.5-megadalton plasmid gives rise to hemolytic recipients. This implicates a gene in hemolysin production at a site other than the 38.5-megadalton plasmid. In a survey of enterococcal bacteriocins, Brock et al. (7) reported that Streptococcus faecalis subsp. zymogenes strain X-14 has bacteriolytic and hemolytic activity. Additional work by Brock and Davie (6) demonstrated that both these lytic activities are the product of a single system. An analysis by Granato and Jackson (13) of the bacteriolytic-hemolytic system ofS. faecalis subsp. zymogenes strain X-14 showed that it is bicomponent in nature, consisting of an activator (A) component and lysin. Although the lysin component has slight activity by itself in some cases (14), maximum bacteriolysis or hemolysis is attained by activating the lysin with the activator; the A component has no activity by itself. Recently there have been several reports (12, 15, 18) that presented evidence favoring the hypothesis that the genes coding for hemolysinbacteriolysin formation in enterococci are borne on plasmids. In addition, Dunny and Clewell, Jacob et al., and Tomura et al. (12, 15, 18) have shown that lysin production is transmissible by conjugation. Based on this information, it should be expected that the hemolysin-bacteriolysin production of S. faecalis subsp. zymogenes strain X-14 is plasmid-borne and transmissible by conjugation. In this paper we confirm the latter notion but present evidence that the hemolysin-bacteriolysin system is not coded entirely on a plasmid. Our data suggest that the production of the A component of the hemolysin requires at least one gene on the plasI Authorized for publication by the Pennsylvania Agricultural Experiment Station, Pennsylvania State University, as paper no. 5203.
mid and at least one gene on one of the other replicons of the cell. MATERIALS AND METHODS Materials. Pronase, lysozyme, and ethidium bromide were purchased from Calbiochem; erythromycin and CsCl (grade 1) were from Sigma; acridine orange was from Allied Chemical; sodium dodecyl sulfate (SDS) was from BDH and Baker; [methyl3H]thymidine and [methyl-14C]thymidine were purchased from the Amersham/Searle Corp.; and penicillin was from Eli Lilly & Co. Media. AC broth (17) was used as a general culture medium. AC agar was prepared by adding 1.5% agar (Difco) to the AC broth. Penassay broth (antibiotic no. 3, Difco) was used in the curing experiments and adjusted to pH 7.8. Unless otherwise stated, horse blood agar was prepared by supplementing AC agar with 5% sterile horse blood (in Alsever solution). For conjugation in broth, Oxoid nutrient broth no. 2 was used. Bacterial strains. Table 1 describes most of the strains of bacteria used and their characteristics. S faecalis strain JH2 was obtained from A. Jacob, and strain DS-5-Cl was obtained from D. B. Clewell. Although strain JH2 was reported by Jacob and Hobbs (16) as being sensitive to kanamycin, it is not as sensitive as some of the other strains used in this investigation and is therefore referred to as being kanamycin resistant (Table 1). S. faecalis subsp. zymogenes strains X-14, X-14/NGBL2, and X-14/ NGBA18 were obtained from R. W. Jackson. For simplicity, strain X-14/NGBL2 will be referred to as L2 and X-14/NGBA18 as A18. Also, we are retaining the original genetic nomenclature of Granato and Jackson (13), which designates the L2 strain as being lysin positive, activator negative and the A18 strain as being lysin negative, activator positive. S. faecalis subsp. liquefaciens strain 3 is a non-proteolytic mutant of S. faecalis subsp. liquefaciens strain
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Subspecies
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TABLE 1. S. faecalis strains used in genetic experiments Relevant phenotypesa Comments, references, and/
Strain no.
or remarks Wild type (references 6 and 7) s s Nitrosoguanidine mutant s + X-14/NGBL2 zymogenes of X-14 s Nitrosoguanidine mutant + 8 8 X-14/NGBA18 zymogenes of X-14 (reference 13) s Cured with sodium doder s X-14-5 zymogenes cyl sulfate Reference 12 DS-5-Cl zymog r r Wild type (reference 16) s JH2 faecalis r r r Spontaneous mutant of JH2Eror faecalis JH2 a Genetic symbols: Hem, hemolysis; Bac, bacteriolysin; A, activator; L, lysin; Ero, erythromycin (10 ml); Kan, kanamycin (80 j.g/ml); 81A, bacteriophage 81A; s, sensitive; r, resistant.
zymogenes
X-14
Hem
Bac
A
L
Ero
Kan
81A
+
+
+
+
8
S
s
itg/
31. It was obtained by ultraviolet light irradiation (1) and is used as an indicator strain for bacteriolysin sensitivity. S. faecalis subsp. zymogenes strain 26C1 was obtained from the Cornell University stock collection. It is used as a plasmidless control for sucrose density gradients. Isolation of plasmid DNA. Cells were grown at 37°C in 25-ml volumes of AC broth supplemented with 250 uCi of [methyl-3H]thymidine or 25 ,uCi of [methyl-14C]thymidine. After growth to 95 Klett units (a Klett-Summerson colorimeter with a no. 54 filter was used), cells were chilled on ice for 10 min, centrifuged at 20,200 x g for 10 min at 4°C, and then washed once with buffered TES solution [0.03 M tris(hydroxymethyl)aminomethane, 0.005 M Na2 ethylenediaminetetraacetic acid, and 0.05 M NaCl at pH 8.0]. Cells were then resuspended in 1.0 ml of 25% (wt/wt) sucrose [in 0.05 M tris(hydroxymethyl)aminomethane, pH 8.0] and lysed according to the method previously described by Clewell et al. (10). When differentially labeled cultures were prepared for mixed lysis, they were mixed during the washing step. Preparation of dye buoyant density gradients was similar to the procedure used by Clewell and Helinski (9). A 1.5-ml volume of the cleared lysate was added to 6.3 ml of a TES buffer containing 3 mg of ethidium bromide and 7.0 g of CsCl. Gradients were centrifuged for 48 h at 15°C in a Beckman 5OTi fixedangle rotor at 42,000 rpm. They were then fractionated (13 drops per fraction) using a Buchler piercing unit with an 18-gauge needle, and 50-,ul samples were spotted onto 1.5-cm filter paper squares (Whatman no. 3, medium flow rate). Fractions containing satellite material were pooled and dialyzed against 0.1 x SSC (0.01 M NaCl, 0.0015 M sodium citrate, pH 7.4). After oven drying at 70°C for 20 min, the filter papers were processed in batches by washing twice with cold 10% (wt/vol) trichloroacetic acid and twice with 70% ethanol. They were then dried again and counted in a cocktail consisting of 5 g of PPO (2,5diphenyloxazole) and 0.3 g of dimethyl-POPOP {1,4bis-[2]-(5-phenyloxazolyl)benzene} per liter of tolu-
ene, using a Packard model 3375 scintillation spectrometer. Sucrose velocity sedimentation. Samples of pooled, dialyzed, satellite material, 0.2 ml in volume, were layered onto 4.6-ml gradients of 5 to 20% (wt/wt) sucrose prepared (using three freeze-thaw cycles) according to the method of Baxter-Gabbard (3). TES buffer was used for neutral gradients. Alkaline gradients were prepared in a solution of 1.0 M NaCl-0.001 M Na2 ethylenediaminetetraacetic acid0.3 M NaOH at pH 12.0 (9). A Beckman SW50.1 rotor was used for centrifugation. Gradients were fractionated with a Buchler piercing unit. Six-drop fractions were then collected onto 1.5-cm filter paper squares, processed, and counted as in the dye buoyant density gradients. Conjugation in broth. A modification of the method of Jacob and Hobbs (16) was used. Using overnight cultures, 0.5 ml of donor and 0.5 ml of recipient were mixed with 5 ml of Oxoid nutrient broth no. 2. After incubation of the mixed culture for 4 h, dilutions were plated onto AC agar containing erythromycin (10 Zg/ml). After 48 h of incubation, the plates were replica plated onto horse blood agar, and the number of hemolytic colonies was enumerated. Bacteriolysin assay for the detection of activator in culture supernatants. For the preparation of supernatants of strains A18, L2, X-14-5, and JH2Eror, cells were grown to 90 Klett units (a no. 54 filter was used) and then removed by centrifugation at 20,200 x g for 10 min. The remaining supernatant was stored on ice until use, a period that was always less than 24 h (13). Preparation of indicator cells for bacteriolysis, and the bacteriolytic assay itself, has been previously described (1, 2). S. faecalis subsp. liquefaciens strain 3 was used as the indicator organism. After growth to 90 Klett units, the cells were washed twice with 0.01 M phosphate in 0.147 M NaCl at pH 6.8 and resuspended in 0.01 M phosphate in 0.147 M NaCl (pH 6.8) at a turbidity of 150 Klett units. In place of the lysin, the L2 supernatant was used in
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one of the tubes, and a mixture containing five parts of the L2 supernatant and one part of either the A18 supernatant, X-14-5 supernatant, or JH2EroF supernatant was used in the remaining tubes. After 3 h of incubation, the percent bacteriolysis was calculated as described by Appelbaum and Zimmerman (1). Curing. Penassay broth, pH 7.8, was supplemented with either acridine orange (6 ug/ml) or SDS (concentrations varied from 0.01 to 0.04%). When acridine orange was used, the broths were inoculated with a loopful of an overnight AC broth culture and incubated for 18 h at 37°C. Dilutions were then plated out onto AC agar and covered with 3.5 ml of 0.75% soft agar overlay. The plates were then incubated for 24 h at 37°C and covered with a second soft agar overlay containing 0.1 ml of inoculum of strain 3 of the indicator organism. Clear zones in the otherwise confluent growth of the indicator strain appeared over colonies of wild-type X14, producing the bacteriolysin, but not over colonies of the non-bacteriolytic, cured derivatives. When SDS was used as a curing agent, a 0.1-ml inoculum taken from an overnight broth culture was used, and the broths were incubated at 37°C. After an 18-h incubation, dilutions of the culture were plated out onto horse blood agar, and both hemolytic-negative (Hem-) and hemolytic-positive (Hem+) colonies were observed. Subcultures of isolated Hem- colonies gave rise to Hem+ revertants. An analysis by sucrose density gradient centrifugation of these Hem- cultures and their Hem+ revertant counterparts showed that these strains still retained their 38.5-megadalton (Mdal) plasmids. On the other hand, stable Hem- strains could be isolated by growing the cells in AC broth (after SDS treatment) at 45°C. These stable Hem- strains had no 38.5-Mdal plasmid. Later it was observed that stable Hem- strains could be isolated more directly by carrying out the SDS treatment at 450C rather than 37°C. Purification of bacterial isolates with new phenotypes. All colonies were restreaked three times after isolation from the particular treatment. Verification as to their streptococcal classification was based on gram-positive reaction, streptococcal morphology, catalase-negative reaction, lysis by streptococcal phage, and growth on selective antibiotics. Sonic oscillation of cells from strains L2 and X14. Cells were grown to 90 Klett units in brain heart infusion broth, then harvested, and washed with 0.01 M phosphate in 0.147 M NaCl at pH 6.8. They were resuspended in one-tenth their original volume and sonically treated on ice for 30 min at 1.2 A using an MSE sonic oscillator. This treatment reduced the viable plate counts by 98%.
RESULTS Identification of plasmid species in strains X-14, L2, A18, and cured derivatives of strain X-14. From strain X-14, an erythromycin-resistant (Ero') mutant (minimal inhibitory concentration > 320 ,ug/ml) was derived spontaneously. This resistant strain (designated X-14
J. BAcTzRUOL.
Ero') was then grown in 0.02% SDS at 37°C, and of 500 colonies examined, 3 appeared to be nonhemolytic. Each of the latter was inoculated into AC broth and incubated at 45°C for 24 h, and from these broths, stable nonhemolytic strains were isolated. One of these stable Hemstrains, designated X-14-5, was used in this study. A profile of a neutral sucrose gradient of plasmid material from a mixed lysate preparation of X-14 and X-14-5 is shown in Fig. 1. Three peaks can be identified in strain X-14. Marker studies using strain DS-5-Cl indicate their sedimentation values to be 60, 40, and 22S. Calculations were based on the formula S,1S2 = DJ D2 (8). (The sedimentation values of the two plasmids of strain DS-5-Cl have been reported as 28 and 588 [10].) Using alkaline sucrose gradients, it was determined that the 60 and 22S peaks are composed of covalently closed circular DNA. An increase in the 40S peak at the expense of the 60S peak indicates that the open circular form of the 60S plasmid sediments at 40S. The mo-
N
0 x
I
N
0 x
0-
CL
It
C-)
FRACTION NUMBER FIG. 1. Neutral sucrose gradient analysis ofDNA from a mixed lysate preparation of 3H-labeled X-14 and "'C-labeled X-14-5. Satellite peak DNA from dye buoyant density gradients (CsCl-ethidium bromide) was pooled and dialyzed against 0.1 x SSC, and a 02-mi sample was layered onto 5 to 20% sucrose gradients. Centrifugation was for 6( min at 48,000 rpm at 15°C. Sedimentation is from right to left.
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lecular weights of the 60 and 22S plasmid were calculated as being 38.5 x 106 and 3.6 x 106, respectively, using the formula s°2,w = 0.O34MD28 (4). Strain X-14-5 does not contain the 38.5-Mdal peak (see Fig. 1). Five other nonhemolytic derivatives of strain X-14 were obtained in four other curing experiments using either SDS or acridine orange. They also had lost the 38.5Mdal peak. The nonhemolytic A18 and L2 mutants (derived by N-methyl-N'-nitro-N-nitrosoguanidine [NTG] treatment) were analyzed on a neutral sucrose gradient; they exhibited a profile that was identical to the X-14 wild type. The material in the 40S region of the neutral sucrose gradient of strain X-14-5 is not composed of covalently closed circular material, since it remains near the top of the gradient in alkaline sucrose sedimentation. By decreasing the centrifugation time, we were unable to recover any new peaks that might have represented a supercoil counterpart of this peak. It could represent fragments of chromosomal material or possibly the linear breakdown products of a very large plasmid, which we are unable to recover in a supercoil conformation at present. Using a lysate preparation of plasmidless JH2Eror (16), we have demonstrated that chromosomal contamination does occur in that portion of the neutral sucrose gradient. This was accomplished by pooling fractions of the dye buoyant density gradient below the chromosomal peak, where a satellite peak would have been expected to appear had it been present. When this material was dialyzed against 0.1 x SSC and analyzed in 5 to 20% neutral sucrose gradients, a heterogeneous peak with an average sedimentation of 40S could be identified. The same results were obtained when another strain, 26C1, identified as plasmidless in our laboratory, was used. Results of cross-streaking. Granato and Jackson (13) were able to show that crossstreaking their nonhemolytic A18 mutant (activator producing) against their nonhemolytic L2 mutant (lysin producing) resulted in a characteristic spur-shaped zone of hemolysis on a blood agar plate. As a matter of fact, because both activator and lysin are extracellular in nature, a zone of hemolysis can be demonstrated by parallel streaking each culture, provided they are close enough together on the agar to allow for overlapping diffusion of both extracellular entities. This latter type of hemolysis on a horse blood agar plate is to be contrasted with our observation. By cross-streaking our nonhemolytic cured strains across an inoculum of L2 on horse blood agar, it was noted that areas of the plate containing the
mixture gave rise to hemolysis after 24 to 48 h of incubation at 370C (Fig. 2). The hemolysis could be differentiated from that described by Granato and Jackson (13), which arose from cross-streaking L2 with strain A18, because a symmetrical zone of hemolysis rather than the characteristic spur formation that they had observed was seen around the area of mixed culture. When strain X-14-5 was streaked parallel to strain L2, at distances varying from 1 mm to 4 cm, lysis was not observed, indicating that cellto-cell contact between the two strains was required (Fig. 3). If cells were taken from hemolytic areas (where cell-to-cell contact had been allowed to occur) and streaked out, hemolytic colonies could be isolated. All of these colonies retaining their hemolytic ability after a second streaking remained hemolytic through three more successive streakings, indicating that either some form of genetic exchange had taken place or that we were observing a mutation. Since strain X-14-5 and the hemolytic derivatives are all erythromycin resistant (over 30 hemolytic isolates were examined), it is assumed that if we were observing genetic exchange, strain X-14-5 was probably the recipient and strain L2 the donor. Those colonies not retaining their hemolytic ability after a second streaking were assumed to be strain L2, since strain L2 colonies will appear hemolytic on a plate containing other hemolytic colonies, that are producing activator. Identical results were observed when strain JH2Eror (an erythromycin-resistant derivative
1067
FIG. 2. Strain X-14-5 and JH2Eror were streaked from left to right across a streak of L2 on horse blood agar, giving rise to areas of hemolysis on the right side of the plate where mixed cultures of either strains X-14-5 and L2 or JH2Eror and L2 occurred.
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FIG. 3. Strain X-14-5, at a distance of 1 mm, was streaked parallel to strain L2. Then X-14-5 was allowed to make contact with L2 at the bottom of the streak. Hemolysis is seen only where contact between the two cultures was made.
of strain JH2) was streaked across strain L2. Strain JH2Eror and the stable hemolytic derivatives could be differentiated from strain L2 by their resistance to erythromycin, kanamycin, and bacteriophage 81A. Strain L2 was clearly the donor and JH2Eror the recipient. It was later determined that the same observations could be made when either brain heart infusion (Difco) or Oxoid nutrient broth no. 2 was used as the blood agar base. To test whether these hemolytic strains had arisen by mutation, strains JH2Eror, X-14-5, and L2 were examined for mutation to Hem+. No mutants were detected when approximately 104 cells of each strain were observed. Furthermore, no hemolysis was observed when strain L2 was streaked across strain L2, X-14-5 across X-14-5, or JH2Eror across JH2Eror. It is interesting to note that in an analogous cross-streaking experiment where L2 was replaced by A18 and X-14-5 was streaked across it, no hemolysis was observed. Conjugation in broth. When strain L2 was used as the donor and either strain X-14-5 or JH2Eror as the recipient, hemolytic organisms could be detected at a frequency of 4.3/103 recipients (3.5/102 donor) and 2.4/108 recipients (2.0/ 108 donor), respectively. Since Brock has observed that strain X-14 (from which L2 was derived) is lysogenic for a bacteriophage, tr-14 (5), transduction cannot completely be ruled out as the mode of genetic transfer; however, we were unable to observe genetic transfer when culture filtrates of strain L2 were used in place of L2 broth culture. Furthermore, Brock noted that when he lysogen-
J. BACTRIOL.
ized another strain of S. faecalis, strain X-46, with the bacteriophage r-14, it did not acquire the hemolytic trait. Transformation was deemed not be the mode of this genetic exchange either, because deoxyribonuclease did not inhibit the reaction. A conjugal cross in broth was run using X-14 as a donor and X-14-5 as the recipient. Although the starting number of X-14 organisms in the mixed culture was 1.6 x 107 and increased to 1.9 x 108 after 4 h of mixed'incubation, a dramatic killing of strain X-14-5 similar to that noted by Tomura et al. was observed (18). The number of X-14-5 organisms dropped from an initial 3.6 x 107 to 6 x 104 organisms/ ml. Eror Hem+ organisms were detected at a rate of 1 x 10-2/recipient (1.67 x 10-5/donor). This killing was not observed in conjugal crosses where L2 was used as donor; in these crosses, both donor and recipient (either X-14-5 or JH2Eror) had increased in numbers after 4 h of mixed incubation. Plasmid profiles of hemolytic derivatives of strains X-14-5 and JH2Eror after cross-streaking of these strains with strain L2. Plasmid material was isolated from two hemolytic transconjugants of strain X-14-5 (designated X14-5A and X-14-5B) and two hemolytic derivatives of strain JH2Eror (designated JH2A and JH2B) using dye buoyant density gradients. The plasmid material was analyzed on 5 to 20% neutral sucrose gradients, and strain L2 was incorporated into the gradient as a marker. All four strains acquired the 38.5-Mdal plasmid. Neither of the JH2Eror transconjugants acquired the cryptic 3.6-Mdal plasmid. Figure 4 shows two of these plasmid profiles, one from a hemolytic derivative of strain X-14-5 and one from a hemolytic derivative of strain JH2Eroe. Failure to detect bacteriolysin activator. Strains X-14-5 and JH2Eror were tested to see if they were producing activator. Table 2 shows that neither strain produces activator at significant levels, since their supernatants fail to elevate lytic activity of L2 supernatant beyond the level that can be reached by the L2 supernatant alone (0 to 2%). When supernatants from strain A18 were added to the L2 supernatants in the same proportions as the X-14-5 and JH2Eror supernatants, the bacteriolytic activity was elevated to 55% bacteriolysis. Assay of sonic extracts of strain L2 for inhibitor of activator. The possibility that L2 may be producing an inhibitor as a result of its mutation was considered. It was reasoned that if this were the case, the substance inhibiting the activator would probably not be a diffusible substance, since the A component from the strain A18 supernatant activates the lysis com-
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designated L2C3, was isolated using 0.01% SDS and incubation at 450C for 48 h. After plating out dilutions of the broth on AC agar, screening for cured derivatives was accomplished by cross-streaking individual colonies from the AC agar across an inoculum of A18 and looking for those isolates that did not give rise to characteristic hemolytic spur formation. (Three such -2 isolates were obtained from the 84 isolates screened.) Two additional genetic markers ~~~~~~~~~~.5were introduced into L2C3. A spontaneous chloramphenicol-resistant mutant (minimal inhibitory concentration, 20 to 40 mg/ml) was isolated in two steps. This derivative was then plated out on a lawn of bacteriophage 81A (a 0 laboratory strain), and a spontaneous resistant mutant was isolated from this lawn. The result(B)N ing strain was designated L2C3 Cmpr pr (chloramphenicol resistant, phage resistant). An analysis of plasmid material of both strains was undertaken by growing L2C3 Cmpr -2 pr on [methyl-3H]thymidine and L2 on [methyl"4C]thymidine. Sucrose density gradient data from the mixed lysate preparation of L2C3 Cmpr Pr and L2 showed that the 38.5-Mdal plasmid was no longer present in strain L2C3 Cmpr 0~ pr, and its plasmid profile was now identical to that of X-14-5 as shown in Fig. 1. Strain L2C3 Cmpr pr was streaked across L2 on a horse blood agar plate as had been done 10 20 30 with X-14-5 and JH2Eror. No zones of hemolysis FRACTION NUMBER appeared in the area of mixed culture after 72 h FIG. 4. Neutral sucrose gradient analysis of two of incubation, thus indicating that L2C3 Cmpr hemolytic transconjugants. Plasmid material was pr is different from X-14-5 and JH2Eror. Neverisolated from dye buoyant density gradients, pooled, theless, when cells were removed from the area and dialyzed against 0.1 x SSC. 3H-labeled material of mixed culture and streaked out on chloramfrom each transconjugant was mixed with equal vol- phenicol agar, 9 out of the 22 isolates examined umes of "4C-labeled material from strain L2, and then 02-ml samples were layered onto the 5 to 20% were Cmpr pr, lysin positive, activator negative, indicating that the plasmid in L2 had been sucrose gradient. (A) X-14-5A; (B) JH2A. transferred to L2C3 Cmpr pr. When L2C3 Cmpr pr was mated in a conjugal ponent in the supernatant of strain L2. If inhib- cross in broth with X-14 Eror (a spontaneous itor is produced by strain L2, it would most mutant previously described in the text), a dralikely be associated with the whole cells. matic of the recipient was observed as killing To test this possibility, sonic extracts from 10 had been observed with the conjugation beml of strain L2 (prepared as described in Ma- tween X-14 and X-14-5. After 4 h of incubation terials and Methods) were added to 10 ml of the of the mixed culture of L2C3 Cmp" Pr and X-14 strain A18 supernatant and incubated for 1 h at Eror, the number of Cmpr pr organisms had 370C. One milliliter of this preparation was dropped from an initial concentration of 107 to added to 5 ml of the L2 supernatant, and the mixture was used in the bacteriolytic assay TABLE 2. Bacteriolysis produced by various lysin against strain 3 indicator cells. As a control, preparations on Streptococcus faecalis subsp. sonic extracts from strain X-14 were used in liquefaciens strain 3 place of sonic extracts from strain L2. No differ% Bacteriolysis mixture Assay ence between the two preparations could be + 55 A18 L2 detected. If an inhibitor is produced by strain 0-2 L2 L2, we were unable to detect it in this assay. 0 A18 Re-introduction of the 38.5-Mdal plasmid 0 JH2Eroe + L2 into cured derivative of strain L2. A derivative 0 X-14-5 + L2 of strain L2 cured of its 38.5-Mdal plasmid, 0
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FRAZIER AND ZIMMERMAN
103 organisms/ml. We examined 19 of these Cmpr pr organisms and found that 1 was Hem+ and 18 were Hem-. All of the Hem- organisms were cross-streaked with strain A18 and also found to be lysin negative. In a confirmatory experiment, L2C3 Cmpr ps was crossed with X-14 Eror; 5 our of 20 Cmp' isolates were found to be Hem+. DISCUSSION The localization on a plasmid of genes coding for hemolysin has been reported by other laboratories (12, 15, 18). In this paper we confirm this observation for X-14 by showing the hemolysin is coded on the 38.5-Mdal plasmid of this strain; loss of the 38.5-Mdal plasmid leads to loss of hemolysin production, as is exemplified by the data associated with the SDS-cured derivative, X-14-5. The nonhemolytic L2 mutant of X-14 that was produced by NTG treatment (13) also contains the 38.5-Mdal plasmid, lending credence to the hypothesis that its loss of activity has resulted from a mutation. That the plasmid coding for hemolysin is transmissible by conjugation has been reported before (12, 15, 18). Our work also demonstrates this. Cross-streaking of strain L2 with strain X14-5 and strain JH2Ero' gives rise to hemolytic derivatives containing the 38.5-Mdal plasmid, and identification of markers on these hemolytic derivatives establishes that strains X-14-5 and JH2Eror were recipients of donor DNA. The fact that the Hem+ derivatives of strain JH2Eror have acquired the 38.5-Mdal plasmid and not the 3.6-Mdal plasmid is further evidence that they are actually derivatives of strain JH2Eror. Were we to postulate that L2 is the recipient of donor DNA, a far more complicated explanation would be necessary to account for the data. For example, one would have to postulate a loss of the 3.6-Mdal plasmid in addition to picking up three new genetic markers (Eror, Kanr, 81Ar) that are identical to those in JH2Eror. In the genetic exchange we observed, the transfer of the 38.5-Mdal plasmid from strain L2 to other Hem- strains (X-14-5 or JH2Eror) gives rise to the unanticipated recovery of Hem+ organisms. These data could be explained if X-14-5 and JH2Eror were activator producers and if the gene for activator production were on the chromosome or a prophage. But Table 2 shows that neither X-14-5 nor JH2Eror are activator producers. Nevertheless, the data indicate that some gene located either on the chromosome or attached as a prophage to the chromosome is essential for active hemolysin production. More specifically, the data seem
J. BACTERIOL.
to indicate that at least one gene on the plasmid and one gene on the chromosome or a prophage are required for the production of active activator. The possible relationship between these two genes may be explained in several ways. Although one gene product must be either the A component itself, or a precursor of the A component, another product could be a control
protein that is involved in turning the gene(s) for activator on or off. Another possibility is that an enzyme, such as a protease, is required for the production of an active activator. This enzyme could cleave a precursor of the activator, giving rise to active activator. A third and least-plausible possibility is that as a result of a chromosomal mutation in L2, or a mutation on the 22S plasmid, a totally unrelated gene product is altered. This substance now completely inhibits the activator or in some way prevents it from carrying out its function in the hemolytic reaction. When the 60S plasmid is transferred to a new cellular environment (devoid of the altered gene product resulting from a mutation on the chromosome or the 22S plasmid) by conjugation, the activator is no longer inhibited. To test these remote possibilities would require that L2 be cured of the cryptic 3.6-Mdal plasmid and then infected with the 38.5-Mdal plasmid to see if it still was unable to produce the activator. If the mutation for inhibitor production were on the chromosome rather than on the plasmid, a conjugal system involving chromosomal transfer would have to be developed before this issue could be resolved genetically. In any event, attempts to demonstrate the possible existence of this inhibitory material produced in strain L2 as a result of its chromosomal or plasmid mutation were negative. We were unable to detect any anti-activator activity in the extracts of strain L2 when they were added to A18 supernatant containing activator. The fact that L2C3 Cmpr pr, when streaked across L2, does not give rise to a hemolytic zone in the area of mixed culture, but that lysinpositive, activator-negative Cmpr pr transconjugants can be isolated from the mixed culture, confirms that the defect is not on the 38.5-Mdal plasmid. If it were, L2C3 Cmpr pr would be expected to behave like JH2Ero' and X-14-5 in such a cross-streaking experiment by giving rise to a zone of hemolysis around the area of mixed culture. Since a conjugal cross using X14 Eror as a donor and L2C3 Cmpr Pr as a recipient gives rise to hemolytic recipients, it appears that in this particular cross something is being transferred in addition to the 38.5-Mdal plasmid. This additional element apparently
VOL. 130, 1977
contains the genetic information that L2 lacks. Since JH2Eror contains no satellite fraction of DNA, and at the same time it contains the gene required for activator function, we must conclude that this gene is associated with the chromosome. But this gene also appears to be transmissible in its own right) as evidenced by the data from the cross of L2C3 Cmpr pr and X-14 Eror. Under these circumstancas, it would appear that our data are most easily explained by assuming that a transmissible prophage carries the gene into a recipient. Less probable is the possibility that strains X-14 Eror, X-14-5, and JH2Eror contain a phage or plasmid that remains stable in the cytoplasm under conditions in which its DNA is linear rather than supercoiled and is thus not isolated as part of the satellite DNA in dye buoyant density gradients. A large amount of evidence has accumulated, suggesting that the hemolysin may be regulated in terms of its level of production as well as its activity. For example, Appelbaum and Zimmerman (1) have reported that glucose or K2HPO4 in excess of 0.5% (wt/vol), when added to growth medium, was inhibitory toward lysin production, whereas increasing concentrations of L-arginine-hydrochloride gave increasing yields of lytic activity. The effects of glucose may have been due to catabolite repression. Davie and Brock (11) reported that the teichoic acid produced by strain X-14 is inhibitory to the lysin activity and probably was responsible for the resistance strain X-14 had to its own lysin. Granato and Jackson later reported that polyglycerophosphate enhances activity of lysin when added to a mixture of A and lysin filtrates. Werth and Jackson (19) were able to detect production of an inhibitor by X-14 and its nonlytic mutants when they were grown in the presence of subinhibitory concentrations of bacitracin, vancomycin, D-cycloserine, and phosphomycin. They later identified this inhibitory substance as phosphotidylserine (Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, P25, p. 145). The regulation of lysin is by no means simple if all of the examples above are involved. It would certainly be feasible that one or more control substances govern the synthesis of the A component.
LOCI OF HEMOLYSIN PRODUCTION LITERATURE CITED
1. Appelbaum, B., and L. N. Zimmerman. 1974. Produc-
2.
3. 4.
5.
6. 7.
8. 9.
10.
11. 12.
13. 14.
15.
16.
17.
18.
ACKNOWLEDGMENT This research was supported by funds from the Pennsylvania Agricultural Experiment Station of The Pennsylvania State University.
1071
19.
tion of hemolysin and bacteriolysin in a synthetic medium by Streptococcus faecalis var. zymogenes. Infect. Immun. 10:991-995. Basinger, S. F., and R. W. Jackson. 1968. Bacteriocin (hemolysin) of Streptococcus zymogenes. J. Bacteriol. 96:1895-1902. Baxter-Gabbard, K. L. 1972. A simple method for the large-scale preparation of sucrose gradients. FEBS Lett. 20:117-119. Bazarral, M., and D. R. Helinski. 1968. Characterization of multiple circular DNA forms of colicinogenic factor El from Proteus mirabilis. Biochemistry 7:3513-3519. Brock, T. D. 1964. Host range of certain virulent and temperate bacteriophages attacking group D streptococci. J. Bacteriol. 88:165-171. Brock, T. D., and J. M. Davie. 1963. Probable identity of a group D hemolysin with a bacteriocine. J. Bacteriol. 86:708-712. Brock, T. D., B. Peacher, and D. Pierson. 1963. Survey of the bacteriocines of enterococci. J. Bacteriol. 86:702-707. Burgi, E., and A. D. Hershey. 1963. Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3:309-321. Clewell, D. B., and D. R. Helinski. 1970. Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event. Biochemistry 9:4428-4440. Clewell, D. B., Y. Yagi, G. M. Dunny, and S. K. Schultz. 1974. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a plasmid determining erythromycin resistance. J. Bacteriol. 117:283289. Davie, J. M., and T. D. Brock. 1966. Effect of teichoic acid on resistance of the membrane lytic agent of Streptococcus zymogenes. J. Bacteriol. 92:1623-1631. Dunny, G. M., and D. B. Clewell. 1975. Transmissible toxin (hemolysin) plasmid in Streptococcus faecalis and its mobilization of a noninfectious drug resistance plasmid. J. Bacteriol. 124:784-790. Granato, P. A., and R. W. Jackson. 1969. Bicomponent nature of lysin from Streptococcus zymogenes. J. Bacteriol. 100:865-868. Jackson, R. W. 1971. Bacteriolysis and inhibition of gram-positive bacteria by components of Streptococcus zymogenes lysin. J. Bacteriol. 105:156-159. Jacob, A. E., G. J. Douglas, and S. J. Hobbs. 1975. Selftransferable plasmids determining the hemolysin and bacteriocin of Streptococcus faecalis var. zymogenes. J. Bacteriol. 121:863-872. Jacob, A. E., and S. J. Hobbs. 1974. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J. Bacteriol. 117:360-372. Rabin, R., and L. N. Zimmerman. 1956. Proteinase biosynthesis by Streptococcus faecalis var. liquefaciens. I. The effect of carbon and nitrogen sources, pH and inhibitors. Can. J. Microbiol. 2:747-756. Tomura, T., T. Hirano, T. Ito, and M. Yoshioka. 1973. Transmission of bacteriocinogenicity by conjugation in group D streptococci. Jpn. J. Microbiol. 17:445-452. Werth, J. M., and R. W. Jackson. 1971. Effects of various drugs on hemolytic activity of Streptococcus zymogenes. J. Bacteriol. 108:844-848.
JOURNAL OF BACTzRIOLOGY, June 1977, p. 1064-1071 Copyright C) 1977 American Society for Microbiology
Genetic Loci of Hemolysin Production in Streptococcus faecalis subsp. zymogenes' MARSHA L. FRAZIER AND LEONARD N. ZIMMERMAN*
Department of Microbiology and Cell Biology, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 18 November 1976
Two plasmids corresponding to molecular weights of 38.5 x 106 and 3.6 x 106 have been identified in Streptococcus faecalis subsp. zymogenes strain X-14. The larger plasmid is required for hemolysin-bacteriolysin production. Strain L2, a nonlytic nitrosoguanidine mutant of strain X-14, still harbors the hemolytic plasmid and produces the lysin component, but not the activator component, of the lytic system. Conjugal transfer of this plasmid from strain L2 to plasmid-free strains and strains cured of the 38.5-megadalton plasmid gives rise to hemolytic recipients. This implicates a gene in hemolysin production at a site other than the 38.5-megadalton plasmid. In a survey of enterococcal bacteriocins, Brock et al. (7) reported that Streptococcus faecalis subsp. zymogenes strain X-14 has bacteriolytic and hemolytic activity. Additional work by Brock and Davie (6) demonstrated that both these lytic activities are the product of a single system. An analysis by Granato and Jackson (13) of the bacteriolytic-hemolytic system ofS. faecalis subsp. zymogenes strain X-14 showed that it is bicomponent in nature, consisting of an activator (A) component and lysin. Although the lysin component has slight activity by itself in some cases (14), maximum bacteriolysis or hemolysis is attained by activating the lysin with the activator; the A component has no activity by itself. Recently there have been several reports (12, 15, 18) that presented evidence favoring the hypothesis that the genes coding for hemolysinbacteriolysin formation in enterococci are borne on plasmids. In addition, Dunny and Clewell, Jacob et al., and Tomura et al. (12, 15, 18) have shown that lysin production is transmissible by conjugation. Based on this information, it should be expected that the hemolysin-bacteriolysin production of S. faecalis subsp. zymogenes strain X-14 is plasmid-borne and transmissible by conjugation. In this paper we confirm the latter notion but present evidence that the hemolysin-bacteriolysin system is not coded entirely on a plasmid. Our data suggest that the production of the A component of the hemolysin requires at least one gene on the plasI Authorized for publication by the Pennsylvania Agricultural Experiment Station, Pennsylvania State University, as paper no. 5203.
mid and at least one gene on one of the other replicons of the cell. MATERIALS AND METHODS Materials. Pronase, lysozyme, and ethidium bromide were purchased from Calbiochem; erythromycin and CsCl (grade 1) were from Sigma; acridine orange was from Allied Chemical; sodium dodecyl sulfate (SDS) was from BDH and Baker; [methyl3H]thymidine and [methyl-14C]thymidine were purchased from the Amersham/Searle Corp.; and penicillin was from Eli Lilly & Co. Media. AC broth (17) was used as a general culture medium. AC agar was prepared by adding 1.5% agar (Difco) to the AC broth. Penassay broth (antibiotic no. 3, Difco) was used in the curing experiments and adjusted to pH 7.8. Unless otherwise stated, horse blood agar was prepared by supplementing AC agar with 5% sterile horse blood (in Alsever solution). For conjugation in broth, Oxoid nutrient broth no. 2 was used. Bacterial strains. Table 1 describes most of the strains of bacteria used and their characteristics. S faecalis strain JH2 was obtained from A. Jacob, and strain DS-5-Cl was obtained from D. B. Clewell. Although strain JH2 was reported by Jacob and Hobbs (16) as being sensitive to kanamycin, it is not as sensitive as some of the other strains used in this investigation and is therefore referred to as being kanamycin resistant (Table 1). S. faecalis subsp. zymogenes strains X-14, X-14/NGBL2, and X-14/ NGBA18 were obtained from R. W. Jackson. For simplicity, strain X-14/NGBL2 will be referred to as L2 and X-14/NGBA18 as A18. Also, we are retaining the original genetic nomenclature of Granato and Jackson (13), which designates the L2 strain as being lysin positive, activator negative and the A18 strain as being lysin negative, activator positive. S. faecalis subsp. liquefaciens strain 3 is a non-proteolytic mutant of S. faecalis subsp. liquefaciens strain
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LOCI OF HEMOLYSIN PRODUCTION
VOL. 130, 1977
Subspecies
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TABLE 1. S. faecalis strains used in genetic experiments Relevant phenotypesa Comments, references, and/
Strain no.
or remarks Wild type (references 6 and 7) s s Nitrosoguanidine mutant s + X-14/NGBL2 zymogenes of X-14 s Nitrosoguanidine mutant + 8 8 X-14/NGBA18 zymogenes of X-14 (reference 13) s Cured with sodium doder s X-14-5 zymogenes cyl sulfate Reference 12 DS-5-Cl zymog r r Wild type (reference 16) s JH2 faecalis r r r Spontaneous mutant of JH2Eror faecalis JH2 a Genetic symbols: Hem, hemolysis; Bac, bacteriolysin; A, activator; L, lysin; Ero, erythromycin (10 ml); Kan, kanamycin (80 j.g/ml); 81A, bacteriophage 81A; s, sensitive; r, resistant.
zymogenes
X-14
Hem
Bac
A
L
Ero
Kan
81A
+
+
+
+
8
S
s
itg/
31. It was obtained by ultraviolet light irradiation (1) and is used as an indicator strain for bacteriolysin sensitivity. S. faecalis subsp. zymogenes strain 26C1 was obtained from the Cornell University stock collection. It is used as a plasmidless control for sucrose density gradients. Isolation of plasmid DNA. Cells were grown at 37°C in 25-ml volumes of AC broth supplemented with 250 uCi of [methyl-3H]thymidine or 25 ,uCi of [methyl-14C]thymidine. After growth to 95 Klett units (a Klett-Summerson colorimeter with a no. 54 filter was used), cells were chilled on ice for 10 min, centrifuged at 20,200 x g for 10 min at 4°C, and then washed once with buffered TES solution [0.03 M tris(hydroxymethyl)aminomethane, 0.005 M Na2 ethylenediaminetetraacetic acid, and 0.05 M NaCl at pH 8.0]. Cells were then resuspended in 1.0 ml of 25% (wt/wt) sucrose [in 0.05 M tris(hydroxymethyl)aminomethane, pH 8.0] and lysed according to the method previously described by Clewell et al. (10). When differentially labeled cultures were prepared for mixed lysis, they were mixed during the washing step. Preparation of dye buoyant density gradients was similar to the procedure used by Clewell and Helinski (9). A 1.5-ml volume of the cleared lysate was added to 6.3 ml of a TES buffer containing 3 mg of ethidium bromide and 7.0 g of CsCl. Gradients were centrifuged for 48 h at 15°C in a Beckman 5OTi fixedangle rotor at 42,000 rpm. They were then fractionated (13 drops per fraction) using a Buchler piercing unit with an 18-gauge needle, and 50-,ul samples were spotted onto 1.5-cm filter paper squares (Whatman no. 3, medium flow rate). Fractions containing satellite material were pooled and dialyzed against 0.1 x SSC (0.01 M NaCl, 0.0015 M sodium citrate, pH 7.4). After oven drying at 70°C for 20 min, the filter papers were processed in batches by washing twice with cold 10% (wt/vol) trichloroacetic acid and twice with 70% ethanol. They were then dried again and counted in a cocktail consisting of 5 g of PPO (2,5diphenyloxazole) and 0.3 g of dimethyl-POPOP {1,4bis-[2]-(5-phenyloxazolyl)benzene} per liter of tolu-
ene, using a Packard model 3375 scintillation spectrometer. Sucrose velocity sedimentation. Samples of pooled, dialyzed, satellite material, 0.2 ml in volume, were layered onto 4.6-ml gradients of 5 to 20% (wt/wt) sucrose prepared (using three freeze-thaw cycles) according to the method of Baxter-Gabbard (3). TES buffer was used for neutral gradients. Alkaline gradients were prepared in a solution of 1.0 M NaCl-0.001 M Na2 ethylenediaminetetraacetic acid0.3 M NaOH at pH 12.0 (9). A Beckman SW50.1 rotor was used for centrifugation. Gradients were fractionated with a Buchler piercing unit. Six-drop fractions were then collected onto 1.5-cm filter paper squares, processed, and counted as in the dye buoyant density gradients. Conjugation in broth. A modification of the method of Jacob and Hobbs (16) was used. Using overnight cultures, 0.5 ml of donor and 0.5 ml of recipient were mixed with 5 ml of Oxoid nutrient broth no. 2. After incubation of the mixed culture for 4 h, dilutions were plated onto AC agar containing erythromycin (10 Zg/ml). After 48 h of incubation, the plates were replica plated onto horse blood agar, and the number of hemolytic colonies was enumerated. Bacteriolysin assay for the detection of activator in culture supernatants. For the preparation of supernatants of strains A18, L2, X-14-5, and JH2Eror, cells were grown to 90 Klett units (a no. 54 filter was used) and then removed by centrifugation at 20,200 x g for 10 min. The remaining supernatant was stored on ice until use, a period that was always less than 24 h (13). Preparation of indicator cells for bacteriolysis, and the bacteriolytic assay itself, has been previously described (1, 2). S. faecalis subsp. liquefaciens strain 3 was used as the indicator organism. After growth to 90 Klett units, the cells were washed twice with 0.01 M phosphate in 0.147 M NaCl at pH 6.8 and resuspended in 0.01 M phosphate in 0.147 M NaCl (pH 6.8) at a turbidity of 150 Klett units. In place of the lysin, the L2 supernatant was used in
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FRAZIER AND ZIMMERMAN
one of the tubes, and a mixture containing five parts of the L2 supernatant and one part of either the A18 supernatant, X-14-5 supernatant, or JH2EroF supernatant was used in the remaining tubes. After 3 h of incubation, the percent bacteriolysis was calculated as described by Appelbaum and Zimmerman (1). Curing. Penassay broth, pH 7.8, was supplemented with either acridine orange (6 ug/ml) or SDS (concentrations varied from 0.01 to 0.04%). When acridine orange was used, the broths were inoculated with a loopful of an overnight AC broth culture and incubated for 18 h at 37°C. Dilutions were then plated out onto AC agar and covered with 3.5 ml of 0.75% soft agar overlay. The plates were then incubated for 24 h at 37°C and covered with a second soft agar overlay containing 0.1 ml of inoculum of strain 3 of the indicator organism. Clear zones in the otherwise confluent growth of the indicator strain appeared over colonies of wild-type X14, producing the bacteriolysin, but not over colonies of the non-bacteriolytic, cured derivatives. When SDS was used as a curing agent, a 0.1-ml inoculum taken from an overnight broth culture was used, and the broths were incubated at 37°C. After an 18-h incubation, dilutions of the culture were plated out onto horse blood agar, and both hemolytic-negative (Hem-) and hemolytic-positive (Hem+) colonies were observed. Subcultures of isolated Hem- colonies gave rise to Hem+ revertants. An analysis by sucrose density gradient centrifugation of these Hem- cultures and their Hem+ revertant counterparts showed that these strains still retained their 38.5-megadalton (Mdal) plasmids. On the other hand, stable Hem- strains could be isolated by growing the cells in AC broth (after SDS treatment) at 45°C. These stable Hem- strains had no 38.5-Mdal plasmid. Later it was observed that stable Hem- strains could be isolated more directly by carrying out the SDS treatment at 450C rather than 37°C. Purification of bacterial isolates with new phenotypes. All colonies were restreaked three times after isolation from the particular treatment. Verification as to their streptococcal classification was based on gram-positive reaction, streptococcal morphology, catalase-negative reaction, lysis by streptococcal phage, and growth on selective antibiotics. Sonic oscillation of cells from strains L2 and X14. Cells were grown to 90 Klett units in brain heart infusion broth, then harvested, and washed with 0.01 M phosphate in 0.147 M NaCl at pH 6.8. They were resuspended in one-tenth their original volume and sonically treated on ice for 30 min at 1.2 A using an MSE sonic oscillator. This treatment reduced the viable plate counts by 98%.
RESULTS Identification of plasmid species in strains X-14, L2, A18, and cured derivatives of strain X-14. From strain X-14, an erythromycin-resistant (Ero') mutant (minimal inhibitory concentration > 320 ,ug/ml) was derived spontaneously. This resistant strain (designated X-14
J. BAcTzRUOL.
Ero') was then grown in 0.02% SDS at 37°C, and of 500 colonies examined, 3 appeared to be nonhemolytic. Each of the latter was inoculated into AC broth and incubated at 45°C for 24 h, and from these broths, stable nonhemolytic strains were isolated. One of these stable Hemstrains, designated X-14-5, was used in this study. A profile of a neutral sucrose gradient of plasmid material from a mixed lysate preparation of X-14 and X-14-5 is shown in Fig. 1. Three peaks can be identified in strain X-14. Marker studies using strain DS-5-Cl indicate their sedimentation values to be 60, 40, and 22S. Calculations were based on the formula S,1S2 = DJ D2 (8). (The sedimentation values of the two plasmids of strain DS-5-Cl have been reported as 28 and 588 [10].) Using alkaline sucrose gradients, it was determined that the 60 and 22S peaks are composed of covalently closed circular DNA. An increase in the 40S peak at the expense of the 60S peak indicates that the open circular form of the 60S plasmid sediments at 40S. The mo-
N
0 x
I
N
0 x
0-
CL
It
C-)
FRACTION NUMBER FIG. 1. Neutral sucrose gradient analysis ofDNA from a mixed lysate preparation of 3H-labeled X-14 and "'C-labeled X-14-5. Satellite peak DNA from dye buoyant density gradients (CsCl-ethidium bromide) was pooled and dialyzed against 0.1 x SSC, and a 02-mi sample was layered onto 5 to 20% sucrose gradients. Centrifugation was for 6( min at 48,000 rpm at 15°C. Sedimentation is from right to left.
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LOCI OF HEMOLYSIN PRODUCTION
lecular weights of the 60 and 22S plasmid were calculated as being 38.5 x 106 and 3.6 x 106, respectively, using the formula s°2,w = 0.O34MD28 (4). Strain X-14-5 does not contain the 38.5-Mdal peak (see Fig. 1). Five other nonhemolytic derivatives of strain X-14 were obtained in four other curing experiments using either SDS or acridine orange. They also had lost the 38.5Mdal peak. The nonhemolytic A18 and L2 mutants (derived by N-methyl-N'-nitro-N-nitrosoguanidine [NTG] treatment) were analyzed on a neutral sucrose gradient; they exhibited a profile that was identical to the X-14 wild type. The material in the 40S region of the neutral sucrose gradient of strain X-14-5 is not composed of covalently closed circular material, since it remains near the top of the gradient in alkaline sucrose sedimentation. By decreasing the centrifugation time, we were unable to recover any new peaks that might have represented a supercoil counterpart of this peak. It could represent fragments of chromosomal material or possibly the linear breakdown products of a very large plasmid, which we are unable to recover in a supercoil conformation at present. Using a lysate preparation of plasmidless JH2Eror (16), we have demonstrated that chromosomal contamination does occur in that portion of the neutral sucrose gradient. This was accomplished by pooling fractions of the dye buoyant density gradient below the chromosomal peak, where a satellite peak would have been expected to appear had it been present. When this material was dialyzed against 0.1 x SSC and analyzed in 5 to 20% neutral sucrose gradients, a heterogeneous peak with an average sedimentation of 40S could be identified. The same results were obtained when another strain, 26C1, identified as plasmidless in our laboratory, was used. Results of cross-streaking. Granato and Jackson (13) were able to show that crossstreaking their nonhemolytic A18 mutant (activator producing) against their nonhemolytic L2 mutant (lysin producing) resulted in a characteristic spur-shaped zone of hemolysis on a blood agar plate. As a matter of fact, because both activator and lysin are extracellular in nature, a zone of hemolysis can be demonstrated by parallel streaking each culture, provided they are close enough together on the agar to allow for overlapping diffusion of both extracellular entities. This latter type of hemolysis on a horse blood agar plate is to be contrasted with our observation. By cross-streaking our nonhemolytic cured strains across an inoculum of L2 on horse blood agar, it was noted that areas of the plate containing the
mixture gave rise to hemolysis after 24 to 48 h of incubation at 370C (Fig. 2). The hemolysis could be differentiated from that described by Granato and Jackson (13), which arose from cross-streaking L2 with strain A18, because a symmetrical zone of hemolysis rather than the characteristic spur formation that they had observed was seen around the area of mixed culture. When strain X-14-5 was streaked parallel to strain L2, at distances varying from 1 mm to 4 cm, lysis was not observed, indicating that cellto-cell contact between the two strains was required (Fig. 3). If cells were taken from hemolytic areas (where cell-to-cell contact had been allowed to occur) and streaked out, hemolytic colonies could be isolated. All of these colonies retaining their hemolytic ability after a second streaking remained hemolytic through three more successive streakings, indicating that either some form of genetic exchange had taken place or that we were observing a mutation. Since strain X-14-5 and the hemolytic derivatives are all erythromycin resistant (over 30 hemolytic isolates were examined), it is assumed that if we were observing genetic exchange, strain X-14-5 was probably the recipient and strain L2 the donor. Those colonies not retaining their hemolytic ability after a second streaking were assumed to be strain L2, since strain L2 colonies will appear hemolytic on a plate containing other hemolytic colonies, that are producing activator. Identical results were observed when strain JH2Eror (an erythromycin-resistant derivative
1067
FIG. 2. Strain X-14-5 and JH2Eror were streaked from left to right across a streak of L2 on horse blood agar, giving rise to areas of hemolysis on the right side of the plate where mixed cultures of either strains X-14-5 and L2 or JH2Eror and L2 occurred.
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FRAZIER AND ZIMMERMAN
FIG. 3. Strain X-14-5, at a distance of 1 mm, was streaked parallel to strain L2. Then X-14-5 was allowed to make contact with L2 at the bottom of the streak. Hemolysis is seen only where contact between the two cultures was made.
of strain JH2) was streaked across strain L2. Strain JH2Eror and the stable hemolytic derivatives could be differentiated from strain L2 by their resistance to erythromycin, kanamycin, and bacteriophage 81A. Strain L2 was clearly the donor and JH2Eror the recipient. It was later determined that the same observations could be made when either brain heart infusion (Difco) or Oxoid nutrient broth no. 2 was used as the blood agar base. To test whether these hemolytic strains had arisen by mutation, strains JH2Eror, X-14-5, and L2 were examined for mutation to Hem+. No mutants were detected when approximately 104 cells of each strain were observed. Furthermore, no hemolysis was observed when strain L2 was streaked across strain L2, X-14-5 across X-14-5, or JH2Eror across JH2Eror. It is interesting to note that in an analogous cross-streaking experiment where L2 was replaced by A18 and X-14-5 was streaked across it, no hemolysis was observed. Conjugation in broth. When strain L2 was used as the donor and either strain X-14-5 or JH2Eror as the recipient, hemolytic organisms could be detected at a frequency of 4.3/103 recipients (3.5/102 donor) and 2.4/108 recipients (2.0/ 108 donor), respectively. Since Brock has observed that strain X-14 (from which L2 was derived) is lysogenic for a bacteriophage, tr-14 (5), transduction cannot completely be ruled out as the mode of genetic transfer; however, we were unable to observe genetic transfer when culture filtrates of strain L2 were used in place of L2 broth culture. Furthermore, Brock noted that when he lysogen-
J. BACTRIOL.
ized another strain of S. faecalis, strain X-46, with the bacteriophage r-14, it did not acquire the hemolytic trait. Transformation was deemed not be the mode of this genetic exchange either, because deoxyribonuclease did not inhibit the reaction. A conjugal cross in broth was run using X-14 as a donor and X-14-5 as the recipient. Although the starting number of X-14 organisms in the mixed culture was 1.6 x 107 and increased to 1.9 x 108 after 4 h of mixed'incubation, a dramatic killing of strain X-14-5 similar to that noted by Tomura et al. was observed (18). The number of X-14-5 organisms dropped from an initial 3.6 x 107 to 6 x 104 organisms/ ml. Eror Hem+ organisms were detected at a rate of 1 x 10-2/recipient (1.67 x 10-5/donor). This killing was not observed in conjugal crosses where L2 was used as donor; in these crosses, both donor and recipient (either X-14-5 or JH2Eror) had increased in numbers after 4 h of mixed incubation. Plasmid profiles of hemolytic derivatives of strains X-14-5 and JH2Eror after cross-streaking of these strains with strain L2. Plasmid material was isolated from two hemolytic transconjugants of strain X-14-5 (designated X14-5A and X-14-5B) and two hemolytic derivatives of strain JH2Eror (designated JH2A and JH2B) using dye buoyant density gradients. The plasmid material was analyzed on 5 to 20% neutral sucrose gradients, and strain L2 was incorporated into the gradient as a marker. All four strains acquired the 38.5-Mdal plasmid. Neither of the JH2Eror transconjugants acquired the cryptic 3.6-Mdal plasmid. Figure 4 shows two of these plasmid profiles, one from a hemolytic derivative of strain X-14-5 and one from a hemolytic derivative of strain JH2Eroe. Failure to detect bacteriolysin activator. Strains X-14-5 and JH2Eror were tested to see if they were producing activator. Table 2 shows that neither strain produces activator at significant levels, since their supernatants fail to elevate lytic activity of L2 supernatant beyond the level that can be reached by the L2 supernatant alone (0 to 2%). When supernatants from strain A18 were added to the L2 supernatants in the same proportions as the X-14-5 and JH2Eror supernatants, the bacteriolytic activity was elevated to 55% bacteriolysis. Assay of sonic extracts of strain L2 for inhibitor of activator. The possibility that L2 may be producing an inhibitor as a result of its mutation was considered. It was reasoned that if this were the case, the substance inhibiting the activator would probably not be a diffusible substance, since the A component from the strain A18 supernatant activates the lysis com-
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designated L2C3, was isolated using 0.01% SDS and incubation at 450C for 48 h. After plating out dilutions of the broth on AC agar, screening for cured derivatives was accomplished by cross-streaking individual colonies from the AC agar across an inoculum of A18 and looking for those isolates that did not give rise to characteristic hemolytic spur formation. (Three such -2 isolates were obtained from the 84 isolates screened.) Two additional genetic markers ~~~~~~~~~~.5were introduced into L2C3. A spontaneous chloramphenicol-resistant mutant (minimal inhibitory concentration, 20 to 40 mg/ml) was isolated in two steps. This derivative was then plated out on a lawn of bacteriophage 81A (a 0 laboratory strain), and a spontaneous resistant mutant was isolated from this lawn. The result(B)N ing strain was designated L2C3 Cmpr pr (chloramphenicol resistant, phage resistant). An analysis of plasmid material of both strains was undertaken by growing L2C3 Cmpr -2 pr on [methyl-3H]thymidine and L2 on [methyl"4C]thymidine. Sucrose density gradient data from the mixed lysate preparation of L2C3 Cmpr Pr and L2 showed that the 38.5-Mdal plasmid was no longer present in strain L2C3 Cmpr 0~ pr, and its plasmid profile was now identical to that of X-14-5 as shown in Fig. 1. Strain L2C3 Cmpr pr was streaked across L2 on a horse blood agar plate as had been done 10 20 30 with X-14-5 and JH2Eror. No zones of hemolysis FRACTION NUMBER appeared in the area of mixed culture after 72 h FIG. 4. Neutral sucrose gradient analysis of two of incubation, thus indicating that L2C3 Cmpr hemolytic transconjugants. Plasmid material was pr is different from X-14-5 and JH2Eror. Neverisolated from dye buoyant density gradients, pooled, theless, when cells were removed from the area and dialyzed against 0.1 x SSC. 3H-labeled material of mixed culture and streaked out on chloramfrom each transconjugant was mixed with equal vol- phenicol agar, 9 out of the 22 isolates examined umes of "4C-labeled material from strain L2, and then 02-ml samples were layered onto the 5 to 20% were Cmpr pr, lysin positive, activator negative, indicating that the plasmid in L2 had been sucrose gradient. (A) X-14-5A; (B) JH2A. transferred to L2C3 Cmpr pr. When L2C3 Cmpr pr was mated in a conjugal ponent in the supernatant of strain L2. If inhib- cross in broth with X-14 Eror (a spontaneous itor is produced by strain L2, it would most mutant previously described in the text), a dralikely be associated with the whole cells. matic of the recipient was observed as killing To test this possibility, sonic extracts from 10 had been observed with the conjugation beml of strain L2 (prepared as described in Ma- tween X-14 and X-14-5. After 4 h of incubation terials and Methods) were added to 10 ml of the of the mixed culture of L2C3 Cmp" Pr and X-14 strain A18 supernatant and incubated for 1 h at Eror, the number of Cmpr pr organisms had 370C. One milliliter of this preparation was dropped from an initial concentration of 107 to added to 5 ml of the L2 supernatant, and the mixture was used in the bacteriolytic assay TABLE 2. Bacteriolysis produced by various lysin against strain 3 indicator cells. As a control, preparations on Streptococcus faecalis subsp. sonic extracts from strain X-14 were used in liquefaciens strain 3 place of sonic extracts from strain L2. No differ% Bacteriolysis mixture Assay ence between the two preparations could be + 55 A18 L2 detected. If an inhibitor is produced by strain 0-2 L2 L2, we were unable to detect it in this assay. 0 A18 Re-introduction of the 38.5-Mdal plasmid 0 JH2Eroe + L2 into cured derivative of strain L2. A derivative 0 X-14-5 + L2 of strain L2 cured of its 38.5-Mdal plasmid, 0
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103 organisms/ml. We examined 19 of these Cmpr pr organisms and found that 1 was Hem+ and 18 were Hem-. All of the Hem- organisms were cross-streaked with strain A18 and also found to be lysin negative. In a confirmatory experiment, L2C3 Cmpr ps was crossed with X-14 Eror; 5 our of 20 Cmp' isolates were found to be Hem+. DISCUSSION The localization on a plasmid of genes coding for hemolysin has been reported by other laboratories (12, 15, 18). In this paper we confirm this observation for X-14 by showing the hemolysin is coded on the 38.5-Mdal plasmid of this strain; loss of the 38.5-Mdal plasmid leads to loss of hemolysin production, as is exemplified by the data associated with the SDS-cured derivative, X-14-5. The nonhemolytic L2 mutant of X-14 that was produced by NTG treatment (13) also contains the 38.5-Mdal plasmid, lending credence to the hypothesis that its loss of activity has resulted from a mutation. That the plasmid coding for hemolysin is transmissible by conjugation has been reported before (12, 15, 18). Our work also demonstrates this. Cross-streaking of strain L2 with strain X14-5 and strain JH2Ero' gives rise to hemolytic derivatives containing the 38.5-Mdal plasmid, and identification of markers on these hemolytic derivatives establishes that strains X-14-5 and JH2Eror were recipients of donor DNA. The fact that the Hem+ derivatives of strain JH2Eror have acquired the 38.5-Mdal plasmid and not the 3.6-Mdal plasmid is further evidence that they are actually derivatives of strain JH2Eror. Were we to postulate that L2 is the recipient of donor DNA, a far more complicated explanation would be necessary to account for the data. For example, one would have to postulate a loss of the 3.6-Mdal plasmid in addition to picking up three new genetic markers (Eror, Kanr, 81Ar) that are identical to those in JH2Eror. In the genetic exchange we observed, the transfer of the 38.5-Mdal plasmid from strain L2 to other Hem- strains (X-14-5 or JH2Eror) gives rise to the unanticipated recovery of Hem+ organisms. These data could be explained if X-14-5 and JH2Eror were activator producers and if the gene for activator production were on the chromosome or a prophage. But Table 2 shows that neither X-14-5 nor JH2Eror are activator producers. Nevertheless, the data indicate that some gene located either on the chromosome or attached as a prophage to the chromosome is essential for active hemolysin production. More specifically, the data seem
J. BACTERIOL.
to indicate that at least one gene on the plasmid and one gene on the chromosome or a prophage are required for the production of active activator. The possible relationship between these two genes may be explained in several ways. Although one gene product must be either the A component itself, or a precursor of the A component, another product could be a control
protein that is involved in turning the gene(s) for activator on or off. Another possibility is that an enzyme, such as a protease, is required for the production of an active activator. This enzyme could cleave a precursor of the activator, giving rise to active activator. A third and least-plausible possibility is that as a result of a chromosomal mutation in L2, or a mutation on the 22S plasmid, a totally unrelated gene product is altered. This substance now completely inhibits the activator or in some way prevents it from carrying out its function in the hemolytic reaction. When the 60S plasmid is transferred to a new cellular environment (devoid of the altered gene product resulting from a mutation on the chromosome or the 22S plasmid) by conjugation, the activator is no longer inhibited. To test these remote possibilities would require that L2 be cured of the cryptic 3.6-Mdal plasmid and then infected with the 38.5-Mdal plasmid to see if it still was unable to produce the activator. If the mutation for inhibitor production were on the chromosome rather than on the plasmid, a conjugal system involving chromosomal transfer would have to be developed before this issue could be resolved genetically. In any event, attempts to demonstrate the possible existence of this inhibitory material produced in strain L2 as a result of its chromosomal or plasmid mutation were negative. We were unable to detect any anti-activator activity in the extracts of strain L2 when they were added to A18 supernatant containing activator. The fact that L2C3 Cmpr pr, when streaked across L2, does not give rise to a hemolytic zone in the area of mixed culture, but that lysinpositive, activator-negative Cmpr pr transconjugants can be isolated from the mixed culture, confirms that the defect is not on the 38.5-Mdal plasmid. If it were, L2C3 Cmpr pr would be expected to behave like JH2Ero' and X-14-5 in such a cross-streaking experiment by giving rise to a zone of hemolysis around the area of mixed culture. Since a conjugal cross using X14 Eror as a donor and L2C3 Cmpr Pr as a recipient gives rise to hemolytic recipients, it appears that in this particular cross something is being transferred in addition to the 38.5-Mdal plasmid. This additional element apparently
VOL. 130, 1977
contains the genetic information that L2 lacks. Since JH2Eror contains no satellite fraction of DNA, and at the same time it contains the gene required for activator function, we must conclude that this gene is associated with the chromosome. But this gene also appears to be transmissible in its own right) as evidenced by the data from the cross of L2C3 Cmpr pr and X-14 Eror. Under these circumstancas, it would appear that our data are most easily explained by assuming that a transmissible prophage carries the gene into a recipient. Less probable is the possibility that strains X-14 Eror, X-14-5, and JH2Eror contain a phage or plasmid that remains stable in the cytoplasm under conditions in which its DNA is linear rather than supercoiled and is thus not isolated as part of the satellite DNA in dye buoyant density gradients. A large amount of evidence has accumulated, suggesting that the hemolysin may be regulated in terms of its level of production as well as its activity. For example, Appelbaum and Zimmerman (1) have reported that glucose or K2HPO4 in excess of 0.5% (wt/vol), when added to growth medium, was inhibitory toward lysin production, whereas increasing concentrations of L-arginine-hydrochloride gave increasing yields of lytic activity. The effects of glucose may have been due to catabolite repression. Davie and Brock (11) reported that the teichoic acid produced by strain X-14 is inhibitory to the lysin activity and probably was responsible for the resistance strain X-14 had to its own lysin. Granato and Jackson later reported that polyglycerophosphate enhances activity of lysin when added to a mixture of A and lysin filtrates. Werth and Jackson (19) were able to detect production of an inhibitor by X-14 and its nonlytic mutants when they were grown in the presence of subinhibitory concentrations of bacitracin, vancomycin, D-cycloserine, and phosphomycin. They later identified this inhibitory substance as phosphotidylserine (Abstr. Annu. Meet. Am. Soc. Microbiol. 1973, P25, p. 145). The regulation of lysin is by no means simple if all of the examples above are involved. It would certainly be feasible that one or more control substances govern the synthesis of the A component.
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1. Appelbaum, B., and L. N. Zimmerman. 1974. Produc-
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ACKNOWLEDGMENT This research was supported by funds from the Pennsylvania Agricultural Experiment Station of The Pennsylvania State University.
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