JOURNAL OF BACTERIOLOGY, Oct. 1976, p. 347-355 Copyright 0 1976 American Society for Microbiology

Vol. 128, No. 1 Printed in U.S.A.

Transformation of Streptococcus sanguis Challis by Plasmid Deoxyribonucleic Acid from Streptococcus faecalis DONALD J. LEBLANC* AND FLORENCE P. HASSELL Laboratory of Microbiology and Immunology, National Institute of Dental Research, Bethesda, Maryland 20014 Received for publication 1 June 1976

Plasmid deoxyribonucleic acid (DNA) from Streptococcus faecalis, strain DS5, was transferred to the Challis strain of Streptococcus sanguis by transformation. Two antibiotic resistance markers carried by the (3 plasmid from strain DS5, erythromycin and lincomycin, were transferred to S. sanguis at a maximum frequency of 1.8 x 10-5/colony-forming unit. Approximately 70% of the covalently closed circular DNA isolated from transformant cultures by dye buoyant density gradients was shown to be hybridizable to ,8 plasmid DNA. Two major differences were observed betwen the 8 plasmid from S. faecalis and the plasmid isolated from transformed S. sanguis: (i) the 3 plasmid from strain DS5 sedimented in velocity gradients at 43S, whereas the covalently closed circular DNA from transformed Challis sedimented at 41S, suggesting a 1.5-Mdal deletion from the 8 plasmid occurred; (ii) although the 43S (8 plasmid remained in the supercoiled configuration for several weeks after isolation, the 41S plasmid was rapidly converted to a linear double-stranded molecule. Attempts to transform S. sanguis with the a plasmid from S. faecalis, strain DS5, were unsuccessful.

The presence of bacterial plasmids in the genus Streptococcus was first reported in 1972 (9).

Subsequently, these extrachomosomal genetic elements have been demonstrated in members of Lancefield groups A (3), D (6, 16, 17), H (12, 15), and N (8, 22). Among strains of Streptococcus faecalis, plasmids have been shown to code for such phenotypic traits as antibiotic resistance (5, 6, 9, 17) and hemolysin and bacteriocin production (13, 16). Certain metabolic properties of streptococci may also be attributed to plasmids, such as lactose fermentation and proteinase production by group N strains (8, 22, 23) and the synthesis of insoluble extracellular polysaccharides by Streptococcus mutans (15). Preliminary evidence, based on curing experiments, suggests that plasmids may be responsible for the production of M proteins by virulent group A streptococci (2). Thus, as in the Enterobacteriaceae (7), streptococcal plasmids appear to affect the pathogenicity, ecology, and taxonomic status of their hosts. In addition to the above phenotypic traits, other characteristics of plasmids in the Enterobacteriaceae are also shared by streptococcal plasmids. Multiple antibiotic resistance (17) and hemolysin and bacteriocin production (13, 16) can be found on conjugative plasmids that mediate their own transfer in S. faecalis. The

phenomenon of plasmid incompatibility has also been demonstrated in this species (17). Furthermore, it is possible to mobilize a nontransmissible plasmid when the host bacterium also harbors a conjugative plasmid (13). Among the group N streptococci it has been possible to transfer plasmid-coded phenotypes by transduction (21, 23). Finally, the occurrence of some form of plasmid transfer across species lines in nature is suggested by the finding that plasmids isolated from two different species, a group D and a group A strain, were of the same size, coded for the same antibiotic resistances, and shared 95% homology with each other (29). We have been interested in the possible role of plasmids in the pathogenicity ecology and taxonomy of the oral streptococci. As a preliminary step we attempted to develop a plasmidtransfer system as a tool for determining phenotypic properties coded by streptococcal plasmids. The transfer of plasmid deoxyribonucleic acid (DNA) between species of streptococci by transformation has, to our knowledge, not been reported. In this communication we describe the transformation of S. sanguis Challis by plasmid DNA isolated from a strain of S. faecalis. Evidence is presented that the transferred DNA is stably maintained in an extrachromosomal state by the transformant but ex347

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hibits altered physical characteristics relative to the transforming plasmid. MATERIALS AND METHODS Bacterial strains and growth conditions. A transformable Challis strain of S. sanguis (NCTC7868) was obtained from R. M. Cole (National Institutes of Health, Bethesda, Md.). The D85 strain of S. faecalis (ATCC 14508), containing three different plasmids (6), was kindly provided by D. B. Clewell (University of Michigan, Ann Arbor, Mich.). Cultures of S. sanguis for use in transformation experiments were grown in Todd-Hewitt broth (Difco) containing 4% sheep blood (blood broth). Transformation medium (TM) consisted of 1% proteose peptone, 0.2% yeast extract, 0.2% D-glucose, pH 7.8, supplemented with 10% horse serum (heat inactivated for 30 min at 56°C). All other cell cultures were grown in brain heart infusion broth (BBL) containing 20 mM -glucose (BHI + glucose). Solid media contained 2% agar (Difco). All incubations were at 37°C. Labeling of DNA and preparation of cell lysates. DNA was labeled by growing cells for 16 to 20 h in 100 ml of BHI + glucose supplemented with 15 pCi of P3H]thymidine (60 to 65 Ci/mmol; Schwarz/Mann) per ml or 0.4 ,uCi of [14C]thymidine (50 to 55 mCi/ mmol; Schwarz/Mann) per ml. The growth medium also contained 20 mM i-Dthreonine to facilitate "spheroplast" formation (1). Labeled cells were harvested by centrifugation and washed twice with 20 ml of 0.01 M tris(hydroxymethyl)aminomethane (Tris)-hydrochloride, pH 8.2. The washed cells were suspended in 50 ml of 0.01 M Tris-hydrochloride, pH 8.2, and 50 ml of a 4-mg/ml solution of lysozyme (Sigma) in 0.01 M Tris-hydrochloride, pH 8.2, was added. After a 60-min incubation at 37°C, the spheroplasts were centrifuged at 8,000 rpm at 4°C in a Sorvall RC2B centrifuge. The pellet was resuspended in 8 ml of 0.01 M Tris-hydrochloride-0.01 M ethylenediaminetetraacetic acid (EDTA), pH 8.2 (TE). After the addition of 1 ml of a 10-mg/ml solution of Pronase (Calbiochem, nuclease free; selfdigested at 37°C for 2 h and heated at 80°C for 5 min) in TE, lysis was affected by 1% sodium dodecyl sulfate (SDS) at 37°C for 30 to 60 min. Lysis was always 95 to 100% for S. faecalis and varied between 70 and 97% for S. sanguis. Extraction of DNA. Plasmid DNA was separated from the bulk of chromosomal DNA by a procedure adapted from Currier and Nester (10). Lysates, prepared as described above, were passed through an 18-gauge needle six to seven times to shear the chromosomal DNA. While stirring slowly on a magnetic stirrer, 1 N NaOH was added until the pH of the lysate was raised to between 12.1 and 12.4. Stirring was continued for 10 min, at which time the pH was quickly lowered to 8.5 by the addition of 2 M Tris-hydrochloride, pH 7.0. Solid NaCl was added to a final concentration of 3%, followed by the addition of an equal volume of NaCl-saturated phenol. Stirring was continued for an additional 5 min, and then phase separation was affected by centrifugation for 15 min at maximum speed in an IEC model CL

J. BACTERIOL.

table-top centrifuge. At this stage only doublestranded DNA, which theoretically consists primarily of covalently closed circular (CCC) plasmid DNA, should be in the aqueous phase. The aqueous phase was further extracted with an equal volume of chloroform-isoamyl alcohol (24:1). After vigorous mixing for 1 min, the phases were again separated by centrifugation. The DNA in the aqueous phase (usually between 9 and 11 ml) was precipitated by the addition of 0.25 ml of 1 M MgCl2, 0.05 ml of 1 M sodium phosphate buffer, pH 6.8, and 7.5 ml of cold 99% ethyl alcohol. After storage at -20°C for 30 min, the precipitate was collected by low-speed centrifugation at 4°C and suspended in 0.1 M EDTA, pH 7.7. The DNA extract was dialyzed against TES buffer (TE + 0.05 M NaCl) for 16 to 20 h at 4°C, and CCC DNA was separated from any relaxed circular or linear plasmid molecules and contaminating chromosomal DNA by dye buoyant density gradient centrifugation. Sedimentation techniques. Velocity gradient analyses were performed in linear neutral sucrose gradients in a buffer consisting of 0.02 M Tris-hydrochloride, pH 7.2, 0.01 M EDTA, and 1.0 M NaCl. For analytical purposes, 0.2-ml samples were layered onto 4.8-ml, 5 to 20% linear gradients and centrifuged at 49,000 rpm for 75 min at 4°C in an SW50.1 rotor in a Beckman L2-65B preparative ultracentrifuge. Gradients were analyzed by collecting 5-drop fractions through the bottom of the tube, directly onto trichloroacetic acid-containing 12.5-cm Whatman GF/B grade filters (14, 20). For preparative neutral sucrose gradients, 1- to 2ml samples were layered onto 36-ml, 10 to 30% linear gradients. Centrifugation was at 20,000 rpm for 16 h at 5°C in a Beckman SW27 rotor. Fractions (25 drops) were collected into tubes, and portions were precipitated with trichloroacetic acid and counted to determine fractions to be pooled for further analysis. Equilibrium density gradient analyses were performed by adding 8.9 g of solid cesium chloride (Schwarz/Mann, optical grade) to 9 ml of sample in TES buffer. Ethidium bromide was added to a final concentration of 300 ,ug/ml, and the refractive index was adjusted to 1.3867 + 0.0004, if necessary. Gradients were formed by centrifugation at 35,000 rpm for 48 to 60 h at 20°C in a Beckman type 50 Ti fixedangle rotor. Fractionation, at 10 drops per fraction, was as for the preparative velocity gradients described above. 14C-labeled simian virus 40 DNA, used as an internal 21S marker in preparative velocity gradients, was prepared as previously described (18). The 14Clabeled 43S, , plasmid DNA, included in analytical gradients, was isolated on preparative neutral sucrose gradients as described below. Preparation of transforming DNA. The a and (8 plasmids from S. faecalis strain DS5 were separated on preparative neutral sucrose gradients using pooled satellite DNA fractions from a dye buoyant density gradient derived from 14C-labeled cell lysates. Fractions from the 28S (a) or 43S (,B) region of the sucrose gradient were pooled and dialyzed against TES buffer. Alternatively, total DS5 plasmid DNA from a dye buoyant density gradient was

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dialyzed as above. Transforming DNA was concentrated by alcohol precipitation and suspended in sterile 0.025 M sodium phosphate buffer, pH 7.5, at a concentration of 10 to 20 ,ug/ml. Transformation reactions. Competent Challis cultures were obtained by the method of Perry and Slade (27). A 10-fold dilution of an overnight blood broth culture of Challis was made in fresh blood broth. Portions (0.1 ml) of the diluted culture were added to 1.8 ml of TM and incubated at 37°C for the times indicated. At the appropriate time, 0.1 ml of transforming DNA was added to cultures in TM, and incubation was continued for 15 min. Control cultures to which sterile 0.025 M phosphate buffer was added in place of transforming DNA were included in all cases. After the 15-min incubation in the presence of DNA, 0.1 ml of a sterile 2-mg/ml solution of deoxyribonuclease (bovine pancreas DNase I, Calbiochem) in 0.02 M MgSO4 was added. After the addition of DNase, the cultures were allowed to incubate for an additional 2 h before plating. Dilutions were spread on BHI + glucose supplemented with the appropriate antibiotic for selection of transformants. Colony-forming units (CFU) per ml of culture were determined on BHI + glucose without antibiotics. All plates were incubated at 37°C for 48 to 72 h before counting. DNA-DNA hybridization. For determining the degree of homology between plasmid DNA from transformed strains and transforming DNA from strain D85, 3H-labeled DNA from the former was concentrated by alcohol precipitation. The precipitate was collected by centrifugation and suspended in 0.01x SSC (SSC is 0.15 M NaCl-0.015 M sodium citrate) and dialyzed against 0.01x SSC. The DNA was sheared by sonic treatment for 30 min at maximum power in a 10 Kc Raytheon sonic oscillator and denatured at 100°C for 3 min. This was reacted with low-specific-activity, 14C-labeled DNA immobilized on membrane filters (type HA; Millipore Corp.) (2 ,ug of '4C-labeled DNA per filter, in 50% formamide [Matheson, Coleman and Bell] and 0.3 M NaCl for 24 h at 37°C [19]). The filters were washed free of unbound DNA, dried, and counted. Controls containing 3H-labeled DNA and filters containing no bound DNA were included in all cases.

RESULTS of parent bacterial strains. Properties Members of three groups of streptococci, H, N and 0, have been shown to be transformable by chromosomal DNA extracted from homologous and heterologous groups and species of streptococci (11, 25, 26). The transformable group H strains appear to be the most versatile recipients, in that DNA extracted from groups A, C, D, G, and H have been successfully used in transformation reactions (25). Thus, the Challis strain of S. sanguis appeared to be a good candidate for establishing a streptococcal plasmid DNA transformation system. Preliminary experiments were carried out to determine if the Challis strain already har-

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bored any plasmids. Cell cultures were incubated for 16 h in the presence of [3H]thymidine. After cell lysis and extraction of DNA as described above, the DNA was banded in dye buoyant density gradients. Generally, only a single peak, corresponding to chromosomal DNA, was observed. Occasionally a second satellite peak appeared at a buoyant density of 1.5920 g/cm3 (Fig. 1A). When examined by velocity gradient techniques this satellite species invariably remained near the top of the gradient, with an apparent sedimentation rate of 6-12S (Fig. 2A). If the radiolabel from the peak fractions in the neutral sucrose gradients were rebanded in ethidium bromide-cesium chloride gradients, this material could no longer be seen at a buoyant density of 1.5920 g/cm3 but always sedimented at the same density as the chromosomal DNA (data not shown). These results suggested that the Challis strain contained no native plasmids or, if it did, they did not behave in a manner typical of plasmids described in other bacterial species.

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FIG. 1. Ethidium bromide-cesium chloride buoyant density gradient centrifugation of DNA from S. sanguis, strain Challis, and S. faecalis, strain DS5. DNA was extracted from cell lysates as described in Materials and Methods. The buoyant densities, cal-

culated from refractive index, of appropriate fractions are indicated by arrows. (A) Challis DNA; total 3H cpm in grad ient was 348,000. (B) DS5 DNA; total "4C cpm in gradient was 259,000.

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20 30 40 50 FRACTION NUMBER

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FIG. 2. Preparative neutral sucrose gradient analysis of Challis and DS5 DNA in pooled fractions from dye buoyant density gradients. Fractions indicated below were pooled from the gradients illustrated in Fig. 1. After extraction ofdye and dialysis to remove cesium chloride, the DNA in each pool was sedimented through preparative velocity gradients as described in Materials and Methods. '4C-labeled component I simian virus 40 DNA was included in all gradients as a 21S internal marker, indicated by the arrows. (A) Challis DNA (buoyant density, 1.5920 g/cm3) (fractions 25 to 29, Fig. 1A); total 3H

in gradient was 24,000. (B) DS5 plasmid DNA (fractions 17 to 22, Fig. IB); gradient contained 104,000 "4C cpm; (C) 1.6040 (fractions 18 to 22, Fig. 1A); total 3H cpm was 9,000. cpm

The choice of a plasmid to be used in developing a plasmid transformation system was based on two criteria: (i) that its physical properties in the native host were well characterized, and (ii) that it carried information for at least one easily selected phenotypic marker. S. faecalis, strain DS5, harbors three plasmids with molecular weights of 6 x 10", 17 x 10", and 34 x 106 (designated pAMa,, pAMP,B, and pAM-y, and abbreviated a, (3, and y, respectively) (6, 13). Two of these fulfill both of the above requirements. The a plasmid has been shown to code for resistance to the antibiotic tetracycline (Tc) (5). The resistance of the DS5 strain to erythromycin (Em) and lincomycin (Ln) has similarly

been shown to be dependent on the presence of the P plasmid (6). The DS5 strain of S. faecalis is resistant to greater than 1 mg of Em or Ln per ml and to more than 200 ,ug of Tc per ml. When the Challis strain was tested on these antibiotics it was found that 1 ,ug of Em or Tc per ml and 5 ,ug of Ln per ml completely inhibited the growth of this organism. Thus, these resistance markers could be readily selected for in Challis, and the a and (8 plasmids were chosen to test the transformability of this strain by plasmid DNA. In the process of isolating the DS5 plasmid DNA for transformation, it was observed that these CCC molecules banded at a buoyant density of 1.6040 g/cm3 in ethidium bromide-cesium chloride gradients (Fig. 1B). To insure that Challis DNA would not interfere with the isolation of the S. faecalis plasmids in this strain after transformation, the following experiment was performed. The plasmid DNA from strain DS5 (fractions 17 to 22, Fig. 1B) as well as radiolabel in the region of buoyant density 1.6040 g/cm3 from Challis DNA (fractions 18 to 22, Fig. 1A) were pooled, dialyzed after extraction of ethidium bromide with NaCl-saturated isopropyl alcohol, and sedimented in neutral sucrose gradients. The plasmid DNA from DS5 produced three major peaks, in neutral sucrose gradients, with sedimentation rates of 57S, 43S, and 28S relative to the 21S marker of simian virus 40 component I DNA (Fig. 2B). These peaks corresponded to the covalently closed species of the y, (3, and a plasmids, respectively (6). The pooled material from the Challis strain, however, sedimented well behind the 21S marker DNA (Fig. 2C). Transformation of S. sanguis with S. faecalis plasmid DNA. Experiments were performed in which total plasmid DNA from a dye buoyant density gradient of a DS5 extract (see Fig. 1B) or 28S (a plasmid) or 43S (p plasmid) DNA from a preparative neutral sucrose gradient (Fig. 2B) was added to competent Challis cultures as described above. After a 2-h incubation period (after the addition of DNase) to allow for phenotypic expression of transferred markers, dilutions of each culture were spread on agar plates containing (i) no antibiotics (to determine CFU/ml), (ii) 1 mg of Em per ml, (iii) 150 ,ug of Tc per ml, or (iv) both Em and Tc. When either total plasmid DNA or the 8 plasmid alone had been added to a competent Challis culture, Em resistance was transferred at a frequency of 10-6/CFU (total plasmid) or 2.3 x 10-7/CFU (,8 plasmid) (Table 1). No Tc-resistant colonies were obtained with any ofthe DNA preparations. Under no circumstances did 0.025

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TABLz 1. Transformation of S. sanguzis Challis by plasmid DNA from S. faecalise Donor DNA

Asetlbotic

CFU/ml

Transformation frequency

Total DS5 plasmid

None Em Tc Em + Tc

6.3 x 108 6.3 x 102

10-6

a plasmid

None Tc

7.5 x 108

p plasmid

None Em

5.7 x 108 1.3x 102

Antibiotic

2.3x 10-'

a CCC donor DNA was employed in all transformation reactions. Colonies were counted after an incubation period of 72 h at 37°C. Transforming DNA was added 2 h after transfer of cultures to TM.

M phosphate buffer controls (no DNA) produce colonies on plates containing either Em or Tc. The time required for development of maximum competence for transformation by the /3 plasmid was determined. ,B plasmid DNA (1 p.g/ml, final concentration) was added at 30min intervals after transfer of Challis cultures to TM, up to 180 min. Plates for the selection of Em-resistant transformants contained 150 ,ug of Em per ml. The remainder of the experiment was carried out as described above except for a zero time sample in which the DNase was added prior to the addition of /3 DNA. Em-resistant colonies began to appear at 60 min, and the cells remained competent up to 150 min of incubation in TM (Table 2). The maximum transformation frequency (1.8 x 10-5/CFU) was obtained at 90 min. This time sequence agrees completely with previous studies using a chromosomal marker (11). Additional experiments were performed in which: (i) high concentrations of a plasmid DNA were added to competent cultures, (ii) a plasmid was added to Challis cultures at different times of incubation in TM, (iii) up to 5 h was allowed for phenotypic expression of the Tc resistance gene(s) prior to selection, or (iv) agar plates containing varying concentrations of Tc (from 10 to 150 ug/ml) were used to select for transformants. Under none of these conditions could the Tc resistance phenotype be transferred to the Challis strain. Evidence that Em-resistant Challis transformants contain the p3 plasmid from strain DS5. As previously mentioned, the /8 plasmid from S. faecalis, strain DS5, imparts to its host cells resistance to both Em and Ln. When 10 Em-resistant Challis transformants were incubated in the presence of Ln, all were found to be

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resistant to at least 150 ug of this antibiotic per ml. The minimal inhibitory concentrations (MIC) for both of these antibiotics in strains Challis, DS5, and DL 101 (an Em-resistant transformant) are shown in Table 3. Whereas growth of S. sanguis, strain Challis, was totally inhibited by less than 1 ,g of Em per ml and less than 5 ,g of Ln per ml the MICs for these two antibiotics in strain DL101 were greater than 1 mg/ml and 250 ,ug/ml, respectively. Several Em-resistant transformants were examined for the presence of plasmid DNA. Dye buoyant density gradients of cell lysates from two of these isolates are illustrated in Fig. 3. Both of these transformants produced a new satellite peak indicative of CCC DNA. The buoyant density of the peak material was 1.6040 g/cm3, identical to plasmid DNA extracted from the donor DS5 strain (Fig. 1B). Final confirmation of the identity of the plasmid in strain DL101 with the / plasmid from DS5 was provided by DNA-DNA hybridization. 3H-labeled DNA from the plasmid band in a dye buoyant density gradient derived from a strain DL101 lysate was reacted with 14C-labeled / TABLE 2. Kinetics of competence development for transformation of strain Challis by , plasmid DNAa Time of incubation in Total CFU/ Transformants transforma- T lCma ml /ml tion medium (min) 0 107 0 30 6.0 x 107 0 60 7.4 x 107 1.3 x 102 90 9.7 x 107 1.7 x 103 120 2.2 x 108 1.7 x 102 1.1 x 108 2.1 x 102 150 0 180 9.0 x 107

Transformation frequency

1.8 1.8 7.8 1.9

x 10-6 x 10-5 x 10-7

x 10-6

a Transformation reactions were carried out as described in the text. Transforming DNA was added at the times indicated above.

TABLE 3. MICs of Em and Ln for strains Challis, DS5, and DL1OZa MIC Strain Em

Ln

1 mg/ml DS5 250 j,g/ml >1 mg/ml DL101 a A 0.25-ml portion of an overnight culture in BHI + glucose was transferred to 5 ml of fresh medium containing the appropriate antibiotic. Incubation was for 48 h at 37°C. Each antibiotic was tested at 1, 5, 10, 50, 100, 150, 250, 500, and 1,000 A.g/ml.

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TABLz 4. Hybridization of DNA from strains DS5, DL101, and Challis to (3 plasmid DNA' cpm hybrid- % Hybridiized to .8 zation cpm pm plasmid _____ 3H-labeled DNA added A

a

0.4 0

O

B

A

B

900 742 716 82 80 DL101 plasmid 1,300 777 654 59 50 32 3 2.5 28 1,100 Challis a Hybridizations were carried out as described in Materials and Methods. The aqueous phase from the chloroform-isoamyl alcohol extraction step described in Materials and Methods was used as the source of Challis DNA. DL101 plasmid DNA was obtained from the satellite band in an ethidium bromide-cesium chloride gradient. 3H-labeled and 14C-labeled ,B plasmid was isolated as described in Materials and Methods. Each filter contained 2 ,ug of 14C-labeled (3 plasmid DNA. A 0.05-jAg portion of 3H-labeled DNA was used for each hybridization reaction.

DS5,8 plasmid

10

B

1.664.

5 6 4

2

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25

30

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35

40

45 T.OP

FIG. 3. Dye buoyant density gradient analysis of DNA from transformed Challis strains. DNA extraction and determination of buoyant density were as described in the legend to Fig. 1. (A) 335,000 3H cpm from strain DL1 01; (B) 330,000 3H cpm from strain DL102.

DNA from DS5 immobilized on nitrocellulose filters. As controls, 3H-labeled Challis DNA and 3H-labeled DS5,8 plasmid DNA were also reacted with immobilized 8 plasmid DNA. Duplicate samples of DL101 plasmid hybridized to the (3 plasmid to the extent of 59 and 50%, respectively (Table 4). When normalized to the 82 and 80% hybridization in the homologous reactions, then approximaely 70% of the DL101 DNA banding at a buoyant density of 1.6040 gl cm3 was homologous to the (8 plasmid. The degree of hybridization between the DNA from strain Challis and 8 plasmid was essentially negligible. Further characterization of plasmid DNA from Em-resistant Challis transformants. Fractions comprising the satellite peaks in Fig. 3 were pooled, and the ethidium bromide was removed by extraction with isopropyl alcohol. After dialysis to remove cesium chloride, the pooled DNA was sedimented through preparative neutral sucrose gradients (Fig. 4). The satellite DNA from the transformant DL101 sedimented as one major peak at approximately 41S and four minor peaks at 52S, 29S, 24S, and one near the top of the gradient (Fig. 4A). In

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FIG. 4. Preparative neutral sucrose gradient analysis of plasmid DNA from transformed Challis strains. Fractions comprising the satellite peaks in Fig. 3 were pooled and analyzed in velocity gradients as in Fig. 2. (A) DL11 plasmid (fractions 18 to 23, Fig. 3A); 7,000 3H cpm in gradient. (B) DL102 plasmid (fractions 17 to 21, Fig. 3B); 18,000 3H cpm in gradient.

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contrast, the DNA from strain DL102 sedimented primarily as a single large peak at 26S (Fig. 4B). The above results suggested that strains DL101 and DL102 may contain plasmids of different sizes. However, when the major species of DNA from each strain (41S from DL101 and 26S from DL102) were sedimented in analytical neutral sucrose gradients, together with 14Clabeled 43S DNA from strain DS5, an interesting difference was clear (Fig. 5). The 26S DL102 DNA again sedimented at the same rate (Fig. 5A). However, approximately 60% of the original 41S species from strain DL101 continued to sediment at the same rate (41S), but the remaining radiolabel was located in the same area of the gradient as the material from strain DL102 (Fig. 5B). Perhaps even more dramatic was the sedimentation of the original 41S DL101 DNA after storage at 4°C for 1 week. In 30

30

20

20

1C

I

I

I0

300.

I

20',

I'

0 -10

0

10~

Inn

I

ro 0 U.

10

30

438

C

0

5

zI

-

-I 5

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20

I

I

15

20

D

30

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4

P

FIG. 5. Velocity gradient analysis of major plasmid species from transformed Challis strains. Fractions from the major peaks in the gradients illustrated in Fig. 4 were pooled, dialyzed, and examined in analytical neutral sucrose gradients as described in Materials and Methods. "4C-labeled, 43S, 18 plasmid DNA (0) (1,000 cpm pergradient) was included as an internal marker in all cases. (A) DL102 plasmid (fractions 42 to 48, Fig. 4B); 1,600 total 3H cpm in gradient. (B) DL10Z plasmid (fractions 30 to 37,

Fig. 4A); 1,100 total 3H cpm in gradient. (C) Same as (B) but after storage of dialyzed pool for 1 week at 40C.

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this case (Fig. WC) the 41S species was almost quantitatively converted to the 26S species. These results were somewhat variable from experiment to experiment in that satellite DNA from DL102 occasionally sedimented as a 418 species, whereas that from DL101 sometimes sedimented at 26S. However, the conversion of 41S to 26S and the stability of the 26S species are reproducible properties of plasmid DNA isolated from Em-resistant Challis transformants. A CCC DNA molecule with a sedimentation rate of 41S in velocity gradients corresponds to a molecular weight of approximately 15.5 x 106. This would represent a deletion from the transforming ,8 plasmid of approximately 1.5 x 106. A plasmid with a molecular weight of 15.5 x 106

would be expected to sediment at 29S in the relaxed circular configuration and at 26S when present as a double-stranded linear molecule. Thus, it would appear that the 418 plasmid in transformed Challis strains is readily converted to a linear double-stranded molecule when extracted from its host cell. The above results suggest that the plasmid from S. faecalis, strain DS5, is stably maintained by S. sanguis, strain Challis, after transformation. Although this plasmid may exist as a CCC duplex in vivo, it does not appear to remain in this configuration after isolation of the DNA.

DISCUSSION The results presented in this communication clearly establish the transformability of S. sanguis, strain Challis, by plasmid DNA from S. faecalis. The transformation does not appear to be dependent on the integration of the transforming DNA into the Challis chromosome but rather on the ability of the transformant to support the autonomous replication of the foreign plasmid. The transferred plasmid can be separated from the host chromosome by dye buoyant density gradient centrifugation, indicating that it is present as a CCC duplex molecule (28). Although the transferred plasmid can be isolated from its new host cell as a CCC, it is rapidly converted to a linear molecule. The instability of the circular DNA is observed even after extensive purification through equilibrium and velocity gradients. In contrast to this behavior, the plasmid remains in the CCC state for several weeks after its isolation from the parent S. faecalis strain. The reason for the instability of CCC DNA isolated from S. sanguis is not known. It seems unlikely that a protein-relaxation complex,

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such as that described for the Col E1 plasmid (4) is involved, since the plasmid isolation procedure employed in this study includes lysis with SDS in the presence of Pronase. Both of these compounds preclude the isolation of the Col E1 relaxation complex (4). Secondly, whereas the Col E1 CCC DNA is converted to a relaxed circle in the presence of the relaxation protein (4), the CCC DNA isolated from transformed strains of S. sanguis appears to be directly converted to the linear form. The occasional appearance of satellite DNA in dye buoyant density gradients from cell lysates of the parent Challis strain suggests that this strain may indeed harbor a plasmid(s). However, it has not been possible to confirm the CCC configuration of this DNA by rebanding in cesium chloride containing ethidium bromide or by alkaline velocity gradient analyses. Similar results have also been obtained with certain strains of Streptococcus mutans (B. M. Chassy, personal communication). Other streptococcal plasmids carrying antibiotic resistance and metabolic markers are currently being tested in the S. sanguis transformation system. The purpose of these studies is twofold: (i) to establish the general applicability of the transformation system to streptococcal plasmids, and (ii) to determine if the instability of CCC DNA isolated from S. sanguis can be extended to plasmids derived from other streptococcal species. The inability to select Tc-resistant transformants in experiments employing the a plasmid from strain DS5 as a donor DNA cannot as yet be explained. It may be that a requires a host replicative function supplied by S. faecalis but not present in S. sanguis. It is also possible that the Tc-resistant phenotype coded by a is not expressed in the Challis strain. Experiments designed to differentiate between these two possibilities are currently in progress. LITERATURE CITED 1. Chassy, B. M. 1976. A gentle method for the lysis of oral streptococci. Biochem. Biophys. Res. Commun. 68:603-608. 2. Cleary, P. P., Z. Johnson, and L. Wannamaker. 1975. Genetic instability of M. protein and serum opacity factor of group A streptococci: evidence suggesting extrachromosomal control. Infect. Immun. 12:109118. 3. Clewell, D. B., and A. E. Franke. 1974. Characterization of a plasmid determining resistance to erythromycin, lincomycin, and vernamycin B, in a strain of Streptococcus pyogenes. Antimicrob. Agents Chemother. 5:534-537. 4. 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.

J. BACTERIOL. 5. Clewell, D. B., Y. Yagi, and B. Bauer. 1975. Plasmiddetermined tetracycline resistance in Streptococcus faecalis: evidence for gene amplification during growth in the presence of tetracycline. Proc. Natl. Acad. Sci. U.S.A. 72:1720-1724. 6. 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. 7. Clowes, R. C. 1972. Molecular structure of bacterial plasmids. Bacteriol. Rev. 33:361-405. 8. Cords, B. R., L. L. McKay, and P. Guerry. 1974. Extrachromosomal elements in group N streptococci. J. Bacteriol. 117:1149-1152. 9. Courvalin, P. M., C. Carlier, and Y. A. Chabbert. 1972. Plasmid-linked tetracycline and erythromycin resistance in group D ((Streptococcus)) Ann. Inst. Pasteur Paris 123:755-759. 10. Currier, T., and E. W. Nester. 1976. Evidence for diverse types of large plasmids in tumor-inducing strains of Agrobacterium. J. Bacteriol. 126:157-165. 11. Davidson, J. R., Jr., W. T. Blevins, and T. W. Feary. 1976. Interspecies transformation of streptomycin resistance in oral streptococci. Antimicrob. Agents Chemother. 9:145-150. 12. Dunny, G. M., N. Birch, G. Hascall, and D. B. Clewell. 1973. Isolation and characterization of plasmid deoxyribonucleic acid from Streptococcus mutans. J. Bacteriol. 114:1362-1364. 13. 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. 14. Furano, A. V. 1971. A very rapid method for washing large numbers of precipitates of proteins and nucleic acids. Anal. Biochem. 43:639-640. 15. Higuchi, M., S. Araya, and M. Higuchi. 1976. Plasmid DNA satellite bands seen in lysates of Streptococcus mutans that form insoluble extracellular polysaccharides. J. Dent. Res. 55:266-271. 16. Jacob, A. E., G. J. Douglas, and S. J. Hobbs. 1975. Self-

transferable plasmids determining the hemolysin and bacteriocin of Streptococcus faecalis var. zymogenes. J. Bacteriol. 121:863-872. 17. 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. 18. Lavi, S., S. Rozenblatt, M. F. Singer, and E. Winocour. 1973. Acquisition of sequences homologous to host DNA by closed circular simian virus 40 DNA. II. Further studies on the serial passage of virus clones. J. Virol. 12:492-500. 19. Lavi, S., and E. Winocour. 1972. Acquisition of sequences homologous to host deoxyribonucleic acid by closed circular simian virus 40 deoxyribonucleic acid. J. Virol. 9:309-316. 20. LeBlanc, D. J., and M. F. Singer. 1974. Localization of replicating DNA of simian virus 40 in monkey kidney cells. Proc. Natl. Acad. Sci. U.S.A. 71:2236-2240. 21. McKay, L. L., and K. A. Baldwin. 1974. Simultaneous loss of proteinase- and lactose-utilizing enzyme activities in Streptococcus lactis and reversal of loss by transduction. Appl. Microbiol. 28:342-346. 22. McKay, L. L., and K. A. Baldwin. 1975. Plasmid distribution and evidence for a proteinase plasmid in Streptococcus lactis C2. Appl. Microbiol. 29:546-548. 23. Molskness, T. A., W. E. Sandine, and L. R. Brown. 1974. Characterization of lac+ transductants of Streptococcus lactis. Appl. Microbiol. 28:753-758. 24. Nakae, M., M. Inoue, and S. Mitsuhashi. 1975. Artifi-

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cial elimination of drug resistance from group A betahemolytic streptococci. Antimicrob. Agents Chemother. 7:719-720. 25. Perry, D., and H. D. Slade. 1962. Transformation of streptococci to streptomycin resistance. J. Bacteriol. 83:443-449. 26. Perry, D., and H. D. Slade. 1964. Intraspecific and interspecific transformation in streptococci. J. Bacteriol. 88:595-601. 27. Perry, D., and H. D. Slade. 1966. Effect of filtrates from transformable and nontransformable streptococci on the transformation of streptococci. J. Bacteriol.

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91:2216-2222. 28. Radloff, R., W. Bauer, and J. Vinograd. 1967. A dyebuoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Natl. Acad. Sci. U.S.A. 57:1514-1521. 29. Yagi, Y., A. E. Franke, and D. B. Clewell. 1975. Plasmid-determined resistance to erythromycin: comparison of strains of Streptococcus faecalis and Streptococcus pyogenes with regard to plasmid homology and resistance inducibility. Antimicrob. Agents Chemother. 7:871-873.