APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1984, p. 342-3460099-2240/84/080342-05$02.00/0Copyright 33 1984, American Society for Microbiology

Transformation of Streptococcus sanguis Challis with Streptococcuslactis Plasmid DNAt

SUSAN K. HARLANDER AND LARRY L. McKAY*

Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota 55108

Received 13 December 1983/Accepted 15 May 1984

Streptococcus lactis plasmid DNA, which is required for the fermentation of lactose (plasmid pLM2001), anda potential streptococcal cloning vector plasmid (pDB101) which confers resistance to erythromycin wereevaluated by transformation into Streptococcus sanguis Challis. Plasmid pLM2001 transformed lactose-negative (Lac-) mutants of S. sanguis with high efficiency and was capable of conferring lactose-metabolizingability to a mutant deficient in Enzyme lIlac, Factor IlIlac, and phospho-Il-galactosidase of the lactosephosphoenolpyruvate-phosphotransferase system. Plasmid pDB1I1 was capable of high-efficiency transforma-tion of S. sanguis to antibiotic resistance, and the plasmid could be readily isolated from transformed strains.However, when 20 pLM2001 Lac' transformants were analyzed by a variety of techniques for the presence ofplasmids, none could be detected. In addition, attempts to cure the Lac' transformants by treatment withacriflavin were unsuccessful. Polyacrylamide gel electrophoresis was used to demonstrate that the transform-ants had acquired a phospho-Io-galactosidase characteristic of that normally produced by S. lactis and not S.sanguis. It is proposed that the genes required for lactose fermentation may have become stabilized in thetransformants due to their integration into the host chromosome. The efficient transformation into andexpression of pLM2001 and pDBlO1 genes in S. sanguis provides a model system which could allow thedevelopment of a system for cloning genes from dairy starter cultures into S. sanguis to examine factorsaffecting their expression and regulation.

The lactic streptococci utilize lactose via the phosphoenol-pyruvate-dependent phosphotransferase system (21). Themetabolic pathway for lactose utilization in the lactic strep-tococci, which is similar to that first observed in Staphylo-coccus aureus (25), involves the lactose-specific compo-nents Factor IlIac and Enzyme Iliac, which are involved inphosphorylating lactose during transport, and the enzymephospho-p-galactosidase (P-3-gal), which cleaves the phos-phorylated lactose to glucose and galactose 6-phosphate.The ability of lactic streptococci to metabolize lactose is

not a stable characteristic, because the genes responsible forlactose metabolism are associated with plasmid DNA (21).Lactose-fermenting ability has been associated with thepresence of a distinct plasmid in several strains of Strepto-coccus lactis and Streptococcus cremoris Bi (21). Loss ofplasmid DNA after treatment with various mutagens hasbeen associated with the simultaneous loss of Enzyme Illac,Factor IIlac, and P-n-gal in Lac- derivatives of S. lactis C2(21).

Since the lactic streptococci are industrially importantmicroorganisms by virtue of their ability to metabolizelactose to lactic acid, it would be advantageous if lactosefermentation could be made a stable characteristic. Theavailability of a plasmid DNA transformation system mighteventually allow the utilization of recombinant DNA tech-niques to achieve this goal. A first step in such studies wouldbe to determine whether lactose-metabolizing genes from S.lactis could be transformed into and expressed in anotherbacterium in which appropriate cloning vectors could beutilized. Competent strains of Streptococcuis sanguiis Challis

* Corresponding author.t Paper no. 13,728 of the Scientific Journal Series of the Minneso-

ta Agricultural Experiment Station. The research was conducted inpart under Minnesota Agricultural Experiment Station Project no.18-62.

have been established as versatile recipients of streptococcalplasmid DNA from groups A, B, D, and N (5, 16, 27). Thiscommunication reports the transformation and expression ofboth an antibiotic resistance gene from a streptococcalcloning vector and the lactose-metabolizing genes from S.lactis in S. sanguis Challis.

MATERIALS AND METHODSBacteria and media. Streptococcal strains used in this

investigation are summarized in Table 1. S. lactis LM0232,C2, and KB21 from our stock clilture collection were main-tained through biweekly transfer at 30°C in M17 broth (28)containing 0.5% lactose. S. lactis LM0232 contains a 22-megadalton plasmid which mediates lactose metabolism inthis organism (14). In S. lactis KB21 the lactose-metaboliz-ing (lac) genes have become integrated into the chromosome(22).The lactose-metabolizing (Lac') parental strain of S.

sanguis Challis 7868 and the lactose-negative (Lac-) deriva-tives designated lac8 and 1ac83 were kindly provided byEdward St. Martin, National Institute of Dental Research,Bethesda, Md. The lac8 strain is defective in the enzyme P-3-gal, and lac83 is defective in P-,B-gal, Enzyme lIlac, and

Factor Ililac of the phosphoenolpyruvate-dependent phos-photransferase system (27). These strains were maintainedby passage in brain heart infusion (BHI; Difco Laboratories,Detroit, Mich.) broth.

S. sanguis SM101, which carries the macrolides-lincomy-cin-streptogramin B resistance plasmid, pDB1l1 (5), waskindly provided by Joseph Ferretti, University of Oklahoma,and was maintained in BHI broth containing 10 ,ug oferythromycin per ml.Ten S. sanguis lac8 Lac' strains designated SH10 to

SH19 and 10 S. sanguis lac83 Lac' strains designated SH20to SH29 were obtained by transformation of plasmidpLM2001 from S. lactis LM0232 into the Lac- Challisstrains. These strains were maintained in M17 lactose broth.

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S. SANGUIS TRANSFORMATION WITH S. LACTIS PLASMID DNA 343

TABLE 1. Bacterial strains used in this investigation

Strain Plasmid Phenotype" Source or reference

S. lactis pLM2001 Lac' 14LM0232

S. Iactis C2 6b Lac' 18S. lactis KB21 5b Lac' 22S. sanguis pDB1l1 MLS 5

ChallisSMio1

S. sanguis None Lac' 27Challis 7868

S. sanguis None Lac- 27Challis lac8

S. sanguis None Lac- 27Challis lac83

S. sanguis None Lac' This publicationChallis lac8SH10-19

S. sanguis None Lac' This publicationChallis lac83SH20-29

a Lac' indicates strain is capable of fermenting lactose; Lac- indicatesstrain is unable to ferment lactose; MLS, macrolides-lincomycin-streptogra-min B.

b S. lactis C2 and KB21 contain six and five plasmid species, respectively.

Isolation and purification of plasmid DNA. The procedureof Hansen and Olsen (12) as modified by Walsh and McKay(29) was used for the preparation of cleared lysates from S.lactis LM0232. Lysis of S. sanguis Challis SM101 wasfacilitated by growing this strain in the presence of DL-threonine (7). Large quantities of plasmid DNA were ob-tained by a combination of the cleared lysate and phenolextraction procedures outlined by Behnke and Ferretti (5).Plasmid DNA from both strains was purified and concentrat-ed by dye-buoyant density gradient centrifugation (20).

Transformation. Competent cultures of S. sanguis Challislac8 and lac83 were obtained by daily subculturing in BHIbroth containing 1% horse serum (24). Purified pDB101 andpLM2001 plasmid DNAs were used to transform Challisstrains by the procedure of Behnke (4). Competence factor,prepared from S. sanguis Challis by the method of Gaustad(10), was added 30 min before the addition of transformingDNA. Dilutions of the culture were plated in soft agaroverlays on various nutrient agar plates. Lac' clones weredetected on either a defined medium (RPMI 1640; KCBiological, Inc., Lenexa, Kans.) containing 0.4% lactose asthe sole carbon source or M17 agar plates containing 0.4%lactose and bromcresol purple as a pH indicator. Eryrtransformants were selected on BHI agar plates containing10 ,ug of erythromycin per ml. Plates were incubated anaero-bically (GasPak; BBL Microbiology Systems, Cockeysville,Md.) at 37°C for 4 to 5 days.

Acriflavin treatment of Lac' transformants. S. lactisstrains LM0232, C2, and KB21 and the 20 S. sanguis Challistransformants (10 Lac' S. sangiuis lac8 transformants and 10Lac' S. sanguis lac83 transformants) were subcultured dailyin BHI broth containing 5 and 7.5 p.g of acriflavin (AF) perml for the S. lactis and S. sanguis strains, respectively. Aftertransfers 5 and 10, samples were plated onto M17 agar platescontaining 0.4% lactose and bromcresol purple and scoredfor the presence of Lac- derivatives (white colonies).

Rapid screening for plasmid DNA. Rapid screening oftransformant clones for the presence of plasmid DNA was

performed by the procedures of LeBlanc and Lee (17),Burdett (6), Behnke and Ferretti (5), Walsh and McKay (29),or Anderson and McKay (3). Attempts to isolate DNA fromselected transformant clones in preparative amounts wasalso performed via the above procedures, followed by dye-buoyant density gradient centrifugation to demonstrate thepresence of plasmid DNA.

Agarose gel electrophoresis. Agarose gel electrophoresiswas performed as outlined by Maniatis et al. (20). Gels werestained with ethidium bromide, photographed, and returnedto the apparatus; electrophoresis was continued in thepresence of the dye by the procedure of Franke and Clewell(9). The resident plasmids in Escherichia coli V517 wereused as mobility reference standards (19).

Preparation of cell extracts and polyacrylamide gel electro-phoresis. S. sanguis Challis strains 7868, lac83, and SH10and S. lactis LM0232 were grown for 18 h in 400 ml ofmodified lactobacillus carrier medium (30) containing either10 mM lactose or 10 mM glucose. Cell extracts wereprepared (18) and examined by polyacrylamide gel electro-phoresis under nondenaturing conditions by the proceduresoutlined by Anderson and Peterson (1). Polyacrylamide gelconcentrations in the running and stacking gels were 16 and3%, respectively. Electrophoresis was performed at a con-stant current (40 mA) at ambient temperature for 4 to 5 h.Immediately after electrophoresis the gel was soaked in asolution of 1 mM o-nitrophenyl-p-D-galactopyranoside-6-phosphate in 200 mM Tris (pH 6.5) for 15 min at 37°C. Thegel was photographed at 5-min intervals for 30 min with aPolaroid MP-4 copy camera (Kodak Contrast Process PanFilm) with a fluorescent light box and no. 47 blue filter.

RESULTSTransformation of S. sanguis with pDBlOl and pLM2001

plasmid DNA. Plasmid pDB101, a potential vector for molec-ular cloning in streptococci, was transformed into S. sanguislac8 and lac83 at frequencies of 1 x 10-3 and 7.6 x 10-4,respectively, making recipient cells resistant to erythromy-cin. The S. sanguis Challis lac8 and lac83 strains wereshown not to harbor any native plasmids, and plasmid DNAcould be readily isolated from the Eryr transformants.

Table 2 demonstrates that pLM2001 plasmid DNA from S.lactis was able to transform both Lac- Challis strains to aLac' phenotype, indicating that the lac genes could beexpressed in S. sanguis. Transformation frequencies variedfrom ca. 4-fold in experiments with lac83 to greater than 35-fold in experiments with lac8. Similar variability has been

TABLE 2. Transformation of S. sanguis Challis with S. lactispLM2001 plasmid DNA

CFU/ml in:Recipient strain

Controls Transformants

S. sanguis Iac8a 1.4 x 108 2.0 x 104S. sanguis lac8a 6.0 x 107 3.1 x 105S. sanguis lac8b 1.6 x 109 6.2 x 107S. sanguis lac83a 1.1 x 108 2.6 x 103S. sanguis lac83a 7.2 x 107 6.5 x 103S. sanguis lac83b 3.7 x 108 4.1 x 104

a 5. sanguis Iac8 and lac83 control cells were enumerated on RPMI 1640agar containing 0.4% glucose; transformants were enumerated on RPMI 1640agar containing 0.4% lactose.

b S. sanguis lac8 and lac83 control cells and transformants were enumerat-ed on M17 agar containing 0.4% glucose and lactose, respectively.

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344 HARLANDER AND McKAY

observed in other laboratories (J. Ferretti and E. St. Martin,personal communication) and may be due to variations inmedium components or temperature of incubation, whichmay alter the time of optimum competence. The frequencyof transformation was consistently at least 10-fold higher inlac8 than in lac83 or when M17 was used as the basal platingmedium. No Eryr or Lac+ transformants were obtained onthe appropriate selective media if Challis recipients wereassayed in the absence of plasmid DNA or if the DNA waspretreated with DNase I before incubation with the bacteria(data not shown).

Analysis of Lac+ transformants for plasmid DNA. TwentyLac+ transformants (10 lac8 and 10 1ac83) and S. sanguisChallis SM101 were subjected to five different small-volume(10 ml) DNA rapid screening procedures to analyze for thepresence of plasmid DNA. No plasmid DNA bands wereever detected in the gels under conditions in which thepresence of pDB101 was consistently demonstrated. PlasmidDNA was not masked by chromosomal DNA in the gel, asplasmid bands were not visible even after continued electro-phoresis of stained gels in the presence of ethidium bromide(data not shown).

Since the plasmids under study might be present at lowcopy number, four transformants of lac8 and lac83 weresubjected to large-volume (400- to 800-ml) plasmid isolationprocedures, followed by dye-buoyant density gradient cen-trifugation. A satellite band in which covalently closedcircular plasmid DNA normally appears was not detectablein any of the gradient tubes. Furthermore, if the appropriatearea of the gradient was removed, dialyzed, and concentrat-ed, plasmid DNA could not be detected by agarose gelelectrophoresis.

Effect of AF treatment on Lac+ transformants. McKay etal. (23) have shown that treatment of lactic streptococci withAF increased the frequency of Lac- variants, presumablyby selective interference with plasmid DNA replication (13).Table 3 shows the effect of successive transfers in the

TABLE 3. Effect of successive transfers in the presence of AFon lactose utilization by Lac' transformants

BacterialNo. of Total no. No. ofBacterial No. of CFU/ml of colonies confirmed % Lac-strain' transfers eaie aexamined Lac-S. lactis 5 1.2 x 109 289 87 30.1C2 10 1.5 x 109 312 197 63.1

S. lactis 5 8.9 x 108 210 42 20.0LM0232 10 1.2 x 109 301 183 60.8

S. lactis 5 1.6 x 109 443 0 0KB21 10 9.8 x 108 459 0 0

S. sanguis 5 1.5 x 109 325 0 0SH10 10 1.0 x 109 228 1 0.4

S. sanguis 5 1.6 x 109 351 1 0.3SH11 10 1.7 x 109 396 1 0.2

S. sanguis 5 5.1 x 108 219 0 0SH20 10 4.8 x 108 321 1 0.3

S. sanguis 5 1.1 x 109 236 0 0SH21 10 8.3 x 108 291 2 0.7a Both the S. sanguis lac8 (SH1O and SH11) and the lac83 (SH20 and SH21)

transformants represent 2 of 10 strains tested. Similar data were obtained withall of the isolates.

A BC D

FIG. 1. Polyacrylamide gel electrophoresis of P-3-gal activity incell extracts of (A) S. sanguis lac83, (B) S. sanguis SH20, (C) S.sanguis 7868, and (D) S. lactis LM0232. Enzyme activity wasdetected as described in the text.

presence of AF on the Lac' transformants. The resultsindicate that under conditions which cause greater than 60%of the cells to become Lac- in control cultures (S. lactis C2and LM0232), the appearance of Lac- variants in all 20 ofthe Lac' S. sanguis transformants was less than 1%. Thisdata is similar to that obtained for S. lactis KB21, in whichthe lac genes have been stabilized by integration into thechromosome (22).

Polyacrylamide gel electrophoresis of cell extracts. St. Mar-tin et al. (27) have demonstrated that the molecular weightsof the P-a-gal from S. sanguis and S. lactis 11454 are 52,000and 40,000, respectively. To demonstrate the acquisition ofthe structural gene for the S. lactis P-3-gal in the Lac' S.sanguis transformants, cell extracts were prepared from S.sanguis 7868, lac83, SH20, and S. lactis LM0232. As demon-strated in Fig. 1, the Lac' S. sanguis transformant (lane B)had clearly acquired the capacity to produce P-,-gal similarto that normally produced by S. lactis LM0232 (lane D),which served as the source of transforming DNA.

DISCUSSIONThe results presented in this communication establish the

transformability of Lac- S. sanguis Challis strains by plas-mid DNA from both S. sanguis SM101 and S. lactis LM0232(Table 2). Plasmid pDB1l1 is capable of autonomous replica-tion in the recipient strains and could be readily isolatedfrom Eryr transformants. The pLM2001 plasmid from S.lactis LM0232, on the other hand, could not be isolated fromtransformant clones by any of the various procedures at-tempted. In addition, when Lac' transformants were suc-cessively cultured under conditions which normally causecuring of S. lactis plasmid DNA, the Lac' phenotype wasmaintained even after 10 consecutive transfers in the pres-ence of AF (Table 3).

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S. SANGUIS TRANSFORMATION WITH S. LACTIS PLASMID DNA

It is known that acridine dyes are capable of eliminatingplasmids from carrier strains of bacteria (13) and that theyhave previously been used to increase the occurrence ofLac- isolates of both S. lactis (15, 23) and S. cremoris Bi (2)due to plasmid curing. Interestingly, certain strains of S.cremoris contain plasmids which code for the lac genes, yetthese plasmids resist curing with AF. However, if thelactose-fermenting ability was transferred from S. cremorisC3 to S. lactis via conjugation and Lac' transconjugantscultured in the presence of AF, Lac- isolates could berecovered at a frequency comparable to that obtained withcuring of S. lactis strains. It was suggested that the stabilityof lactose metabolism to acridine dyes in S. cremoris may bethe result of decreased permeability of S. cremoris to thesedyes compared with S. lactis (26). It may be that S. sanguislac8 and lac83 have low permeability to AF; thus, lactosemetabolism would be a stable trait in these strains via amechanism similar to that seen in S. cremoris.However, the failure to isolate plasmid DNA from the S.

sanguis Lac' transformants indicates that the mechanism ofresistance to AF curing in the lac8 and lac83 transformants ismore complex than a reduced permeability to AF. Klaen-hammer et al. (14) have demonstrated that the lactoseplasmid from S. lactis C2 was selectively lost during DNAisolation procedures which involved lysozyme treatments ofgreater than 20 min. The authors suggested that the labilityof the Lac plasmid is due to endogenous nuclease activityduring the lysozyme treatment period. Since several of theprocedures (5, 6, 17) which were used to isolate plasmidDNA from the Lac' S. sanguis transformants involvedlysozyme treatments of at least 20 min, additional DNAisolation procedures applicable to dairy streptococci (3, 29)which involve shorter incubation times in lysozyme wereattempted. In addition, the nuclease inhibitor diethylpyro-carbonate was added before cell lysis when the method ofBehnke and Ferretti (5) was used, and lysozyme treatmentwas reduced to 15 min. Plasmid DNA was not detectable inthe Lac' transformants by any of the above procedures.Since these techniques have been used to isolate streptococ-cal plasmids with a wide range of physical characteristicsfrom numerous strains, it can be proposed that the plasmidstransformed into the Lac- S. sanguis strains are no longerpresent as autonomously replicating elements.

St. Martin et al. (27) have observed that when unfraction-ated plasmid DNA from S. lactis 11454, which contains sixplasmids, including a 32-megadalton lactose plasmid, wastransformed into S. sanguis Challis lac83, no plasmid DNAcould be detected in the transformants and the lactose traitwas stably maintained. These investigators suggested thatthe lac genes from S. lactis may have become integrated intothe S. sanguis chromosome. Inability to recover plasmidDNA in transconjugants of strains of S. cremoris has beenpreviously reported by Snook and McKay (26). In addition,Gibson et al. (11) have observed the transfer of erythromycinand lincomycin resistance coded for by the pAMP1 plasmidfrom Streptococcus faecalis DR1501(,3) into Lactobacilluscasei DR1003, yet no plasmid DNA could be isolated fromlysates of the transconjugants.

In another gram-positive organism, Bacillus subtilis, for-eign DNA which has been cloned into a vector plasmid maybecome excised and incorporated into the chromosome if theforeign DNA possesses a region which is homologous withthe chromosome. It is also possible to favor integration byusing vectors which cannot replicate in B. subtilis (8). Itseems reasonable to propose that a region of homologybetween the pLM2001 lactose plasmid and the S. sanguis

chromosome could exist. In addition, the pLM2001 plasmidmay be incapable of replication in S. sanguis, and due to thestrong selective pressure used to screen Lac' transform-mants, integration into the host chromosome may be fa-vored. Although the data suggest integration of the lac genesinto the chromosome, the fate of these genes must beverified by direct hybridization of 32P-labeled probes con-taining the lac genes from pLM2001 with digests of chromo-somal DNA from the Lac+ transformants.The P-n-gal activity in the Lac+ transformants of S.

sanguis was the same as in the donor strain and could beclearly distinguished from the P-p-gal activity normallyfound in S. sanguis. The conversion of S. sanguis Challislac83 to a Lac' phenotype indicates that the structural genecoding for P-4-gal and presumably those coding for EnzymeIIlac and Factor Illiac have been transferred from S. lactis toS. sanguis.The demonstration of an efficient transformation system

and expression of metabolically important genes for dairyfermentations in S. sanguis Challis may allow the develop-.ment of a system for cloning genes from lactic acid bacteriain S. sanguis. This would allow examination of the expres-sion and regulation of genes important in dairy and foodfermentation processes.

ACKNOWLEDGMENTSWe thank Joseph Ferretti for many helpful discussions during this

project.This research was supported in part by Dairy Research Incorpo-

rated, Rosemont, Ill.

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3. Anderson, D. G., and L. L. McKay. 1983. Simple and rapidmethod for isolating large plasmid DNA from lactic streptococ-ci. Appl. Environ. Microbiol. 46:549-552.

4. Behnke, D. 1981. Plasmid transformation of Streptococcussanguis (Challis) occurs by circular and linear molecules. Mol.Gen. Genet. 182:490-497.

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