(+)-Lactate Dehydrogenase of Streptococcus mutans - Infection and ...

Vol. 59, No. 11

INFECTION AND IMMUNITY, Nov. 1991, p. 3930-3934 0019-9567/91/113930-05$02.00/0 Copyright C) 1991, American Society for Microbiology

DNA Sequence and In Vitro Mutagenesis of the Gene Encoding the Fructose-1,6-Diphosphate-Dependent L-(+)-Lactate Dehydrogenase of Streptococcus mutans M. J. DUNCAN* AND J. D. HILLMAN Department of Molecular Genetics, Forsyth Dental Center, 140 The Fenway, Boston, Massachusetts 02115

Received 10 June 1991/Accepted 14 August 1991 Previously, the fructose-1,6-diphosphate-dependent L-(+)-lactate dehydrogenase gene of Streptococcus mutans JH1000 was cloned into Escherichia coli (J. D. Hillman, M. J. Duncan, and K. P. Stashenko, Infect. Immun. 58:1290-1295, 1990). In the present study, the nucleotide sequence of 1.29 kb of S. mutans DNA which contained the promoter and protein-coding region of the gene was determined. In vitro disruption of the gene was achieved by deletion of the promoter and a major portion of the protein-coding sequence. Subsequently, a tetracycline resistance gene from S. mutans was inserted at the deletion site as a marker for selection. In addition, evidence from Southern hybridization showed that S. mutans JH1000 contained a single copy of the lactate dehydrogenase gene.

gene in Escherichia coli (4). In this report, we present the DNA sequence of the cloned gene. We describe the in vitro construction of a deletion mutation and the insertion of an S. mutans tetracycline resistance gene (13) as a marker for selection. We also provide evidence that S. mutans JH1000 (the parent of the bacteriocin-producing strain) contains one copy of the LDH gene.

Our objective is to construct an effector strain of Streptococcus mutans to use in replacement therapy for the prevention of dental caries. Replacement therapy exploits interactions between microorganisms, specifically, when the colonization of a site by one organism inhibits the growth of a potential pathogen at that site. Mutans streptococci are opportunistic pathogens, and replacement therapy is a means of establishing a less virulent population in an individual. The replacing strain must satisfy several criteria to be effective and safe. It must not cause disease or predispose the host to other diseases; it must prevent the colonization or growth of the pathogen; and finally, the organism must persistently colonize its ecological niche. One goal of our work, to isolate a strain which could superinfect and permanently colonize the human oral cavity, was realized with the isolation of S. mutans JH1005 (8). The hyperproduction of a small bacteriocinlike molecule by this strain is responsible for its unique colonizing properties in vivo (5, 6). The etiology of caries is the erosion of the tooth surface by acidic products of sugar fermentations by mutans streptococci. Thus, our work has also focused on the isolation of lactate dehydrogenase (LDH) mutants which produce less acid (7). An S. mutans strain which is both defective in LDH and overproduces bacteriocin would satisfy the essential criteria for an effector strain. However, we have been unable to isolate an LDH mutant of the bacteriocin producer by conventional chemical mutagenesis. Therefore, we decided to use recombinant DNA techniques to construct a strain in which the LDH mutation and bacteriocin hyperproduction were combined. In this way, we also can guarantee that the effector is specifically defective in LDH and does not harbor cryptic mutations potentially generated by chemical mutagenesis. In addition, a nonreverting LDH mutation can be constructed by deleting significant portions of the proteincoding DNA sequence. We have cloned the wild-type copy of the S. mutans LDH *

MATERIALS AND METHODS

Chemicals, media, bacterial strains, and plasmids. Chemicals and antibiotics were from Sigma Chemical Co., St. Louis, Mo. Bacteria were grown overnight at 37°C in brain heart infusion (Difco Laboratories, Detroit, Mich.) or LuriaBertani broth (LB) or on plates of the same media containing 1.5% agar. Glucose tetrazolium plates were prepared by the method of Miller (12). As required, ampicillin and tetracycline were added to plates and media at 25 and 10 ,ug/ml, respectively. S. mutans JH1000 serotype c (6) and E. coli MC1061 (1) have been described previously. Plasmid pVA981 was generously supplied by F. L. Macrina. DNA sequencing. Sequencing reactions were done with Sequenase Version 2.0 (United States Biochemical Corp., Cleveland, Ohio) according to the manufacturer's protocol with minor adjustments to the dideoxynucleotide concentrations. 2',3'-Dideoxynucleotide triphosphates and 2'-deoxynucleotide phosphates were purchased from Pharmacia P-L Biochemicals, Piscataway, N.J. 35S-labeled (a-thio)dATP was from Dupont, NEN Research Products, Boston, Mass. The pBR322 SalI site (counterclockwise) sequencing primer was obtained from New England BioLabs, Inc., Beverly, Mass. Subsequent primers were obtained from The Midland Certified Reagent Company, Midland, Tex. Primer A (CT GCTGGTGCACCCC) corresponded to positions 503 to 517 of the DNA sequence; primer B (GAACATGGTGACTC AG) corresponded to positions 793 to 808; and primer C (GA ACTAGAATCAGTACG) corresponded to positions 409 to 425. Sequencing gel conditions were those described by Lane et al. (9). DNA and protein homology searches were performed with DNAsis and Prosis (Hitachi) software, respectively.

Corresponding author. 3930

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384 43

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TTT GAA AAA GCC GT? GGC GAC GCT CTT GAT CTT AGT CAT GCA CTT GCT Phe Glu Lys Ala Val Gly Asp Ala Leu Asp Leu Ser His Ala Lou Ala

432 59

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TTT ACT TCA CCA AAA AAA ATC TAT GCA GCT AAA TAT GAA GAC TGT GCG Phe Thr Ser Pro Lys Lys Ile Tyr Ala Ala Lys Tyr Glu Asp Cys Ala

480 75

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528 91

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GAA ACT CGC CTC GAT CTC GTT GGT AAA AAT CT? GCC ATC AAT AAA TCA Glu Thr Arg Leu Asp Leu Val Gly Lys Asn Leu Ala Ile Asn Lys Ser

576 107

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ATT GTT ACT CAA GTT ATA GAA TCA GGC TTT AAT GGT ATC TTC CTC GTT Ile Val Thr Gln Val Ile Glu Ser Gly Phe Asn Gly Ile Phe Leu Val

624 123

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672 139

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720 155

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768 171

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816 187

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864 203

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912 219

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960 235

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1008 251

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1056 267

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1104 283

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1152 299

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1201 316

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FIG. 1. Nucleotide sequence of the S. mutans LDH gene. The coding strand is depicted 5' to 3'; nucleotides and amino acids are numbered to the left and right of each line. Translation of the LDH protein begins at nucleotide 256 and ends at nucleotide 1239. In the DNA 5' to the LDH protein-coding region, potential ribosome binding (RBS) and -10 and -35 consensus sequence sites of a promoter are indicated. The underlined sequence is the complement of the 17-mer probe used to identify the LDH clone in E. coli (3). A proposed rho-independent terminator is indicated by arrows. The GenBank accession number is described in the text. 3931

3932

INFECT. IMMUN.

DUNCAN AND HILLMAN

Recombinant DNA methods. Restriction enzymes and T4 ligase were obtained from New England BioLabs, Inc., and were used with the supplied buffer or according to the conditions specified by the supplier. Chromosomal DNA was isolated from S. mutans JH1000 cells which had grown overnight at 37°C in 1 liter of brain heart infusion broth containing 20 mM DL-threonine. After centrifugation, the cell pellet was resuspended in 10 ml of 25 mM sodium phosphate buffer (pH 6.4) with 20% glucose. Mutanolysin was added to 1 mg/ml, and incubation at 55°C was performed for 20 min. Two milliliters of 0.5 M EDTA (pH 8.0) was added, and the incubation continued for a further 5 min. Sodium lauryl sarcosine was added to a final concentration of 0.5%. Chromosomal DNA was purified on a cesium chloride-ethidium bromide density gradient. DNA fragments from restriction enzyme digestions were excised from 0.6% agarose separating gels and extracted by the freeze-squeeze method (14) and then by ethanol precipitation. Agarose gels (1%) were routinely used for restriction enzyme analysis. Southern hybridization. The Enhanced Chemiluminescence (ECL) gene detection system from Amersham International PLC (Amersham, England) was used. The supplier's protocols were used without modification for hybridization and for DNA probe preparation from SeaPlaque low-melting-temperature agarose (FMC Corp., Rockland, Maine). Nitrocellulose filters were exposed to Hyperfilm-ECL (Amersham). Nucleotide sequence accession number. The GenBank accession number for the S. mutans fructose-1,6-diphosphatedependent LDH gene is M72545 (description LDH SM). RESULTS DNA sequence of S. mutans fructose-1,6-diphosphate-dependent LDH. The nucleotide sequence and the predicted amino acid sequence of the LDH gene are shown in Fig. 1. Deletion analysis of the original clone (plO-5) indicated that the LDH gene was contained within approximately 1.2 kb of DNA (4). Subclone 4W6 contained the smallest DNA insert (approximately 2.0 kb) in vector pBR322 that still retained LDH activity. Sequencing was begun with this clone by using the 15-mer pBR322 SalI site primer (counterclockwise) from New England BioLabs. Subsequent primers used base sequences obtained from the results of the previous step. Sequence data revealed that while retaining LDH activity and the active site, subclone 4W6 lacked the LDH promoter and the first 95 nucleotides of the LDH-coding sequence. Presumably, LDH activity was promoted by sequences within the tetracycline gene of pBR322. Thereafter, sequencing reactions were done with p10-5, which contained the full insert. The G+C content of the LDH gene was 39.43%. The gene promoter region contained a potential -35 region, TTAG AA, at nucleotides 199 to 204. A possible -10 region, TAA TAAATT, occurred at nucleotides 228 to 237. A putative ribosome binding site, AAGGAGA, was located between nucleotides 239 and 245, 10 bases upstream from the ATG translation initiation codon. The sequence complementary to the 17-mer probe used to identify the LDH clone in E. coli (4) was found at positions 268 to 284. The HpaI, HindIII, and PstI sites of the published restriction map (4) were all confirmed by the DNA sequence, as was the size of the gene. An extensive hairpin loop immediately following the termination codon TAA encompasses bases 1245 to 1264 and 1269 to 1288 and contains a 20-base inverted repeat (with two

Hpal

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FIG. 2. Inactivation of the S. mutans LDH gene. El, S. mutans chromosomal DNA; M, S. mutans LDH gene; _, pBR322; EJ, S. mutans chromosomal DNA containing Tcr gene. Numbers indicate the length of DNA in kilobases. Tetracycline (Tc') and ampicillin (Amp') resistance markers are indicated.

mismatches) separated by a 4-base loop. This structure resembles a rho-independent terminator region. In vitro mutagenesis of S. mutans LDH gene. The strategy used to inactivate the S. mutans LDH gene is shown in Fig. 2. Clone plO-5 DNA was digested with HpaI, and the largest (ca. 7.5 kb) of the three resulting fragments was excised from a preparative gel. This fragment contained all of pBR322, approximately 1.3 kb of S. mutans flanking chromosomal DNA 5' to the LDH gene, 1.5 kb of S. mutans DNA 3' to the LDH gene, and 392 bases of the LDH carboxy-terminal sequence. The LDH promoter and 850 bases of coding sequence, including the active site, were deleted. Thus, there is no possibility of this mutation reverting to wild type in vivo. To identify and select this mutation in E. coli, and ultimately in S. mutans, we inserted the S. mutans tetracycline resistance gene from pVA981 (13) which is also expressed in E. coli. pVA981 was digested with HinclI, and the 4.0-kb fragment containing the tetracycline resistance gene was excised from a preparative gel. The blunt-ended 7.5-kb

VOL. 59, 1991

LACTATE DEHYDROGENASE GENE OF S. MUTANS A

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HpaI fragment from plO-5 was ligated to the blunt-ended 4.0-kb HincII fragment from pVA981. Calcium-treated E. coli MC1061 cells were transformed with the ligation mix, and ampicillin- and tetracycline-resistant clones were selected on LB plates. After overnight incubation at 37°C, approximately 1.7 x 104 transformants were obtained per jig of input DNA. All the transformants grew as red colonies on glucose tetrazolium plates, indicating decreased acid production, hence decreased LDH activity. MC1061 containing the parental clone p10-5 grew as white colonies on this indicator medium (4). Plasmid DNAs from five transformants were analyzed by restriction enzyme digestion. As expected after blunt-ended ligation, the tetracycline gene was found in both orientations according to the published restriction map (13). Fructose-1,6-diphosphate-dependent LDH activity could not be detected in cell extracts prepared from two of the mutants tested (data not shown). S. mutans JH1000 contains one LDH gene. We demonstrated by Southern hybridization (15) of restriction enzymedigested JH1000 DNA that this strain contains a single LDH gene. Figure 3A shows the relevant restriction enzyme sites in clone p10-5 insert DNA. The hybridization probe was the 0.5-kb HpaI fragment containing the DNA sequence of the LDH active site. JH1000 chromosomal DNA was digested with two sets of 6-base cutting restriction enzymes. For the

first set, DNA was digested with MluI (Fig. 3B, lane b), with HindIII (Fig. 3B, lane c), and with both enzymes in a double digest (Fig. 3B, lane d). According to the map, each enzyme cuts once on either side of the probe. Thus, in the double digest a single band of approximately 1.7 kb should hybridize, as shown in Fig. 3B, lane d. In the second set of digests, we used SpeI, which cuts twice within the insert to yield a single hybridization band of ca. 3.4 kb (Fig. 3B, lane e). EcoRV cut three times in the insert, and since one site was within the probe sequence, two hybridizing fragments of 4.4 and 2.1 kb were obtained with this enzyme (Fig. 3B, lane f). In the SpeI-EcoRV double digest, the 2.1-kb EcoRV hybridizing fragment, which does not contain an SpeI site, remained unchanged. The larger EcoRV hybridizing fragment, which does contain an SpeI site, yielded a smaller 0.75-kb hybridizing band (Fig. 3B, lane g). This hybridization pattern is consistent with the presence of one copy of the LDH gene. DISCUSSION The DNA sequence of the S. mutans fructose-1,6-diphosphate-dependent LDH gene confirmed our earlier results concerning its size and restriction map (4). Furthermore, the mole percent guanosine-plus-cytosine content of the gene

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INFECT. IMMUN.

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tion of a linear DNA fragment containing the disrupted gene. A similar approach was used successfully (10) to inactivate the S. mutans spaP gene encoding the major surface protein antigen P1. To date, our attempts to carry out the substitution have not succeeded, and we are trying to optimize electroporation conditions. ACKNOWLEDGMENTS Bruce Paster and Gayle Fraser for advice and assisWe thank tance with DNA sequencing and Denise Guerrero and Mary Ann

Cugini for manuscript preparation. This work was supported by Public Health Service grant DE 04529 from the National Institute of Dental Research.

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FIG. 4. Optimally aligned amino acid sequences of S. mutans and L. casei L-(+)-LDHs. Numbers refer to amino acid residues. S. m, S. mutans; L. c., L. casei; :, precise homology; *, genetic equivalent resulting from a single nucleotide base change in a codon.

was 39.43%, which correlates well with the values given for DNA from the genus Streptococcus (2). Searches (11) of the National Biomedical Research Foundation Protein Identification Resource and The European Molecular Biology Laboratory Swiss-Prot data bases with Hitachi Prosis software revealed maximum homology to five L-(+)-LDHs, all from bacterial sources. These included Lactobacillus casei and the activities from four Bacillus spp. Figure 4 compares the amino acid sequences of the enzymes from S. mutans and L. casei (3). There is 64.9% homology in a 322-amino-acid overlap between these proteins on the basis of precise matches. Homology increases to 80% when taking into consideration these matches together with genetic equivalents resulting from single base changes in codons. Complete homology was observed in all the proteins for the five amino acids centered about the active site histidine (residue 181 in the S. mutans enzyme). The cloned S. mutans LDH gene was irreversibly inactivated by an extensive deletion encompassing the promoter and a significant amount of the protein-coding sequence. The glucose tetrazolium medium growth phenotype of E. coli clones obtained after transformation with the mutant LDH gene, and the absence of enzyme activity in their extracts, confirmed that the gene was completely disabled. Finally, we established that S. mutans JH1000 contains one copy of the LDH gene in its chromosome. JH1000 DNA was digested with two sets of restriction enzymes chosen specifically to be most informative from the restriction mapping analysis. The digested DNA was probed with a fragment containing the active site of the cloned LDH gene. The results showed unequivocally that S. mutans JH1000 contains a single active site of the cloned LDH gene, a crucial finding for the final step in our strategy to construct an effector strain. We wish to substitute the LDH mutant gene for the wild-type chromosomal allele of the bacteriocin-producing S. mutans strain. This will be accomplished by electropora-

REFERENCES 1. Casadaban, M. J., and S. N. Cohen. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179-207. 2. Ferretti, J. J., and R. Curtiss III. 1987. Mole percent guanosine plus cytosine (G+C) of streptococci, p. 295-296. In J. J. Ferretti and R. Curtiss III (ed.), Streptoccocal genetics. American Society for Microbiology, Washington, D.C. 3. Hensel, R., U. Mayr, and C.-Y. Yang. 1983. The complete primary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei. Eur. J. Biochem. 134:503-511. 4. Hillman, J. D., M. J. Duncan, and K. P. Stashenko. 1990. Cloning and expression of the gene encoding the fructose-1,6diphosphate-dependent L-(+)-lactate dehydrogenase of Streptococcus mutans. Infect. Immun. 58:1290-1295. 5. Hillman, J. D., A. L. Dzuback, and S. W. Andrews. 1987. Colonization of the human oral cavity by a Streptococcus mutans mutant producing increased bacteriocin. J. Dent. Res.

66:1092-1094. 6. Hillman, J. D., K. P. Johnson, and B. I. Yaphe. 1984. Isolation of a Streptococcus mutans strain producing a novel bacteriocin. Infect. Immun. 44:141-144. 7. Hillman, J. D., and S. S. Socransky. 1987. Replacement therapy for the prevention of dental disease. Adv. Dent. Res. 1:119-125. 8. Hillman, J. D., B. I. Yaphe, and K. P. Johnson. 1985. Colonization of the oral cavity by a strain of Streptococcus mutans. J. Dent. Res. 64:1272-1274. 9. Lane, D. J., B. Pace, G. J. Olsen, D. A. Stahl, M. L. Sogin, and N. R. Pace. 1985. Rapid determination of 16S ribosomal RNA sequence for phylogenetic analyses. Proc. Natl. Acad. Sci. USA 82:6955-6959. 10. Lee, S. F., A. Progulske-Fox, G. W. Erdos, D. A. Piacentini, G. Y. Ayakawa, P. J. Crowley, and A. S. Bleiweis. 1989. Construction and characterization of isogenic mutants of Streptococcus mutans deficient in major surface protein antigen P1 (1/11). Infect. Immun. 57:3306-3313. 11. Lipman, D. J., and W. R. Pearson. 1985. Rapid and sensitive protein similarity searches. Science 227:1435-1441. 12. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 13. Tobian, J. A., M. L. Cline, and F. L. Macrina. 1984. Characterization and expression of a cloned tetracycline resistance determinant from the chromosome of Streptococcus mutans. J. Bacteriol. 160:556-563. 14. Sealy, P. G., and E. M. Southern. 1989. Electrophoresis of DNA, p. 39-76. In D. Rickwood and B. D. Hames (ed.), Gel electrophoresis of nucleic acids. IRL press, Oxford. 15. Southern, E. M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98:503-517.