Gene, 116 (1992) 3:5-42 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0378-1119/92/$05.00
35
GENE 06483
In vivo inactivation of the Streptococcus mutans recA gene mediated by PCR amplification and cloning of a recA DNA fragment (Streptococci; re~.:ombination; RecA; bacterial virulence factors; oral pathogens)
Robert G. Quivey Jr. and Roberta C. Faustoferri Department of Dental Research and Cariology Center. University of Rochester School of Medicine and Dentistry. Rociwster. NY 14642 (USA)
Received by R.E. Yasbin: 29 April 1991; Revised/Accepted: 2 January/10 February 1992; Received at publishers: 5 March 1992
SUMMARY
The inactivation of the RecA protein in pathogenic oral streptococci would facilitate genetic analysis of potential virulence factors in these strains. Comparison of recA nucleotide (nt) sequences from a number of bacteria has suggested that two regions of highly conserved RecA amino acid (aa) sequence could be used as a basis for synthesizing degenerate oligodeoxyribonucleotide primers with which to amplify recA homologues from the streptococci. Accordingly, primer mixtures were used to amplify a 693-bp fragment of the Streptococcus mutans chromosome by PCR. The amplified fragment was cloned and its identity confirmed via hybridization to an Escherichia coli recA gene probe and by nt sequence determination. The recA homologue fragment from S. mutans GS-5 was 63% and 75% homologous to the deduced aa sequences of the E. coli and Bacillus subtilis RecA enzymes, respectively. The S. mutans recA fragment was mutagenized in vitro via insertional inactivation and returned to the chromosome using allelic exchange. The resulting strains of S. mutans were shown to be substantially more sensitive to UV irradiation than the wild-type strain. Further, the ability to incorporate linear markers into the chromosome was abolished in putative S. mutans recA strains, thus indicating the functional inactivation of RecA in these microorganisms.
INTRODUCTION
The enzyme primarily responsible for the homologous recombination of chromosomal DNA in bacteria is the RecA protein (for review see Miller and Kokjohn, 1990). The gene encoding the recombinase, recA, has been cloned
Correspmldence to: Dr. R.G. Quivey, Department of Dental Research, University of Rochester School of Medicine and Dentistry, Box 611,601 EImwood Ave., Rochester, NY 14642 (USA) Tel. (716)275-1435; Fax (716)473-2679.
Abbreviations: A., Anabaena; aa, amino acid(s); Ap, ampiciilin; Aq., Aquaspiriilum; B., Bacillus; Bop'., Bordetella; BHI, Brain Heart Infusion (medium); bp, base pair(s); BSA, bovine serum albumin; Denhardt's solution, 0.02% Ficoll/0.02% polyvinylpyrrolidone-40/0.02% BSA; dNTP, deoxyribonucleoside triphosphate; E., Escherichia; Er, erythromy-
from numerous bacteria and their nt sequences determined. The goals of several of these studies have been the use of the cloned gene to generate site-directed RecA-deficient mutant strains with which to facilitate further genetic manipulations and to probe the role of homologous DNA recombination in bacterial virulence (Goldberg and
cin; fif, gene encoding fructosyltransferase; GTF, glucosyltransferase; gt.f, gene encoding GTF; kb, kilobase(s) or 1000 bp; Kin, kanamycin; LB, Luria-Bertani (medium); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ori, origin of DNA replication; P., Proteus; PCR, polymerase chain reaction; Is., Pseudomonas; RecA, recombination enzyme; recA, gene encoding RecA; R, resistance/resistant; S., Streptococcus; SDS, sodium dodecyl sulfate; Se., Serratia; SSC, 0.15 M NaCI/0.015 M Na3,citrate pH 7.6; T., Thiobacillus; Taq, Thermus aquaticus; TBE buffer, 89mM Tris.HCl/89 mM boric acid/9 mM Na2'EDTA pH 8: Tc, tetracycline; UV, ultraviolet; wt, wild type.
36 Mekanlanos, 1986; Koomey and Falkow, 1987; Venkatesh et al.. 1990). Recently, evidence has been provided that suggests that RecA protein may play a role in the high-frequency recombination between the g@? and gtfC genes in S. mutans (&da and Kuramitsu, 1988; Sun et al., 1991). The gtf genes (B, C and D) encode glucosyltransferase enzymes that are responsible for the synthesis of extracellular glucan polymers in dental plaque and are thought to be virulence factors in the etiology of dental caries. Mutants of S. mutans defective in the ability to produce water-insoluble polymers (formed by the action of the B and C gene products) appear to be noncariogenic in experimental animals (Clark et al., 1978; Tanzer and Freedman, 1978; Tanzer et al., 1985; Munro et al., 19!>1).The genes encoding the GTF enzymes have been cloned, and their nt sequences have indicated that the B and C genes are highly homologous to each other (Shiroza et al., 1987; Ueda et al., 1988, respectively). Recombination between homologous regions of the gfl genes results in the apparent inactivation of the GTF-I and GTF-SI proteins, leaving only a functionally active GTFS protein from the g@ gene (Ueda and Kuramitsu, 1988). Strains exhibiting only the GTF-S activity appear to be no longer cariogenic in rats (Munro et al., 1991). The role of RecA is unknown, but data indicate that the recombination between homologous regions of the gtf genes is not precise (Sun et al., 1991). Thus, recombination results in strains containing different products, thereby suggesting the absence of a site-specific recombinase for the gg genes. In order to pursue observations of g/@-C recombination and to extend our abilities to understand the genetic regulation of virulence factors in the oral bacteria, we have investigated the possibility of isolating site-directed mutations in the vecA-homologue from Srveprococcus mutans. Previous reports have suggested some difficulty in cloning complete n&homologues from Gram - organisms (Rhee and Morrison, 1988; Marrero and Yasbin, 1988), even though the enzyme from apparently distant sources can complement vecA-defective strains of E. coli (Murphy et al., 1990). The polymerase chain reaction (Saiki et al., 1985) appeared to offer the ability to amplify a substantial partion of the gene without necessarily amplifying regulatory regions associated with the coding region. Recently, we have used degenerate mixtures of oligos to amplify a gene fragment encoding the catalytic subunit of the streptococCal membrane-bound, proton-translocating ATPase (I-I + ATPase) (Quivey et al., 1991). Similarly, an examination of the published nt sequences from a number of recA homologues suggested that oligos based on highly conserved aa sequences could be used to amplify a substantial portion of the streptococcal recA homologue.
RESULTS AND DISCUSSION
(a) PCR amplification of the Streptococcus mutuns recA homologue The invariant regions in RecA proteins, shown boxed in Fig. 1, provided the basis for the synthesis of degenerate oligos to be used in priming the PCR amplification of the streptococcal homologue. The forward primer, Val-GluIle-Tyr-Gly-Pro-Glu, was based on the aa sequence starting at Va164of the E. cofi protein. The complementary or reverse primer, Gly-Ala-Trp-Tyr-Ser-Tyr, was based on the aa sequence starting at Gly286 of the E. coli protein (Sancar et al., 1980). At positions of 2-nt degeneracy, both nt were included in the synthetic mix; at positions of 3- or 4-nt degeneracy, deoxyinosine was inserted. The resulting oligo mixtures contained four different primers in the forward direction and eight in the reverse direction. Both primers contained restriction sites that had been incorporated into the oligo sequences (see legend for Fig. 2). The expected size for the PCR products, utilizing the E. coli recA gene as a template, was approx. 690 bp. The products of both the S. mutuns and E. co& amplifications were in the 700.,bp range, indicating a successful amplification using the degenerate primers described above (Fig. 2). The primers used to direct PCR amplification contained a Hind111 site in the forward primer and an SstI site in the reverse primer. However, attempts to clone the S. mutarts-derived fragment hybridizing to the E. coli probe failed when the amplified DNA was digested with HindIII+SstI to take advantage of the programmed restriction sites. Subsequently, the amplified, putative S. mutans recA DNA fragment was cloned into the vector pCRlOO0 (InVitrogen, La Jolla, CA) by taking advantage of the 3’adenosines placed on blunt-ended DNA fragments by Tuq
64 + ii. coli EIYGPESSCKTTLT B. sublilis EVYGPESSGKTTVA S. mwcesceus EIYGPESSGKTTLT A. variclbills EIYGPESSGKTTVA R mirabilis EIYGPES~GKTTLT Ps. aeruginosaEIYGPESSGKTTLT T. ferroridans EIYGPESSGKTTLT Bar.perfussis B IYGPESSGKTTLT Aq. mognetotacticumE IYGPESSGKTTLA Synechococcus sp. EIYGPESSGKTTVA
Fig. 3. Alignmcnl of highly conserved aa from bacterial RecA and RccE (E).subrilis) protein sequences. The boxes contain sequences that are completely conserved. Numbers indicate the position of the aa in the E. coli protein. Sources of the sequences were as follows: E. co/i (Sancar et al., i 980); f3. subtilis(Stranathan et al., 1990); Bor. pertussis (Favre and Viret, 1990); A. variabilis (Owttrim and Coleman, 1989); Aq. tnagneto~acticum (Berson et al., 1990); Ps. aerugirzosaPA0 (San0 and Kageyman, 1987); P. tnirabilis (Akaboshi et al., 1989); T. ferrooxidatts (Ramesar et al., 1989); S. marcescetts (Ball et al., 1990); S~~echococcus sp. (Murphy et al., 1990).
37
A
B 1
12
2
3
C 4
S
1
2
3
4
S
345
bp
---- 3 0 5 4
--4072 -- 3054 --2036 --1636 --1018 --1018 -- 506
Fig. 2. PCR amplification of recA.homologue from $. mutans and hybridization of E. coil and S. muums amplified recA fragments to S. mutans GS-5 chromosomal DNA. (Panel A) Electrophoretogram of PCR-amplified recA fragments from E. coli and S, mutans and cloned S. mutans PCR-amplified recA fragment. Lanes: I, 693-bp PCR-amplified fragment of E. coil recA from plasmid pDRI453 carrying the intact E. coil recA gene (Sancar and Rupp, 1979); 2, PCR-amplified S. mutans chromosomal DNA; 3, HindIll-digested pRQ201, containing a clone of the S. mutans 693-bp PCR-amplified fragment; 4, HindlII-digested chromosomal DNA; 5, one-kb DNA ladder (Life Technologies/BRL, Gaithersburg, MD). (Panel B) Hybridization ofradio-labeled E. coil recA fragment to the blot shown in panel A. (Panel C) Hybridization of radio-labeled S. mutans recA fragment to the blot shown in panel A. Methods. Genomic DNA from S. mutans was prepared as described previously (Chassy, 1976). PCR (Saiki et al., 1985) amplification of DNA was accomplished using a GeneAmp T M kit (Perkin Elmer/Cetus, Norwich. CT). Final reaction concentration of reagents was as follows: 200 mM dNTPs/4 mM MgCl2/ 50 mM KCI/10 mM Tris.HCI pH 8.3/1% (w/v) BSA/5 .,nits of Taq polymerase/1 unit of Perfect Match Enhancer (Stratagene, La Jolla, CA). For amplification of S. mutans G S-5 genomic DNA, 10 sg of DNA was added to each reaction with 5 pmol of each primer. Amplification of plasmid pDR 1453 was similar except that ! ng of plasmid was used as template and Perfect Match Enhancer was not included in the reaction. The forward primer sequence, based on the information shown in Fig. 2, was 5'-GGCCAGTAAGCTTGGTIGARATITAYGGICCIGA, where the singly underlined sequence corresponds to the conserved RecA aa sequence V-E-I-Y-G-P-E and the doubly underlined sequence is a programmed HindIII site. The reverse primer sequence was 5'-GTAGGTCCAGAGCTCTAISWRTACCAIGCIGC, where the singly underlined sequence corresponds to the conserved RecA aa sequence G-A-W-Y-S-Y and the doubly underlined sequence represents a programmed SstI site. Uncommon nt in the sequences shown here were defined as follows: A or G = R; A or T = W, T or C = Y; G or C = S. Amplification was carried out in a programmable heater (Perkin Elmer/Cetus, Norwich, CT) using the following cycling regime: 2 min at 94°C for denaturation; 2 min at 55°C for primer annealing; and 2 min at 72°C for strand synthesis. Typically, 35 cycles were completed. Plasmid pRQ201 was constructed by cloning the putative, amplified S. mutans recA DNA fragment into plasmid pCRI000 (lnVitrogen, La Jolla, CA). Plasmid DNA was prepared using an alkaline lysate procedure as before (Quivey et al., 1991). DNAs were separated on 1% agarose gels in TBE buffer (Peacock and Dingman, 1968) and transferred to nitrocellulose membranes by capillary transfer. Gels containing genomic DNA fragments were acid-treated as described by Wahl et al. (1979) and denatured as described by Southern (1975). Following membranetransfer, the DNAs were cross-linked to membranes via UV irradiation (Reed and Mann, 1985) using a Stratalinker-1800 as recommended by the manufacturer (Stratagene, La Jolla, CA). The film was exposed to the hybridized blot for I h. In order to reduce the possibility of false-positive hybridization between probe-vector sequences and chromosomal DNA, amplified recA fragments from plasmid pDRI453 (representing the E. coil recA gene) and S. muums chromosome were purified from agarose gels as described (Heery et al., 1990) and radioactively labeled for use as probes via random-hexamer priming with [~.3.,p ]dATP (Feinberg and Vogelstein, 1983; 1984), using commercially available kits (BRL, Gaithersburg, MD). Hybridizations were done at 55°C in solutions containing 5 x Denhardt's solution (Denhardt, 1966)/5 x SSC/100#g/ml denatured salmon sperm DNA. Following overnight incubation, blots were washed as follows: two 15 min washes, each at room temperature, in I x SSC buffer, containing 1 x Denhardt's solution, followed by two additional 55°C washes for 30 min each in ! x SSC, containing 0.1% SDS. Blots containing hybridized material were air-dried, mounted on Whatman 3 mm paper (VWR Scientific, Rochester, NY), over-layed with Kodak X-Omat AR X-ray film (Eastman Kodak Co., Rochester, NY), placed in film cassettes with intensifying screens (Cronex, DuPont and Co., Wilmington, DE) and stored at -70°C until development. All restriction enzymes, T4 DNA ligase and agarose were purchased from Life Technologies/BRL (Gaithersburg, MD). Common reagents were purchased from the Sigma Chemical Co. (St. Louis, MO). [a-32P]dATP used in DNA probe-labeling was obtained from Amersham (Arlington Heights, IL) at a specific activity of 3000 Ci/mmol.
p o l y m e r a s e (Clark, 1988). H i n d l l l digestion o f o n e resulting clone, referred to as p R Q 2 0 1 , indicated an internal H i n d I I I site in the amplified fragment. T h u s , the digestion o f the p l a s m i d p R Q 2 0 1 D N A w i t h H i n d I I I (lane 3 o f Fig. 2, p a n e l s B a n d C ) s h o w e d t w o b a n d s , b o t h o f w h i c h hybrid-
ized to the S. r o m a n s recA probe. T h e upper b a n d a p p a r ently c o n s i s t e d o f vector plus insert D N A with the lower b a n d c o n s i s t i n g o f an insert f r a g m e n t that is b o u n d e d o n o n e side by the internal H i n d I I I site a n d on the other e n d by the p r o g r a m m e d H i n d I I I site provided for by the for-
38 10
20
30
40
50
60
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.
*
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GGG CCG GAA TCT TCT GGT AAG ACA ACT GTC GCT CTT CAT GCT GCT GCT CAG GCG CAA AAA GAT GGC GGT ATT GCC GCT G P E S S G K T T V A L H A A A Q A Q K D G G I A A> 80
90
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100
110
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120
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130
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140
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150
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TTC ATT GAT GCA GAA CAT GCC CTT GAT CCA GCC TAT GCT GCT GCT CTT GGC GTT AAT ATT GAT GAG CTT TTG CTT TCA F I D A E H A L D P A Y A A A L G V N 7 D E L L L S> 160
170
180
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190
200
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210
220
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CAA CCA GAT TCA GGA GAA CAG GGT CTT GAA ATT GCA GGG AAA TTG ATT GAT TCT GGC GCT GTT GAT TTA GTT GTT GTT Q P D S G E Q G L E I A G K L I D S G A V D L V V V> 240
250
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GAC TCA GTG GCA GCT TTA GTA CCA CGT GCG GAG ATT GAC GGA GAT ATT GGT AAT AGT CAT GTT GGC TTA CAA G~A CGC D S V A A L V P R A E I D G D I G N S H V G L Q A R> 320
330
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350
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360
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ATG ATG AGT CAA GCG ATG CGT AAA TTA TCA GCT TCA ATC AAT AAA ACA AAA ACC ATT GCT ATT TTT ATT AAT CAA TTG M M S Q A M R K L S A S I N K T K T I A I F I N Q L> 400
410
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420
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430
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440
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450
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460
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CGG GAA AAA GTT GGT ATT ATG TTT GGT AAT CCA GAA ACA ACC CCT GGC GGG CGT GCC TTG AAG TTT TAT TCT TCT GTG R E K V G I M F G N P E T T P G G R A L K F Y S S V>
470
480
490
500
510
520
530
540
CGT CTT GAT GTC CGC GGC AAT ACT CAA ATT AAA GGA ACC GGG GAA CAA AAA GAC AGC AAT ATT GGT AAA GAG ACC AKA R L D V R G N T Q I K G T G E Q K D S N I G K E T K> 550
560
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ATT AAA GTT GTT AAA AAT AAA GTT GCT CCA CCA TTT AAG GAA GCT TTT GTA GAA ATT ATA TAT GGT GAA GGC ATT TCT K V V K N K V A P P F K E A F V E I I Y G E G I S> 630
640
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CGT ACA GGT GAA TTA GTT AAG ATT GCC AGT GAT TTG GGA ATT ATC CAA AAA CGT GGC GCC TGG TAT ACC R T G E L V K I A S D L G I I Q K R G A W Y T>
Fig. 3. Nucleotide sequence of the cloned 693-bp recA homologue from S mutans GS-5, and the deduced aa sequence. The nt sequence of the amplified and cloned recA fragment from S. muums GS-5 was determined via double-stranded DNA sequencing from CsCI density-gradient-purified templates as described previously (Tabor and Richardson, 1987) using the Sequenase 2.0 kit from US Biochemicals (Clevehmd, OH). The nt sequence data reported in this paper appears in the EMBL, GenBank and DDBJ Nueleotide Sequence Databases under the accession No. M61897.
1 S. mutanB B. sub,ills E. ¢011
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Fig. 4, Deduced aa sequence ofthe cloned, amplified S, muta,s recA-homologue, shown with the homologues from E. coli and B. subtilis Rec ~, Conserved aa sequences are boxed.
39 (b) Cloning and nt sequence of the Streptococcus mutans recA gene fragment The nt sequence of the cloned fragment, shown in Fig. 3, confirmed the similarity of the S. mutans recA fragment to the homologous regions found in E. coli (Sancar et al., 1980) and B. subtilis (Strathanan et al., 1990). Fig. 4 shows the deduced aa sequence alignments of homologous regions from the S. mutans, E. coil and the B. subtilis recA genes. Of the 230 aa shown, S. mutans and E. coli are identical at 145 (63% homologous), whereas S. mutans and B. subtilis are conserved at 173 residues (75% homolo-
ward PCR primer. The arrangement of Hindlll sites was later confirmed by nt sequence determination (see below). Hybridization of the S. mutans genomic DNA amplification products to the E. coil recA probe suggested that the amplification product of the streptococcal DNA was indeed an S. mutans recA-homologue (Fig. 2). Interestingly, the E. coil recA probe did not hybridize to that portion of the amplified S. mutans DNA still bound to the vector under the washing stringencies used, indicating that the nt sequences were dissimilar.
/acZ'
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pRQ202 ~Er" Km R ~ H l m l l l l lacZ' ~ DigestwRh Pst
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transform S. muW.ns, select Er g
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ErR
recA Iinearizedplasmtd1 1
S. mutanschromosome X
Fig. 5. Insertional inactivation scheme for the generation of in vivo recA mutations in S. mutans UAI59. Fill-in reactions were performed with T4 DNA polymerase and used according to the manufacturer's (BRL/LTI, Gaithersburg, MD) instructions. Transformation of S. mutans UA ! 59 was accomplished using a previously described protocol (Perry and Kuramitsu, 1981). Transformations were performed in the dark. Symbols: for all plasmids, the stippled arrow represents the ~-complementation region of the lacZ gene of E. coli; the construction of pTS 19E has been described (Aoki et al., 1986); the construction of pRQ201 and pRQ202 is described in sections b and c, respectively;the solid bar in plasmids pRQ201 and pRQ202 represents the S. mutans recA PCR fragment described in section b. The arrow associated with the solid bar indicates the direction of transcription ('sense strand') for the PCR fragment. Antibiotic resistance genes carried on the various plasmids and plasmid origins of replication are indicated on the maps as open bars or open arrows.
40 gous). Of the differing residues between B. subtilis and S. mutans, many are unaltered in their respective charges, suggesting a high degree of structural conservation between the two enzymes. (c) Insertionai inactivation Streptococcus mutans
of
the
recA
gene
Probed with pRO201
Probed with pTSIgE
I 1 2 3 4 5
6
I 7 8 9
I 10 11 12
in
The nt sequence of the cloned fragment indicated the presence of a unique HincII at bp 251. Thus, the strategy to construct a recA strain of S. mutans consisted of the insertion of an Er R element from plasmid pTSl9E (Aoki et al., 1986). The 1.8-bp B a m H I fragment containing the Er a marker was excised from pTS 19E and cloned into the Hincll site of pRQ201 as shown schematically in Fig. 5. The resulting plasmid, pRQ202, was digested with Pstl and used to transform S. mutans UA159 to Er R. Strain UA159 was selected for these experiments because it has been shown to be transformable with plasmid and chromosomal markers and has been shown to be highly cariogenic in animal models (Murchison etal., 1986). Chromosomal DNA from Er R transformants was examined for integration of the Er e marker and alteration of the HindIII. digestion pattern via Southern (1975) blotting. Fig. 6 shows the results of the hybridization of plasmids pTS 19E (containing the Er R marker) and pRQ201 (containing the putative recA gene fragment clone) to Hindlll-digested chromosomal DNA prepared from the prototroph and two potentially mutant strains. The autoradiogram resulting from the Southern blot shows the wt chromosome with two distinctive Hindlll bands at approx. 1600 and 4000 bp that hybridize strongly to pRQ201, containing the recA gone fragment. The two putative mutant strains showed the loss of the 1600-bp band and the appearance of a strongly hybridizing band at approx. 6000 bp. When digests were probed with pTSI9E containing the Er e marker, the wt DNA showed no homology with the probe (Fig. 6, lane 10) and the 1600-bp band seen with the E. coil recA probe was not visible; whereas Er e marker homology was seen with the 4000-bp and 6000-bp bands in the putative mutant strains. We concluded from this experiment that the recA allele and Er e marker were physically linked as a result of recombination of the insertionally-disrupted recA fragment into the chromosomes of these strains via allelic exchange. To further characterize the putative mutant strains, one organism, designated URI00, was selected for study. Sensitivity to UV light has been used previously to characterize, among other bacteria, putative recombination-defective strains of Streptococcus faecalis (Yagi and Clewell, 1980) and Streptococcus sanguis (Dance-Moore and Volpe, 1985). Strains UA159 and URI00 were irradiated in a programmable UV light source for varying times and plated on rich medium for enumeration of viable bacteria. The results of irradiation, shown in Fig. 7, were quite similar to those
bp 12216
m
6108
m
3054--2036-1636 -1018
---
506 ----
Fig. 6. Southernhybridization ofwt and putativerecA mutationsprobed with pRQ201 and pTS 19E. Plasmid and chromosomal DNAs were prepared as described above in the legend to Fig. 2. 10#g of chromosomal DNAs were digested with Hindlll, Plasmid DNAs (i pg) were digested as follows: pRQ201 with HindIll and pTSlgE with BamHI, according to the manufacturer(BRL/LTI, Gaithvrsburg, MD). Blotting,hybridization conditions, washing conditions and autoradiography following hybridization wereas describedin Fig. 2, with the exceptionthat finalwashes were as follows: three 20-rain washes at room temperaturewith 2 x SSC containing0. 1% SDS followedby one 20-minwash at 45°C with 2 x SSC containing 0.1% SDS, Lanes: I, one-kb ladder; 2, HindIII-digested pRQ201; 4, Hindlll-digested S. muums UAI59 genomic DNA; 5o HindlIl.digested S. mutans UR 100 genomic DNA; 6, HindIIi digested S. mutans UR200 genomic DNA; 8, BamHI-digested pTSi9E~ 10, HindIii-digested S. mutans UAI59 genomic DNA: 11, HindIll-digested S. mutmls URI00 genomicDNA: 12, HindIII-digestedS. mutansUR200 genomic DNA; 3, 7, 9, empty. shown earlier for S. sanguis (Daneo-Moore and Volpe, 1985) and indicated clearly that the putative recA mutant, URI00, was more sensitive to UV irradiation than the wt, strain. These results strongly suggested that URI00 was impaired in its ability to repair thymidine-dimers formed as a result of UV irradiation, in turn suggesting a loss of functionally active RecA protein (reviewed in Taylor, 1988). The role of RecBCD complex-homologues in UV damage repair for the streptococci is currently unknown. In order to characterize URI00 for its ability to incorporate chromosomal DNA, transformation experiments were performed using the protocol of Perry and Kuramitsu (1981). Chromosomal DNA was prepared from a strain containing a Tc e marker in the fif gone of S. mutans (obtained from R.A. Burne, University of Rochester) and used to transform UA159 and the putative recA mutant strain UR100. Plasmid pSA3 (Dao and Ferretti, 1985) DNA was
41 100
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formation with plasmid D N A was essentially unaltered in the mutant strain as compared to the wt. However, the frequency of plasmid transformation was substantially lower than that previously reported for strain UA159 (Murchison et al., 1986), and may reflect some differences in handling of the strains or in post-transformation expression time in nonselective medium, prior to plating on selective medium. Nevertheless, we conclude from the inability to incorporate a linear chromosomal marker, that the putative mutant strain UR100, is indeed defective in the RecA protein and that the ability to recombine homologous DNAs in this strain is absent.
"
o'12s Iso 17s
(d) Conclusions
Exposure Time (s) Fig. 7. UV survival of S. mutans strains UAI59 and URi00. Strains UAI59 and URI00 were grown to mid.logarithmic phase in BH! (Difco, Detroit, MI). The cells were sonicated briefly to dechain the organisms (4 x 30 s pulses at 400 W, Braun-Sonic 1510 Braun Biotech, Allentown, PA). Dechained cells were verifiedvisuallyby phase microscopy. Aliquots of cells (1 ml) were transferred into sterile dishes (10 x 30 mm, Falcon) and were irradiated pairwise for varying times as shown in a programmable UV light source (Stratalinker-1800, Stratagene, La Jolla, CA). The source delivers 3000#W/cm-"(Stratagene, La Jolla, CA). Followingirradiation, 0.1 ml aliquots were diluted in 1.0 ml dilution-tubes containing fresh BH! and plated on BHI-agar medium for the outgrowth of colonyforming units. also used to transform the respective strains to TCR. The results, shown in Table I as colony-forming units, indicated that recombination of the chromosomal marker was completely abolished in the putative recA strain. Transformation frequencies with the wt strain were comparable to those reported previously (Murchison et al., 1986). Trans-
(1) The clone of a PCR-amplified fragment of S. m u t a , s GS-5 D N A has been shown by DNA hybridization, nt sequence determination and alignment of deduced aa sequences with known RecA proteins to encode a portion, estimated to be approx. 65 % as compared to E. coil, of the full-length coding region of the recA homologue from S. mutans GS-5. (2) The clone of the S. mutans recA gene fragment has permitted its mutagenesis via insertional inactivation with a 1.8-kb Er R marker. The physical linkage ofthe Er R marker with recA DNA, as shown by Southern blotting along with the behavior of the mutant strains, with regard to UV sensitivity and inability to incorporate chromosomal DNA, strongly indicates that the RecA enzyme activity in these strains has been eliminated or at least, greatly diminished. (3) The construction of recA strains of S. mutans will facilitate genetic regulation studies and the further analysis of the role of RecA in the recombination of the gtf genes. Furthermore, the clone will provide a probe for DNA fragments containing the 5' end ofthe recA gene with attendant regulatory regions.
TABLE I Transformation of S. t;mtans strains
ACKNOWLEDGEMENTS
Strain u
DNA s o u r c e b
Markerc
Transformant frequencyd
UA 159 UR100
S. mutans chr. S. mutans chr.
fff::Tca ftf::Tca
700 0
UAI59 URI00
pSA3 plasmid pSA3 plasmid
TcR TcR
30 20
" Strain UAI59 (Murchison et al., 1986); strain URI00 was constructed in this study (see section e). b chr, chromosomal (genomic) DNA. c Chromosomal DNA containing a TcR marker in the fif gene was obtained from R.A. Burne (Department of Dental Research, University of Rochester). The DNA was prepared as described (Chassy, 1976). d Transformation was essentially as described by Perry and Kuramitsu (1981) using 10 Fg of' DNA/transformation. Transformant frequency is reported as the number of colony-forming units per transformation experiment.
We thank Cynthia Luisi-DeLuca for plasmid pDR1453 and relevant E. coil strains. We also thank Patricia Noonan for assistance with the manuscript preparation. This work was supported by NIH DEO-7003-05 as part of the Rochester Caries Research Center, by BRSG PHS $7RR0540327 and in part by DEO-7777. REFERENCES Akaboshi, E., Yip, M.L.R. and Howard-Flanders, P.: Nucleotide sequence of the recA gene of Proteus mirabilis. Nucleic Acids Res. 17 (1989) 4390. Aoki, H., Shiroza, T., Hayakawa, M., Sate, S. and Kuramitsu, H.K.: Cloning era Streptococcusmutans glucosyltransferasegene coding for insoluble glucan synthesis. Infect. lmmun. 53 (1986)587-594. Ball, T.K., Wasmuth, C.R., Braunagel, S.C. and Benedik, M.J.: Exprcs-
42 sion of Serratia marcescens extraceUular proteins requires recA. J. Bacteriol. 172 (1990) 342-349. Berson, A.E., Peters, M.R. and Waleh, N.S.: Nucleotide sequence of recA gene of Aquaspirilhon magnetotacticum. Nucleic Acids Res. 18 (1990) 675. Chassy, B.M.: A gentle method for the lysis oforal streptococci. Biochem. Biophys. Res. Commun. 68 (1976) 603-608. Clark, J.M.: Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16 (1988) 9677-9686. Clark, W.B., Bammann, L.L. and Gibbons, R.J.: Ability of Streptococcus mutans and a glucosyltransferase defective mutant to colonize rodents and attach to hydroxyapatite surfaces. Infect. Immun. 21 (1978)681684. Dance-Moore, L. and Volpe, A.: Recombination-deficient Streptococcus sanguis, Infect. lmmun. 48 (1985) 584-586. Dao, M.L. and Ferretti, J.J,: Streptococcus-Escherichia coil shuttle vector pSA3 and its use in the cloning of streptococcal genes. Appl. Environ. Mierobiol. 49 (1985) i 15-119. Denhardt, D.T.: A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23 (1966)641646. Favre, D. and Viret, J.F.: Nuclcotide sequence of the recA gent of Bordetellapertussis. Nucleic Acids Res. 18 (1990)4243. Feinberg, A.P. and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132 (1983) 6-13. Feinberg, A.P. and Vogelstein, B.: Addendum: a technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137 (1984) 266-267. Goldberg, i. and Mekanlanos, J.J.: Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant. J. Bacteriol. 165 (1986) 715-722. Heery, D.M., Gannon, F. and Powell, R.: A simple method for subcloning DNA fragments from gel slices, Trends Gcnct. 6 (1990) 173. Koomey, J.M. and Fa!kow, S.: Cloning of the recA gone of Ne~:'seria gom~rrlweae and construction of gonococcal recA mutants. J. Bacteriol. 169 (1987) 790..'N5. Mart ~ro, R and Yasbin, R.E.: Cloning of the Bacillus sl~btilis recE gene and functional expression of recE÷ in B. subtilis. J. Bacteriol. 170 (1988) 33~-344. Miller, R,V, and Kokjohn, T.A.: General microbiology of recA: environm~'ntal and evolutionary significance, Annu. Rev, Microbiol, 44 (1990) 365-394. Munro, C., Michalek, S.M, and Macrina, F.L.: Cariogcnicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by all¢lic exchange, Infect, Immun. 59 (1991) 2316-2323. Murchison, H,H,, Barrett, J.F., Cardineau, G.A. and Curtiss Ill, R.: Transformation of Streptococcus mutans with chromosomal and shuttie plasmid (pYA629) DNAs. Infect. Immun, 54 (1986) 273-282. Murphy, R.C,, Gasparich, G,E., Bryant, D.A. and Porter, R,D.: Nuclcotide sequence and further characterization of the Synechococcus sp. strain PCC 7002 recA gene: complemcntation of a cyanobaeterial recA mutation by the Escherichia coli recA gone, J. Bacteriol, 172 (1990) 967-976, Owttrim, G.W. and Coleman, J,R,: Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene, J, Bacteriol. 171 (1989) 5713-5719. Peacock, A.C, and Dingman, C.W.: Molecular weight estimation and separation ofribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistq, 7 (1968)668-674. Perry, D. and Kuramitsu, H.K.: Genetic transformation of Streptococcus mutans. Infect. Immun. 32 (1981) 1295-1297.
Quivey Jr., R.G., Faustoferri, R.C., Belli, W.A. and Flores, J,S.: Polymerase chain reaction amplification, cloning, sequence determination and homologies of streptococcal ATPase-encoding DNAs. Gene 97 (1991) 63-68. Ramesar, R.S., Abratt, V., Woods, D.R. and Rawlings, D.E.: Nucleotide sequence and expression of a cloned Thiobacillus ,ferrooxidans recA gene in Escherichia coll. Gene 78 (1989) 1-8. Reed, K.C. and Mann, D.A.: Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13 (1985) 7207-7221. Rhee, D.K. and Morrison, D.A.: Genetic transformation ir~Streptococcus pneumoniae: molecular cloning and characterization of~recP, a gene required for genetic recombination, J. Bacteriol. 170 (1988)630-637. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N.: Enzymatic amplification of g-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230 (1985) 1350-1354. Sancar, A. and Rupp, W.D.: Physical map of the recA gene. Prec. Natl. Acad. Sci. USA 76 (1979) 3144-3148. Sancar, A., Stachelck, C., Konigsberg, W. and Rupp, W.D.: Sequences of the recA gene and protein. Prec. Natl. Acad. Sci. USA 77 (1980) 261 !-2615. Sane, Y. and Kageyama, M.: The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO. Mol. Gen. Genet. 208 (1987) 412-419. Shiroza, T., Ueda, S. and Kuramitsu, H.K.: Sequence analysis ofthe gt.lB gene from Streptococcus mutans. J. Bacteriol. 169 (1987) 4263-4270. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98 (1975) 503-517. Stranethan, M.C., Bayles, K.W. and Yasbin, R.E.: The nucleotide sequence of the recE÷ gen¢ of Bacillus subtilis. Nucleic Acids Res. 18 (1990) 4249. Sun, S., Dance-Moore, L., Marri, L., Hantman, M. and Shockman, G.D.: Genetic and physiological studies of variants of Streptococcus mutans GS-5 that produce nonmucoid colonies on sucrose-containing media. In: Dunny, G.M., Cleary, P.P. and McKay, L.L. (Eds.), Genetics and Molecular Biology of Streptococci, Lactococci, and Enterococci. Am. Soc. Microt~iol.,Washington, DC, 1991, pp. 256-260. Tabor, S. and Richardson, C.C.: DNA sequence analysis with a modified bacteriophage T7 DNA polymcrase. Prec. Natl. Acad. Sci. USA 84 (! 987) 4767-477 I. Tanzer, J.M. and Freedman, M.L.: Genetic alterations of Streptococcus mutans" virulence. Adv. Expt. Med. Biol. 107 (1978) 661-672. Tanzer, J.M,, Freedman, M.L. and Fitzgerald, R.J.: Virulence of mutants defective in glucosyltransferase, dextran-mediated aggregation, or dextranase activity. In: Mergenhagen, S.E. and Rosan, B. (Eds.), Molecular Basis of Oral Microbial Adhesion. Am. Soc. Microbiol., Washington, DC, 1985, pp. 204-21 !, Taylor, A.F,: RecBCD enzyme of Escherichia coli. In: Kueherlapati, R. and Smith, G.R, (Eds,), Genetic Recombination. Am. Soc. Microbiol., Washington, DC, 1988, pp. 231-263. Ueda, $. and Kuramitsu, H.K.: Molecular basis for the spontaneous generation of colonization.defectivemutants of Streptococcus mutans. Mol. Microbiol. 2 (1988) 135-140. Ueda, S., Shiroza, T. and Kuramitsu, H.K.: Sequence analysis of the gtfC gene from Streptococcus mutans G S-5. Gene 69 (198~,) 101-109. Vcnkatesh, "I'.V., Reddy, M,A. and Das, H,K.: Cloning and characterization of the Azotobacter vinelamlii recA gene and construction of a recA deletion mutant. Mol. Gen. Genet. 224 (1990) 482-486. Wahl, G.M,, Stern, M. and Stark, G.R.: Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl,paperand rapid hybridization by using dextran sulfate. Prec. Natl. Acad. Sci. USA 76 (1979) 3683-3687. Yagi, Y. and Clewell, D.B.: Recombination-deficient mutant of Streptococcusfaecalis. J. Baeterioi. 143 (1980) 966-970.
35
GENE 06483
In vivo inactivation of the Streptococcus mutans recA gene mediated by PCR amplification and cloning of a recA DNA fragment (Streptococci; re~.:ombination; RecA; bacterial virulence factors; oral pathogens)
Robert G. Quivey Jr. and Roberta C. Faustoferri Department of Dental Research and Cariology Center. University of Rochester School of Medicine and Dentistry. Rociwster. NY 14642 (USA)
Received by R.E. Yasbin: 29 April 1991; Revised/Accepted: 2 January/10 February 1992; Received at publishers: 5 March 1992
SUMMARY
The inactivation of the RecA protein in pathogenic oral streptococci would facilitate genetic analysis of potential virulence factors in these strains. Comparison of recA nucleotide (nt) sequences from a number of bacteria has suggested that two regions of highly conserved RecA amino acid (aa) sequence could be used as a basis for synthesizing degenerate oligodeoxyribonucleotide primers with which to amplify recA homologues from the streptococci. Accordingly, primer mixtures were used to amplify a 693-bp fragment of the Streptococcus mutans chromosome by PCR. The amplified fragment was cloned and its identity confirmed via hybridization to an Escherichia coli recA gene probe and by nt sequence determination. The recA homologue fragment from S. mutans GS-5 was 63% and 75% homologous to the deduced aa sequences of the E. coli and Bacillus subtilis RecA enzymes, respectively. The S. mutans recA fragment was mutagenized in vitro via insertional inactivation and returned to the chromosome using allelic exchange. The resulting strains of S. mutans were shown to be substantially more sensitive to UV irradiation than the wild-type strain. Further, the ability to incorporate linear markers into the chromosome was abolished in putative S. mutans recA strains, thus indicating the functional inactivation of RecA in these microorganisms.
INTRODUCTION
The enzyme primarily responsible for the homologous recombination of chromosomal DNA in bacteria is the RecA protein (for review see Miller and Kokjohn, 1990). The gene encoding the recombinase, recA, has been cloned
Correspmldence to: Dr. R.G. Quivey, Department of Dental Research, University of Rochester School of Medicine and Dentistry, Box 611,601 EImwood Ave., Rochester, NY 14642 (USA) Tel. (716)275-1435; Fax (716)473-2679.
Abbreviations: A., Anabaena; aa, amino acid(s); Ap, ampiciilin; Aq., Aquaspiriilum; B., Bacillus; Bop'., Bordetella; BHI, Brain Heart Infusion (medium); bp, base pair(s); BSA, bovine serum albumin; Denhardt's solution, 0.02% Ficoll/0.02% polyvinylpyrrolidone-40/0.02% BSA; dNTP, deoxyribonucleoside triphosphate; E., Escherichia; Er, erythromy-
from numerous bacteria and their nt sequences determined. The goals of several of these studies have been the use of the cloned gene to generate site-directed RecA-deficient mutant strains with which to facilitate further genetic manipulations and to probe the role of homologous DNA recombination in bacterial virulence (Goldberg and
cin; fif, gene encoding fructosyltransferase; GTF, glucosyltransferase; gt.f, gene encoding GTF; kb, kilobase(s) or 1000 bp; Kin, kanamycin; LB, Luria-Bertani (medium); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ori, origin of DNA replication; P., Proteus; PCR, polymerase chain reaction; Is., Pseudomonas; RecA, recombination enzyme; recA, gene encoding RecA; R, resistance/resistant; S., Streptococcus; SDS, sodium dodecyl sulfate; Se., Serratia; SSC, 0.15 M NaCI/0.015 M Na3,citrate pH 7.6; T., Thiobacillus; Taq, Thermus aquaticus; TBE buffer, 89mM Tris.HCl/89 mM boric acid/9 mM Na2'EDTA pH 8: Tc, tetracycline; UV, ultraviolet; wt, wild type.
36 Mekanlanos, 1986; Koomey and Falkow, 1987; Venkatesh et al.. 1990). Recently, evidence has been provided that suggests that RecA protein may play a role in the high-frequency recombination between the g@? and gtfC genes in S. mutans (&da and Kuramitsu, 1988; Sun et al., 1991). The gtf genes (B, C and D) encode glucosyltransferase enzymes that are responsible for the synthesis of extracellular glucan polymers in dental plaque and are thought to be virulence factors in the etiology of dental caries. Mutants of S. mutans defective in the ability to produce water-insoluble polymers (formed by the action of the B and C gene products) appear to be noncariogenic in experimental animals (Clark et al., 1978; Tanzer and Freedman, 1978; Tanzer et al., 1985; Munro et al., 19!>1).The genes encoding the GTF enzymes have been cloned, and their nt sequences have indicated that the B and C genes are highly homologous to each other (Shiroza et al., 1987; Ueda et al., 1988, respectively). Recombination between homologous regions of the gfl genes results in the apparent inactivation of the GTF-I and GTF-SI proteins, leaving only a functionally active GTFS protein from the g@ gene (Ueda and Kuramitsu, 1988). Strains exhibiting only the GTF-S activity appear to be no longer cariogenic in rats (Munro et al., 1991). The role of RecA is unknown, but data indicate that the recombination between homologous regions of the gtf genes is not precise (Sun et al., 1991). Thus, recombination results in strains containing different products, thereby suggesting the absence of a site-specific recombinase for the gg genes. In order to pursue observations of g/@-C recombination and to extend our abilities to understand the genetic regulation of virulence factors in the oral bacteria, we have investigated the possibility of isolating site-directed mutations in the vecA-homologue from Srveprococcus mutans. Previous reports have suggested some difficulty in cloning complete n&homologues from Gram - organisms (Rhee and Morrison, 1988; Marrero and Yasbin, 1988), even though the enzyme from apparently distant sources can complement vecA-defective strains of E. coli (Murphy et al., 1990). The polymerase chain reaction (Saiki et al., 1985) appeared to offer the ability to amplify a substantial partion of the gene without necessarily amplifying regulatory regions associated with the coding region. Recently, we have used degenerate mixtures of oligos to amplify a gene fragment encoding the catalytic subunit of the streptococCal membrane-bound, proton-translocating ATPase (I-I + ATPase) (Quivey et al., 1991). Similarly, an examination of the published nt sequences from a number of recA homologues suggested that oligos based on highly conserved aa sequences could be used to amplify a substantial portion of the streptococcal recA homologue.
RESULTS AND DISCUSSION
(a) PCR amplification of the Streptococcus mutuns recA homologue The invariant regions in RecA proteins, shown boxed in Fig. 1, provided the basis for the synthesis of degenerate oligos to be used in priming the PCR amplification of the streptococcal homologue. The forward primer, Val-GluIle-Tyr-Gly-Pro-Glu, was based on the aa sequence starting at Va164of the E. cofi protein. The complementary or reverse primer, Gly-Ala-Trp-Tyr-Ser-Tyr, was based on the aa sequence starting at Gly286 of the E. coli protein (Sancar et al., 1980). At positions of 2-nt degeneracy, both nt were included in the synthetic mix; at positions of 3- or 4-nt degeneracy, deoxyinosine was inserted. The resulting oligo mixtures contained four different primers in the forward direction and eight in the reverse direction. Both primers contained restriction sites that had been incorporated into the oligo sequences (see legend for Fig. 2). The expected size for the PCR products, utilizing the E. coli recA gene as a template, was approx. 690 bp. The products of both the S. mutuns and E. co& amplifications were in the 700.,bp range, indicating a successful amplification using the degenerate primers described above (Fig. 2). The primers used to direct PCR amplification contained a Hind111 site in the forward primer and an SstI site in the reverse primer. However, attempts to clone the S. mutarts-derived fragment hybridizing to the E. coli probe failed when the amplified DNA was digested with HindIII+SstI to take advantage of the programmed restriction sites. Subsequently, the amplified, putative S. mutans recA DNA fragment was cloned into the vector pCRlOO0 (InVitrogen, La Jolla, CA) by taking advantage of the 3’adenosines placed on blunt-ended DNA fragments by Tuq
64 + ii. coli EIYGPESSCKTTLT B. sublilis EVYGPESSGKTTVA S. mwcesceus EIYGPESSGKTTLT A. variclbills EIYGPESSGKTTVA R mirabilis EIYGPES~GKTTLT Ps. aeruginosaEIYGPESSGKTTLT T. ferroridans EIYGPESSGKTTLT Bar.perfussis B IYGPESSGKTTLT Aq. mognetotacticumE IYGPESSGKTTLA Synechococcus sp. EIYGPESSGKTTVA
Fig. 3. Alignmcnl of highly conserved aa from bacterial RecA and RccE (E).subrilis) protein sequences. The boxes contain sequences that are completely conserved. Numbers indicate the position of the aa in the E. coli protein. Sources of the sequences were as follows: E. co/i (Sancar et al., i 980); f3. subtilis(Stranathan et al., 1990); Bor. pertussis (Favre and Viret, 1990); A. variabilis (Owttrim and Coleman, 1989); Aq. tnagneto~acticum (Berson et al., 1990); Ps. aerugirzosaPA0 (San0 and Kageyman, 1987); P. tnirabilis (Akaboshi et al., 1989); T. ferrooxidatts (Ramesar et al., 1989); S. marcescetts (Ball et al., 1990); S~~echococcus sp. (Murphy et al., 1990).
37
A
B 1
12
2
3
C 4
S
1
2
3
4
S
345
bp
---- 3 0 5 4
--4072 -- 3054 --2036 --1636 --1018 --1018 -- 506
Fig. 2. PCR amplification of recA.homologue from $. mutans and hybridization of E. coil and S. muums amplified recA fragments to S. mutans GS-5 chromosomal DNA. (Panel A) Electrophoretogram of PCR-amplified recA fragments from E. coli and S, mutans and cloned S. mutans PCR-amplified recA fragment. Lanes: I, 693-bp PCR-amplified fragment of E. coil recA from plasmid pDRI453 carrying the intact E. coil recA gene (Sancar and Rupp, 1979); 2, PCR-amplified S. mutans chromosomal DNA; 3, HindIll-digested pRQ201, containing a clone of the S. mutans 693-bp PCR-amplified fragment; 4, HindlII-digested chromosomal DNA; 5, one-kb DNA ladder (Life Technologies/BRL, Gaithersburg, MD). (Panel B) Hybridization ofradio-labeled E. coil recA fragment to the blot shown in panel A. (Panel C) Hybridization of radio-labeled S. mutans recA fragment to the blot shown in panel A. Methods. Genomic DNA from S. mutans was prepared as described previously (Chassy, 1976). PCR (Saiki et al., 1985) amplification of DNA was accomplished using a GeneAmp T M kit (Perkin Elmer/Cetus, Norwich. CT). Final reaction concentration of reagents was as follows: 200 mM dNTPs/4 mM MgCl2/ 50 mM KCI/10 mM Tris.HCI pH 8.3/1% (w/v) BSA/5 .,nits of Taq polymerase/1 unit of Perfect Match Enhancer (Stratagene, La Jolla, CA). For amplification of S. mutans G S-5 genomic DNA, 10 sg of DNA was added to each reaction with 5 pmol of each primer. Amplification of plasmid pDR 1453 was similar except that ! ng of plasmid was used as template and Perfect Match Enhancer was not included in the reaction. The forward primer sequence, based on the information shown in Fig. 2, was 5'-GGCCAGTAAGCTTGGTIGARATITAYGGICCIGA, where the singly underlined sequence corresponds to the conserved RecA aa sequence V-E-I-Y-G-P-E and the doubly underlined sequence is a programmed HindIII site. The reverse primer sequence was 5'-GTAGGTCCAGAGCTCTAISWRTACCAIGCIGC, where the singly underlined sequence corresponds to the conserved RecA aa sequence G-A-W-Y-S-Y and the doubly underlined sequence represents a programmed SstI site. Uncommon nt in the sequences shown here were defined as follows: A or G = R; A or T = W, T or C = Y; G or C = S. Amplification was carried out in a programmable heater (Perkin Elmer/Cetus, Norwich, CT) using the following cycling regime: 2 min at 94°C for denaturation; 2 min at 55°C for primer annealing; and 2 min at 72°C for strand synthesis. Typically, 35 cycles were completed. Plasmid pRQ201 was constructed by cloning the putative, amplified S. mutans recA DNA fragment into plasmid pCRI000 (lnVitrogen, La Jolla, CA). Plasmid DNA was prepared using an alkaline lysate procedure as before (Quivey et al., 1991). DNAs were separated on 1% agarose gels in TBE buffer (Peacock and Dingman, 1968) and transferred to nitrocellulose membranes by capillary transfer. Gels containing genomic DNA fragments were acid-treated as described by Wahl et al. (1979) and denatured as described by Southern (1975). Following membranetransfer, the DNAs were cross-linked to membranes via UV irradiation (Reed and Mann, 1985) using a Stratalinker-1800 as recommended by the manufacturer (Stratagene, La Jolla, CA). The film was exposed to the hybridized blot for I h. In order to reduce the possibility of false-positive hybridization between probe-vector sequences and chromosomal DNA, amplified recA fragments from plasmid pDRI453 (representing the E. coil recA gene) and S. muums chromosome were purified from agarose gels as described (Heery et al., 1990) and radioactively labeled for use as probes via random-hexamer priming with [~.3.,p ]dATP (Feinberg and Vogelstein, 1983; 1984), using commercially available kits (BRL, Gaithersburg, MD). Hybridizations were done at 55°C in solutions containing 5 x Denhardt's solution (Denhardt, 1966)/5 x SSC/100#g/ml denatured salmon sperm DNA. Following overnight incubation, blots were washed as follows: two 15 min washes, each at room temperature, in I x SSC buffer, containing 1 x Denhardt's solution, followed by two additional 55°C washes for 30 min each in ! x SSC, containing 0.1% SDS. Blots containing hybridized material were air-dried, mounted on Whatman 3 mm paper (VWR Scientific, Rochester, NY), over-layed with Kodak X-Omat AR X-ray film (Eastman Kodak Co., Rochester, NY), placed in film cassettes with intensifying screens (Cronex, DuPont and Co., Wilmington, DE) and stored at -70°C until development. All restriction enzymes, T4 DNA ligase and agarose were purchased from Life Technologies/BRL (Gaithersburg, MD). Common reagents were purchased from the Sigma Chemical Co. (St. Louis, MO). [a-32P]dATP used in DNA probe-labeling was obtained from Amersham (Arlington Heights, IL) at a specific activity of 3000 Ci/mmol.
p o l y m e r a s e (Clark, 1988). H i n d l l l digestion o f o n e resulting clone, referred to as p R Q 2 0 1 , indicated an internal H i n d I I I site in the amplified fragment. T h u s , the digestion o f the p l a s m i d p R Q 2 0 1 D N A w i t h H i n d I I I (lane 3 o f Fig. 2, p a n e l s B a n d C ) s h o w e d t w o b a n d s , b o t h o f w h i c h hybrid-
ized to the S. r o m a n s recA probe. T h e upper b a n d a p p a r ently c o n s i s t e d o f vector plus insert D N A with the lower b a n d c o n s i s t i n g o f an insert f r a g m e n t that is b o u n d e d o n o n e side by the internal H i n d I I I site a n d on the other e n d by the p r o g r a m m e d H i n d I I I site provided for by the for-
38 10
20
30
40
50
60
70
.
*
*
*
*
*
*
GGG CCG GAA TCT TCT GGT AAG ACA ACT GTC GCT CTT CAT GCT GCT GCT CAG GCG CAA AAA GAT GGC GGT ATT GCC GCT G P E S S G K T T V A L H A A A Q A Q K D G G I A A> 80
90
*
*
100
110
*
120
*
130
*
140
*
150
*
*
TTC ATT GAT GCA GAA CAT GCC CTT GAT CCA GCC TAT GCT GCT GCT CTT GGC GTT AAT ATT GAT GAG CTT TTG CTT TCA F I D A E H A L D P A Y A A A L G V N 7 D E L L L S> 160
170
180
*
*
*
190
200
*
210
220
*
*
*
230 *
CAA CCA GAT TCA GGA GAA CAG GGT CTT GAA ATT GCA GGG AAA TTG ATT GAT TCT GGC GCT GTT GAT TTA GTT GTT GTT Q P D S G E Q G L E I A G K L I D S G A V D L V V V> 240
250
260
270
280
290
300
310
*
*
*
*
*
*
*
.
GAC TCA GTG GCA GCT TTA GTA CCA CGT GCG GAG ATT GAC GGA GAT ATT GGT AAT AGT CAT GTT GGC TTA CAA G~A CGC D S V A A L V P R A E I D G D I G N S H V G L Q A R> 320
330
*
340
*
350
*
360
*
370
*
380
*
390
*
.
ATG ATG AGT CAA GCG ATG CGT AAA TTA TCA GCT TCA ATC AAT AAA ACA AAA ACC ATT GCT ATT TTT ATT AAT CAA TTG M M S Q A M R K L S A S I N K T K T I A I F I N Q L> 400
410
*
420
*
430
*
440
*
450
*
460
*
*
CGG GAA AAA GTT GGT ATT ATG TTT GGT AAT CCA GAA ACA ACC CCT GGC GGG CGT GCC TTG AAG TTT TAT TCT TCT GTG R E K V G I M F G N P E T T P G G R A L K F Y S S V>
470
480
490
500
510
520
530
540
CGT CTT GAT GTC CGC GGC AAT ACT CAA ATT AAA GGA ACC GGG GAA CAA AAA GAC AGC AAT ATT GGT AAA GAG ACC AKA R L D V R G N T Q I K G T G E Q K D S N I G K E T K> 550
560
*
570
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ATT AAA GTT GTT AAA AAT AAA GTT GCT CCA CCA TTT AAG GAA GCT TTT GTA GAA ATT ATA TAT GGT GAA GGC ATT TCT K V V K N K V A P P F K E A F V E I I Y G E G I S> 630
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Fig. 3. Nucleotide sequence of the cloned 693-bp recA homologue from S mutans GS-5, and the deduced aa sequence. The nt sequence of the amplified and cloned recA fragment from S. muums GS-5 was determined via double-stranded DNA sequencing from CsCI density-gradient-purified templates as described previously (Tabor and Richardson, 1987) using the Sequenase 2.0 kit from US Biochemicals (Clevehmd, OH). The nt sequence data reported in this paper appears in the EMBL, GenBank and DDBJ Nueleotide Sequence Databases under the accession No. M61897.
1 S. mutanB B. sub,ills E. ¢011
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Fig. 4, Deduced aa sequence ofthe cloned, amplified S, muta,s recA-homologue, shown with the homologues from E. coli and B. subtilis Rec ~, Conserved aa sequences are boxed.
39 (b) Cloning and nt sequence of the Streptococcus mutans recA gene fragment The nt sequence of the cloned fragment, shown in Fig. 3, confirmed the similarity of the S. mutans recA fragment to the homologous regions found in E. coli (Sancar et al., 1980) and B. subtilis (Strathanan et al., 1990). Fig. 4 shows the deduced aa sequence alignments of homologous regions from the S. mutans, E. coil and the B. subtilis recA genes. Of the 230 aa shown, S. mutans and E. coli are identical at 145 (63% homologous), whereas S. mutans and B. subtilis are conserved at 173 residues (75% homolo-
ward PCR primer. The arrangement of Hindlll sites was later confirmed by nt sequence determination (see below). Hybridization of the S. mutans genomic DNA amplification products to the E. coil recA probe suggested that the amplification product of the streptococcal DNA was indeed an S. mutans recA-homologue (Fig. 2). Interestingly, the E. coil recA probe did not hybridize to that portion of the amplified S. mutans DNA still bound to the vector under the washing stringencies used, indicating that the nt sequences were dissimilar.
/acZ'
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Fig. 5. Insertional inactivation scheme for the generation of in vivo recA mutations in S. mutans UAI59. Fill-in reactions were performed with T4 DNA polymerase and used according to the manufacturer's (BRL/LTI, Gaithersburg, MD) instructions. Transformation of S. mutans UA ! 59 was accomplished using a previously described protocol (Perry and Kuramitsu, 1981). Transformations were performed in the dark. Symbols: for all plasmids, the stippled arrow represents the ~-complementation region of the lacZ gene of E. coli; the construction of pTS 19E has been described (Aoki et al., 1986); the construction of pRQ201 and pRQ202 is described in sections b and c, respectively;the solid bar in plasmids pRQ201 and pRQ202 represents the S. mutans recA PCR fragment described in section b. The arrow associated with the solid bar indicates the direction of transcription ('sense strand') for the PCR fragment. Antibiotic resistance genes carried on the various plasmids and plasmid origins of replication are indicated on the maps as open bars or open arrows.
40 gous). Of the differing residues between B. subtilis and S. mutans, many are unaltered in their respective charges, suggesting a high degree of structural conservation between the two enzymes. (c) Insertionai inactivation Streptococcus mutans
of
the
recA
gene
Probed with pRO201
Probed with pTSIgE
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6
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The nt sequence of the cloned fragment indicated the presence of a unique HincII at bp 251. Thus, the strategy to construct a recA strain of S. mutans consisted of the insertion of an Er R element from plasmid pTSl9E (Aoki et al., 1986). The 1.8-bp B a m H I fragment containing the Er a marker was excised from pTS 19E and cloned into the Hincll site of pRQ201 as shown schematically in Fig. 5. The resulting plasmid, pRQ202, was digested with Pstl and used to transform S. mutans UA159 to Er R. Strain UA159 was selected for these experiments because it has been shown to be transformable with plasmid and chromosomal markers and has been shown to be highly cariogenic in animal models (Murchison etal., 1986). Chromosomal DNA from Er R transformants was examined for integration of the Er e marker and alteration of the HindIII. digestion pattern via Southern (1975) blotting. Fig. 6 shows the results of the hybridization of plasmids pTS 19E (containing the Er R marker) and pRQ201 (containing the putative recA gene fragment clone) to Hindlll-digested chromosomal DNA prepared from the prototroph and two potentially mutant strains. The autoradiogram resulting from the Southern blot shows the wt chromosome with two distinctive Hindlll bands at approx. 1600 and 4000 bp that hybridize strongly to pRQ201, containing the recA gone fragment. The two putative mutant strains showed the loss of the 1600-bp band and the appearance of a strongly hybridizing band at approx. 6000 bp. When digests were probed with pTSI9E containing the Er e marker, the wt DNA showed no homology with the probe (Fig. 6, lane 10) and the 1600-bp band seen with the E. coil recA probe was not visible; whereas Er e marker homology was seen with the 4000-bp and 6000-bp bands in the putative mutant strains. We concluded from this experiment that the recA allele and Er e marker were physically linked as a result of recombination of the insertionally-disrupted recA fragment into the chromosomes of these strains via allelic exchange. To further characterize the putative mutant strains, one organism, designated URI00, was selected for study. Sensitivity to UV light has been used previously to characterize, among other bacteria, putative recombination-defective strains of Streptococcus faecalis (Yagi and Clewell, 1980) and Streptococcus sanguis (Dance-Moore and Volpe, 1985). Strains UA159 and URI00 were irradiated in a programmable UV light source for varying times and plated on rich medium for enumeration of viable bacteria. The results of irradiation, shown in Fig. 7, were quite similar to those
bp 12216
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Fig. 6. Southernhybridization ofwt and putativerecA mutationsprobed with pRQ201 and pTS 19E. Plasmid and chromosomal DNAs were prepared as described above in the legend to Fig. 2. 10#g of chromosomal DNAs were digested with Hindlll, Plasmid DNAs (i pg) were digested as follows: pRQ201 with HindIll and pTSlgE with BamHI, according to the manufacturer(BRL/LTI, Gaithvrsburg, MD). Blotting,hybridization conditions, washing conditions and autoradiography following hybridization wereas describedin Fig. 2, with the exceptionthat finalwashes were as follows: three 20-rain washes at room temperaturewith 2 x SSC containing0. 1% SDS followedby one 20-minwash at 45°C with 2 x SSC containing 0.1% SDS, Lanes: I, one-kb ladder; 2, HindIII-digested pRQ201; 4, Hindlll-digested S. muums UAI59 genomic DNA; 5o HindlIl.digested S. mutans UR 100 genomic DNA; 6, HindIIi digested S. mutans UR200 genomic DNA; 8, BamHI-digested pTSi9E~ 10, HindIii-digested S. mutans UAI59 genomic DNA: 11, HindIll-digested S. mutmls URI00 genomicDNA: 12, HindIII-digestedS. mutansUR200 genomic DNA; 3, 7, 9, empty. shown earlier for S. sanguis (Daneo-Moore and Volpe, 1985) and indicated clearly that the putative recA mutant, URI00, was more sensitive to UV irradiation than the wt, strain. These results strongly suggested that URI00 was impaired in its ability to repair thymidine-dimers formed as a result of UV irradiation, in turn suggesting a loss of functionally active RecA protein (reviewed in Taylor, 1988). The role of RecBCD complex-homologues in UV damage repair for the streptococci is currently unknown. In order to characterize URI00 for its ability to incorporate chromosomal DNA, transformation experiments were performed using the protocol of Perry and Kuramitsu (1981). Chromosomal DNA was prepared from a strain containing a Tc e marker in the fif gone of S. mutans (obtained from R.A. Burne, University of Rochester) and used to transform UA159 and the putative recA mutant strain UR100. Plasmid pSA3 (Dao and Ferretti, 1985) DNA was
41 100
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formation with plasmid D N A was essentially unaltered in the mutant strain as compared to the wt. However, the frequency of plasmid transformation was substantially lower than that previously reported for strain UA159 (Murchison et al., 1986), and may reflect some differences in handling of the strains or in post-transformation expression time in nonselective medium, prior to plating on selective medium. Nevertheless, we conclude from the inability to incorporate a linear chromosomal marker, that the putative mutant strain UR100, is indeed defective in the RecA protein and that the ability to recombine homologous DNAs in this strain is absent.
"
o'12s Iso 17s
(d) Conclusions
Exposure Time (s) Fig. 7. UV survival of S. mutans strains UAI59 and URi00. Strains UAI59 and URI00 were grown to mid.logarithmic phase in BH! (Difco, Detroit, MI). The cells were sonicated briefly to dechain the organisms (4 x 30 s pulses at 400 W, Braun-Sonic 1510 Braun Biotech, Allentown, PA). Dechained cells were verifiedvisuallyby phase microscopy. Aliquots of cells (1 ml) were transferred into sterile dishes (10 x 30 mm, Falcon) and were irradiated pairwise for varying times as shown in a programmable UV light source (Stratalinker-1800, Stratagene, La Jolla, CA). The source delivers 3000#W/cm-"(Stratagene, La Jolla, CA). Followingirradiation, 0.1 ml aliquots were diluted in 1.0 ml dilution-tubes containing fresh BH! and plated on BHI-agar medium for the outgrowth of colonyforming units. also used to transform the respective strains to TCR. The results, shown in Table I as colony-forming units, indicated that recombination of the chromosomal marker was completely abolished in the putative recA strain. Transformation frequencies with the wt strain were comparable to those reported previously (Murchison et al., 1986). Trans-
(1) The clone of a PCR-amplified fragment of S. m u t a , s GS-5 D N A has been shown by DNA hybridization, nt sequence determination and alignment of deduced aa sequences with known RecA proteins to encode a portion, estimated to be approx. 65 % as compared to E. coil, of the full-length coding region of the recA homologue from S. mutans GS-5. (2) The clone of the S. mutans recA gene fragment has permitted its mutagenesis via insertional inactivation with a 1.8-kb Er R marker. The physical linkage ofthe Er R marker with recA DNA, as shown by Southern blotting along with the behavior of the mutant strains, with regard to UV sensitivity and inability to incorporate chromosomal DNA, strongly indicates that the RecA enzyme activity in these strains has been eliminated or at least, greatly diminished. (3) The construction of recA strains of S. mutans will facilitate genetic regulation studies and the further analysis of the role of RecA in the recombination of the gtf genes. Furthermore, the clone will provide a probe for DNA fragments containing the 5' end ofthe recA gene with attendant regulatory regions.
TABLE I Transformation of S. t;mtans strains
ACKNOWLEDGEMENTS
Strain u
DNA s o u r c e b
Markerc
Transformant frequencyd
UA 159 UR100
S. mutans chr. S. mutans chr.
fff::Tca ftf::Tca
700 0
UAI59 URI00
pSA3 plasmid pSA3 plasmid
TcR TcR
30 20
" Strain UAI59 (Murchison et al., 1986); strain URI00 was constructed in this study (see section e). b chr, chromosomal (genomic) DNA. c Chromosomal DNA containing a TcR marker in the fif gene was obtained from R.A. Burne (Department of Dental Research, University of Rochester). The DNA was prepared as described (Chassy, 1976). d Transformation was essentially as described by Perry and Kuramitsu (1981) using 10 Fg of' DNA/transformation. Transformant frequency is reported as the number of colony-forming units per transformation experiment.
We thank Cynthia Luisi-DeLuca for plasmid pDR1453 and relevant E. coil strains. We also thank Patricia Noonan for assistance with the manuscript preparation. This work was supported by NIH DEO-7003-05 as part of the Rochester Caries Research Center, by BRSG PHS $7RR0540327 and in part by DEO-7777. REFERENCES Akaboshi, E., Yip, M.L.R. and Howard-Flanders, P.: Nucleotide sequence of the recA gene of Proteus mirabilis. Nucleic Acids Res. 17 (1989) 4390. Aoki, H., Shiroza, T., Hayakawa, M., Sate, S. and Kuramitsu, H.K.: Cloning era Streptococcusmutans glucosyltransferasegene coding for insoluble glucan synthesis. Infect. lmmun. 53 (1986)587-594. Ball, T.K., Wasmuth, C.R., Braunagel, S.C. and Benedik, M.J.: Exprcs-
42 sion of Serratia marcescens extraceUular proteins requires recA. J. Bacteriol. 172 (1990) 342-349. Berson, A.E., Peters, M.R. and Waleh, N.S.: Nucleotide sequence of recA gene of Aquaspirilhon magnetotacticum. Nucleic Acids Res. 18 (1990) 675. Chassy, B.M.: A gentle method for the lysis oforal streptococci. Biochem. Biophys. Res. Commun. 68 (1976) 603-608. Clark, J.M.: Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 16 (1988) 9677-9686. Clark, W.B., Bammann, L.L. and Gibbons, R.J.: Ability of Streptococcus mutans and a glucosyltransferase defective mutant to colonize rodents and attach to hydroxyapatite surfaces. Infect. Immun. 21 (1978)681684. Dance-Moore, L. and Volpe, A.: Recombination-deficient Streptococcus sanguis, Infect. lmmun. 48 (1985) 584-586. Dao, M.L. and Ferretti, J.J,: Streptococcus-Escherichia coil shuttle vector pSA3 and its use in the cloning of streptococcal genes. Appl. Environ. Mierobiol. 49 (1985) i 15-119. Denhardt, D.T.: A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23 (1966)641646. Favre, D. and Viret, J.F.: Nuclcotide sequence of the recA gent of Bordetellapertussis. Nucleic Acids Res. 18 (1990)4243. Feinberg, A.P. and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132 (1983) 6-13. Feinberg, A.P. and Vogelstein, B.: Addendum: a technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137 (1984) 266-267. Goldberg, i. and Mekanlanos, J.J.: Cloning of the Vibrio cholerae recA gene and construction of a Vibrio cholerae recA mutant. J. Bacteriol. 165 (1986) 715-722. Heery, D.M., Gannon, F. and Powell, R.: A simple method for subcloning DNA fragments from gel slices, Trends Gcnct. 6 (1990) 173. Koomey, J.M. and Fa!kow, S.: Cloning of the recA gone of Ne~:'seria gom~rrlweae and construction of gonococcal recA mutants. J. Bacteriol. 169 (1987) 790..'N5. Mart ~ro, R and Yasbin, R.E.: Cloning of the Bacillus sl~btilis recE gene and functional expression of recE÷ in B. subtilis. J. Bacteriol. 170 (1988) 33~-344. Miller, R,V, and Kokjohn, T.A.: General microbiology of recA: environm~'ntal and evolutionary significance, Annu. Rev, Microbiol, 44 (1990) 365-394. Munro, C., Michalek, S.M, and Macrina, F.L.: Cariogcnicity of Streptococcus mutans V403 glucosyltransferase and fructosyltransferase mutants constructed by all¢lic exchange, Infect, Immun. 59 (1991) 2316-2323. Murchison, H,H,, Barrett, J.F., Cardineau, G.A. and Curtiss Ill, R.: Transformation of Streptococcus mutans with chromosomal and shuttie plasmid (pYA629) DNAs. Infect. Immun, 54 (1986) 273-282. Murphy, R.C,, Gasparich, G,E., Bryant, D.A. and Porter, R,D.: Nuclcotide sequence and further characterization of the Synechococcus sp. strain PCC 7002 recA gene: complemcntation of a cyanobaeterial recA mutation by the Escherichia coli recA gone, J. Bacteriol, 172 (1990) 967-976, Owttrim, G.W. and Coleman, J,R,: Regulation of expression and nucleotide sequence of the Anabaena variabilis recA gene, J, Bacteriol. 171 (1989) 5713-5719. Peacock, A.C, and Dingman, C.W.: Molecular weight estimation and separation ofribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistq, 7 (1968)668-674. Perry, D. and Kuramitsu, H.K.: Genetic transformation of Streptococcus mutans. Infect. Immun. 32 (1981) 1295-1297.
Quivey Jr., R.G., Faustoferri, R.C., Belli, W.A. and Flores, J,S.: Polymerase chain reaction amplification, cloning, sequence determination and homologies of streptococcal ATPase-encoding DNAs. Gene 97 (1991) 63-68. Ramesar, R.S., Abratt, V., Woods, D.R. and Rawlings, D.E.: Nucleotide sequence and expression of a cloned Thiobacillus ,ferrooxidans recA gene in Escherichia coll. Gene 78 (1989) 1-8. Reed, K.C. and Mann, D.A.: Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 13 (1985) 7207-7221. Rhee, D.K. and Morrison, D.A.: Genetic transformation ir~Streptococcus pneumoniae: molecular cloning and characterization of~recP, a gene required for genetic recombination, J. Bacteriol. 170 (1988)630-637. Saiki, R.K., Scharf, S., Faloona, F., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N.: Enzymatic amplification of g-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230 (1985) 1350-1354. Sancar, A. and Rupp, W.D.: Physical map of the recA gene. Prec. Natl. Acad. Sci. USA 76 (1979) 3144-3148. Sancar, A., Stachelck, C., Konigsberg, W. and Rupp, W.D.: Sequences of the recA gene and protein. Prec. Natl. Acad. Sci. USA 77 (1980) 261 !-2615. Sane, Y. and Kageyama, M.: The sequence and function of the recA gene and its protein in Pseudomonas aeruginosa PAO. Mol. Gen. Genet. 208 (1987) 412-419. Shiroza, T., Ueda, S. and Kuramitsu, H.K.: Sequence analysis ofthe gt.lB gene from Streptococcus mutans. J. Bacteriol. 169 (1987) 4263-4270. Southern, E.M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98 (1975) 503-517. Stranethan, M.C., Bayles, K.W. and Yasbin, R.E.: The nucleotide sequence of the recE÷ gen¢ of Bacillus subtilis. Nucleic Acids Res. 18 (1990) 4249. Sun, S., Dance-Moore, L., Marri, L., Hantman, M. and Shockman, G.D.: Genetic and physiological studies of variants of Streptococcus mutans GS-5 that produce nonmucoid colonies on sucrose-containing media. In: Dunny, G.M., Cleary, P.P. and McKay, L.L. (Eds.), Genetics and Molecular Biology of Streptococci, Lactococci, and Enterococci. Am. Soc. Microt~iol.,Washington, DC, 1991, pp. 256-260. Tabor, S. and Richardson, C.C.: DNA sequence analysis with a modified bacteriophage T7 DNA polymcrase. Prec. Natl. Acad. Sci. USA 84 (! 987) 4767-477 I. Tanzer, J.M. and Freedman, M.L.: Genetic alterations of Streptococcus mutans" virulence. Adv. Expt. Med. Biol. 107 (1978) 661-672. Tanzer, J.M,, Freedman, M.L. and Fitzgerald, R.J.: Virulence of mutants defective in glucosyltransferase, dextran-mediated aggregation, or dextranase activity. In: Mergenhagen, S.E. and Rosan, B. (Eds.), Molecular Basis of Oral Microbial Adhesion. Am. Soc. Microbiol., Washington, DC, 1985, pp. 204-21 !, Taylor, A.F,: RecBCD enzyme of Escherichia coli. In: Kueherlapati, R. and Smith, G.R, (Eds,), Genetic Recombination. Am. Soc. Microbiol., Washington, DC, 1988, pp. 231-263. Ueda, $. and Kuramitsu, H.K.: Molecular basis for the spontaneous generation of colonization.defectivemutants of Streptococcus mutans. Mol. Microbiol. 2 (1988) 135-140. Ueda, S., Shiroza, T. and Kuramitsu, H.K.: Sequence analysis of the gtfC gene from Streptococcus mutans G S-5. Gene 69 (198~,) 101-109. Vcnkatesh, "I'.V., Reddy, M,A. and Das, H,K.: Cloning and characterization of the Azotobacter vinelamlii recA gene and construction of a recA deletion mutant. Mol. Gen. Genet. 224 (1990) 482-486. Wahl, G.M,, Stern, M. and Stark, G.R.: Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl,paperand rapid hybridization by using dextran sulfate. Prec. Natl. Acad. Sci. USA 76 (1979) 3683-3687. Yagi, Y. and Clewell, D.B.: Recombination-deficient mutant of Streptococcusfaecalis. J. Baeterioi. 143 (1980) 966-970.
