Construction and Analysis of a Streptococcus parasanguis recA Mutant: Homologous Recombination Is Not Required for Adhesion in an In Vitro Tooth Surface Model

Abstract

PCR was used to amplify an internal region of the recA gene from Streptococcus parasanguis FW213. The PCR fragment was used as a probe to recover the entire streptococcal recA gene from an S. parasanguis genomic library, and the sequence of the gene was determined. The deduced product of the S. parasanguis recA gene showed a high degree of amino acid identity with other prokaryotic RecA proteins. The cloned recA sequence was disrupted in vitro by insertional mutagenesis, and the mutated allele was then introduced into the S. parasanguis chromosome by homologous recombination. Results of Southern hybridizations confirmed the replacement of the wild-type recA gene with the mutated allele. The recA mutant strain was considerably more sensitive to UV light than the parental strain, and this phenotype was consistent with a mutation in recA. The S. parasanguis recA mutant showed no reduction in its ability to adhere in the in vitro tooth surface model, saliva-coated hydroxylapatite (SHA), or in its ability to express the fimbria-associated adhesin Fap1. These results demonstrate that in vitro attachment of S. parasanguis FW213 to SHA and expression of Fap1 are recA independent.

The sanguis group of streptococci are the primary colonizers of the tooth surface in humans and constitute a major component of dental plaque (16), the biofilm associated with caries (tooth decay) and periodontal disease. In addition, when these oral streptococci gain access to the bloodstream, they can successfully colonize the heart tissue, becoming a major cause of subacute bacterial endocarditis (3). Adherence of oral streptococci to host surfaces is a critical first step in the colonization process, which is facilitated by multiple adhesins expressed on the bacterial cell surface (17). These adhesins are considered to be important factors in determining the success of oral streptococcal colonization and survival within the human host.

Results of studies on Streptococcus parasanguis FW213 indicate that attachment to the tooth surface is mediated by peritrichous surface fimbriae (11, 14, 15). Fimbria-specific polyclonal antiserum inhibits by more than 90% binding of S. parasanguis to an in vitro tooth surface model, i.e., saliva-coated hydroxylapatite (SHA) (11). Wild-type, fimbriated S. parasanguis binds well to SHA, but afimbriated mutants do not (15). The genes encoding two fimbria-associated adhesions, FimA and Fap1, have been cloned and characterized. The fimA gene encodes a 36-kDa lipoprotein that blocks the adherence of S. parasanguis to SHA (13, 22). FimA is an important virulence factor in S. parasanguis endocarditis and may promote adherence to fibrin in cardiac vegetations (4). The fap1 gene encodes a high-molecular-weight protein that is involved in adhesion to SHA and appears to be important in the assembly of fimbriae (31).

The RecA protein of Escherichia coli plays a pivotal role in both homologous recombination (5) and DNA repair (29). RecA is required by some pathogenic bacteria for expression of virulence factors, particularly if a recombination step is involved in such expression. Some examples in which RecA is required for expression of virulence traits include adherence and colonization factors of Vibrio cholerae (19) and the different pilus types in Neisseria gonorrhoeae (18).

A recombination-deficient strain of S. parasanguis would be useful for further genetic studies with this organism, e.g., in complementation analyses. Such a mutant also would be useful to assess the role of homologous recombination in the expression of S. parasanguis adherence factors. For example, the genetic locus encoding the fimbria-associated adhesin Fap1 contains extensive repeat regions (GenBank accession no. AF100426), and recombination may play a role in the expression of Fap1.

Accumulating evidence indicates that the recA gene is ubiquitous in prokaryotes and that analogous RecA proteins function similarly in different bacterial species (21, 23). Interspecific complementation of an E. coli recA mutant has been used to identify the recA genes of many gram-negative bacteria, but this approach has not been successful with gram-positive bacteria (8). The PCR has been used successfully to amplify recA sequences from several gram-positive bacteria and mycoplasms (7, 9). In the present study, a PCR-amplified DNA fragment internal to the recA gene was used as a probe to recover the entire recA gene from an S. parasanguis FW213 genomic library. Once identified, the cloned recA gene was disrupted in vitro, and the mutated allele was introduced into the chromosome to create an S. parasanguis recA mutant. The mutant was assessed for RecA function as well as for its ability to adhere to SHA and to express the fimbria-associated adhesin Fap1.

MATERIALS AND METHODS

Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are listed in Table 1. S. parasanguis FW213 (formerly called Streptococcus sanguis) (6) is the parent strain in which a recA mutation was constructed. FW213 possesses peritrichous fimbriae and is adherent to SHA (14). The insertionally inactivated recA strain, VT1354, was constructed in this study. Streptococcal strains were grown statically in the presence of 5% CO₂ at 37°C in Todd-Hewitt broth (TH broth; Difco Laboratories, Detroit, Mich.). Tetracycline (15 μg ml⁻¹) and/or kanamycin (120 μg ml⁻¹) was added as required. E. coli JM109 (32) was used for plasmid propagation. E. coli was grown on Luria-Bertani medium (24), and when required for plasmid selection, ampicillin (100 μg ml⁻¹), tetracycline (15 μg ml⁻¹), and/or kanamycin (25 μg ml⁻¹) was included. Agar was added to a final concentration of 1.5% to prepare solid medium.

TABLE 1.

Bacterial strains and plasmids used in this study

Strain or plasmid	Relevant characteristics	Source or reference
Strains
S. parasanguis
FW213	Wild type	6
VT1354	FW213 recA::aphA-3; Kmr	This study
E. coli JM109	Host strain for cloning	32; ATCCa
Plasmids
pT7Blue	PCR cloning vector; Apr	Novagen
pVT1185	640-bp PCR-derived recA fragment ligated into “T” cloning site of pT7 Blue; Apr	This study
pVT1148	pVT1185 with kanamycin resistance gene inserted at EcoRV site of recA fragment; Apr Kmr	This study
pVA981	Replicates in E. coli but not in streptococci; Tcr	26
pVT1290	2.2-kb insert from pVT1148 containing disrupted recA fragment ligated into pVA981; Kmr Tcr	This study
pVT1356	3.1-kb HindIII-EcoRI fragment containing entire recA gene ligated into HindIII-EcoRI sites of pT7Blue; Apr	This study

^aATCC, American Type Culture Collection. 

The plasmid pT7Blue T-Vector (Novagen, Madison, Wis.) was used for cloning gel-purified PCR products. pVT1185 was generated by ligating a 640-bp PCR product containing internal S. parasanguis recA sequences into the “T” cloning site of pT7Blue. The cloned recA sequence was disrupted in pVT1185 at a unique EcoRV site by restriction endonuclease digestion followed by the cloning of a 1.5-kb fragment containing a gene encoding a streptococcal type III 3′-5′-aminoglycoside phosphotransferase (aphA-3 [28]) into the EcoRV site by blunt-end ligation. The plasmid selected after this procedure, pVT1148, contained aphA-3 inserted into the recA gene in the opposite orientation. The vector used to deliver the mutated recA sequence into the S. parasanguis chromosome was plasmid pVA981 (26), a 7.1-kb pBR325 derivative carrying a tetracycline resistance (Tc^r) determinant active in both E. coli and streptococci. The mutated recA sequence was excised from pVT1148 and cloned into the EcoRI site of pVA981 by blunt-end ligation, creating pVT1290.

DNA manipulations.

DNA manipulations and other molecular biology techniques were carried out essentially as described previously (24). DNA probes used in Southern or colony hybridization analyses were radiolabeled with [α-³²P]dCTP by nick translation with a commercially available kit (Gibco BRL, Grand Island, N.Y.). Procedures for filter hybridizations were performed as suggested by the membrane manufacturer (Amersham Corp., Arlington Heights, Ill.) and included high-stringency washes. The Puregene DNA isolation kit (Gentra Systems, Inc., Minneapolis, Minn.) was used for the isolation of S. parasanguis genomic DNA.

Southern hybridization analysis of chromosomal DNA digested with several restriction endonucleases was used to construct a physical map of the region surrounding the S. parasanguis recA gene. A ca. 3.1-kb EcoRI-HindIII fragment, containing the recA gene, was identified by hybridization with the labeled pVT1185 probe. Subsequently, a partial S. parasanguis genomic library containing 2.3- to 4.4-kb EcoRI-HindIII fragments was constructed. Purified fragments were ligated into pT7Blue, and the resulting gene bank was transformed into E. coli JM109 cells. Clones carrying the recA gene were identified by colony hybridization analysis (24). The plasmid hybridizing to the pVT1185 probe was designated pVT1356.

DNA sequencing was carried out at the Vermont Cancer Center, DNA Analysis Facility, at the University of Vermont. Nucleotide and protein similarity searches were done with the BLAST, BLASTN, and BLASTX programs (1) via the GenomeNet WWW server. Pairwise and multiple protein sequence alignments were done with the ALIGN (30) and CLUSTALW (25) programs, respectively. Predicted RecA protein sequences from Streptococcus pneumoniae, Lactococcus lactis, Bacillus subtilis, E. coli, Streptococcus mutans, and Streptococcus pyogenes were derived from nucleotide sequences with accession numbers Z17307, M88106, X52132, V00328, M61879, and U21934, respectively.

Oligonucleotides and PCR conditions.

Conserved domains in gram-positive RecA proteins provided the basis for the synthesis of degenerate oligonucleotide primers to be used in a PCR. The DNA sequence of the coding strand primer, based on the conserved RecA protein domain 76-Glu-Ile-Tyr-Gly-Pro-Glu-Ser-Ser-Gly-84 (numbering corresponds to S. pneumoniae RecA sequence), is 5′-GAAATCTA(C,T)GG(A,T,G)CC(A,G)GA(A,G)TCTTCT-3′. The DNA sequence of the complementary strand primer, based on the conserved RecA protein domain 282-Gly-Glu-Gly-Ile-Ser-(Lys,Arg)-Thr-288, is 5′-GG(G,T)GAAGG(C,T,A)ATTTCTCGTAC-3′. DNA amplification was performed in a Perkin-Elmer 9600 thermocycler (Perkin-Elmer Cetus, Norwalk, Conn.) with the GeneAmp PCR reagent kit (Perkin-Elmer Cetus) according to the manufacturer’s directions. Approximately 0.5 μg of S. parasanguis FW213 genomic DNA was used as a template. The PCR conditions were as follows: one cycle at 94°C for 3 min followed by 35 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C. The amplification reaction was completed with an additional cycle of 3 min at 72°C. The PCR amplification product was purified from a 2% (wt/vol) agarose gel with the QIAquick gel extraction kit (Qiagen, Inc.) as described by the supplier.

Electrotransformation of E. coli and S. parasanguis.

E. coli cells were prepared for electroporation as previously described (2). Cells (50 μl) plus DNA were electroporated in a 0.2-cm-gap-size cuvette in a Bio-Rad gene pulser set at 2.5 kV, 25 μF, and 200 Ω. Immediately following electroporation, 360 μl of SOC medium (24) was added to the cuvette. Cells were transferred to a 1.5-ml microcentrifuge tube and incubated at 37°C for 1 h. Cells were then spread on Luria-Bertani agar plates supplemented with appropriate antibiotics and incubated overnight at 37°C.

Electrotransformation of S. parasanguis cells with plasmid DNA was performed as previously described (12). Following electroporation, 350 μl of recovery medium was added immediately to the cuvette. Cells were transferred to a 1.5-ml microcentrifuge tube and incubated at 37°C in 5% CO₂ for 2 h. The cells were then spread on TH agar plates supplemented with appropriate antibiotics and incubated as described above for 24 to 48 h.

UV light sensitivity test.

Overnight cultures of S. parasanguis FW213 and VT1354 were diluted 1:50 in 10 ml of TH broth and incubated statically at 37°C in 5% CO₂ to mid-logarithmic growth phase. Cells were diluted 1:50 in phosphate-buffered saline, and chains of the microorganism were disrupted by sonication (four times for 15 s each time) at 80 to 85 W in a Bronson sonifier with an ultrasonic cuphorn. Two-milliliter aliquots of cells were transferred to sterile 35-mm-diameter by 10-mm-depth petri dishes and irradiated under a germicidal lamp (15 W, model G15T8; General Electric) at a distance of 25 cm for various periods of time. During irradiation, cells were mixed gently with a stir bar. Immediately following irradiation, cells were spread at appropriate dilutions on TH agar plates and incubated at 37°C in 5% CO₂. Nonirradiated cells served as a control. After 2 days of incubation, UV sensitivity was evaluated by colony counting.

Adhesion of S. parasanguis to SHA.

Preparation of hydroxylapatite beads, clarification of saliva, preparation of SHA beads, and the adhesion assay used have been described previously (11). Data were obtained from two independent one-point adhesion assays (15) performed in triplicate.

Quantification of Fap1.

Surface expression of the fimbria-associated protein Fap1 was determined for S. parasanguis strains by a whole-bacterial-cell enzyme-linked immunosorbent assay (10) as described previously (31). Anti-Fap1 mouse monoclonal antibody, MAb F51 (11), was used as the primary antibody, followed by incubation with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.). Color development was quantified by measurement of absorbance at 490 nm with an EL311 Automated microplate reader (BIO-TEK, Instruments, Inc., Winooski, Vt.).

SDS-polyacrylamide gel electrophoresis and immunoblot analysis.

S. parasanguis cultures were grown to late-logarithmic phase (5 × 10⁸ cells ml⁻¹), and cells from 1-ml aliquots were harvested by centrifugation. Bacterial pellets were resuspended in 100 μl of sample buffer (62.5 mM Tris-HCl [pH 6.8], 10% glycerol, 2% sodium dodecyl sulfate [SDS], 5% β-mercaptoethanol), and 15 μl samples were analyzed by SDS–7.5% polyacrylamide gel electrophoresis (20). Size-separated proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, N.H.) and examined by Western blotting (27). The membranes were blocked with a 5% (wt/vol) nonfat milk solution, probed with MAb F51, and then incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibody as described previously (31). Antibody conjugates were detected with a chemiluminescence system as described by the manufacturer (NEN Life Science Products, Boston, Mass.).

Nucleotide sequence accession number.

The nucleotide sequence GenBank accession no. is AF069745.

RESULTS AND DISCUSSION

Isolation of the S. parasanguis recA gene.

An S. parasanguis recA mutant was required to facilitate further genetic studies and to assess the role of recombination in the expression of adherence factors in this organism. The cloning and characterization of the recA gene of S. parasanguis was the first step in the construction of such a mutant.

First, degenerate oligonucleotide primers were used in a PCR to amplify an internal fragment of the S. parasanguis recA gene. The predicted amino acid sequences of previously characterized recA genes from five gram-positive bacteria, B. subtilis, L. lactis, S. mutans, S. pneumoniae, and S. pyogenes, were aligned. Two well-conserved domains, approximately 196 amino acids apart, were used to design degenerate oligonucleotide primers. PCR with this primer pair and S. parasanguis genomic DNA resulted in amplification of a 640-bp PCR product. The PCR product was cloned, and the nucleotide base sequences of both strands were determined. The DNA fragment contained an uninterrupted open reading frame encoding a sequence of amino acids that has 98% and 66% identity with the corresponding regions of the S. pneumoniae and E. coli RecA proteins, respectively. This result indicated that an internal portion of the S. parasanguis recA gene had been cloned successfully.

Next, a restriction site map of the S. parasanguis recA region (Fig. 1) was constructed by Southern hybridization analyses and from DNA sequence data obtained from the cloned internal recA fragment. It was determined that the S. parasanguis recA gene resided on a ca. 3.1-kb HindIII-EcoRI fragment (Fig. 1), and subsequently, a partial genomic library of 2.3- to 4.4-kb HindIII-EcoRI fragments was constructed and pVT1356 was identified by colony hybridization. Results of DNA sequence analyses confirmed that pVT1356 contained the entire coding region of S. parasanguis recA as well as regions upstream and downstream of the gene.

FIG. 1.

Restriction map of the S. parasanguis FW213 recA locus. The bar below the map corresponds to the 640-bp PCR-derived recA fragment that was used as a probe. The arrow represents the recA coding region as subsequently derived from DNA sequencing. The boxed EcoRV site indicates the position at which the recA gene was disrupted. Restriction enzymes used are as follows: B, BamHI; E, EcoRI; H, HindIII; R, EcoRV; and S, SphI.

The S. parasanguis recA gene encodes a putative polypeptide composed of 381 amino acid residues with a molecular mass of approximately 41 kDa. The sequence of the deduced S. parasanguis RecA protein was compared to its counterparts from various gram-positive bacteria as well as from E. coli (data not shown). The high level of identity among the aligned amino acid sequences clearly indicates that the proteins are related. Overall, the deduced amino acid sequence of the S. parasanguis recA gene is most similar to the S. pneumoniae RecA sequences, with 90% of the residues being identical. S. parasanguis RecA also has a high degree of similarity with the RecA protein from the gram-negative bacterium E. coli, with 57% of the residues being identical.

Construction of a recA mutant in S. parasanguis.

The isolation of a well-characterized recA mutant could most easily be obtained by disruption of the cloned gene with a selectable marker and reintroduction of the mutated sequence into the chromosome of S. parasanguis. This was done with an efficient gene replacement protocol developed in this laboratory (12). The plasmid used for the recA allelic replacement experiments was pVT1290. Since pVT1290 is unable to replicate in streptococcal cells, recombination with homologous chromosomal DNA must occur for the generation of a recA-deficient strain. Plasmid pVT1290 was transformed into wild-type S. parasanguis FW213 by electroporation. It was expected that transformants arising by allelic replacement would be Km^r and Tc^s; transformants arising from a single crossover event would contain the entire plasmid integrated into the chromosome at the recA region. Such derivatives would be both Km^r and Tc^r and would carry a wild-type copy and a mutant copy of recA. Therefore, Km^r transformants were tested on TH agar plates containing tetracycline. Km^r Tc^s derivatives were tentatively scored as having resulted from an allelic replacement event.

Southern hybridization analysis was used to verify the allelic replacement event. DNA was prepared from several Km^r Tc^s transformants and from the wild-type strain FW213 and digested with BamHI. The DNA fragments were separated by electrophoresis, transferred to a nylon membrane, and hybridized with ³²P-labeled pVT1185, which carries the 640-bp internal recA fragment. DNA from two of the transformants gave a hybridization pattern indicative of allelic replacement (Fig. 2A, lanes 2 and 3). The recA probe hybridized with a single 2.0-kb BamHI restriction fragment in the wild-type strain (Fig. 2A, lanes 1 and 4); the 2.0-kb fragment was absent in the two putative recA mutants and replaced by a band at 3.5 kb.

FIG. 2.

Southern hybridization analysis of the recA region of S. parasanguis wild-type strain, FW213, and of two putative recA-deficient derivatives. Chromosomal DNA from each strain was digested with BamHI and fractionated on a 0.75% agarose gel. DNA fragments were transferred to Hybond-N⁺ membranes (Amersham) and hybridized either to pVT1185 (A) or to the kanamycin resistance determinant aphA-3 (B), which had been ³²P labeled. Lanes 1 and 4 contain FW213 DNA; lanes 2 and 3 contain DNA from two putative recA-deficient derivatives. Sizes in kilobases are noted at the left.

The Southern blot was then stripped and probed with the Km^r determinant aphA-3. The aphA-3 probe hybridized with the new 3.5-kb fragment in the two putative recA mutants (Fig. 2B, lanes 2 and 3) but not with the wild-type strain (Fig. 2B, lanes 1 and 4). Probing with aphA-3 confirmed that the increase in size of the 2.0-kb fragment in the Km^r Tc^s transformants was due to the insertion of the kanamycin resistance determinant at the recA locus. These results confirmed that the disrupted recA allele was successfully integrated into the chromosome of S. parasanguis FW213 in place of the wild-type recA allele.

Phenotypic characterization of the S. parasanguis recA mutant.

One transformant, designated VT1354, was chosen for study. Since recA mutants are expected to be significantly more sensitive to DNA-damaging agents than their isogenic parental strain, VT1354 was tested for its sensitivity to UV irradiation. A UV survival curve was constructed for FW213 and VT1354 (Fig. 3). The resistance to UV exposure of VT1354 was significantly lower than that of the parental strain. At each UV-light dose tested, VT1354 was approximately 1,000-fold more sensitive to UV irradiation than FW213. This increased sensitivity of VT1354 to UV light is consistent with a recA mutant phenotype.

FIG. 3.

Survival of S. parasanguis FW213 and VT1354 after exposure to UV light. Bacteria were irradiated with UV light for increasing amounts of time, and the percentage of surviving cells was determined by comparison with cells that had not been irradiated. Results shown are the averages from two independent experiments.

Expression of the fimbria-associated adhesin Fap1 in the S. parasanguis recA mutant.

In N. gonorrhoeae the recA gene is involved in the expression of pilus antigenic variation and in phase transitions (18), and in V. cholerae recA is required for the expression of adherence and colonization factors (19). Many of the adherence- and virulence-associated factors of the streptococci are cell surface polypeptides containing amino acid repeat blocks; several of these adherence factors are high-molecular-mass cell wall proteins (17). In some cases, the repeated sequence blocks within these proteins have been implicated in binding to substrates within the mammalian host. Although the repeat sequences within the genes coding for these proteins could serve as potential sites for homologous recombination events, it is not known whether expression of these streptococcal adherence and colonization factors is affected by recA.

Recently, the fimbria-associated adhesin Fap1 was identified in S. parasanguis FW213 (31). This adhesin is also a high-molecular-mass streptococcal surface protein that contains extensive amino acid repeat blocks. Fap1 is involved in adhesion of S. parasanguis to an in vitro tooth surface model, SHA, and also appears to be important in the expression of fimbriae, surface structures that mediate attachment to the tooth surface by S. parasanguis FW213. Because of the presence of repeat regions in Fap1 and because recA plays a role in the expression of adherence- and virulence-associated factors in other bacteria, we asked whether the recA gene of S. parasanguis FW213 was required for the expression of Fap1 or for the adherence of S. parasanguis to SHA.

Three independent measures were employed to determine whether inactivation of the recA gene affected production of Fap1. First, relative immunoreactivities of S. parasanguis FW213 or recA mutant VT1354 were compared in a whole-bacterial-cell enzyme-linked immunosorbent assay with the anti-Fap1 MAb, MAb F51. Experiments were performed in quadruplicate, and standard deviations did not exceed 5% of the mean. Reactivity of MAb F51 with wild-type or with recA mutant cells did not differ significantly (data not shown), suggesting that the recA mutation did not alter the cell surface expression of the Fap1 fimbria-associated adhesin.

It was shown previously that fap1 encodes a 200-kDa protein (31). Immunoblot analysis with MAb F51 was used to compare the abilities of the wild-type and recA mutant cells to synthesize the 200-kDa protein. The MAb detected a 200-kDa protein in both the wild-type and recA-deficient strains (Fig. 4). Furthermore, the amounts of the Fap1 protein produced by the wild-type strain and by its recA derivative seem to be roughly equivalent. This result suggests that production of Fap1 is unaffected in the S. parasanguis recA mutant.

FIG. 4.

Expression of Fap1 in S. parasanguis FW213 and recA-deficient derivative VT1354. For Western immunoblot analysis whole-cell lysates of FW213 and VT1354 were probed with MAb F51. Lane 1, FW213; Lane 2, VT1354. Sizes (in kilodaltons) of prestained molecular mass markers (Gibco BRL) are indicated on the left.

Wild-type, fimbriated S. parasanguis cells bind in the in vitro tooth surface model, SHA. An adhesion assay was performed to assess the effect of the recA mutation on the ability of S. parasanguis to bind SHA. No difference was found between wild-type and mutant strains. Approximately 46% of input cells of both S. parasanguis FW213 and S. parasanguis VT1354 adhered to SHA (data not shown), indicating that inactivation of recA did not affect the ability of S. parasanguis cells to adhere to SHA.

On the basis of the above data, it appears that recA does not affect production and surface expression of the fimbria-associated adhesin Fap1. Neither does it appear to play a significant role in adherence of S. parasanguis to SHA. Thus, homologous recombination does not appear to play a role in the in vitro expression of Fap1. The question of whether there is functional significance to the repeat blocks of Fap1 awaits further investigation.

Conclusions.

The entire recA gene from the oral bacterium S. parasanguis FW213 was cloned, and its nucleotide base sequence was determined. The deduced S. parasanguis recA gene product, RecA, showed a high level of amino acid identity with RecA proteins from other prokaryotic species. A defined mutation within the cloned S. parasanguis recA gene was constructed in vitro and reintroduced into the streptococcal chromosome by transformation. An S. parasanguis recA mutant, obtained by allelic replacement, behaved similarly to recA mutants of other prokaryotic species, suggesting that the functional activities of the recA gene products are conserved as well. Finally, recA is not required for the in vitro expression of the fimbria-associated adhesin Fap1 or for adherence of S. parasanguis to SHA. We anticipate that the recA-deficient strain described here will be a valuable tool in future genetic studies of S. parasanguis FW213.