JOURNAL OF BACTERIOLOGY, Apr. 1992, p. 2729-2732 0021-9193/92/082729-04$02.00/0 Copyright © 1992, American Society for Microbiology Vol. 174, No. 8 Degenerate Oligonucleotide Primers for Enzymatic Amplification of recA Sequences from Gram-Positive Bacteria and Mycoplasmas KEVIN DYBVIG,l,2* SUSAN K. HOLLINGSHEAD,2 DAVID G. HEATH,3 DON B. CLEWELL,3'4 FEI SUN,2 AND ANN WOODARD2 Departments of Comparative Medicine' and Microbiology,2 University ofAlabama at Birmingham, Birmingham, Alabama 35294, and Department of Biologic and Materials Sciences, School of Dentistry,3 and Department of Microbiology and Immunology, School of Medicine,4 The University of Michigan, Ann Arbor, Michigan 48109 Received 18 October 1991/Accepted 3 February 1992 RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulnonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms. The recA gene product is a key protein involved in DNA recombination and repair. In Escherichia coli, it is required for all pathways of homologous recombination and is essen- tial for the initiation of the SOS response after DNA damage (3, 12, 21, 26). The recA gene from a variety of gram- negative bacteria has been studied (18), but little is known about recA in gram-positive bacteria other than Bacillus subtilis. In B. subtilis, RecA protein apparently regulates a global network analogous to the SOS response of E. coli (13, 14). The B. subtilis recA gene (formerly referred to as recE) has been cloned, and its sequence has extensive homology with those of gram-negative bacterial recA genes (25). Using the B. subtilis recA gene as a hybridization probe, we have recently cloned the recA gene from the mycoplasma Acholeplasma laidlawii (8). Although mycoplasmas (class Mollicutes) lack cell walls, they are phylogenetically related to gram-positive bacteria (16, 27). The predicted amino acid sequence of the entire A. laidlawii recA gene product has 68% identity with the corresponding amino acids in the B. subtilis RecA protein, suggesting that these proteins are functionally very similar. The initial goal of the present study was to isolate the recA gene from the murine pathogen Mycoplasma pulmonis. It has previously been shown that this organism is capable of incorporating DNA into its chromosome by homologous recombination (15). However, attempts in our laboratory to detect recA-like sequences in M. pulmonis by using the B. subtilis and A. laidlawii recA genes as hybridization probes were not successful. Therefore, an alternative approach for isolation of recA sequences, involving the use of the poly- merase chain reaction (PCR), was explored. Because of the relative ease of this approach, the goal of this study was expanded to include recA sequences from several gram- positive bacteria. The organisms used in this study were A. laidlawii 8195 (24), M. pulmonis KD735 (7), Mycoplasma mycoides subsp. mycoides GM9 (6), Streptococcus mutans UA96 (1), Strep- * Corresponding author. tococcus pyogenes D471 (23), Streptococcus pneumoniae 851 (obtained from S. Lacks), Lactococcus lactis subsp. lactis MG1363 (10), Enterococcus faecalis OGIX (11), E. faecalis ATCC 29212, and Staphylococcus aureus ATCC 29213. All organisms were grown at 37°C. A. laidlawii and M. pulmonis were grown in mycoplasma medium (5), and gram-positive bacteria were grown in brain heart infusion broth (Difco Laboratories, Detroit, Mich.). Mycoplasmal DNA was isolated by lysing cells with sodium dodecyl sulfate (SDS) and extracting DNA with phenol as described elsewhere (4). For gram-positive bacteria, cells (10 ml) were centrifuged and resuspended in 200 ,ul of buffer consisting of 10 mM Tris (pH 8.0) and 50 mM EDTA. A 200-,l volume of nonionic detergent solution (0.45% Tween 20, 0.45% Noni- det P-40) containing 24 ,ug of proteinase K was added, and the mixture was incubated at 56°C for 1 h. Lysis was completed by the addition of 40 ,ul of a 10% (wt/vol) solution of SDS, and DNA was isolated by following the protocol used for mycoplasmal DNA. The recA genes from B. subtilis (25), A. laidlawii (8), and E. coli (22) were aligned to identify conserved regions that could serve as candidate sites for binding of PCR primers. Conserved regions chosen for study encoded amino acid residues 14 to 20, 90 to 96, 117 to 123, 187 to 192, and 199 to -369 -246 1 2 3 4 56 7 8 9 FIG. 1. Ethidium bromide-stained 2% agarose gel of DNAs amplified with recA primers. Lanes: 1, no template DNA; 2, M. pulmonis; 3, E. faecalis; 4, S. pneumoniae; 5, S. aureus; 6, L. lactis; 7, S. mutans; 8, A. laidlawii. Lane 9 contains as a marker the 123-bp molecular weight ladder available from GIBCO/BRL Life Technol- ogies Inc., Gaithersburg, Md. The sizes of two of the marker DNAs (in base pairs) are shown in the right margin. 2729