MolGenGenet (1987) 210:203 210 Complementation of Bacillus subtilis polA mutants by DNA polymerase I from Streptococcus pneumoniae Susana Martinez 1, Paloma Lopez 1, Manuel Espinosa 1, and Sanford A. Lacks 2 1 Centro de Investigaciones Biologicas, C.S.I.C., E-28006-Madrid, Spain 2 Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA Summary. The polA gene of Streptococcus pneumoniae cloned in the recombinant plasmid pSM22 is expressed in Bacillus subtilis. Extracts of B. subtilis polA mutants con- taining pSM22 showed 6 times more DNA polymerase ac- tivity than extracts of wild-type cells without the plasmid. Complete complementation of the B. subtilis polA5 and polA59 mutations with respect to in vivo resistance to UV irradiation and methyl methanesulfonate was observed when four copies of the pneumococcal polA gene were pres- ent in each cell. Ectopic integration of the polA gene togeth- er with a cat marker into the chromosome of B. subtilis gave chromosomal insertions containing single and double doses of the pneumococcal polA gene. Correlation with gene dosage was observed for both chloramphenicol acetyltrans- ferase and DNA polymerase activities measured in vitro. Depending on the number of copies of the S. pneumoniae polA gene present, restoration of DNA repair functions in polA mutants of B. subtilis was either partial or complete. Key words: DNA repair - UV resistance - Ectopic integra- tion - Gene dosage - Heterospecific gene expression lated (Gass and Cozzarelli 1973; Laipis and Ganesan 1972), but from the biochemical and genetic points of view, both the poIA gene and its product remain poorly characterized (Kornberg 1980). In the gram-positive Streptococcus pneu- moniae no genetic studies of the polA gene have yet been performed. However, the pneumococcal polA gene has been recently cloned (Martinez et al. 1986) in the S. pneumoniae cloning system (Stassi et al. 1981). The broad-host-range multicopy plasmid pLSI (Lacks et al. 1986) was used as vector, and a recombinant plasmid, pSM22, which contains the intact pneumococcal polA gene, was isolated (Martinez et al. 1986). Transfer ofpSM22 to B. subtilis and expression in this host of the exonuclease activity of the pneumococcal Pol I was demonstrated (Martinez et al. 1986). To see whether the pneumococcal Pol I could substitute functionally for the corresponding B. subtilis protein, we have transferred pSM22 to polA mutants of B. subtilis. To- tal complementation of the polA mutations was observed in vivo. However, when only one copy of the S. pneumoniae gene was integrated into the chromosome of the B. subtilis polA mutants, only partial complementation was detected. Introduction One way to gain insight into the functional equivalence of heterospecific products in bacteria is by complementa- tion analysis of a foreign gene in a mutant recipient. The important role of DNA polymerase I (Pol I) enzymes in excision repair of damaged DNA (Kornberg 1980) lends particular interest to complementation studies of these pro- teins. Pol I appears to be the predominant DNA polymeriza- tion activity in bacteria. Investigation of prokaryotic Pol I has been carried out mainly in Escherichia coli, although the enzyme has been detected in other bacterial species (Kornberg 1980). The polA gene of E. coli has been cloned in lambda (Kelley et al. 1977; Spanos and Sedgwick 1984) and subcloned in a low copy number plasmid (Spanos and Sedgwick 1984). The sequence of the wild-type polA gene has been determined (Joyce et al. 1982) and the base chan- ges of several alleles established (Joyce et al. 1985). In the gram-positive Bacillus subtilis, Pol I has been purified (Kornberg 1980) and various polA mutants have been iso- Offprint requests to ." S.A. Lacks Materials and methods Bacterial strains and plasmids. Bacterial strains used are listed in Table 1. Most of the experiments were carried out with B. subtilis YB965 because this strain had been cured of prophages. The following plasmids were employed: pLS 1 (Lacks et al. 1986), pSM22 (Martinez et al. 1986), pJS3 (Ballester et al. 1986) and pHV452 [a gift of S.D. Ehrlich, (Niaudet et al. 1984)]. From the last plasmid, a spontaneous deleted derivative called pHV452A1 was isolated. Restric- tion analysis of pHV452AI showed a deletion of about 2.5 kb in the X region of the B. subtilis chromosomal insert in the parental plasmid (Niaudet et al. 1984). Growth and transformation of cultures. Cultures of S. pneu- moniae and B. subtilis were grown and transformed with chromosomal or plasmid DNA using published procedures (Garcia et al. 1978; Lopez et al. 1982). Chromosomal trans- formants of S. pneumoniae were selected in agar medium containing 0.2% maltose or 0.2% sucrose plus chloram- phenicol at 2.5 gg/ml. Chromosomal transformants of B. subtilis were selected for Trp + in minimal medium (Espi- nosa et al. 1984) and for Cm r with chloramphenicol at 5 gg/ ml. Plasmid transformants of S. pneumoniae and B. subtilis were selected for TC with tetracycline (Tc) at 1 and 50 gg/ml