An operon that confers UV resistance by evoking the SOS mutagenic response in streptococcal conjugative transposon Tn5252

Abstract

Streptococcus pneumoniae Rx1 is capable of repairing lesions caused by DNA-damaging agents in an error-free manner but lacks a UV-inducible error-prone repair system due to the absence of chromosomally encoded UmuDC-like proteins. We have identified an operon-like structure 8 kb from the left end of the pneumococcal conjugative transposon Tn5252 that confers SOS function in the host cells. DNA sequence analysis of this region revealed the presence of four open reading frames (ORFs). The deduced amino acid sequence of one of them, ORF13, which is capable of encoding a protein of 49.7 kDa, showed significant homology to UmuC, MucB, and other proteins involved in the SOS response. The carboxy-terminal region of another, ORF14, which is predicted to encode a 26-kDa polypeptide, shared similarity with UmuD- and MucA-like proteins that carry the amino acid residues recognized by the activated RecA* protein for proteolytic cleavage. The presence of plasmids carrying subcloned DNA from this region was found to restore UV-inducible mutagenic repair of chromosomal DNA in Escherichia coli cells defective in error-prone repair as well as in pneumococcus and Enterococcus faecalis UV202. Mutations within ORF13 abolished UV-induced mutagenesis but did not affect the conjugal transposition of the element.

FIG. 1

Restriction map and the predicted gene organization of the uvr operon of the 47.5-kb Tn5252. The EcoRI site at the right end is about 8.5 kb from the left end of the element. Relevant restriction sites are shown. Thin line, chromosomal DNA; box, transposon DNA; crosshatched box, the 4.5-kb DNA containing the uvr genes; black boxes, direct repeats of insertion sequence-like sequences. The location of the cat is shown. The vertical lines in the transposon DNA indicate EcoRI sites. Subclones derived from the 4.5-kb EcoRI fragment of DNA shown in the lower panel with relevant restriction endonuclease sites and a nested set of deletion derivatives obtained following exonuclease III and S1 treatments were used to determine the sequence data from both the strands. The directions and lengths of the potential ORFs are shown at bottom.

FIG. 2

Multiple sequence alignment of the predicted product from ORF13 and its homologs ORFU from Lactococcus lactis plasmid pNP10 (5), uvrA of E. faecalis (19), UV-damage repair protein of B. subtilis (accession no. Z99115), rumB of IncJ plasmid R391 from E. coli (12), samB from S. typhimurium (18), mucB of R46 plasmid from S. typhimurium (17), and the chromosomal umuC from S. typhimurium (26) and E. coli (20). Conserved amino acids are shaded in black and conservative substitutions are shaded in gray.

FIG. 3

Effect of UV irradiation on survival of three organisms. (A) S. pneumoniae strains: □, SP1311; ■, SP1317; ○, SP1402; ●, SP1405; ▵, SP1323; ◊, SP1324. (B) E. faecalis strains: ×, JH2-2; □, UV202; ●, SF5002; ■, SF5004. (C) E. coli strains: ×, AB1157; □, RM1140; ◊, RM1140(pSE117); ⧫, RM1140(pKM101); ■, RM1140(pSJ142); ▵, RM1140(pAT29). The UV survival curves were obtained as indicated in Materials and Methods, and the numbers are averages obtained from at least two independent experiments.

FIG. 4

Physical analysis of Em^r transformants carrying the insertion of pVA891 within ORF13 in Tn5252. Autoradiogram showing Southern hybridization of ³²P-labeled pDR6 to ClaI- (A) and HindIII- (B) digested chromosomal DNA from SP1291 (SP1000 carrying a deletion within ORF13) (lane 1), SP1000 (Rx1::Tn5252) (lane 2), and S. pneumoniae Rx1 (lane 3). The indicated sizes correspond to the standards in lane M, which consist of a set of calibrated fragments from pSK(+) or derivative plasmids, all of which react with the probe.