INTRODUCTION

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Biological mechanisms to repair UV-damaged DNA are widespread among many, if not all, organisms (25). Escherichia coli is the best-studied model prokaryote to elucidate the various pathways leading to the repair of the genetic material and survival of the cells (25). Besides nucleotide excision repair involving the products of the uvrA, uvrB, and uvrC genes, error-prone repair in association with the umuDC gene products is also known to occur (9). The latter process, the SOS response that results in the increase of replication errors, is triggered by the activated RecA* protein, which facilitates the autocleavage of the UmuD protein to yield the active UmuD' C-terminal fragment as with the LexA repressor (3). The processed UmuD'C complex is thought to bind to DNA polymerases, altering the fidelity of nucleotide incorporation at abasic sites leading to increased mutagenesis (25). The human pathogen Streptococcus pneumoniae also carries the genes involved in the excision repair of UV-damaged DNA (6, 16). However, the SOS response, which results in mutagenic repair of UV-damaged DNA, has been shown to be absent in this organism (6).

We have been studying the biology of the conjugative transposon Tn5252, which was originally detected in the chromosome of S. pneumoniae BM6001 (1). It is a 47-kb element carrying a chloramphenicol resistance determinant and seems to have a propensity to accumulate a variety of heterologous DNA segments and disseminate them into a number of streptococcal species (1). The element is capable of self-transfer, and it site specifically integrates in the genome of the host cells (30). The entire transposon has been cloned in fragments in E. coli, and a detailed physical map has been obtained (31). Mutational, DNA sequencing, and functional analyses of the genes and their products revealed the clustering of many of the transfer-related genes at the terminal regions of the element (10). However, about 20 kb of DNA in the central region of Tn5252 seem apparently devoid of transfer functions. DNA sequencing analysis showed that the deduced amino acid sequence of two of the four open reading frames (ORFs) within an operon-like structure at the left terminal region of the transposon showed significant similarities to UmuDC homologs from a number of gram-positive and gram-negative bacteria. Here we present our studies relating to the genetic and physical characterization of this operon, which confers resistance to UV light by error-prone repair.

	MATERIALS AND METHODS

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Bacterial strains, media, transformation, and conjugation. Bacterial strains and plasmids used in this study are listed in Table 1. The growth of streptococcal cultures, conjugation, competence regimen, and plating techniques have been described previously (1). Recombinant plasmids were generated in recombination-deficient E. coli JM109 by transformation in accordance with the method of Hanahan (8). Bacterial cells carrying pAT29 or derivatives were grown in media containing spectinomycin (150 µg/ml). The antibiotics used for UV-induced mutagenesis were optochin (5 µg/ml) and fusidic acid (20 µg/ml). For scoring of streptococcal transconjugants, optochin (20 µg/ml), erythromycin (5 µg/ml), and streptomycin (200 µg/ml) were used.

DNA procedures. DNA restriction and modification enzymes were used according to the recommendations of the suppliers. Chromosomal DNAs from streptococci were isolated as described previously (1). Plasmid DNA from E. coli was prepared by using the Wizard Prep kit (Promega). DNA hybridizations were done by the method of Southern (27) with a GeneScreen Plus membrane (Du Pont, New England Nuclear) as support.

DNA sequencing and computer analysis of DNA sequences. Relevant DNA fragments were subcloned into the pBluescript plasmid vectors SK(+) and KS(+) (Stratagene). Apart from the universal primers, synthetic oligonucleotide primers used in DNA sequencing were made at the Oklahoma State University Core Facility. The dideoxy chain termination DNA sequencing was performed at the Core Facility by using a 373 Strech automatic sequencer (Perkin-Elmer, Applied Biosystems Division) with fluorescent chain terminators. Sequence assembly and analyses were performed by using MacVector 3.5 software.

UV irradiation. Cells were grown to early exponential phase (approximately 10⁸ CFU/ml) in an appropriate broth at 37°C and harvested by centrifugation at 5,000 × g in a Sorvall RC-5B centrifuge (DuPont Instruments). The cells were washed twice and resuspended in prechilled phosphate-buffered saline buffer containing 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄ (pH 7.4). Three milliliters of the cell suspension was placed in a sterile plastic petri dish (60 by 15 mm) and exposed to various doses of UV radiation from a germicidal bulb (model XX-15F; Spectronics Corporation) with a peak emission of 256 nm. Doses of UV radiation were determined with a UVX radiometer (Ultra Violet Products). Survivors were counted by plating appropriate dilutions of cells on agar plates. All manipulations and incubations subsequent to UV irradiation were carried out in the dark or under amber light in order to minimize photoreactivation.

UV-induced mutagenesis. Appropriate dilutions of E. coli cells were plated immediately following UV irradiation on minimal medium plating lacking histidine to score for revertants. UV-treated S. pneumoniae cells were diluted 20-fold in CATPG medium (1), grown to 2 × 10⁸ CFU/ml at 37°C in the dark, and plated on appropriate selective plates to score for survivors and drug-resistant mutants. When needed, the cells were stored at 80°C in 10% glycerol. The following day, the cells were thawed in an ice water bath, diluted, and plated.

Nucleotide sequence accession number. The sequence reported here was assigned GenBank accession no. L29324.

	RESULTS

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Features of the nucleotide sequence. Previously, it had been shown that the genes encoding DNA processing functions, such as site-specific recombination and DNA relaxation during conjugal transfer of Tn5252, were present at the left terminal region of the element (10). To identify other possible transfer-related genes nearby, the DNA sequence of a 3.3-kb fragment of DNA on the right side of the DNA relaxase (28) was determined. For DNA sequencing, a 4.55-kb EcoRI fragment from this region was cloned into pSK+ to create pDR6. Fig. 1 shows the organization of the region in Tn5252 reported here. The G+C content of the sequenced region was 33.6%. The sequence revealed the presence of four ORFs, ORF14, ORF13, ORF12, and ORF11, transcribed in the same orientation as a single unit with a putative ribosome-binding site (RBS) placed upstream of each at the appropriate distance. A gram-positive consensus promoter-like sequence (7) was noted about 140 bases upstream of the translational start site of ORF14. Twenty-one bases upstream of the 35 region, a 14-bp inverted imperfect repeat (12 of 14) was present. In the RBS region of ORF14, another inverted imperfect repeat (17 of 21) with a G value of 14.7 kcal/mol was located. ORF12 and ORF11, which have the capacity to encode proteins with molecular masses of 11 and 11.7 kDa, respectively, were found to overlap by eight bases. ORF14 and ORF13 encoded proteins with molecular masses of 26 and 49.7 kDa, respectively.

View larger version (9K): [in this window] [in a new window]   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.

View larger version (113K): [in this window] [in a new window]   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.

UV-induced killing. To determine whether the operon carrying ORF13 was involved in the enhancement of survival of the host cell upon UV treatment, the 4.55-kb EcoRI fragment carrying this region was cloned into the Streptococcus-E. coli shuttle multicopy plasmid pAT29 to create pSJ142. The recombinant plasmid was introduced into E. faecalis UV202 cells by electroporation and into S. pneumoniae SP1311 (UV^s) and E. coli AB1157 and RM1140 via transformation. Comparison of UV sensitivity levels at 20 J/m² consistently showed a twofold increase in UV protection of SF5002 carrying Tn5252 with a single copy of ORF13, indicating the involvement of the element in the protection. At this dose, UV resistance increased about 10-fold in SF5004 cells carrying pSJ142 compared to that of the host cells without the plasmid (Fig. 3b), even though at lower doses this level of protection was less evident. On the other hand, in the highly UV-sensitive pneumococcal strain SP1311, the presence of pSJ142 conferred about 3,000-fold protection, whereas no significant protection was detectable when ORF13 was present as a single copy within Tn5252 as a part of the chromosome (Fig. 3A). When a 0.5-kb XbaI fragment internal to ORF13 was deleted from within pSJ142, the UV survival disappeared, indicating a definitive role for ORF13 in the observed protection. Similar results were observed with E. coli host cells, indicating that the streptococcal UV-resistant genes could complement the function in gram-negative bacteria (Fig. 3C).

View larger version (14K): [in this window] [in a new window]   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.

SOS response. Even though pneumococci are capable of dark repair of UV-damaged DNA (6, 16), the distinct lack of an SOS system involving error-prone repair has been documented (6). To determine whether the operon containing ORF13 and conferring UV resistance was due to error-prone polymerization of DNA, pneumococcal cells carrying pSJ142 were exposed to various doses of UV and screened for the appearance of mutations in various genetic loci. The results are given in Table 2. For both the markers (resistance to optochin and to fusidic acid) a significant number of mutants appeared compared to the cells not exposed to UV. For reasons that are not clear, the number of optochin-resistant mutants obtained was 20-fold higher than those of the fusidic acid resistance.

The role of ORF13 in the conjugal transfer of Tn5252. To assess the relevance of ORF13 in the conjugal transfer of the element, a pneumococcal mutant strain was created. The E. coli plasmid pVA891 carries a streptococcal erythromycin resistance (Em^r) determinant that is expressed in pneumococci when the plasmid is integrated into the chromosome (14). The 0.5-kb XbaI fragment in ORF13 in pSJ142 was replaced with XbaI-digested pVA891. The resulting ligated molecule was linearized with PstI that cleaves at the vector pAT29, which is part of pSJ142. The digested DNA was introduced into competent SP1000 cells carrying Tn5252 (1). Due to the flanking homology provided by ORF13 DNA, the heterologous pVA891 was expected to be inserted in this homology-dependent event (10).

View larger version (67K): [in this window] [in a new window]   FIG. 4.   Physical analysis of Emr 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.

	DISCUSSION

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We have determined the nucleotide sequence of a 3.3-kb DNA fragment at the right end of the DNA relaxase gene, the product of which is thought to be involved in the site-specific nicking of the circularized transposon molecule prior to its conjugal transfer. The sequence data revealed the presence of an operon-like region containing consensus promoter-independent and rho-independent transcription terminator-like sequences. Four ORFs were found, two of which were significantly similar to the proteins involved in error-prone repair of UV-damaged DNA. Homology alignment of ORF13 protein of Tn5252 with UmuC homologs from other systems indicated that, while the proteins originating from gram-negative bacteria were more similar to each other, those from gram-positive sources formed a distinct group, indicating the evolutionary divergence of the two types. Plasmids carrying this segment of transposon DNA were able to confer UV-induced mutagenic response and survival following exposure to UV in gram-positive as well as gram-negative bacteria devoid of error-prone repair capability. Deletion of a 0.5-kb DNA fragment from within ORF13 led to the abolition of the observed SOS mutagenic response, demonstrating the involvement of this region of DNA in the repair of UV-damaged genetic material by introduction of mutations. This is the first demonstration of UV-induced SOS response and mutability in pneumococci.

Two ORFs, encoding proteins of about 46 and 15 kDa, have been noted in the most-studied operons of gram-negative bacteria, umu and muc, whereas four ORFs are present within the SOS operon of Tn5252. The role(s), if any, of the products of ORF11 and ORF12 in SOS mutagenesis remains unknown at present. The imp operon of the IncI plasmid TP110 (13) has also been shown to carry an ORF capable of encoding a 9.5-kDa protein with unknown function in addition to the UmuD and MucB homologs. Further studies should establish the role(s) of these proteins in SOS response in pneumococcus.

According to the current model for SOS response, drawn mostly from studies in E. coli, the activated RecA* protein stimulates the autocleavage of the LexA as well as the UmuD to UmuD'. By a mechanism not currently understood, the UmuCD'₂ complex then enables the DNA polymerase to continue through the DNA lesions in an error-prone manner (25). The finding that the SOS-related gene products of Tn5252 could be processed in such a way to restore the UV-induced mutability in E. coli cells demonstrates that the structural and mechanistic details of this class of proteins are probably conserved in a wide range of bacterial species. It was also intriguing that the highest similarity to the ORF14 protein, the homolog of UmuD, was the repressor of the fructosyltransferase gene of S. mutans which carries the conserved residues found in the LexA family of proteins and has been implicated as a virulence factor in the development of dental caries.

Surprisingly, plasmids are rare in clinical isolates of S. pneumoniae even though they have the capacity to receive and maintain plasmids from other streptococci via transformation under laboratory conditions. The recent emergence and dissemination in pneumococci of resistance to multiple antibiotics have been chiefly due to the conjugative transposons (4, 22, 23). These novel elements seem to have functionally replaced plasmids in this species. The chloramphenicol resistance determinant in Tn5252 has been shown to be flanked by direct repeats of an insertion sequence-like element (21) which very often leads to its spontaneous "curing" (21). The remaining cryptic element is transfer proficient (1, 21). The ability of Tn5252-like elements to persist under natural conditions even in the absence of antibiotic selection is probably due to other genes not related to the conjugal transfer which may enhance the survival of their hosts. The presence of genes involved in the SOS response in Tn5252 is an example supporting this notion.