Cloning and genetic analysis of the UV resistance determinant (uvr) encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pAD1.

摘要

The conjugative pheromone-responsive plasmid pAD1 (59.6 kb) of Enterococcus faecalis encodes a UV resistance determinant (uvr) in addition to the hemolysin-bacteriocin determinant. pAD1 enhances the UV resistance of wild-type E. faecalis FA2-2 and E. faecalis UV202, which is a UV-sensitive derivative of E. faecalis JH2-2. A 2.972-kb fragment cloned from between 27.7 and 30.6 kb of the pAD1 map conferred UV resistance function on UV202. Sequence analysis showed that the cloned fragment contained three open reading frames designated uvrA, uvrB, and uvrC. The uvrA gene is located on the pAD1 map between 28.1 and 29.4 kb. uvrB is located between 30.1 and 30.3 kb, and uvrC is located between 30.4 and 30.6 kb on the pAD1 map. The uvrA, uvrB, and uvrC genes encode sequences of 442, 60, and 74 amino acids, respectively. The deduced amino acid sequence of the uvrA-encoded protein showed 20% homology of the identical residues with the E. coli UmuC protein. Tn917 insertion mutagenesis and deletion mutant analysis of the cloned fragment showed that uvrA conferred UV resistance. A palindromic sequence, 5'-GAACNGTTC-3', which is identical to the consensus sequence found within the putative promoter region of the Bacillus subtilis DNA damage-inducible genes, was located within the promoter region of uvrA. Two uvrA transcripts of different lengths (i.e., 1.54 and 2.14 kb) which terminate at different points downstream of uvrA were detected in UV202 carrying the deletion mutant containing uvrA. The longer transcript, 2.14 kb, was not detected in UV202 carrying the deletion mutant containing both uvrA and uvrB, which suggests that uvrB encodes a terminator for the uvrA transcript. The uvrA transcript was not detected in any significant quantity in UV202 carrying the cloned fragment containing uvrA, uvrB, and uvrC; on the other hand, the 1.54-kb uvrA transcript was detected in the strain exposed to mitomycin C, which suggests that the UvrC protein functions as a regulator of uvrA.