Molecular analysis of the recA gene and SOS box of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7

The recA gene of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7 was isolated by a PCR-based method and sequenced. The complete nucle otide sequence consists of 1089 bp encoding a polypeptide of 363 amino acid s which is most closely related to the RecA proteins from species of Rhizob iaceae and Rhodospirillaceae. A recA-deficient strain of R. palustris no. 7 was obtained by gene replacement. As expected, this strain exhibited incre ased sensitivity to DNA-damaging agents. Transcriptional fusions of the rec A promoter region to lacZ confirmed that the R. palustris no. 7 recA gene i s inducible by DNA damage. Primer extension analysis of recA mRNA located t he recA gene transcriptional start. A sequential deletion of the fusion pla smid was used to delimit the promoter region of the recA gene. A gel mobili ty shift assay demonstrated that a DNA-protein complex is formed at this pr omoter region. This DNA-protein complex was not formed when protein extract s from cells treated with DNA-damaging agents were used, indicating that th e binding protein is a repressor. Comparison of the minimal R. palustris no . 7 recA promoter region with the recA promoter sequences from other alpha- Proteobacteria revealed the presence of the conserved sequence GAACA-N-6-G( A/T)AC. Site-directed mutations that changed this consensus sequence abolis hed the DNA-damage-mediated expression of the R. palustris recA gene, confi rming that this sequence is the SOS box of R. palustris and probably plays the same role in other alpha-Proteobacteria.