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

Valérie Dumay

Molecular Microbiology and Genetics Lab, Research Institute of Innovative Technology for the Earth, 9–2 Kizugawadai, Kizu-cho, Soraku-gun, Kyoto 619–0292, Japan

ABSTRACT

The recA gene of the purple non-sulfur bacterium Rhodopseudomonas palustris no. 7 was isolated by a PCR-based method and sequenced. The complete nucleotide sequence consists of 1089 bp encoding a polypeptide of 363 amino acids which is most closely related to the RecA proteins from species of Rhizobiaceae and Rhodospirillaceae. A recA-deficient strain of R. palustris no. 7 was obtained by gene replacement. As expected, this strain exhibited increased sensitivity to DNA-damaging agents. Transcriptional fusions of the recA promoter region to lacZ confirmed that the R. palustris no. 7 recA gene is inducible by DNA damage. Primer extension analysis of recA mRNA located the recA gene transcriptional start. A sequential deletion of the fusion plasmid was used to delimit the promoter region of the recA gene. A gel mobility shift assay demonstrated that a DNA-protein complex is formed at this promoter region. This DNA-protein complex was not formed when protein extracts from cells treated with DNA-damaging agents were used, indicating that the binding protein is a repressor. Comparison of the minimal R. palustris no. 7 recA promoter region with the recA promoter sequences from other -Proteobacteria revealed the presence of the conserved sequence GAACA-N₆-G(A/T)AC. Site-directed mutations that changed this consensus sequence abolished the DNA-damage-mediated expression of the R. palustris recA gene, confirming that this sequence is the SOS box of R. palustris and probably plays the same role in other -Proteobacteria.

Author for correspondence: Hideaki Yukawa. Tel: + 81 774 75 2308. Fax: + 81 774 75 2321. e-mail: yukawa@rite.or.jp

Present address: E. C. Slater Institute, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.

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