IDENTIFICATION OF THE RHODOBACTER-SPHAEROIDES SOS BOX

Gel-mobility shift assays with crude cell extracts of Rhodobacter spha eroides, which belongs to the alpha group of the proteobacteria, have shown that a protein binds to the promoter of its recA gene, resulting in two retardation bands. Analysis of the minimal region of the R. sp haeroides recA gene required for the formation of the DNA-protein comp lexes, revealed the presence of the motifs GTTCN(7)GATC and GAACN(7)GA AC, which are centred at positions -21 and +8 from the transcriptional starting point respectively. Using PCR mutagenesis, we have demonstra ted that these two motifs are required for the formation of both DNA-p rotein complexes in vitro as well as for the DNA damage-mediated induc ibility of the recA gene in vivo. Furthermore, the level of the recA g ene expression in the constitutive mutants is the same as that achieve d by the wild-type cells after DNA damage, indicating that the binding protein must be a repressor. The motif GTTCN(7)GTTC is also present u pstream of the R. sphaeroides uvrA promoter, which in vitro specifical ly binds to a protein and whose expression is DNA damage inducible. Mu tagenesis of this motif abolishes both the binding of this protein to the uvrA promoter and the DNA damage-mediated expression of this gene. The fact that the recA and uvrA wild-type promoters compete with each other for the retardation band formation, but not with their mutant d erivatives in any of these motifs, indicates that the same repressor b inds to the operator of both genes. All these results lead us to propo se the sequence GTTCN(7)GTTC as the SOS box of R. sphaeroides, This is the first SOS box known whose sequence is a direct repeat and not a p alindrome.