Gel-mobility shift assays with crude cell extracts of Rhodobacter sphaeroides, 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. sphaeroides recA gene required for the formation of the DNA-protein complexes, revealed the presence of the motifs GTTCN7GATC and GAACN7GAAC, which are centred at positions -21 and +8 from the transcriptional starting point respectively. Using PCR mutagenesis, we have demonstrated that these two motifs are required for the formation of both DNA-protein complexes in vitro as well as for the DNA damage-mediated inducibility of the recA gene in vivo. Furthermore, the level of the recA gene expression in the constitutive mutants is the same as that achieved by the wild-type cells after DNA damage, indicating that the binding protein must be a repressor. The motif GTTCN7GTTC is also present upstream of the R. sphaeroides uvrA promoter, which in vitro specifically binds to a protein and whose expression is DNA damage inducible. Mutagenesis 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 derivatives in any of these motifs, indicates that the same repressor binds to the operator of both genes. All these results lead us to propose the sequence GTTCN7GTTC as the SOS box of R. sphaeroides. This is the first SOS box known whose sequence is a direct repeat and not a palindrome.

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The uvrB gene of Pseudomonas aeruginosa has been isolated from a genomic library by complementation of an Escherichia coli uvrB mutant. The complete nucleotide sequence of P. aeruginosa uvrB consists of 2,013 bp, encoding a polypeptide of 670 amino acids. A P. aeruginosa SOS consensus region, which functions as a binding site for the LexA repressor molecule, is not present in the upstream region of the uvrB gene isolated. By transcriptional fusions with a reporter gene, it has been demonstrated that, contrary to what happens with the homologous gene of E. coli, the P. aeruginosa uvrB gene is not DNA damage inducible. Nevertheless, the UvrB protein must be functional in P. aeruginosa cells because a uvrB-defective mutant is extremely sensitive to UV radiation.

The effect of plasmid pKM101 on the survival of Escherichia coli AB1157, growing in minimal medium, in the presence of a 4-quinolone DNA gyrase inhibitor was investigated. The presence of this plasmid decreased susceptibility to the quinolone ciprofloxacin, whereas mucAB genes present in a multicopy plasmid did not. The same effect of pKM101 was detected in a recA430 mutant, confirming that it was not really related to the SOS response. In contrast, when survival assays were performed under amino acid starvation conditions, pKM101 did not confer protection against ciprofloxacin. All of these results indicated that the synthesis of a product(s), different from MucAB, which was encoded by the plasmid pKM101 increased the rate of survival of the AB1157 strain in the presence of quinolone. To identify the gene(s) responsible for this phenotype, several plasmid derivatives carrying different portions of pKM101 were constructed. The 2.2-kb region containing korB, traL, korA, and traM genes was sufficient to decrease susceptibility to quinolone. This plasmidic fragment also made the AB1157 host strain grow more slowly (the Slo phenotype). Moreover, the suppression of the Slo phenotype by addition of adenine to the cultures abolished the decreased susceptibility to quinolone. These results are evidence that the protection against quinolone conferred by this region of pKM101 in strain AB1157 is a direct consequence of the slow growth rate.pKM101 is a 35.4-kb self-transmissible broad-host-range IncN plasmid, which was derived from plasmid R46 (17) by a spontaneous deletion of 14 kb (2, 12). This plasmid has been extensively studied because it contains the mucAB genes (18), which are analogous to the umuDC genes whose products are involved in error-prone repair of DNA damage that can occur as a consequence of the SOS response. The ability of pKM101 to increase bacterial mutability has resulted in its introduction into Salmonella typhimurium strains used in the Ames test, enhancing the sensitivity of this system to detect chemicals as mutagens (16).Besides mucAB, other genes of pKM101 that participate in plasmid replication, stable maintenance, and host range have been described. The conjugal transfer system of pKM101 consists of a mobilization gene cluster, providing functions required for DNA processing and mobilization, and a cluster of tra genes involved in the synthesis of the pilus and putative mating pore (23). Near this second cluster, the kilA and kilB genes were described to encode potentially lethal products whose lethality was prevented by the products of two other genes, korA and korB (24). In addition, the kilA gene, which is defined to a region between two Tn5 insertions, was also thought to retard cell growth of certain Escherichia coli strains on defined minimal medium (24), and this phenomenon had previously been called the Slo phenotype (11,22). More recently, this cluster of tra genes has been sequenced, and, on the basis of DNA sequence information, the kilB (renamed traE), korA, and korB genes have bee...

The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli-like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides, indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.