A. Pérez-Oseguera, M.A. Ramírez-Romero, M.A. Cevallos
Centro de Investigación sobre Fijación de Nitrógeno, A. P. A.P. 565-A., UNAM Cuernavaca, Morelos, México
As a member of the repABC plasmid family, the replicator region of the R. etli symbiotic plasmid, contains three genes (repA, repB and repC) and a conserved region sequence between repB and repC (Ramírez-Romero et al. 1997).
A genetic analysis of the repABC genes showed that they are organized in an operon. The repA and repB genes encode proteins required in plasmid stability, and in plasmid copy-number control. Also, it has been shown that RepA acts as a trans-acting incompatibility factor (Ramirez-Romero et al. 2000).
Little is known about the regulation of the repABC genes. To understand the transcriptional characteristics of this operon: the transcriptional start site was identified. The -10 and -35 hexameric elements of the repABC promoter were localized by mutational analysis, and the negative transcriptional regulator and its target site was identified by genetic and biochemical analysis.
Several repA::gusA fusions were constructed to identify the minimal region of DNA necessary for the expression of the operon. The results indicate that the promoter region is contained within 82 bp upstream of rep A. The transcription start site of the repABC operon was found 57 bp upstream of the initiation codon oí rep A. Upstream of this site, a sequence similar to the -35 and -10 boxes of the Escherichia coli cr70 promoter consensus was recognized. A mutagenesis analysis of the putative -35 and -10 boxes showed that they were correctly identified.
The P-glucuronidase activity of the repA::gusA fusion was repressed in the presence of RepA but not with RepB and RepC, indicating that RepA is the negative transcription regulator.
To demonstrate that RepA interacts with the sequences located upstream of the repA gene, two strategies were followed. First, a DMS footprinting analysis in vitro of R. etli strains containing the repA::gusA, in the presence or absence of RepA was performed. The results of these experiments suggest that the putative binding site for RepA is one inverted repeat sequence of 14 nucleotides located upstream of repA. Second, gel mobility shift assays were performed with a purified RepA hexahistidine derivative and with a 160 bp region upstream of repA, as target DNA. These experiments showed that RepA is able to bind to this DNA, and that this activity has an absolute requirement of ATP or ADP, suggesting that ATP hydrolysis is not necessary for DNA binding.
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