Analysis of the Ros repressor of Agrobacterium virC and virD operons: molecular intercommunication between plasmid and chromosomal genes

Abstract

The virulence genes of the Agrobacterium tumefaciens Ti plasmid are regulated both positively and negatively. The products of the genes of the virC and virD operons play an important role in host specificity and T-DNA processing. These operons are transcribed in opposite directions and therefore bear diametrically oriented promoters. These promoters are positively regulated by the VirG protein, which is believed to be activated through phosphorylation by a histidine kinase encoded by the virA gene. The virC and virD operons are also regulated by a 15.5-kDa repressor protein encoded by the ros chromosomal gene. A mutation in ros causes the constitutive expression of virC and virD in the complete absence of the VirG protein. It appears, therefore, that the Ros repressor interacts with the regulatory region of these operons. The Ros repressor is shown here to bind to an upstream sequence (Ros box) comprising 40 bp bearing a 9-bp inverted repeat, TATATTTCA/TGTAATATA, in the promoter region of these operons. The affinity for this sequence is specific and tenacious, since the addition of at least a 20,000-fold excess of competitor DNA failed to remove the Ros protein coding sequence from the Ros box. DNase I footprint analysis showed that the Ros box overlaps the binding site of VirG (Vir box). This result suggests that virC and virD transcription is modulated by Ros and VirG proteins.