ChvD, a chromosomally encoded ATP-binding cassette transporter-homologous protein involved in regulation of virulence gene expression in Agrobacterium tumefaciens

A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the Agrobacterium tumefaciens VirB8 protein was carried out. This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon scre en as a potential regulator of virG expression, chvD, In this report, the c loning of the entire chvD gene is described and the gene is sequenced and c haracterized. Insertion of a promoterless lacZ gene into the chvD locus gre atly attenuated virulence and vir gene expression. Compared to that of the wild-type strain, growth of the chvD mutant was reduced in rich, but not mi nimal, medium. Expression of chvD, as monitored by expression of beta -gala ctosidase activity from the chvD-lacZ fusion, occurred in both rich and min imal media as well as under conditions that induce virulence gene expressio n. The ChvD protein is highly homologous to a family of ATP-binding cassett e transporters involved in antibiotic export from bacteria and has two comp lete Walker box motifs, Molecular genetic analysis demonstrated that disrup tion of either Walker A box, singly, does not inactivate this protein's eff ect on virulence but that mutations in both Walker A boxes renders it incap able of complementing a chvD mutant strain. Constitutive expression of virG in the chvD mutant strain restored virulence, supporting the hypothesis th at ChvD controls virulence through effects on virG expression.