The phenolic vir gene inducer ferulic acid is O-demethylated by the VirH2 protein of an Agrobacterium tumefaciens Ti plasmid

Some or possibly all Ti plasmids of Agrobacterium tumefaciens encode a bicistronic operon designated virH,which encodes two proteins, VirH1 and VirH2, that resemble a family of cytochrome P450-type monooxygenases. Expression of this operon is induced by a family of phenolic compounds that induce all other operons within the vir regulon, We hypothesized that either or both of these proteins might metabolize some or all of these phenolic compounds. We therefore tested induction of a vir promoter by a variety of phenolic compounds in isogenic strains that express or lack virH1 and virH2, Although some compounds were equally effective inducers regardless of the virH status, other compounds induced vir expression far more effectively in the virH mutant than in the virH-proficient host, For all tested compounds, VirH2 appeared to be solely responsible for this effect, One such compound, ferulic acid, was chosen for biochemical analysis, Ferulic acid was degraded by a VirH-proficient host but not by a VirH mutant. The wild-type strain released large amounts of a more hydrophilic compound into the cell supernatant. This compound was tested by mass spectroscopy, nuclear magnetic resonance and UV spectroscopy and found to consist of caffeic acid, This indicates that wild-type strains convert virtually all added ferulic acid to caffeic acid, and that VirH2 is essential for this O-demethylation reaction, Ferulic acid was far more toxic than caffeic acid to the wildtype strain, although the wild-type strain was more resistant to ferulic acid than was the virH mutant, Caffeic acid was slowly removed from the broth, suggesting further metabolic reactions.