VSRA, A 2ND 2-COMPONENT SENSOR REGULATING VIRULENCE GENES OF PSEUDOMONAS-SOLANACEARUM

The wilt-inducing phytopathogen Pseudomonas solanacearum produces seve ral extracellular virulence factors, both polysaccharides (EPS I) and proteins (EXPs), which are independently regulated by a LysR-type tran scriptional regulator, PhcA, and a histidine kinase sensor, VsrB. Here we characterize a third locus, vsrA, which is also required for norma l production of EPS I, some EXPs and wilt disease. Analysis of eps:: l acZ reporters in vsrA mutants showed that, like vsrB and phcA, vsrA is required for maximal expression (transcription) of eps, which contain s some of the genes necessary for production of EPS I. Unlike vsrB and phcA mutants, however, eps transcription (and EPS I production) by vs rA mutants varies from 3 to 17% of wild-type levels, depending on grow th conditions. inactivation of vsrA also causes a dramatic reduction i n production of three species of EXPs (28 kDa, 48 kDa, and 66 kDa), an d an apparent increase in production of a few other EXPs. Unlike most other EPS-deficient P. solanacearum strains, vsrA mutants caused almos t no disease symptoms when 10(4) cells were stem-inoculated into tomat o plants. This correlated with a greater than 10-fold reduction in the ir ability to grow in planta. vsrA was cloned from a P. solanacearum g enomic library by complementation of the vsrA mutant and was further s ubcloned on a 2.3 kb DNA fragment. PhoA fusion analysis and subcellula r localization of the vsrA gene product in Escherichia coli maxicells suggest that it is a 53 kDa membrane-associated protein. Analysis of t he nucleotide sequence of vsrA revealed a 502 residue open reading fra me with homology to the histidine kinase domain of sensors in the two- component regulator by P. solanacearum is simultaneously controlled by dual two-component sensors.