VsrA, a second two‐component sensor regulating virulence genes of Pseudomonas solanacearum

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Abstract
Summary: The wilt‐inducing phytopathogen Pseudomonas solanacearum produces several extracellular virulence factors, both polysaccharides (EPS I) and proteins (EXPs), which are independently regulated by a LysR‐type transcriptional regulator, PhcA, and a histidine kinase sensor, VsrB. Here we characterize a third locus, vsrA, which is also required for normal production of EPS I, some EXPs and wilt disease. Analysis of eps::lacZ reporters in vsrA mutants showed that, like vsrB and phcA, vsrA is required for maximal expression (transcription) of eps, which contains some of the genes necessary for production of EPS I. Unlike vsrB and phcA mutants, however, eps transcription (and EPS I production) by vsrA mutants varies from 3 to 17% of wild‐type levels, depending on growth conditions. Inactivation of vsrA also causes a dramatic reduction in production of three species of EXPs (28kDa, 48kDa, and 66kDa), and an apparent increase in production of a few other EXPs. Unlike most other EPS‐deficient P. solanacearum strains, vsrA mutants caused almost no disease symptoms when 104 cells were stem‐inoculated into tomato plants. This correlated with a greater than 10‐fold reduction in their ability to grow in plants. vsrA was cloned from a P. solanacearum genomic library by complementation of the vsrA mutant and was further subcloned on a 2.3kb DNA fragment. PhoA fusion analysis and subcellular localization of the vsrA gene product in Escherichia coli maxicells suggest that it is a 53 kDa membrane‐associated protein. Analysis of the nucleotide sequence of vsrA revealed a 502 residue open reading frame with homology to the histidine kinase domain of sensors in the two‐component regulator family. This discovery shows that EPS I production by P. solanacearum is simultaneously controlled by dual two‐component sensors.