Activation of Bvg-Repressed Genes in Bordetella pertussis by RisA Requires Cross Talk from Noncooperonic Histidine Kinase RisK

The two-component response regulator RisA, encoded by open reading frame BP3554 in the Tohama I genomic sequence, is a known activator of genes, a set of genes whose expression is increased under the same environmental conditions (known as modulation) that result in repression of the virulence regulon. Here we demonstrate that RisA is phosphorylated and that RisA phosphorylation is required for activation of genes. An adjacent histidine kinase gene, , is truncated by frameshift mutation in but not in or Neither deletion of ' or nor phenotypic modulation with MgSO affected levels of phosphorylated RisA (RisA∼P) in However, RisA phosphorylation did require the histidine kinase encoded by BP3223, here named RisK (cognate histidine kinase of RisA). RisK was also required for expression of the genes. This requirement could be obviated by the introduction of the phosphorylation-mimicking RisA mutant, indicating that an active conformation of RisA, but not phosphorylation , is crucial for activation. Interestingly, expression of genes is still modulated by MgSO in cells harboring the RisA mutation, suggesting that the activated RisA senses additional signals to control expression in response to environmental stimuli. In , the BvgAS two-component system activates the expression of virulence genes by binding of BvgA∼P to their promoters. Expression of the reciprocally regulated genes requires RisA and is also repressed by the Bvg-activated BvgR. RisA is an OmpR-like response regulator, but RisA phosphorylation was not expected because the gene for its presumed, cooperonic, histidine kinase is inactivated by mutation. In this study, we demonstrate phosphorylation of RisA by a noncooperonic histidine kinase. We also show that RisA phosphorylation is necessary but not sufficient for activation but, importantly, is not affected by BvgAS status. Instead, we propose that expression is controlled by BvgAS through its regulation of BvgR, a cyclic di-GMP (c-di-GMP) phosphodiesterase.