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 theBordetella pertussis Tohama I genomic sequence, is a known activator ofvrg genes, a set of genes whose expression is increased under the same environmental conditions (known as modulation) that result in repression of thebvgAS virulence regulon. Here we demonstrate that RisA is phosphorylatedin vivo and that RisA phosphorylation is required for activation ofvrg genes. An adjacent histidine kinase gene,risS , is truncated by frameshift mutation inB. pertussis but not inBordetella bronchiseptica orBordetella parapertussis . Neither deletion ofrisS ′ orbvgAS nor phenotypic modulation with MgSO4 affected levels of phosphorylated RisA (RisA∼P) inB. pertussis . 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 thevrg genes. This requirement could be obviated by the introduction of the phosphorylation-mimicking RisAD60E mutant, indicating that an active conformation of RisA, but not phosphorylationper se , is crucial forvrg activation. Interestingly, expression ofvrg genes is still modulated by MgSO4 in cells harboring the RisAD60E mutation, suggesting that the activated RisA senses additional signals to controlvrg expression in response to environmental stimuli.IMPORTANCE InB. pertussis , the BvgAS two-component system activates the expression of virulence genes by binding of BvgA∼P to their promoters. Expression of the reciprocally regulatedvrg 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 RisAin vivo by a noncooperonic histidine kinase. We also show that RisA phosphorylation is necessary but not sufficient forvrg activation but, importantly, is not affected by BvgAS status. Instead, we propose thatvrg expression is controlled by BvgAS through its regulation of BvgR, a cyclic di-GMP (c-di-GMP) phosphodiesterase.