AbstractThe whooping cough agentBordetella pertussisregulates the production of its virulence factors by the BvgA/S system. Phosphorylated BvgA activates the virulence-activated genes (vags) and represses the expression of the virulence-repressed genes (vrgs) via the activation of thebvgRgene. In modulating conditions, with MgSO4, the BvgA/S system is inactive, and thevrgs are expressed. Here, we show that the expression of almost allvrgs depends on RisA, another transcriptional regulator. We also show that somevags are surprisingly no longer modulated by MgSO4in therisA−background. RisA also regulates the expression of other genes, including chemotaxis and flagellar operons, iron-regulated genes, and genes of unknown function, which may or may not be controlled by BvgA/S. We identified RisK as the likely cognate RisA kinase and found that it is important for expression of most, but not all RisA-regulated genes. This was confirmed using the phosphoablative RisAD60N and the phosphomimetic RisAD60E analogues. Thus the RisA regulon adds a new layer of complexity toB. pertussisvirulence gene regulation.