Stoichiometry and perturbation studies of the LiaFSR system of Bacillus subtilis

The response regulator/histidine kinase pair LiaRS of Bacillus subtilis, together with its membrane-bound inhibitor protein LiaF, constitutes an envelope stress-sensing module that is conserved in Firmicutes bacteria. LiaR positively autoregulates the expression of the liaIH-liaGFSR operon from a strictly LiaR-dependent promoter (P(liaI) ). A comprehensive perturbation analysis revealed that the functionality of the LiaFSR system is very susceptible to alterations of its protein composition and amounts. A genetic analysis indicates a LiaF:LiaS:LiaR ratio of 18:4:1. An excess of LiaS over LiaR was subsequently verified by quantitative Western analysis. This stoichiometry, which is crucial to maintain a functional Lia system, differs from any other two-component system studied to date, in which the response regulator is present in excess over the histidine kinase. Moreover, we demonstrate that LiaS is a bifunctional histidine kinase that acts as a phosphatase on LiaR in the absence of a suitable stimulus. An increased amount of LiaR - both in the presence and in the absence of LiaS - leads to a strong induction of P(liaI) activity due to phosphorylation of the response regulator by acetyl phosphate. Our data demonstrate that LiaRS, in contrast to other two-component systems, is non-robust with regard to perturbations of its stoichiometry.