The general stress response of Bacillus subtilis and close relatives provides the cell with protection from a variety of stresses. The upstream component of the environmental stress signal transduction cascade is activated by the RsbT kinase that switches binding partners from a 25 S macromolecular complex, the stressosome, to the RsbU phosphatase. Once the RsbU phosphatase is activated by interacting with RsbT, the alternative sigma factor, B , directs transcription of the general stress regulon. Previously, we demonstrated that the N-terminal domain of RsbU mediates the binding of RsbT. We now describe residues in N-RsbU that are crucial to this interaction by experimentation both in vitro and in vivo. Furthermore, crystal structures of the N-RsbU mutants provide a molecular explanation for the loss of interaction. Finally, we also characterize mutants in RsbT that affect binding to both RsbU and a simplified, binary model of the stressosome and thus identify overlapping binding surfaces on the RsbT "switch."In Gram-positive bacteria such as Bacillus subtilis, and the human pathogens Listeria monocytogenes and Staphylococcus aureus, exposure to physical or energy stress results in the transcription of the general stress regulon (1, 2). In B. subtilis the general stress regulon comprises ϳ150 genes (3, 4), the protein products of which confer multiple resistances onto the cell. The general stress regulon is controlled by the alternative sigma factor, B (5-9). The availability of B for forming transcriptionally competent complexes with RNA polymerase is regulated by a set of signal transduction proteins termed regulators of sigma B (Rsb). In unstressed cells B is held in a complex with its anti-sigma factor RsbW and is unable to direct RNA polymerase to the promoters of the general stress genes. RsbV is the anti-anti-sigma factor specific to RsbW, and the RsbV-driven release of B from the RsbW-B complex allows B to displace the housekeeping sigma factor, A , from RNA polymerase holoenzyme (10). RsbW is also a protein kinase that is specific for RsbV, and phosphorylated RsbV (RsbV-P), which cannot form a complex with RsbW, accumulates in unstressed cells (11). Hence, it is the phosphorylation state of RsbV that determines the amount of freely available B for directing the transcription of the general stress regulon.To activate B during stress, RsbV-P must be dephosphorylated. In B. subtilis this is achieved by one of two phosphatases depending on the type of stress that is being experienced by the cell. A decrease in ATP concentration leads to the activation of RsbP (12, 13), either via an interaction with RsbQ or as a direct consequence of the product of the RsbQ hydrolase activity on RsbP. Environmental stresses such as heat or salt shock or ethanol treatment (14 -16) result instead in the activation of RsbU. Like RsbP, RsbU belongs to the type 2C serine/threonine phosphatase family, and RsbU is activated by a physical interaction with its partner protein, RsbT (16,17). RsbT is a member of the GHKL family of ki...