Two Surfaces of a Conserved Interdomain Linker Differentially Affect Output from the RST Sensing Module of the Bacillus subtilis Stressosome

Abstract

The stressosome is a 1.8-MDa cytoplasmic complex that conveys environmental signals to the B stress factor of Bacillus subtilis. A functionally irreducible complex contains multiple copies of three proteins: the RsbRA coantagonist, RsbS antagonist, and RsbT serine-threonine kinase. Homologues of these proteins are coencoded in different genome contexts in diverse bacteria, forming a versatile sensing and transmission module called RST after its common constituents. However, the signaling pathway within the stressosome itself is not well defined. The N-terminal, nonheme globin domains of RsbRA project from the stressosome and are presumed to channel sensory input to the C-terminal STAS domains that form the complex core. A conserved, 13-residue ␣-helical linker connects these domains. We probed the in vivo role of the linker using alanine scanning mutagenesis, assaying stressosome output in B. subtilis via a B -dependent reporter fusion. Substitutions at four conserved residues increased output 4-to 30-fold in unstressed cells, whereas substitutions at four nonconserved residues significantly decreased output. The periodicity of these effects supports a model in which RsbRA functions as a dimer in vivo, with the linkers forming parallel paired helices via a conserved interface. The periodicity further suggests that the opposite, nonconserved faces make additional contacts important for efficient stressosome operation. These results establish that the linker influences stressosome output under steady-state conditions. However, the stress response phenotypes of representative linker substitutions provide less support for the notion that the N-terminal globin domain senses acute environmental challenge and transmits this information via the linker helix.

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

“…2D), the difference in activation ("nhaA2p-lacZ + activity with stress" − "nhaA2p-lacZ + activity without stress") relative to the WT ["relative stress activation" (RSA)] rather than the absolute nhaA2p-lacZ + activity was compared. The same approach (measurement of RSA) has been used before in the Bacillus subtilis GSR to discriminate between the contribution of sensory proteins to the basal activity level and their roles in stress activation (31,32). As shown in Fig.…”

Activation of the General Stress Response of Bacillus subtilis by Visible Light

“…It is possible that the correct mutations to disrupt signal sensing in N‐RsbRA simply remain to be identified. However, available data are also consistent with the stress signal entering the stressosome elsewhere, such as directly in the C‐terminal STAS domains of the RsbR proteins . In such a model, the N‐terminal domains of the RsbR proteins could function as modulators of the basal activity of the stressosome .…”

“…Mutations in the globin‐like N‐terminal domain of RsbRA (N‐RsbRA) can affect the basal output level of the GSR in unstressed cells, but none have been identified with significant effects on stress induction of the GSR . Similarly, effects of mutations in the linker between N‐RsbRA and the C‐terminal STAS domain are mostly confined to the basal level of GSR activation in unstressed cells . It is possible that the correct mutations to disrupt signal sensing in N‐RsbRA simply remain to be identified.…”

Simulations of stressosome activation emphasize allosteric interactions between RsbR and RsbT

“…Finally, the cellular automaton enables qualitative analysis of RsbR mutations. Amino-acid substitutions on certain positions of RsbRA result either in elevated or reduced pre-stress output while maintaining a wild type stress response (Table 2) [31,32]. Since the stress response is unaffected, the protein interactions of the stressosome are not involved and thus the truth table remains unchanged.…”