title: 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 σ(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 α-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 σ(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.
authors.affiliation: Departments of Microbiology, University of California, Davis, California, USA.
authors.forename: Tatiana A
authors.initials: TA
authors.lastname: Gaidenko
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Bie
authors.forename: Enoch P
authors.initials: EP
authors.lastname: Baldwin
authors.forename: Chester W
authors.initials: CW
authors.lastname: Price
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D012808:Sigma Factor / Q000378:metabolism; D015398:Signal Transduction; D013312:Stress, Physiological
chemical_list: D001426:Bacterial Proteins; D050505:Mutant Proteins; D010750:Phosphoproteins; C119218:RsbR protein, Bacillus subtilis; C079011:SigB protein, Bacteria; D012808:Sigma Factor; C119219:RsbT protein, Bacillus subtilis; D017346:Protein Serine-Threonine Kinases