Mutational analysis of Escherichia coli σ28 and its target promoters reveals recognition of a composite −10 region, comprised of an 'extended −10' motif and a core −10 element

σ²⁸ controls the expression of flagella-related genes and is the most widely distributed alternative σ factor, present in motile Gram-positive and Gram-negative bacteria. The distinguishing feature of σ²⁸ promoters is a long −10 region (GCCGATAA). Despite the fact that the upstream GC is highly conserved, previous studies have not indicated a functional role for this motif. Here we examine the functional relevance of the GCCG motif and determine which residues in σ²⁸ participate in its recognition. We find that the GCCG motif is a functionally important composite element. The upstream GC constitutes an extended −10 motif and is recognized by R91, a residue in Domain 3 of σ²⁸. The downstream CG is the upstream edge of −10 region of the promoter; two residues in Region 2.4, D81 and R84, participate in its recognition. Consistent with their role in base-specific recognition of the promoter, R91, D81 and D84 are universally conserved in σ²⁸ orthologues. σ²⁸ is the second Group 3 σ shown to use an extended −10 region in promoter recognition, raising the possibility that other Group 3 σs will do so as well.     

Dissection of recognition determinants of Escherichia coli σ32 suggests a composite −10 region with an 'extended −10' motif and a core −10 element

σ³² controls expression of heat shock genes in Escherichia coli and is widely distributed in proteobacteria. The distinguishing feature of σ³² promoters is a long −10 region (CCCCATNT) whose tetra-C motif is important for promoter activity. Using alanine-scanning mutagenesis of σ³² and in vivo and in vitro assays, we identified promoter recognition determinants of this motif. The most downstream C (−13) is part of the −10 motif; our work confirms and extends recognition determinants of −13C. Most importantly, our work suggests that the two upstream Cs (−16, −15) constitute an 'extended −10' recognition motif that is recognized by K130, a residue universally conserved in β- and γ-proteobacteria. This residue is located in the α-helix of σDomain 3 that mediates recognition of the extended −10 promoter motif in other σs. K130 is not conserved in α- and δ-/ε-proteobacteria and we found that σ³² from the α-proteobacterium Caulobacter crescentus does not need the extended −10 motif for high promoter activity. This result supports the idea that K130 mediates extended −10 recognition. σ³² is the first Group 3 σ shown to use the 'extended −10' recognition motif.     

Use of cell wall stress to characterize σ22 (AlgT/U) activation by regulated proteolysis and its regulon in Pseudomonas aeruginosa

MucA sequesters extracytoplasmic function (ECF) σ²² ( algT/U encoded) from target promoters including P algD for alginate biosynthesis. We have shown that cell wall stress (e.g. d -cycloserine) is a potent inducer of the algD operon. Here we showed that MucB, encoded by the algT-mucABCD operon, interacts with MucA in the sigma–sequestration complex. We hypothesized that AlgW protease (a DegS homologue) is activated by cell wall stress to cleave MucA and release σ²². When strain PAO1 was exposed to d -cycloserine, MucA was degraded within just 10 min, and σ²² was activated. However, in an algW mutant, MucA was stable with no increased σ²² activity. Studies on a yaeL mutant, defective in an RseP/YaeL homologue, suggest that YaeL protease cleaves MucA only after cleavage by AlgW. A defect in mucD , encoding a periplasmic HtrA/DegP homologue, caused MucA instability, suggesting MucD degrades cell wall stress signals. Overall, these data indicate that cell wall stress signals release σ²² by regulated intramembrane proteolysis (RIP). Microarray analyses identified genes of the early and late cell wall stress stimulon, which included genes for alginate production. The subset of genes in the σ²² regulon was then determined, which included gene products predicted to contribute to recovery from cell wall stress.