A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σ^T and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ₂ and σ₄, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ₂ and σ₄ in the SL domain to open about a flexible connector loop and bind anti‐σ factor.     

The H‐NS‐like protein StpA represses the RpoS (σ38) regulon during exponential growth of Salmonella Typhimurium

StpA is a paralogue of the nucleoid‐associated protein H‐NS that is conserved in a range of enteric bacteria and had no known function in Salmonella Typhimurium. We show that 5% of the Salmonella genome is regulated by StpA, which contrasts with the situation in Escherichia coli where deletion of stpA only had minor effects on gene expression. The StpA‐dependent genes of S. Typhimurium are a specific subset of the H‐NS regulon that are predominantly under the positive control of σ³⁸ (RpoS), CRP‐cAMP and PhoP. Regulation by StpA varied with growth phase; StpA controlled σ³⁸ levels at mid‐exponential phase by preventing inappropriate activation of σ³⁸ during rapid bacterial growth. In contrast, StpA only activated the CRP‐cAMP regulon during late exponential phase. ChIP‐chip analysis revealed that StpA binds to PhoP‐dependent genes but not to most genes of the CRP‐cAMP and σ³⁸ regulons. In fact, StpA indirectly regulates σ³⁸‐dependent genes by enhancing σ³⁸ turnover by repressing the anti‐adaptor protein rssC. We discovered that StpA is essential for the dynamic regulation of σ³⁸ in response to increased glucose levels. Our findings identify StpA as a novel growth phase‐specific regulator that plays an important physiological role by linking σ³⁸ levels to nutrient availability.     

Conformational flexibility of σ70 in anti‐terminator loading

In promoter DNA, the preferred distance of the ?10 and ?35 elements for interacting with RNA polymerase‐bound σ⁷⁰ is 17 bp. However, the Devi et al. paper in this issue of Molecular Microbiology demonstrates that when the C‐terminal domain of σ⁷⁰, including the 3.2 linker, is not attached to the core enzyme, distances between 0 and 3 bp can be accommodated. This attests to the great flexibility of the 3.2 linker. The particularly stable complex with the 2 bp separation may lend itself to structural studies of an early elongation complex containing σ⁷⁰.     

PDZ domains of RseP are not essential for sequential cleavage of RseA or stress‐induced σE activation in vivo

The Escherichia coli σ^E extracytoplasmic stress response monitors and responds to folding stress in the cell envelope. A protease cascade directed at RseA, a membrane‐spanning anti‐σ that inhibits σ^E activity, controls this critical signal‐transduction system. Stress cues activate DegS to cleave RseA; a second cleavage by RseP releases RseA from the membrane, enabling its rapid degradation. Stress control of proteolysis requires that RseP cleavage is dependent on DegS cleavage. Recent in vitro and structural studies found that RseP cleavage requires binding of RseP PDZ‐C to the newly exposed C‐terminal residue (Val148) of RseA, generated by DegS cleavage, explaining dependence. We tested this mechanism in vivo. Neither mutation in the putative PDZ ligand‐binding regions nor even deletion of entire RseP PDZ domains had significant effects on RseA cleavage in vivo, and the C‐terminal residue of DegS‐processed RseA also little affected RseA cleavage. Indeed, strains with a chromosomal rseP gene deleted for either PDZ domain and strains with a chromosomal rseA V148 mutation grew normally and exhibited almost normal σ^E activation in response to stress signals. We conclude that recognition of the cleaved amino acid by the RseP PDZ domain is not essential for sequential cleavage of RseA and σ^E stress response in vivo.