Regulation of RNA polymerase sigma subunit synthesis in Escherichia coli: intracellular levels of four species of sigma subunit under various growth conditions.

By a quantitative Western immunoblot analysis, the intracellular levels of two principal sigma subunits, sigma 70 (sigma D, the rpoD gene product) and sigma 38 (sigma S, the rpoS gene product), and of two minor sigma subunits, sigma 54 (sigma N, the rpoN gene product) and sigma 28 (sigma F, the rpoF gene product), were determined in two Escherichia coli strains, W3110 and MC4100. The results indicated that the levels of sigma 54 and sigma 28 are maintained at 10 and 50%, respectively, of the level of sigma 70 in both strains growing at both exponential and stationary phases, but in agreement with the previous measurement for strain MC4100 (M. Jishage and A. Ishihama, J. Bacteriol. 177:6832-6835, 1995), the level of sigma 38 was undetectable at the exponential growth phase but increased at 30% of the level of sigma 70 at the stationary phase. Stress-coupled change in the intracellular level was observed for two sigma subunits: (i) the increase in sigma 38 level and the decrease in sigma 28 level upon exposure to heat shock at the exponential phase and (ii) the increase in sigma 38 level under high-osmolality conditions at both the exponential and stationary phases.     

A master regulator sigmaB governs osmotic and oxidative response as well as differentiation via a network of sigma factors in Streptomyces coelicolor

The differentiating bacterium Streptomyces coelicolor harbours some 66 sigma factors, which support its complex life cycle. sigma(B), a functional homologue of sigma(S) from Escherichia coli, controls both osmoprotection and differentiation in S. coelicolor A3(2). Microarray analysis revealed sigma(B)-dependent induction of more than 280 genes by 0.2 M KCl. These genes encode several sigma factors, oxidative defence proteins, chaperones, systems to provide osmolytes, cysteine, mycothiol, and gas vesicle. sigma(B) controlled induction of itself and its two paralogues (sigma(L) and sigma(M)) in a hierarchical order of sigma(B)-->sigma(L)-->sigma(M), as revealed by S1 mapping and Western blot analyses. The phenotype of each sigma mutant suggested a sequential action in morphological differentiation; sigma(B) in forming aerial mycelium, sigma(L) in forming spores and sigma(M) for efficient sporulation. sigma(B) was also responsible for the increase in cysteine and mycothiol, the major thiol buffer in actinomycetes, upon osmotic shock, revealing an overlap between protections against osmotic and oxidative stresses. Proteins in sigB mutant were more oxidized (carbonylated) than the wild type. These results support a hypothesis that sigma(B) serves as a master regulator that triggers other related sigma factors in a cascade, and thus regulates differentiation and osmotic and oxidative response in S. coelicolor.     

Synthesis and structure-activity relationships of N-(3-phenylpropyl)-N'-benzylpiperazines: Potent ligands for sigma1 and sigma2 receptors

Ten N-(3-phenylpropyl)-N'-benzylpiperazines having different substituents on the benzyl moiety were synthesized and evaluated for sigma(1) and sigma(2) receptor binding. The sigma(1) affinities were 0.37-2.80nM, sigma(2) affinities were 1.03-34.3nM, and selectivities, as sigma(2)/sigma(1) affinity ratios, ranged from 1.4 to 52. Three compounds tested in a phenytoin shift binding assay profiled as probable sigma(1) antagonists. Quantitative structure-activity relationships depended on pi(x), MR or E(s) and Hammett sigma values. The hydrophobicity term is negative for sigma(1) binding but positive for sigma(2) binding, indicating a major difference between the pharmacophores.     

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR

Rhodobacter sphaeroides sigma(E) is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R.sphaeroides, sigma(E) activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits sigma(E) activity. We found that formation of the sigma(E):ChrR complex inhibits the ability of sigma(E) to form a stable complex with core RNA polymerase. Since the sigma(E):ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in sigma(E) that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of sigma(E) that either increased or decreased the sensitivity of sigma(E) for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of sigma(E) to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the sigma(70) superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of sigma(E) interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.