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Concept: Sigma factor

170

The alternate sigma factor sigH of Mycobacterium tuberculosis is expressed under stress and acts as a major regulator of several genes, including some other sigma factors and redox systems. While it is auto-regulated by its own promoter at the transcriptional level, its regulation at the post-translational level is through its cognate protein, an anti-sigma factor, RshA. Hither before RshA was believed to be a zinc-associated anti-sigma factor (ZAS) and the binding of RshA to SigH is redox dependent. Here, we show that RshA coordinates a [2Fe-2S] cluster using cysteines as ligands and native RshA has more affinity to [2Fe-2S] cluster than to zinc. Furthermore, we used amide hydrogen deuterium exchange mass spectrometry (HDX-MS), followed by site-directed mutagenesis in SigH and RshA, to elucidate the interaction mechanism of RshA and SigH and the potential role of metal ion clustering in SigH regulation. Three regions in SigH, comprising of residues 1-25, 58-69, 90-111, 115-132 and 157-196 and residues 35-57 of RshA show decreased deuterium exchange and reflect decreased solvent accessibility upon complexation with SigH. Of the three RshA mutants, created based on the HDX results, the RsHA E37A mutant shows stronger interaction with SigH, relative to WT RshA, while the H49A mutant abolishes interactions and the C(53)XXC(56)AXXA mutant has no effect on complexation with SigH. The D22A, D160A and E162 SigH mutants show significantly decreased binding to RshA and the E168A mutant completely abolished interactions with RshA, indicating that the SigH-RshA interaction is mediated by salt bridges. In addition, SigH-RshA interaction does not require clustering of metal ions. Based on our results, we propose a molecular model of the SigH-RshA interaction.

Concepts: Gene, Gene expression, Mass spectrometry, Hydrogen, Atom, Metal, Zinc, Sigma factor

12

In cells, specific regulators often compete for limited amounts of a core enzymatic resource. It is typically assumed that competition leads to partitioning of core enzyme molecules among regulators at constant levels. Alternatively, however, different regulatory species could time share, or take turns utilizing, the core resource. Using quantitative time-lapse microscopy, we analyzed sigma factor activity dynamics, and their competition for RNA polymerase, in individual Bacillus subtilis cells under energy stress. Multiple alternative sigma factors were activated in ∼1-hr pulses in stochastic and repetitive fashion. Pairwise analysis revealed that two sigma factors rarely pulse simultaneously and that some pairs are anti-correlated, indicating that RNAP utilization alternates among different sigma factors. Mathematical modeling revealed how stochastic time-sharing dynamics can emerge from pulse-generating sigma factor regulatory circuits actively competing for RNAP. Time sharing provides a mechanism for cells to dynamically control the distribution of cell states within a population. Since core molecular components are limiting in many other systems, time sharing may represent a general mode of regulation.

Concepts: DNA, Gene expression, Enzyme, RNA, RNA polymerase, Bacillus, Bacillus subtilis, Sigma factor

4

At the beginning of the transcription process, the RNAP core enzyme requires a sigma-factor to recognize the genomic location where the process initiates. Although the critical role of sigma-factors has been long appreciated and characterized for many individual promoters, we do not yet have a genome-scale assessment of their function.

Concepts: Gene, Gene expression, Promoter, Transcription, Enzyme, Escherichia coli, RNA polymerase, Sigma factor

2

The ability of bacteria to adapt to stress depends on the conditional expression of specific sets of genes. Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma (σ) factors that regulate functions important for survival under conditions eliciting cell envelope stress. Of these, four have been studied in detail: σ M , σ W , σ X and σ V . These four σ factors recognize overlapping sets of promoters, although the sequences that determine this overlapping recognition are incompletely understood. A major role in promoter selectivity has been ascribed to the core -10 and -35 promoter elements. Here, we demonstrate that a homopolymeric T-tract motif, proximal to the -35 element, functions in combination with the core promoter sequences to determine selectivity for ECF sigma factors. This motif is most critical for promoter activation by σ V , and contributes variably to activation by σ M , σ X and σ W . We propose that this motif, which is a feature of the deduced promoter consensus for a subset of ECF σ factors from many species, imparts intrinsic DNA curvature to influence promoter activity. The differential effect of this region among ECF σ factors thereby provides a mechanism to modulate the nature and extent of regulon overlap.

Concepts: DNA, Gene, Gene expression, Promoter, Bacteria, RNA polymerase, Bacillus, Sigma factor

2

A core component of the α-proteobacterial general stress response (GSR) is the extracytoplasmic function (ECF) sigma factor EcfG, exclusively present in this taxonomic class. Half of the completed α-proteobacterial genome sequences contain two or more copies of genes encoding σ(EcfG) -like sigma factors, with the primary copy typically located adjacent to genes coding for a cognate anti-sigma factor (NepR) and two-component response regulator (PhyR). So far, the widespread occurrence of additional, non-canonical σ(EcfG) copies has not satisfactorily been explained. This study explores the hierarchical relation between Rhizobium etli σ(EcfG1) and σ(EcfG2) , canonical and non-canonical σ(EcfG) proteins, respectively. Contrary to reports in other species, we find that σ(EcfG1) and σ(EcfG2) act in parallel, as nodes of a complex regulatory network, rather than in series, as elements of a linear regulatory cascade. We demonstrate that both sigma factors control unique yet also shared target genes, corroborating phenotypic evidence. σ(EcfG1) drives expression of rpoH2, explaining the increased heat sensitivity of an ecfG1 mutant, while katG is under control of σ(EcfG2) , accounting for reduced oxidative stress resistance of an ecfG2 mutant. We also identify non-coding RNA genes as novel σ(EcfG) targets. We propose a modified model for GSR regulation in R. etli, in which σ(EcfG1) and σ(EcfG2) function largely independently. Based on a phylogenetic analysis and considering the prevalence of α-proteobacterial genomes with multiple σ(EcfG) copies, this model may also be applicable to numerous other species.

Concepts: DNA, Gene, Gene expression, Virus, Genome, RNA, Non-coding RNA, Sigma factor

1

Streptomyces are of great biological and industrial significance due to their complex morphological development and ability to produce numerous secondary metabolites. However, the intrinsic biochemical mechanisms underlying morphogenesis and secondary metabolism are rarely revealed, partially because of the limited availability of the biochemical tools in Streptomyces. Here we provided series of integrative vectors with various affinity tags, including single tags 3×FLAG, 3×HA, 3×Strep-tag II, 18×His, 13×Myc, and dual tags, all of which were driven from a strong constitutive promoter ermEp*. Using a sigma factor SigT from S. coelicolor as a model, we successfully expressed and immuno-detected SigT fused with all tags. Moreover, after SigT was N-terminally tagged with 3×FLAG and C-terminally tagged with 18×His, we isolated SigT-interactive proteins from the S. coelicolor lysate based on the tandem affinity purification (TAP). Particularly, among the proteins purified, the SigT cognate anti-sigma factor RstA ranked the top with the most total independent spectra. These data suggested the feasibility of these affinity tags in Streptomyces, which will be widely employed to explore the biochemical mechanisms to further understand the dynamic and elaborate regulation in this genus.

Concepts: DNA, Protein, Gene expression, Promoter, Molecular biology, Metabolism, Species, Sigma factor

1

Sigma factors are multi-domain subunits of bacterial RNA polymerase (RNAP) that play critical roles in transcription initiation, including the recognition and opening of promoters as well as the initial steps in RNA synthesis. This review focuses on the structure and function of the major sigma-70 class that includes the housekeeping sigma factor (Group 1) that directs the bulk of transcription during active growth, and structurally-related alternative sigma factors (Groups 2-4) that control a wide variety of adaptive responses such as morphological development and the management of stress. A recurring theme in sigma factor control is their sequestration by anti-sigma factors that occlude their RNAP-binding determinants. Sigma factors are then released through a wide variety of mechanisms, often involving branched signal transduction pathways that allow the integration of distinct signals. Three major strategies for sigma release are discussed: regulated proteolysis, partner-switching, and direct sensing by the anti-sigma factor.

Concepts: DNA, Gene, Gene expression, Promoter, Enzyme, Transcription factor, RNA polymerase, Sigma factor

0

It has been firmly established that organic osmolytes (compatible solutes) of halophilicBacteriaandArchaeahave positive effects on conformation and activity of proteins, and may therefore improve their functional production. In particular, the amino acid derivative ectoine is known for its conformational stabilization, aggregation suppression, and radical protection properties. The natural producer and industrial production strainHalomonas elongataaccumulates ectoine in the cytoplasm, and as a result offers a unique stabilizing environment for recombinant proteins. For the construction of broad hoast range vector systems with fluorescent reporter proteins, we chose the salt-inducible promoter region of the ectoine gene cluster (promA). A closer inspection of the genetic background revealed that its combination of sigma 38 (σ38) and sigma 70 (σ70) promoters was followed by a weak ribosomal binding site (RBS). This inspired a systematic approach for the construction of apromA-based vector series with a synthetic RBS region using the RBS Calculator v2.0, which resulted in a greatly improved salt-dependent expression-even in a deletion construct lacking the σ38promoter. To expand the application range of this expression system, we looked further into the possible export of recombinant proteins into the periplasm. Bothsecandtatleader sequences fromH. elongataproved to be suitable for directed periplasmic transport into an extreme environment of freely selectable ionic strength.

Concepts: DNA, Protein, Gene, Gene expression, Promoter, Amino acid, RNA polymerase, Sigma factor

0

Global changes in bacterial gene expression can be orchestrated by the coordinated activation/deactivation of alternative sigma (σ) factor subunits of RNA polymerase. Sigma factors themselves are regulated in myriad ways, including via anti-sigma factors. Here, we have determined the solution structure of anti-sigma factor CsfB, responsible for inhibition of two alternative sigma factors, σGand σE, during spore formation by Bacillus subtilis. CsfB assembles into a symmetrical homodimer, with each monomer bound to a single Zn2+ion via a treble-clef zinc finger fold. Directed mutagenesis indicates that dimer formation is critical for CsfB-mediated inhibition of both σGand σE, and we have characterized these interactions in vitro. This work represents an advance in our understanding of how CsfB mediates inhibition of two alternative sigma factors to drive developmental gene expression in a bacterium.

Concepts: DNA, Gene, Cell nucleus, Gene expression, Bacteria, Transcription, RNA polymerase, Sigma factor

0

We assessed the occurrence of phenotypic variation in Azospirillum brasilense strains Sp7, Cd, Sp245, Az39 and phv2 during growth in rich media, screening for variants altered in colony pigmentation or extracellular polysaccharide (EPS) production. Previous studies showed that EPS-overproducing variants of Sp7 appear frequently following starvation or growth in minimal medium. In contrast, no such variants were detected during growth in rich media in the tested strains except for few variants of phv2. Regarding alteration in colony pigmentation (from pink to white in strain Cd and from white to pink in the others), strain Sp7 showed a relatively high frequency of variation (0.009% to 0.026%). Strain Cd showed a lower frequency of alteration in pigmentation (0 to 0.008%), and this type of variation was not detected in the other strains. In A. brasilense, carotenoid synthesis is controlled by two RpoE sigma factors and their cognate ChrR anti-sigma factors, the latter acting as negative regulators of carotenoid synthesis. Here, all tested (n=28) pink variants of Sp7 carried mutations in one of the anti-sigma factor genes, chrR1. Our findings indicate that, in A. Brasilense, phenotypic variation is strain- and environment-dependent and support the central role of ChrR1 in regulation of carotenoid production.

Concepts: DNA, Gene expression, Evolution, DNA repair, Phenotype, White, Sigma factor, Anti-sigma factors