Cognate Anti-sigma Factor Research Articles

Molecular regulation of Mycobacterium tuberculosis Sigma factor H with Anti-sigma factor RshA under stress condition

Mycobacterium tuberculosis is the causative agent of tuberculosis, the leading fatal infectious disease that claims millions of lives every year. M. tuberculosis regulates its stress condition response using its regulatory protein, Sigma Factor H, which binds with its cognate anti-sigma factor RshA in normal conditions, forming a complex inhibiting transcription. During oxidative stress, SigH is released from the complex and binds to RNA Polymerase (RNAP) to initiate transcription. Thus, it is important to understand the molecular conformational state of SigH in complex with different protein partners under different cellular or environmental contexts. This work intends to analyze the SigH-RshA complex, which revealed the variation in SigH shown during complex formation with RNAP and RshA, respectively. Previously, Hydrogen Deuterium Exchange-Mass Spectrometry (HDX-MS) analysis of SigH-RshA interaction provided a detailed insight into the critical residues participating in the interaction. The HDX-MS data were used to dock RshA on the open conformation of SigH from the SigH-RNAP complex structure (PDB: 5ZX2), and closed conformation was obtained from protein modelling. The docking revealed that closed conformation of SigH complexing with RshA in terms of HDX-MS data revealed a major structural shift in SigH while interacting with two different binding partners, RshA and RNAP, under variable environmental conditions. This structural shift of SigH with RshA and RNAP has significance in understanding the stress response of M. tuberculosis, and SigH could prove to be a potential drug target.

Computational and cellular exploration of the protein-protein interaction between Vibrio fischeri STAS domain protein SypA and serine kinase SypE

ABSTRACT Anti-sigma factor antagonists SpoIIAA and RsbV from Bacillus subtilis are the archetypes for single-domain STAS proteins in bacteria. The structures and mechanisms of these proteins along with their cognate anti-sigma factors have been well studied. SpoIIAA and RsbV utilize a partner-switching mechanism to regulate gene expression through protein-protein interactions to control the activity of their downstream anti-sigma factor partners. The Vibrio fischeri STAS domain protein SypA is also proposed to employ a partner-switching mechanism with its partner SypE, a serine kinase/phosphatase that controls SypA’s phosphorylation state. However, this regulation appears opposite to the canonical pathway, with SypA being the more downstream component rather than SypE. Here we explore the commonalities and differences between SypA and the canonical single-domain STAS proteins SpoIIAA and RsbV. We use a combination of AlphaFold 2 structure predictions and computational modeling to investigate the SypA-SypE binding interface. We then test a subset of our predictions in V.fischeri by generating and expressing SypA variants. Our findings suggest that, while SypA shares many sequence and structural traits with anti-sigma factor antagonist STAS domain proteins, there are significant differences that may account for SypA’s distinct regulatory output.

Analysis of Whole-Genome Sequences for the Prediction of Penicillin Resistance and β-Lactamase Activity in Bacillus anthracis.

Penicillin (PEN) is a low-cost option for anthrax treatment, but naturally occurring resistance has been reported. β-Lactamase expression (bla1, bla2) in Bacillus anthracis is regulated by a sigma factor (SigP) and its cognate anti-sigma factor (RsiP). Mutations leading to truncation of RsiP were previously described as a basis for PEN resistance. Here, we analyze whole-genome sequencing (WGS) data and compare the chromosomal sigP-bla1 regions from 374 B. anthracis strains to determine the frequency of mutations, identify mutations associated with PEN resistance, and evaluate the usefulness of WGS for predicting PEN resistance. Few (3.5%) strains contained at least 1 of 11 different mutations in sigP, rsiP, or bla1. Nine of these mutations have not been previously associated with PEN resistance. Four strains showed PEN resistance (PEN-R) by conventional broth microdilution, including 1 strain with a novel frameshift in rsiP. One strain that carries the same rsiP frameshift mutation as that found previously in a PEN-R strain showed a PEN-susceptible (PEN-S) phenotype and exhibited decreased bla1 and bla2 transcription. An unexpectedly small colony size, a reduced growth rate, and undetectable β-lactamase activity levels (culture supernatant and cell lysate) were observed in this PEN-S strain. Sequence analysis revealed mutations in genes associated with growth defects that may contribute to this phenotype. While B. anthracis rsiP mutations cannot be exclusively used to predict resistance, four of the five strains with rsiP mutations were PEN-R. Therefore, the B. anthracis sigP-bla1 region is a useful locus for WGS-based PEN resistance prediction, but phenotypic testing remains essential. IMPORTANCE Determination of antimicrobial susceptibility of B. anthracis is essential for the appropriate distribution of antimicrobial agents for postexposure prophylaxis (PEP) and treatment of anthrax. Analysis of WGS data allows for the rapid detection of mutations in antimicrobial resistance (AMR) genes in an isolate, but the presence of a mutation in an AMR gene does not always accurately predict resistance. As mutations in the anti-sigma factor RsiP have been previously associated with high-level penicillin resistance in a limited number of strains, we investigated WGS assemblies from 374 strains to determine the frequency of mutations and performed functional antimicrobial susceptibility testing. Of the five strains that contained mutations in rsiP, only four were PEN-R by functional antimicrobial susceptibility testing. We conclude that while sequence analysis of this region is useful for AMR prediction in B. anthracis, genetic analysis should not be used exclusively and phenotypic susceptibility testing remains essential.

Development of Series of Affinity Tags in Streptomyces

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.

Translational Control of the SigR-Directed Oxidative Stress Response in Streptomyces via IF3-Mediated Repression of a Noncanonical GTC Start Codon.

ABSTRACTThe major oxidative stress response in Streptomyces is controlled by the sigma factor SigR and its cognate antisigma factor RsrA, and SigR activity is tightly controlled through multiple mechanisms at both the transcriptional and posttranslational levels. Here we show that sigR has a highly unusual GTC start codon and that this leads to another level of SigR regulation, in which SigR translation is repressed by translation initiation factor 3 (IF3). Changing the GTC to a canonical start codon causes SigR to be overproduced relative to RsrA, resulting in unregulated and constitutive expression of the SigR regulon. Similarly, introducing IF3* mutations that impair its ability to repress SigR translation has the same effect. Thus, the noncanonical GTC sigR start codon and its repression by IF3 are critical for the correct and proper functioning of the oxidative stress regulatory system. sigR and rsrA are cotranscribed and translationally coupled, and it had therefore been assumed that SigR and RsrA are produced in stoichiometric amounts. Here we show that RsrA can be transcribed and translated independently of SigR, present evidence that RsrA is normally produced in excess of SigR, and describe the factors that determine SigR-RsrA stoichiometry.

Genetic evidence that multiple proteases are involved in modulation of heat-induced activation of the sigma factor SigI in Bacillus subtilis.

The Bacillus subtilis sigI-rsgI operon encodes the heat-inducible sigma factor SigI and its cognate anti-sigma factor RsgI. The heat-activated SigI positively regulates expression of sigI itself and genes involved in cell wall homeostasis and heat resistance. It remains unknown which protease(s) may contribute to degradation of RsgI and heat-induced activation of SigI. In this study, we found that transcription of sigI from its σI-dependent promoter under heat stress was downregulated in a strain lacking the heat-inducible sigma factor SigB. Deletion of protease-relevant clpP, clpC or rasP severely impaired sigI expression during heat stress, whereas deletion of clpE partially impaired sigI expression. Complementation of mutations with corresponding intact genes restored sigI expression. In a null mutant of rsgI, SigI was activated and sigI expression was strongly upregulated during normal growth and under heat stress. In this rsgI mutant, further inactivation of rasP or clpE did not affect sigI expression, whereas further inactivation of clpP or clpC severely or partially impaired sigI expression. Spx negatively influenced sigI expression during heat stress. Possible implications are discussed. Given that clpC, clpP and spx are directly regulated by SigB, SigB appears to control sigI expression under heat stress via ClpC, ClpP and Spx.

Glucose Induces ECF Sigma Factor Genes, sigX and sigM, Independent of Cognate Anti-sigma Factors through Acetylation of CshA in Bacillus subtilis.

Extracytoplasmic function (ECF) σ factors have roles related to cell envelope and/or cell membrane functions, in addition to other cellular functions. Without cell-surface stresses, ECF σ factors are sequestered by the cognate anti-σ factor, leading to inactivation and the resultant repression of regulons due to the inhibition of transcription of their own genes. Bacillus subtilis has seven ECF σ factors including σX and σM that transcribe their own structural genes. Here, we report that glucose addition to the medium induced sigX and sigM transcription independent of their anti-σ factors. This induction was dependent on an intracellular acetyl-CoA pool. Transposon mutagenesis searching for the mutants showing no induction of sigX and sigM revealed that the cshA gene encoding DEAD-box RNA helicase is required for gene induction. Global analysis of the acetylome in B. subtilis showed CshA has two acetylated lysine residues. We found that in a cshA mutant with acetylation-abolishing K to R exchange mutations, glucose induction of sigX and sigM was abolished and that glucose addition stimulated acetylation of CshA in the wild type strain. Thus, we present a model wherein glucose addition results in a larger acetyl-CoA pool, probably leading to increased levels of acetylated CshA. CshA is known to associate with RNA polymerase (RNAP), and thus RNAP with acetylated CshA could stimulate the autoregulation of sigX and sigM. This is a unique model showing a functional link between nutritional signals and the basal transcription machinery.

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis.

BackgroundBacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR.ResultsRpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains.ConclusionIn S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0357-0) contains supplementary material, which is available to authorized users.

HPr antagonizes the anti-σ 70 activity of Rsd in Escherichia coli

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) is a multicomponent system that participates in a variety of physiological processes in addition to the phosphorylation-coupled transport of numerous sugars. In Escherichia coli and other enteric bacteria, enzyme IIA(Glc) (EIIA(Glc)) is known as the central processing unit of carbon metabolism and plays multiple roles, including regulation of adenylyl cyclase, the fermentation/respiration switch protein FrsA, glycerol kinase, and several non-PTS transporters, whereas the only known regulatory role of the E. coli histidine-containing phosphocarrier protein HPr is in the activation of glycogen phosphorylase. Because HPr is known to be more abundant than EIIA(Glc) in enteric bacteria, we assumed that there might be more regulatory mechanisms connected with HPr. The ligand fishing experiment in this study identified Rsd, an anti-sigma factor known to complex with σ(70) in stationary-phase cells, as an HPr-binding protein in E. coli. Only the dephosphorylated form of HPr formed a tight complex with Rsd and thereby inhibited complex formation between Rsd and σ(70). Dephosphorylated HPr, but not phosphorylated HPr, antagonized the inhibitory effect of Rsd on σ(70)-dependent transcriptions both in vivo and in vitro, and also influenced the competition between σ(70) and σ(S) for core RNA polymerase in the presence of Rsd. Based on these data, we propose that the anti-σ(70) activity of Rsd is regulated by the phosphorylation state-dependent interaction of HPr with Rsd.

DegS and RseP Homologous Proteases Are Involved in Singlet Oxygen Dependent Activation of RpoE in Rhodobacter sphaeroides

Singlet oxygen (1O2) is the main agent of photooxidative stress and is generated by photosensitizers as (bacterio)chlorophylls. It leads to the damage of cellular macromolecules and therefore photosynthetic organisms have to mount an adaptive response to 1O2 formation. A major player of the photooxidative stress response in Rhodobacter sphaeroides is the alternative sigma factor RpoE, which is inactivated under non-stress conditions by its cognate anti-sigma factor ChrR. By using random mutagenesis we identified RSP_1090 to be required for full activation of the RpoE response under 1O2 stress, but not under organic peroxide stress. In this study we show that both RSP_1090 and RSP_1091 are required for full resistance towards 1O2. Moreover, we revealed that the DegS and RseP homologs RSP_3242 and RSP_2710 contribute to 1O2 resistance and promote ChrR proteolysis. The RpoE signaling pathway in R. sphaeroides is therefore highly similar to that of Escherichia coli, although very different anti-sigma factors control RpoE activity. Based on the acquired results, the current model for RpoE activation in response to 1O2 exposure in R. sphaeroides was extended.

Canonical and non‐canonical EcfG sigma factors control the general stress response in Rhizobium etli

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 σEcfG-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 σEcfG copies has not satisfactorily been explained. This study explores the hierarchical relation between Rhizobium etli σEcfG1 and σEcfG2, canonical and non-canonical σEcfG proteins, respectively. Contrary to reports in other species, we find that σEcfG1 and σEcfG2 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. σEcfG1 drives expression of rpoH2, explaining the increased heat sensitivity of an ecfG1 mutant, while katG is under control of σEcfG2, accounting for reduced oxidative stress resistance of an ecfG2 mutant. We also identify non-coding RNA genes as novel σEcfG targets. We propose a modified model for GSR regulation in R. etli, in which σEcfG1 and σEcfG2 function largely independently. Based on a phylogenetic analysis and considering the prevalence of α-proteobacterial genomes with multiple σEcfG copies, this model may also be applicable to numerous other species.

Identification of disulphide stress–responsive extracytoplasmic function sigma factors in Rothia mucilaginosa

Rothia mucilaginosa is known as a member of commensal bacterial flora in the oral cavity and has received attention as a potential opportunistic pathogen. We previously determined the genomic sequence of R. mucilaginosa DY-18, a clinical strain with biofilm-like structures isolated from an infected root canal of a tooth with persistent apical periodontitis. We found that the DY-18 genome had only two sigma factor genes that encoded the primary and extracytoplasmic function (ECF) sigma factors. Genomic analysis on the available database of R. mucilaginosa ATCC 25296 (a type strain for R. mucilaginosa) revealed that ATCC 25296 has three sigma factors: one primary sigma factor and two ECF sigma factors, one of which was highly homologous to that of DY-18. ECF sigma factors play an important role in the response to environmental stress and to the production of virulence factors. Therefore, we first examined gene-encoding sigma factors on R. mucilaginosa genome in silico. The homologous ECF sigma factors found in strains DY-18 and ATCC 25296 formed a distinct SigH (SigR) clade in a phylogenetic tree and their cognate anti-sigma factor has a HXXXCXXC motif known to respond against disulphide stress. Quantitative reverse transcription polymerase chain reaction (PCR) and microarray analysis showed that the transcriptional levels of sigH were markedly up-regulated under disulphide stress in both strains. Microarray data also demonstrated that several oxidative-stress-related genes (thioredoxin, mycothione reductase, reductase and oxidoreductase) were significantly up-regulated under the diamide stress. On the basis of these results, we conclude that the alternative sigma factor SigH of R. mucilaginosa is a candidate regulator in the redox state.

Identification and Characterization of the Cognate Anti-Sigma Factor and Specific Promoter Elements of a T. tengcongensis ECF Sigma Factor

Extracytoplasmic function (ECF) σ factors, the largest group of alternative σ factors, play important roles in response to environmental stresses. Tt-RpoE1 is annotated as an ECF σ factor in Thermoanaerobacter tengcongensis. In this study, we revealed that the Tt-tolB gene located downstream of the Tt-rpoE1 gene encoded the cognate anti-σ factor, which could inhibit the transcription activity of Tt-RpoE1 by direct interaction with Tt-RpoE1 via its N-terminal domain. By in vitro transcription assay, the auto-regulation ability of Tt-RpoE1 was determined, and band shift assay showed that Tt-RpoE1 preferred to bind a fork-junction promoter DNA. With truncation or base-specific scanning mutations, the contribution of the nucleotides in −35 and −10 regions to interaction between Tt-RpoE1 and promoter DNA was explored. The promoter recognition pattern of Tt-RpoE1 was determined as 5′ tGTTACN16CGTC 3′, which was further confirmed by in vitro transcription assays. This result showed that the Tt-RpoE1-recognized promoter possessed a distinct −10 motif (−13CGTC−10) as the recognition determinant, which is distinguished from the −10 element recognized by σ70. Site-directed mutagenesis in Region 2.4 of Tt-RpoE1 indicated that the “D” residue of DXXR motif was responsible for recognizing the −12G nucleotide. Our results suggested that distinct −10 motif may be an efficient and general strategy used by ECF σ factors in adaptive response regulation of the related genes.

Structural basis for sigma factor mimicry in the general stress response of Alphaproteobacteria

Reprogramming gene expression is an essential component of adaptation to changing environmental conditions. In bacteria, a widespread mechanism involves alternative sigma factors that redirect transcription toward specific regulons. The activity of sigma factors is often regulated through sequestration by cognate anti-sigma factors; however, for most systems, it is not known how the activity of the anti-sigma factor is controlled to release the sigma factor. Recently, the general stress response sigma factor in Alphaproteobacteria, σ(EcfG), was identified. σ(EcfG) is inactivated by the anti-sigma factor NepR, which is itself regulated by the response regulator PhyR. This key regulator sequesters NepR upon phosphorylation of its PhyR receiver domain via its σ(EcfG) sigma factor-like output domain (PhyR(SL)). To understand the molecular basis of the PhyR-mediated partner-switching mechanism, we solved the structure of the PhyR(SL)-NepR complex using NMR. The complex reveals an unprecedented anti-sigma factor binding mode: upon PhyR(SL) binding, NepR forms two helices that extend over the surface of the PhyR(SL) subdomains. Homology modeling and comparative analysis of NepR, PhyR(SL), and σ(EcfG) mutants indicate that NepR contacts both proteins with the same determinants, showing sigma factor mimicry at the atomic level. A lower density of hydrophobic interactions, together with the absence of specific polar contacts in the σ(EcfG)-NepR complex model, is consistent with the higher affinity of NepR for PhyR compared with σ(EcfG). Finally, by reconstituting the partner switch in vitro, we demonstrate that the difference in affinity of NepR for its partners is sufficient for the switch to occur.

Proteolytic regulation of alginate overproduction in Pseudomonas aeruginosa

Pseudomonas aeruginosa, a Gram-negative bacterium, is a significant opportunistic pathogen associated with skin and soft tissue infections, nosocomial pneumonia and sepsis. In addition, it can chronically colonize the lungs of cystic fibrosis (CF) patients. Overproduction of the exopolysaccharide called alginate provides P. aeruginosa with a selective advantage and facilitates survival in the CF lung. The in vitro phenotype of alginate overproduction observed on solid culture media is referred to as mucoid. Expression of the alginate machinery and biosynthetic enzymes are controlled by the extracytoplasmic sigma factor, σ(22) (AlgU/T). The key negative regulator of both σ(22) activity and the mucoid phenotype is the cognate anti-sigma factor MucA. MucA sequesters σ(22) to the inner membrane inhibiting the sigma factor's transcriptional activity. The well-studied mechanism for transition to the mucoid phenotype is mutation of mucA, leading to loss of MucA function and therefore activation of σ(22) . Recently, regulated intramembrane proteolysis (RIP) has been recognized as a mechanism whereby proteolysis of the anti-sigma factor MucA leads to active σ(22) allowing P. aeruginosa to respond to environmental stress conditions by overproduction of alginate. The goal of this review is to illuminate the pathways leading to RIP that have been identified and proposed.

Sequestration‐based bistability enables tuning of the switching boundaries and design of a latch

Natural biological systems have evolved a diverse array of switches to realize their strategies for environmental response and development. Emerging applications of synthetic biology have begun to exploit such switches to achieve increasingly sophisticated designed behaviors. However, not all switch architectures allow facile design of the switching and memory properties. Furthermore, not all designs are built from components for which large families of variants exist, a requirement for building many orthogonal switch variants. Therefore, there is a critical need from genetic engineers for scalable strategies that yield custom bistable switches. Here, we use a sigma factor and its cognate anti-sigma factor to experimentally verify that ultrasensitivity from sequestration combined with positive feedback is sufficient to build a bistable switch. We show that sequestration allows us to predictably tune the switching boundaries, and we can easily tune our switch to function as a set-reset latch that can be toggled between two states by a pulse of inducer input.

An extracytoplasmic function sigma factor cotranscribed with its cognate anti-sigma factor confers tolerance to NaCl, ethanol and methylene blue in Azospirillum brasilense Sp7

Azospirillum brasilense, a plant-growth-promoting rhizobacterium, is exposed to changes in its abiotic environment, including fluctuations in temperature, salinity, osmolarity, oxygen concentration and nutrient concentration, in the rhizosphere and in the soil. Since extra-cytoplasmic function (ECF) sigma factors play an important role in stress adaptation, we analysed the role of ECF sigma factor (also known as RpoE or σ(E)) in abiotic stress tolerance in A. brasilense. An in-frame rpoE deletion mutant of A. brasilense Sp7 was carotenoidless and slow-growing, and was sensitive to salt, ethanol and methylene blue stress. Expression of rpoE in the rpoE deletion mutant complemented the defects in growth, carotenoid biosynthesis and sensitivity to different stresses. Based on data from reverse transcriptase-PCR, a two-hybrid assay and a pull-down assay, we present evidence that rpoE is cotranscribed with chrR and the proteins synthesized from these two overlapping genes interact with each other. Identification of the transcription start site by 5' rapid amplification of cDNA ends showed that the rpoE-chrR operon was transcribed by two promoters. The proximal promoter was less active than the distal promoter, whose consensus sequence was characteristic of RpoE-dependent promoters found in alphaproteobacteria. Whereas the proximal promoter was RpoE-independent and constitutively expressed, the distal promoter was RpoE-dependent and strongly induced in response to stationary phase and elevated levels of ethanol, salt, heat and methylene blue. This study shows the involvement of RpoE in controlling carotenoid synthesis as well as in tolerance to some abiotic stresses in A. brasilense, which might be critical in the adaptation, survival and proliferation of this rhizobacterium in the soil and rhizosphere under stressful conditions.

Cross-interaction of anti-σH factor RshA with BldG, an anti-sigma factor antagonist in Streptomyces griseus

Stress-response sigma factor σ(H) is negatively regulated by its cognate anti-sigma factor RshA in Streptomyces griseus. As the overexpression of RshA in the wild-type strain confers a distinctive bald phenotype (deficiency in aerial mycelium formation and streptomycin production), RshA is supposed to associate with not only σ(H) but also another regulatory element that plays a crucial role in the developmental control of S. griseus. Here, we show that an anti-sigma factor antagonist BldG associates with RshA and negatively regulates its activity. The bald phenotype conferred by the overexpression of rshA was restored to the wild-type phenotype by the coexpression with bldG. The in vivo and in vitro protein interaction analyses demonstrated the specific association between RshA and BldG. A bldG mutant exhibited a distinctive bald phenotype and was defective in the σ(H) -dependent transcription activities. The positive regulatory role of BldG regarding the σ(H) activity was verified by an in vitro transcriptional analysis, in which the inhibition of σ(H) -dependent transcription by RshA was abolished by the addition of BldG in a dose-responsive manner. Overall, evidence suggests that BldG serves as a master switch for both stress-response and developmental gene expression based on its association with multiple anti-sigma factors in S. griseus.

RseA, the SigE specific anti‐sigma factor of Mycobacterium tuberculosis, is inactivated by phosphorylation‐dependent ClpC1P2 proteolysis

Central to the response of Mycobacterium tuberculosis to environmental stress is the regulation of genes under the control of alternative sigma factors. Sigma E of M. tuberculosis plays an important role in the intracellular life of the bacterium and regulates several genes which are important for maintaining the integrity of the cell envelope stress. This makes it important to understand how SigE is activated under stress. Here we elucidate the mechanisms regulating interaction of SigE with its cognate anti-sigma factor RseA. Cysteines 70 and 73 are required for redox-dependent interaction of RseA with SigE. Under surface stress, PknB-dependent phosphorylation of RseA on T39 is required for its cleavage by ClpC1P2 thereby activating the SigE regulon. Rv2745c (MSMEG_2694), a transcriptional regulator, activates the clp regulon in response to vancomycin-induced stress. Taken together with the previous report that Rv2745c is activated by SigE, our study uncovers a positive feedback loop that activates the sigE regulon under envelope stress.

The third pillar of bacterial signal transduction: classification of the extracytoplasmic function (ECF) σ factor protein family

The ability of a bacterial cell to monitor and adaptively respond to its environment is crucial for survival. After one- and two-component systems, extracytoplasmic function (ECF) sigma factors - the largest group of alternative sigma factors - represent the third fundamental mechanism of bacterial signal transduction, with about six such regulators on average per bacterial genome. Together with their cognate anti-sigma factors, they represent a highly modular design that primarily facilitates transmembrane signal transduction. A comprehensive analysis of the ECF sigma factor protein family identified more than 40 distinct major groups of ECF sigma factors. The functional relevance of this classification is supported by the sequence similarity and domain architecture of cognate anti-sigma factors, genomic context conservation, and potential target promoter motifs. Moreover, this phylogenetic analysis revealed unique features indicating novel mechanisms of ECF-mediated signal transduction. This classification, together with the web tool ECFfinder and the information stored in the Microbial Signal Transduction (MiST) database, provides a comprehensive resource for the analysis of ECF sigma factor-dependent gene regulation.