Anti-σ Factor Research Articles

Structural basis for regulation of a CBASS-CRISPR-Cas defense island by a transmembrane anti-σ factor and its ECF σ partner.

How CRISPR-Cas and cyclic oligonucleotide-based antiphage signaling systems (CBASS) are coordinately deployed against invaders remains unclear. We show that a locus containing two CBASS and one type III-B CRISPR-Cas system, regulated by the transmembrane anti-σ DdvA and its cognate extracytoplasmic function (ECF) σ DdvS, can defend Myxococcus xanthus against a phage. Cryo-electron microscopy reveals DdvA-DdvS pairs assemble as arrow-shaped transmembrane dimers. Each DdvA periplasmic domain adopts a separase/craspase-type tetratricopeptide repeat (TPR)-caspase HetF-associated with TPR (TPR-CHAT) architecture with an incomplete His-Cys active site, lacking three α-helices conserved among CHAT domains. Each active site faces the dimer interface, raising the possibility that signal-induced caspase-like DdvA autoproteolysis in trans precedes RseP-mediated intramembrane proteolysis and DdvS release. Nuclear magnetic resonance reveals a DdvA cytoplasmic CHCC-type zinc-bound three-helix bundle that binds to DdvS σ2 and σ4 domains, undergoing σ4-induced helix extension to trap DdvS. Altogether, we provide structural-mechanistic insights into membrane anti-σ-ECF σ regulation of an antiviral CBASS-CRISPR-Cas defense island.

Filament formation activates protease and ring nuclease activities of CRISPR Lon-SAVED

To combat phage infection, type III CRISPR-Cas systems utilize cyclic oligoadenylates (cAn) signaling to activate various auxiliary effectors, including the CRISPR-associated Lon-SAVED protease CalpL, which forms a tripartite effector system together with an anti-σ factor, CalpT, and an ECF-like σ factor, CalpS. Here, we report the characterization of the Candidatus Cloacimonas acidaminovorans CalpL-CalpT-CalpS. We demonstrate that cA4 binding triggers CalpL filament formation and activates it to cleave CalpT within the CalpT-CalpS dimer. This cleavage exposes the CalpT C-degron, which targets it for further degradation by cellular proteases. Consequently, CalpS is released to bind to RNA polymerase, causing growth arrest in E.coli. Furthermore, the CalpL-CalpT-CalpS system is regulated by the SAVED domain of CalpL, which is a ring nuclease that cleaves cA4 in a sequential three-step mechanism. These findings provide key mechanistic details for the activation, proteolytic events, and regulation of the signaling cascade in the type III CRISPR-Cas immunity.

Insight into the autoproteolysis mechanism of the RsgI9 anti-σ factor from Clostridium thermocellum.

Clostridium thermocellum is a potential microbial platform to convert abundant plant biomass to biofuels and other renewable chemicals. It efficiently degrades lignocellulosic biomass using a surface displayed cellulosome, a megadalton sized multienzyme containing complex. The enzymatic composition and architecture of the cellulosome is controlled by several transmembrane biomass-sensing RsgI-type anti-σ factors. Recent studies suggest that these factors transduce signals from the cell surface via a conserved RsgI extracellular (CRE) domain (also called a periplasmic domain) that undergoes autoproteolysis through an incompletely understood mechanism. Here we report the structure of the autoproteolyzed CRE domain from the C. thermocellum RsgI9 anti-σ factor, revealing that the cleaved fragments forming this domain associate to form a stable α/β/α sandwich fold. Based on AlphaFold2 modeling, molecular dynamics simulations, and tandem mass spectrometry, we propose that a conserved Asn-Pro bond in RsgI9 autoproteolyzes via a succinimide intermediate whose formation is promoted by a conserved hydrogen bond network holding the scissile peptide bond in a strained conformation. As other RsgI anti-σ factors share sequence homology to RsgI9, they likely autoproteolyze through a similar mechanism.

Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV β-scaffold in signal transduction.

Past, Present, and Future of Extracytoplasmic Function σ Factors: Distribution and Regulatory Diversity of the Third Pillar of Bacterial Signal Transduction.

Responding to environmental cues is a prerequisite for survival in the microbial world. Extracytoplasmic function σ factors (ECFs) represent the third most abundant and by far the most diverse type of bacterial signal transduction. While archetypal ECFs are controlled by cognate anti-σ factors, comprehensive comparative genomics efforts have revealed a much higher abundance and regulatory diversity of ECF regulation than previously appreciated. They have also uncovered a diverse range of anti-σ factor-independent modes of controlling ECF activity, including fused regulatory domains and phosphorylation-dependent mechanisms. While our understanding of ECF diversity is comprehensive for well-represented and heavily studied bacterial phyla-such as Proteobacteria, Firmicutes, and Actinobacteria (phylum Actinomycetota)-our current knowledge about ECF-dependent signaling in the vast majority of underrepresented phyla is still far from complete. In particular, the dramatic extension of bacterial diversity in the course of metagenomic studies represents both a new challenge and an opportunity in expanding the world of ECF-dependent signal transduction.

Essential autoproteolysis of bacterial anti-σ factor RsgI for transmembrane signal transduction.

Autoproteolysis has been discovered to play key roles in various biological processes, but functional autoproteolysis has been rarely reported for transmembrane signaling in prokaryotes. In this study, an autoproteolytic effect was discovered in the conserved periplasmic domain of anti-σ factor RsgIs from Clostridium thermocellum, which was found to transmit extracellular polysaccharide-sensing signals into cells for regulation of the cellulosome system, a polysaccharide-degrading multienzyme complex. Crystal and NMR structures of periplasmic domains from three RsgIs demonstrated that they are different from all known proteins that undergo autoproteolysis. The RsgI-based autocleavage site was located at a conserved Asn-Pro motif between the β1 and β2 strands in the periplasmic domain. This cleavage was demonstrated to be essential for subsequent regulated intramembrane proteolysis to activate the cognate SigI, in a manner similar to that of autoproteolysis-dependent activation of eukaryotic adhesion G protein-coupled receptors. These results indicate the presence of a unique prevalent type of autoproteolytic phenomenon in bacteria for signal transduction.

The Prc and CtpA proteases modulate cell-surface signaling activity and virulence in Pseudomonas aeruginosa

Cell-surface signaling (CSS) is a signal transfer system of Gram-negative bacteria that produces the activation of an extracytoplasmic function σ factor (σECF) in the cytosol in response to an extracellular signal. Activation requires the regulated and sequential proteolysis of the σECF-associated anti-σ factor, and the function of the Prc and RseP proteases. In this work, we have identified another protease that modulates CSS activity, namely the periplasmic carboxyl-terminal processing protease CtpA. CtpA functions upstream of Prc in the proteolytic cascade and seems to prevent the Prc-mediated proteolysis of the CSS anti-σ factor. Importantly, using zebrafish embryos and the A549 lung epithelial cell line as hosts, we show that mutants in the rseP and ctpA proteases of the human pathogen Pseudomonas aeruginosa are considerably attenuated in virulence while the prc mutation increases virulence likely by enhancing the production of membrane vesicles.

Insight into the Global Negative Regulation of Iron Scavenger 7-HT Biosynthesis by the SigW/RsiW System in Pseudomonas donghuensis HYS

7-Hydroxytropolone (7-HT) is a unique iron scavenger synthesized by Pseudomonas donghuensis HYS that has various biological activities in addition to functioning as a siderophore. P. donghuensis HYS is more pathogenic than P. aeruginosa toward Caenorhabditis elegans, an observation that is closely linked to the biosynthesis of 7-HT. The nonfluorescent siderophore (nfs) gene cluster is responsible for the orderly biosynthesis of 7-HT and represents a competitive advantage that contributes to the increased survival of P. donghuensis HYS; however, the regulatory mechanisms of 7-HT biosynthesis remain unclear. This study is the first to propose that the ECF σ factor has a regulatory effect on 7-HT biosynthesis. In total, 20 ECF σ factors were identified through genome-wide scanning, and their responses to extracellular ferrous ions were characterized. We found that SigW was both significantly upregulated under high-iron conditions and repressed by an adjacent anti-σ factor. RNA-Seq results suggest that the SigW/RsiW system is involved in iron metabolism and 7-HT biosynthesis. Combined with the siderophore phenotype, we also found that SigW could inhibit siderophore synthesis, and this inhibition can be relieved by RsiW. EMSA assays proved that SigW, when highly expressed, can directly bind to the promoter region of five operons of the nfs cluster to inhibit the transcription of the corresponding genes and consequently suppress 7-HT biosynthesis. In addition, SigW not only directly negatively regulates structural genes related to 7-HT synthesis but also inhibits the transcription of regulatory proteins, including of the Gac/Rsm cascade system. Taken together, our results highlight that the biosynthesis of 7-HT is negatively regulated by SigW and that the SigW/RsiW system is involved in mechanisms for the regulation of iron homeostasis in P. donghuensis HYS. As a result of this work, we identified a novel mechanism for the global negative regulation of 7-HT biosynthesis, complementing our understanding of the function of ECF σ factors in Pseudomonas.

Intrinsically disordered protein regions are required for cell wall homeostasis in Bacillus subtilis.

Intrinsically disordered protein regions (IDRs) have been implicated in diverse nuclear and cytoplasmic functions in eukaryotes, but their roles in bacteria are less clear. Here, we report that extracytoplasmic IDRs in Bacillus subtilis are required for cell wall homeostasis. The B. subtilis σI transcription factor is activated in response to envelope stress through regulated intramembrane proteolysis (RIP) of its membrane-anchored anti-σ factor, RsgI. Unlike canonical RIP pathways, we show that ectodomain (site-1) cleavage of RsgI is constitutive, but the two cleavage products remain stably associated, preventing intramembrane (site-2) proteolysis. The regulated step in this pathway is their dissociation, which is triggered by impaired cell wall synthesis and requires RsgI's extracytoplasmic IDR. Intriguingly, the major peptidoglycan polymerase PBP1 also contains an extracytoplasmic IDR, and we show that this region is important for its function. Disparate IDRs can replace the native IDRs on both RsgI and PBP1, arguing that these unstructured regions function similarly. Our data support a model in which the RsgI-σI signaling system and PBP1 represent complementary pathways to repair gaps in the PG meshwork. The IDR on RsgI senses these gaps and activates σI, while the IDR on PBP1 directs the synthase to these sites to fortify them.

The Burkholderia cenocepacia iron starvation σ factor, OrbS, possesses an on-board iron sensor.

Burkholderia cenocepacia is an opportunistic pathogen that causes severe infections of the cystic fibrosis (CF) lung. To acquire iron, B. cenocepacia secretes the Fe(III)-binding compound, ornibactin. Genes for synthesis and utilisation of ornibactin are served by the iron starvation (IS) extracytoplasmic function (ECF) σ factor, OrbS. Transcription of orbS is regulated in response to the prevailing iron concentration by the ferric uptake regulator (Fur), such that orbS expression is repressed under iron-sufficient conditions. Here we show that, in addition to Fur-mediated regulation of orbS, the OrbS protein itself responds to intracellular iron availability. Substitution of cysteine residues in the C-terminal region of OrbS diminished the ability to respond to Fe(II) in vivo. Accordingly, whilst Fe(II) impaired transcription from and recognition of OrbS-dependent promoters in vitro by inhibiting the binding of OrbS to core RNA polymerase (RNAP), the cysteine-substituted OrbS variant was less responsive to Fe(II). Thus, the cysteine residues within the C-terminal region of OrbS contribute to an iron-sensing motif that serves as an on-board ‘anti-σ factor’ in the presence of Fe(II). A model to account for the presence two regulators (Fur and OrbS) that respond to the same intracellular Fe(II) signal to control ornibactin synthesis and utilisation is discussed.

The structure of the Clostridium thermocellum RsgI9 ectodomain provides insight into the mechanism of biomass sensing.

Clostridium thermocellum is actively being developed as a microbial platform to produce biofuels and chemicals from renewable plant biomass. An attractive feature of this bacterium is its ability to efficiently degrade lignocellulose using surface-displayed cellulosomes, large multi-protein complexes that house different types of cellulase enzymes. Clostridium thermocellum tailors the enzyme composition of its cellulosome using nine membrane-embedded anti-σ factors (RsgI1-9), which are thought to sense different types of extracellular carbohydrates and then elicit distinct gene expression programs via cytoplasmic σ factors. Here we show that the RsgI9 anti-σ factor interacts with cellulose via a C-terminal bi-domain unit. A 2.0Å crystal structure reveals that the unit is constructed from S1C peptidase and NTF2-like protein domains that contain a potential binding site for cellulose. Small-angle X-ray scattering experiments of the intact ectodomain indicate that it adopts a bi-lobed, elongated conformation. In the structure, a conserved RsgI extracellular (CRE) domain is connected to the bi-domain via a proline-rich linker, which is expected to project the carbohydrate-binding unit ~160 Å from the cell surface. The CRE and proline-rich elements are conserved in several other C. thermocellum anti-σ factors, suggesting that they will also form extended structures that sense carbohydrates.

Signal Peptidase-Mediated Cleavage of the Anti-σ Factor RsiP at Site 1 Controls σP Activation and β-Lactam Resistance in Bacillus thuringiensis.

ABSTRACTIn Bacillus thuringiensis, β-lactam antibiotic resistance is controlled by the extracytoplasmic function (ECF) σ factor σP. σP activity is inhibited by the anti-σ factor RsiP. In the presence of β-lactam antibiotics, RsiP is degraded and σP is activated. Previous work found that RsiP degradation requires cleavage of RsiP at site 1 by an unknown protease, followed by cleavage at site 2 by the site 2 protease RasP. The penicillin-binding protein PbpP acts as a sensor for β-lactams. PbpP initiates σP activation and is required for site 1 cleavage of RsiP but is not the site 1 protease. Here, we describe the identification of a signal peptidase, SipP, which cleaves RsiP at a site 1 signal peptidase cleavage site and is required for σP activation. Finally, many B. anthracis strains are sensitive to β-lactams yet encode the σP-RsiP signal transduction system. We identified a naturally occurring mutation in the signal peptidase cleavage site of B. anthracis RsiP that renders it resistant to SipP cleavage. We find that B. anthracis RsiP is not degraded in the presence of β-lactams. Altering the B. anthracis RsiP site 1 cleavage site by a single residue to resemble B. thuringiensis RsiP results in β-lactam-dependent degradation of RsiP. We show that mutation of the B. thuringiensis RsiP cleavage site to resemble the sequence of B. anthracis RsiP blocks degradation by SipP. The change in the cleavage site likely explains many reasons why B. anthracis strains are sensitive to β-lactams.

Evolution of a σ–(c-di-GMP)–anti-σ switch

Filamentous actinobacteria of the genus Streptomyces have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The Streptomyces venezuelae RsiG-(c-di-GMP)2-WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)3S(X)2R(X)3Q(X)3D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a Rubrobacter radiotolerans (RsiG)2-(c-di-GMP)2-WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium R. radiotolerans, the (RsiG)2-(c-di-GMP)2-WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors.

Resonance assignments of the cytoplasmic domain of ECF sigma factor W pathway protein YsdB from Bacillus subtilis.

Bacterial sigma (σ) factor, along with RNA polymerase core enzyme, initiates gene transcription from specific promoter regions and therefore regulates clusters of genes in response to a particular situation. The extracytoplasmic function (ECF) σ factors are a class of alternative σ factors that are often associated with environmental signal transduction across the bacterial membrane, in which external signal triggers the release of active σ from the membrane-anchored anti-σ factor. Gram-positive model organism Bacillus subtilis (B. subtilis) has seven ECF σ factors: σM, σV, σX, σW, σY, σZ and σYlaC. Although all these ECF σ factors were found to be involved in B. subtilis antibiotic resistance, σW is among the most studied and considered to play a pivotal role in responding to antimicrobial stresses. σW is under tight control and remains deactivated until exposure to external stimuli, after which proteases PrsW and RasP cleave the specific anti-sigma factor-RsiW to release and activate σW. Membrane anchored protein YsdB is a negative regulator of this activation, possibly via its direct interaction with PrsW and/or RsiW. Importantly, YsdB is well conserved among Bacilli, including pathogenic bacteria like Bacillus cereus. In this study, we describe the chemical shift assignments of the cytoplasmic domain of YsdB (29-130) of B. subtilis in solution as a basis for further interaction studies and structure determination. The near-complete assignment and the solution structure that will follow could provide a further understanding in σW regulation.

Coevolutionary Analysis Reveals a Conserved Dual Binding Interface between Extracytoplasmic Function σ Factors and Class I Anti-σ Factors.

Extracytoplasmic function σ factors (ECFs) belong to the most abundant signal transduction mechanisms in bacteria. Among the diverse regulators of ECF activity, class I anti-σ factors are the most important signal transducers in response to internal and external stress conditions. Despite the conserved secondary structure of the class I anti-σ factor domain (ASDI) that binds and inhibits the ECF under noninducing conditions, the binding interface between ECFs and ASDIs is surprisingly variable between the published cocrystal structures. In this work, we provide a comprehensive computational analysis of the ASDI protein family and study the different contact themes between ECFs and ASDIs. To this end, we harness the coevolution of these diverse protein families and predict covarying amino acid residues as likely candidates of an interaction interface. As a result, we find two common binding interfaces linking the first alpha-helix of the ASDI to the DNA-binding region in the σ4 domain of the ECF, and the fourth alpha-helix of the ASDI to the RNA polymerase (RNAP)-binding region of the σ2 domain. The conservation of these two binding interfaces contrasts with the apparent quaternary structure diversity of the ECF/ASDI complexes, partially explaining the high specificity between cognate ECF and ASDI pairs. Furthermore, we suggest that the dual inhibition of RNAP- and DNA-binding interfaces is likely a universal feature of other ECF anti-σ factors, preventing the formation of nonfunctional trimeric complexes between σ/anti-σ factors and RNAP or DNA.IMPORTANCE In the bacterial world, extracytoplasmic function σ factors (ECFs) are the most widespread family of alternative σ factors, mediating many cellular responses to environmental cues, such as stress. This work uses a computational approach to investigate how these σ factors interact with class I anti-σ factors-the most abundant regulators of ECF activity. By comprehensively classifying the anti-σs into phylogenetic groups and by comparing this phylogeny to the one of the cognate ECFs, the study shows how these protein families have coevolved to maintain their interaction over evolutionary time. These results shed light on the common contact residues that link ECFs and anti-σs in different phylogenetic families and set the basis for the rational design of anti-σs to specifically target certain ECFs. This will help to prevent the cross talk between heterologous ECF/anti-σ pairs, allowing their use as orthogonal regulators for the construction of genetic circuits in synthetic biology.

C-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

SummaryStreptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Evaluation of specificity determinants in Mycobacterium tuberculosis σ/anti-σ factor interactions

Extra Cytoplasmic Function (ECF) σ factor/regulatory protein (anti-σ factor) pairs govern environment mediated changes in gene expression in bacteria. The release of the ECF σ factor from an inactive σ/anti-σ factor complex is triggered by specific environmental stimuli. The free σ factor then associates with the RNA polymerase and drives the expression of genes in its target regulon. Multiple ECF σ/anti-σ pairs ensure calibrated changes in the expression profile by correlating diverse environmental stimuli with changes in the intracellular levels of different ECF σ factors. Specificity in σ/anti-σ factor interaction is thus essential for accurate signal transduction. Here we describe experiments to evaluate interactions between different M. tuberculosis σ and anti-σ proteins in vitro. The interaction parameters suggest that cross-talk between non-cognate σ/anti-σ pairs is likely. The sequence and conformational determinants that govern interaction specificity in a σ/anti-σ complex are not immediately evident due to substantial structural conservation. Sequence-structure analysis of all σ/anti-σ pairs suggest that conserved residues are not the primary determinants of σ/anti-σ interactions-a finding that suggests a potential route to set tolerance limits in interaction specificity. Non-specific σ/anti-σ interactions are likely to be biologically significant as it can contribute to heterogeneity in cellular responses in a bacterial population under less stringent requirements. This finding is relevant for synthetic biology approaches to engineer bacteria using σ/anti-σ transcription initiation modules for diverse applications in biotechnology.

Mutations of the Bacillus subtilis YidC1 (SpoIIIJ) insertase alleviate stress associated with σM-dependent membrane protein overproduction.

In Bacillus subtilis, the extracytoplasmic function σ factor σM regulates cell wall synthesis and is critical for intrinsic resistance to cell wall targeting antibiotics. The anti-σ factors YhdL and YhdK form a complex that restricts the basal activity of σM, and the absence of YhdL leads to runaway expression of the σM regulon and cell death. Here, we report that this lethality can be suppressed by gain-of-function mutations in yidC1 (spoIIIJ), which encodes the major YidC membrane protein insertase in B. subtilis. B. subtilis PY79 YidC1 (SpoIIIJ) contains a single amino acid substitution in a functionally important hydrophilic groove (Q140K), and this allele suppresses the lethality of high σM. Analysis of a library of YidC1 variants reveals that increased charge (+2 or +3) in the hydrophilic groove can compensate for high expression of the σM regulon. Derepression of the σM regulon induces secretion stress, oxidative stress and DNA damage responses, all of which can be alleviated by the YidC1Q140K substitution. We further show that the fitness defect caused by high σM activity is exacerbated in the absence of the SecDF protein translocase or σM-dependent induction of the Spx oxidative stress regulon. Conversely, cell growth is improved by mutation of specific σM-dependent promoters controlling operons encoding integral membrane proteins. Collectively, these results reveal how the σM regulon has evolved to up-regulate membrane-localized complexes involved in cell wall synthesis, and to simultaneously counter the resulting stresses imposed by regulon induction.

Activation of the Extracytoplasmic Function σ Factor σP by β-Lactams in Bacillus thuringiensis Requires the Site-2 Protease RasP.

Bacteria can utilize alternative σ factors to regulate sets of genes in response to changes in the environment. The largest and most diverse group of alternative σ factors are the extracytoplasmic function (ECF) σ factors. σP is an ECF σ factor found in Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis Previous work showed that σP is induced by ampicillin, a β-lactam antibiotic, and required for resistance to ampicillin. However, it was not known how activation of σP is controlled or what other antibiotics may activate σP Here, we report that activation of σP is specific to a subset of β-lactams and that σP is required for resistance to these β-lactams. We demonstrate that activation of σP is controlled by the proteolytic destruction of the anti-σ factor RsiP and that degradation of RsiP requires multiple proteases. Upon exposure to β-lactams, the extracellular domain of RsiP is cleaved by an unknown protease, which we predict cleaves at site-1. Following cleavage by the unknown protease, the N terminus of RsiP is further degraded by the site-2 intramembrane protease RasP. Our data indicate that RasP cleavage of RsiP is not the rate-limiting step in σP activation. This proteolytic cascade leads to activation of σP, which induces resistance to β-lactams likely via increased expression of β-lactamases.IMPORTANCE The discovery of antibiotics to treat bacterial infections has had a dramatic and positive impact on human health. However, shortly after the introduction of a new antibiotic, bacteria often develop resistance. The bacterial cell envelope is essential for cell viability and is the target of many of the most commonly used antibiotics, including β-lactam antibiotics. Resistance to β-lactams is often dependent upon β-lactamases. In B. cereus, B. thuringiensis, and some B. anthracis strains, the expression of some β-lactamases is inducible. This inducible β-lactamase expression is controlled by activation of an alternative σ factor called σP Here, we show that β-lactam antibiotics induce σP activation by degradation of the anti-σ factor RsiP.

Structural basis for the recognition of MucA by MucB and AlgU in Pseudomonas aeruginosa.

Alginate production in Pseudomonas aeruginosa is regulated by the alternate σ factor AlgU, which in turn is regulated by the MucABCD system. The anti-σ factor MucA binds AlgU in the cytoplasm and prevents AlgU from binding to the RNA polymerase for transcription. MucB binds MucA in the periplasm and inhibits proteolysis of MucA and subsequent release of AlgU. In this work, we report crystal structures of MucA in complex with AlgU and MucB. A structure of MucB alone reveals the structural changes required for MucA recognition. A unique disulfide bond is identified in MucB, and mutation of this disulfide bond results in a shift from monomer to MucB dimers or tetramers. As MucB tetramers have previously been shown to be unable to bind MucA, this suggests a redox-sensitive stress response mechanism in MucB. The AlgU-MucA structure reveals a conserved σ factor/anti-σ factor complex, but AlgU lacks a disulfide bond conserved in many other σ factors. Our structures reveal the molecular basis for MucA recognition by MucB in the periplasm and AlgU in the cytoplasm, thus providing an important step in understanding the mechanisms that regulate a key signal transduction pathway involved in P.aeruginosa pathogenesis. DATABASE: The atomic coordinates and structure factors for MucAcyto -AlgU, MucB, and MucAperi -MucB have been deposited in the Protein Data Bank (PDB) with the accession code 6IN7, 6IN8, and 6IN9, respectively.