Anti-sigma Factor Research Articles

Multidrug tolerance conferred by loss-of-function mutations in anti-sigma factor RshA of Mycobacterium abscessus.

Low-level drug resistance in noncanonical pathways can constitute steppingstones toward acquisition of high-level on-target resistance mutations in the clinic. To capture these intermediate steps in Mycobacterium abscessus (Mab), we performed classic mutant selection experiments with moxifloxacin at twofold its minimum inhibitory concentration (MIC) on solid medium. We found that low-level resistance emerged reproducibly as loss-of-function mutations in RshA (MAB_3542c), an anti-sigma factor that negatively regulates activity of SigH, which orchestrates a response to oxidative stress in mycobacteria. Since oxidative stress is generated in response to many antibiotics, we went on to show that deletion of rshA confers low to moderate resistance-by measure of MIC-to a dozen agents recommended or evaluated for the treatment of Mab pulmonary infections. Interestingly, this moderate resistance was associated with a wide range of drug tolerance, up to 1,000-fold increased survival of a ΔrshA Mab mutant upon exposure to several β-lactams and DNA gyrase inhibitors. Consistent with the putative involvement of the SigH regulon, we showed that addition of the transcription inhibitor rifabutin (RBT) abrogated the high-tolerance phenotype of ΔrshA to representatives of the β-lactam and DNA gyrase inhibitor classes. In a survey of 10,000 whole Mab genome sequences, we identified several loss-of-function mutations in rshA as well as non-synonymous polymorphisms in two cysteine residues critical for interactions with SigH. Thus, the multidrug multiform resistance phenotype we have uncovered may not only constitute a step toward canonical resistance acquisition during treatment but also contribute directly to treatment failure.

Distinct impacts of each anti-anti-sigma factor ortholog of the chlamydial Rsb partner switching mechanism on development in Chlamydia trachomatis.

Partner switching mechanisms (PSMs) are signal transduction systems comprised of a sensor phosphatase (RsbU), an anti-sigma factor (RsbW, kinase), an anti-anti-sigma factor (RsbV, the RsbW substrate), and a target sigma factor. Chlamydia spp. are obligate intracellular bacterial pathogens of animals that undergo a developmental cycle transitioning between the infectious elementary body (EB) and replicative reticulate body (RB) within a host cell-derived vacuole (inclusion). Secondary differentiation events (RB to EB) are transcriptionally regulated, in part, by the housekeeping sigma factor (σ66) and two late-gene sigma factors (σ54 and σ28). Prior research supports that the PSM in Chlamydia trachomatis regulates availability of σ66. Pan-genome analysis revealed that PSM components are conserved across the phylum Chlamydiota, with Chlamydia spp. possessing an atypical arrangement of two anti-anti-sigma factors, RsbV1 and RsbV2. Bioinformatic analyses support RsbV2 as the homolog to the pan-genome-conserved RsbV with RsbV1 as an outlier. This, combined with in vitro data, indicates that RsbV1 and RsbV2 are structurally and biochemically distinct. Reduced levels or overexpression of RsbV1/RsbV2 did not significantly impact C. trachomatis growth or development. In contrast, overexpression of a non-phosphorylatable RsbV2 S55A mutant, but not overexpression of an RsbV1 S56A mutant, resulted in a 3 log reduction in infectious EB production without reduction in genomic DNA (total bacteria) or inclusion size, suggesting a block in secondary differentiation. The block was corroborated by reduced production of σ54/28-regulated late proteins and via transmission electron microscopy.IMPORTANCEChlamydia trachomatis is the leading cause of reportable bacterial sexually transmitted infections (STIs) and causes the eye infection trachoma, a neglected tropical disease. Broad-spectrum antibiotics used for treatment can lead to microbiome dysbiosis and increased antibiotic resistance development in other bacteria, and treatment failure for chlamydial STIs is a recognized clinical problem. Here, we show that disruption of a partner switching mechanism (PSM) significantly reduces infectious progeny production via blockage of reticulate body to elementary body differentiation. We also reveal a novel PSM expansion largely restricted to the species infecting animals, suggesting a role in pathogen evolution. Collectively, our results highlight the chlamydial PSM as a key regulator of development that could be a potential target for novel therapeutics.

Effects of a SigH mutation on tigecycline resistance and the SigH-RshA interaction in Mycobacteroides abscessus

Abstract Mycobacteroides abscessus is an opportunistic pathogen ubiquitous in the environment owing to its ability to remain viable on nutrient-poor surfaces. It is a frequent cause of infections in the cosmetic industry, with patients being infected through cutaneous invasive procedures such as tattooing, piercings and cosmetic surgeries. In immunocompromised hosts, infections can be severe and difficult to treat as M. abscessus has many intrinsic and acquired resistances to different classes of antibiotics. Tigecycline, a tetracycline derivative introduced two decades ago, is a broad-spectrum antibiotic with activity on bacteria that are resistant to many existing antibiotics. Unfortunately, tigecycline-resistant strains of M. abscessus have been reported in recent years. This study aimed to investigate the resistance mechanism of CL7, a tigecycline-resistant, spontaneous mutant derived from M. abscessus ATCC 19977. CL7 notably had a 7 amino-acid truncation in the SigH protein, a sigma factor (transcriptional activator) responsible for mycobacterial responses to environmental stress. RNA sequencing showed that sigH and other genes were up-regulated in CL7 (as compared to ATCC 19977). The gene set enrichment analysis demonstrated that the SigH regulon was significantly over-represented among these genes up-regulated in CL7. A bacterial-2-hybrid assay was performed to investigate the effect of the mutation on the SigH interaction with RshA, the anti-sigma factor that inhibits SigH intracellularly. The results, supported by RNA sequencing, showed that the interaction between RshA and the mutant SigH was impeded. This reduced interaction could lead to a decreased inhibition of SigH by RshA, causing the up-regulation of the sigH gene. Coupled with the RNA polymerase, SigH would then up-regulate genes under its regulation, leading to tigecycline resistance. In general, this study enhances our understanding of tigecycline resistance mechanisms in M. abscessus, and contributes to the development of novel antibacterial therapies and diagnostic tools for managing M. abscessus infections.

Adaptation mechanisms of Clostridioides difficile to auranofin and its impact on human gut microbiota

Auranofin (AF), a former rheumatoid polyarthritis treatment, gained renewed interest for its use as an antimicrobial. AF is an inhibitor of thioredoxin reductase (TrxB), a thiol and protein repair enzyme, with an antibacterial activity against several bacteria including C. difficile, an enteropathogen causing post-antibiotic diarrhea. Several studies demonstrated the effect of AF on C. difficile physiology, but the crucial questions of resistance mechanisms and impact on microbiota remain unaddressed. We explored potential resistance mechanisms by studying the impact of TrxB multiplicity and by generating and characterizing adaptive mutations. We showed that if mutants inactivated for trxB genes have a lower MIC of AF, the number of TrxBs naturally present in clinical strains does not impact the MIC. All stable mutations isolated after AF long-term exposure were in the anti-sigma factor of σB and strongly affect physiology. Finally, we showed that AF has less impact on human gut microbiota than vancomycin.

Partner-switching components PmgA and Ssr1600 regulate high-light acclimation in Synechocystis sp. PCC 6803.

Photomixotrophic growth A (PmgA) is a pleiotropic regulator essential for growth under photomixotrophic and prolonged high-light (HL) conditions in the cyanobacterium Synechocystis sp. PCC 6803. The overall similarity with the antisigma factor of the bacterial partner-switching system indicates that PmgA exerts a regulatory function via phosphorylation of its target proteins. In this study, we performed an in vitro phosphorylation assay and protein-protein interaction analysis and found that PmgA interacts with 4 antisigma antagonist homologs, Ssr1600, Slr1856, Slr1859, and Slr1912, but specifically phosphorylates Ssr1600. Phenotypic analyses using the set of gene disruption and overexpression strains of pmgA and ssr1600 revealed that phosphorylation by PmgA is essential for the accumulation of Ssr1600 protein in vivo. The ssr1600-disrupted mutant showed similar phenotypes as those previously reported for the pmgA-disrupted mutant, namely, no obvious phenotype just after the shift to HL, but higher chlorophyll content, 5-aminolevulinic acid synthesis activity, and psaAB transcript levels than those in the wild type after 6 h. These findings indicate that the phosphorylated form of Ssr1600 works as the output of the partner-switching system to coordinately repress chlorophyll biosynthesis and accumulation of photosystem I during HL acclimation.

The conserved σD envelope stress response monitors multiple aspects of envelope integrity in corynebacteria.

The cell envelope fortifies bacterial cells against antibiotics and other insults. Species in the Mycobacteriales order have a complex envelope that includes an outer layer of mycolic acids called the mycomembrane (MM) and a cell wall composed of peptidoglycan and arabinogalactan. This envelope architecture is unique among bacteria and contributes significantly to the virulence of pathogenic Mycobacteriales like Mycobacterium tuberculosis. Characterization of pathways that govern envelope biogenesis in these organisms is therefore critical in understanding their biology and for identifying new antibiotic targets. To better understand MM biogenesis, we developed a cell sorting-based screen for mutants defective in the surface exposure of a porin normally embedded in the MM of the model organism Corynebacterium glutamicum. The results revealed a requirement for the conserved σD envelope stress response in porin export and identified MarP as the site-1 protease, respectively, that activate the response by cleaving the membrane-embedded anti-sigma factor. A reporter system revealed that the σD pathway responds to defects in mycolic acid and arabinogalactan biosynthesis, suggesting that the stress response has the unusual property of being induced by activating signals that arise from defects in the assembly of two distinct envelope layers. Our results thus provide new insights into how C. glutamicum and related bacteria monitor envelope integrity and suggest a potential role for members of the σD regulon in protein export to the MM.

Role of the sigma factor AlgU in regulating growth, virulence, motility, exopolysaccharide production, and environmental stress adaptation of Pseudomonas syringae pv. actinidiae QSY6

The extracytoplasmic function (ECF) sigma factor AlgU is involved in the regulation of various virulence-related pathways in Pseudomonas syringae, especially alginate biosynthesis and motility, and the role of AlgU differs among P. syringae pathovars. However, to date, the mechanism of its regulation in virulence of P. syringae pv. actinidiae (Psa) is still unclear. ECF sigma factors are a class of alternative sigma factors that typically function with anti-sigma factors as part of cell-surface signaling systems. Under non-inducing conditions, AlgU remains inhibited by anti-sigma factors such as MucA and MucB. To investigate the function of AlgU in Psa, mutant strains lacking algU or lacking algU with mucA and mucB genes, as well as complementary and overexpression strains of algU were generated, respectively. The results showed that AlgU was highly conserved among P. syringae pathovars and positively regulated growth rate, pathogenicity, and resistance to osmotic and oxidative stress of Psa QSY6. While AlgU did not affect the motility and exopolysaccharide production of Psa, its abundant expression enhanced the swimming ability of QSY6 and reduced its production of extracellular polysaccharides. Furthermore, AlgU regulates a number of virulence-related factors, including the Hrp system, the type VI secretion system, and flagellar synthesis. Specifically, AlgU induced the expression of hrpL and hrpRS in vivo, and repressed the transcription of hrpL and tssC in vitro, while promoting the expression of hrpS, fliC, and tssJ. This study contributes to a better understanding of the mechanisms of virulence regulation of AlgU in Psa.

Bacillus subtilis uses the SigM signaling pathway to prioritize the use of its lipid carrier for cell wall synthesis.

Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acid precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria.

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.

A single point mutation in the Listeria monocytogenes ribosomal gene rpsU enables SigB activation independently of the stressosome and the anti-sigma factor antagonist RsbV.

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsUG50C) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the ΔsigB, ΔrsbV, and ΔrsbR1 mutant strains. While the introduction of rpsUG50C in the ΔsigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsUG50C in the ΔrsbV and the ΔrsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsUG50C mutants. Interestingly, all these rpsUG50C mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsUG50C mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsUG50C mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function.

Abstract 1836 Characterization of anti-Sigma factor proteins from Bacteroides thetaiotaomicron

Abstract 1836 Characterization of anti-Sigma factor proteins from Bacteroides thetaiotaomicron

Dual membrane-spanning anti-sigma factors regulate vesiculation in Bacteroides thetaiotaomicron

Bacteroidota are abundant members of the human gut microbiota that shape the enteric landscape by modulating host immunity and degrading dietary- and host-derived glycans. These processes are mediated in part by Outer Membrane Vesicles (OMVs). Here, we developed a high-throughput screen to identify genes required for OMV biogenesis and its regulation in Bacteroides thetaiotaomicron (Bt). We identified a family of Dual membrane-spanning anti-sigma factors (Dma) that control OMV biogenesis. We conducted molecular and multiomic analyses to demonstrate that deletion of Dma1, the founding member of the Dma family, modulates OMV production by controlling the activity of the ECF21 family sigma factor, Das1, and its downstream regulon. Dma1 has a previously uncharacterized domain organization that enables Dma1 to span both the inner and outer membrane of Bt. Phylogenetic analyses reveal that this common feature of the Dma family is restricted to the phylum Bacteroidota. This study provides mechanistic insights into the regulation of OMV biogenesis in human gut bacteria.

The regulatory network comprising ArcAB-RpoS-RssB influences motility in Vibrio cholerae.

The diarrheal disease cholera is caused by the versatile and responsive bacterium Vibrio cholerae, which is capable of adapting to environmental changes. Among others, the alternative sigma factor RpoS activates response pathways, including regulation of motility- and chemotaxis-related genes under nutrient-poor conditions in V. cholerae. Although RpoS has been well characterised, links between RpoS and other regulatory networks remain unclear. In this study, we identified the ArcAB two-component system to control rpoS transcription and RpoS protein stability in V. cholerae. In a manner similar to that seen in Escherichia coli, the ArcB kinase not only activates the response regulator ArcA but also RssB, the anti-sigma factor of RpoS. Our results demonstrated that, in V. cholerae, RssB is phosphorylated by ArcB, which subsequently activates RpoS proteolysis. Furthermore, ArcA acts as a repressor of rpoS transcription. Additionally, we determined that the cysteine residue at position 180 of ArcB is crucial for signal recognition and activity. Thus, our findings provide evidence linking RpoS response to the anoxic redox control system ArcAB in V. cholerae.

The Transposition of Insertion Sequences in Sigma-Factor- and LysR-Deficient Mutants of Deinococcus geothermalis.

Some insertion sequence (IS) elements were actively transposed using oxidative stress conditions, including gamma irradiation and hydrogen peroxide treatment, in Deinococcus geothermalis, a radiation-resistant bacterium. D. geothermalis wild-type (WT), sigma factor gene-disrupted (∆dgeo_0606), and LysR gene-disrupted (∆dgeo_1692) mutants were examined for IS induction that resulted in non-pigmented colonies after gamma irradiation (5 kGy) exposure. The loss of pigmentation occurred because dgeo_0524, which encodes a phytoene desaturase in the carotenoid pathway, was disrupted by the transposition of IS elements. The types and loci of the IS elements were identified as ISDge2 and ISDge6 in the ∆dgeo_0606 mutant and ISDge5 and ISDge7 in the ∆dgeo_1692 mutant, but were not identified in the WT strain. Furthermore, 80 and 100 mM H2O2 treatments induced different transpositions of IS elements in ∆dgeo_0606 (ISDge5, ISDge6, and ISDge7) and WT (ISDge6). However, no IS transposition was observed in the ∆dgeo_1692 mutant. The complementary strain of the ∆dgeo_0606 mutation showed recovery effects in the viability assay; however, the growth-delayed curve did not return because the neighboring gene dgeo_0607 was overexpressed, probably acting as an anti-sigma factor. The expression levels of certain transposases, recognized as pivotal contributors to IS transposition, did not precisely correlate with active transposition in varying oxidation environments. Nevertheless, these findings suggest that specific IS elements integrated into dgeo_0524 in a target-gene-deficient and oxidation-source-dependent manner.

Mechanism of anion exchange and small-molecule inhibition of pendrin

Pendrin (SLC26A4) is an anion exchanger that mediates bicarbonate (HCO3−) exchange for chloride (Cl−) and is crucial for maintaining pH and salt homeostasis in the kidney, lung, and cochlea. Pendrin also exports iodide (I−) in the thyroid gland. Pendrin mutations in humans lead to Pendred syndrome, causing hearing loss and goiter. Inhibition of pendrin is a validated approach for attenuating airway hyperresponsiveness in asthma and for treating hypertension. However, the mechanism of anion exchange and its inhibition by drugs remains poorly understood. We applied cryo-electron microscopy to determine structures of pendrin from Sus scrofa in the presence of either Cl−, I−, HCO3− or in the apo-state. The structures reveal two anion-binding sites in each protomer, and functional analyses show both sites are involved in anion exchange. The structures also show interactions between the Sulfate Transporter and Anti-Sigma factor antagonist (STAS) and transmembrane domains, and mutational studies suggest a regulatory role. We also determine the structure of pendrin in a complex with niflumic acid (NFA), which uncovers a mechanism of inhibition by competing with anion binding and impeding the structural changes necessary for anion exchange. These results reveal directions for understanding the mechanisms of anion selectivity and exchange and their regulations by the STAS domain. This work also establishes a foundation for analyzing the pathophysiology of mutations associated with Pendred syndrome.

Structure of the SigE regulatory network in Mycobacterium tuberculosis.

SigE is one of the main regulators of mycobacterial stress response and is characterized by a complex regulatory network based on two pathways, which have been partially characterized in conditions of surface stress. The first pathway is based on the induction of sigE transcription by the two-component system MprAB, while the second is based on the degradation of SigE anti-sigma factor RseA by ClpC1P2, a protease whose structural genes are induced by ClgR. We characterized the dynamics of the SigE network activation in conditions of surface stress and low pH in Mycobacterium tuberculosis. Using a series of mutants in which the main regulatory nodes of the network have been inactivated, we could explore their hierarchy, and we determined that MprAB had a key role in the network activation in both stress conditions through the induction of sigE. However, while in conditions of surface stress the absence of MprAB totally abrogated sigE induction, under low pH conditions it only resulted in a small delay of the induction of sigE. In this case, sigE induction was due to SigH, which acted as a MprAB backup system. The ClgR pathway, leading to the degradation of the SigE anti-sigma factor RseA, was shown to be essential for the activation of the SigE network only following surface stress, where it showed an equal hierarchy with the MprAB pathway.

A unique sigma/anti-sigma system in the actinomycete Actinoplanes missouriensis

Bacteria of the genus Actinoplanes form sporangia that contain dormant sporangiospores which, upon contact with water, release motile spores (zoospores) through a process called sporangium dehiscence. Here, we set out to study the molecular mechanisms behind sporangium dehiscence in Actinoplanes missouriensis and discover a sigma/anti-sigma system with unique features. Protein σSsdA contains a functional sigma factor domain and an anti-sigma factor antagonist domain, while protein SipA contains an anti-sigma factor domain and an anti-sigma factor antagonist domain. Remarkably, the two proteins interact with each other via the anti-sigma factor antagonist domain of σSsdA and the anti-sigma factor domain of SipA. Although it remains unclear whether the SipA/σSsdA system plays direct roles in sporangium dehiscence, the system seems to modulate oxidative stress responses in zoospores. In addition, we identify a two-component regulatory system (RsdK-RsdR) that represses initiation of sporangium dehiscence.

The Regulatory Functions of the Multiple Alternative Sigma Factors RpoE, RpoHI, and RpoHII Depend on the Growth Phase in Rhodobacter sphaeroides.

Bacterial growth, under laboratory conditions or in a natural environment, goes through different growth phases. Some gene expressions are regulated with respect to the growth phase, which allows bacteria to adapt to changing conditions. Among them, many gene transcriptions are controlled by RpoHI or RpoHII in Rhodobacter sphaeroides. In a previous study, it was proven that the alternative sigma factors, RpoE, RpoHI, and RpoHII, are the major regulators of oxidative stress. Moreover, the growth of bacteria reached a stationary phase, and following the outgrowth, rpoE, rpoHI, and rpoHII mRNAs increased with respect to the growth phase. In this study, we demonstrated the regulatory function of alternative sigma factors in the rsp_0557 gene. The gene rsp_0557 is expressed with respect to the growth phase and belongs to the RpoHI/RpoHII regulons. Reporter assays showed that the antisigma factor ChrR turns on or over the RpoE activity to regulate rsp_0557 expression across the growth phase. In the exponential phase, RpoHII and sRNA Pos19 regulate the expression of rsp_0557 to an appropriate level under RpoE control. In the stationary phase, RpoHI and Pos19 stabilize the transcription of rsp_0557 at a high level. During outgrowth, RpoHI negatively regulates the transcription of rsp_0557. Taken together, our data indicate that these regulators are recruited by cells to adapt to or survive under different conditions throughout the growth phase. However, they still did not display all of the regulators involved in growth phase-dependent regulation. More research is still needed to learn more about the interaction between the regulators and the process of adapting to changed growth conditions and environments.

T3SS chaperone of the CesT family is required for secretion of the anti-sigma factor BtrA in Bordetella pertussis

ABSTRACT Bordetella pertussis is a Gram-negative, strictly human re-emerging respiratory pathogen and the causative agent of whooping cough. Similar to other Gram-negative pathogens, B. pertussis produces the type III secretion system, but its role in the pathogenesis of B. pertussis is enigmatic and yet to be elucidated. Here, we combined RNA-seq, LC-MS/MS, and co-immunoprecipitation techniques to identify and characterize the novel CesT family T3SS chaperone BP2265. We show that this chaperone specifically interacts with the secreted T3SS regulator BtrA and represents the first non-flagellar chaperone required for the secretion of an anti-sigma factor. In its absence, secretion but not production of BtrA and most T3SS substrates is severely impaired. It appears that the role of BtrA in regulating T3SS extends beyond its activity as an antagonist of the sigma factor BtrS. Predictions made by artificial intelligence system AlphaFold support the chaperone function of BP2265 towards BtrA and outline the structural basis for the interaction of BtrA with its target BtrS. We propose to rename BP2265 to BtcB for the Bordetella type III chaperone of BtrA. In addition, the absence of the BtcB chaperone results in increased expression of numerous flagellar genes and several virulence genes. While increased production of flagellar proteins and intimin BipA translated into increased biofilm formation by the mutant, enhanced production of virulence factors resulted in increased cytotoxicity towards human macrophages. We hypothesize that these phenotypic traits result indirectly from impaired secretion of BtrA and altered activity of the BtrA/BtrS regulatory node.

Bacterial SEAL domains undergo autoproteolysis and function in regulated intramembrane proteolysis

Gram-positive bacteria use SigI/RsgI-family sigma factor/anti-sigma factor pairs to sense and respond to cell wall defects and plant polysaccharides. In Bacillus subtilis, this signal transduction pathway involves regulated intramembrane proteolysis (RIP) of the membrane-anchored anti-sigma factor RsgI. However, unlike most RIP signaling pathways, site-1 cleavage of RsgI on the extracytoplasmic side of the membrane is constitutive and the cleavage products remain stably associated, preventing intramembrane proteolysis. The regulated step in this pathway is their dissociation, which is hypothesized to involve mechanical force. Release of the ectodomain enables intramembrane cleavage by the RasP site-2 protease and activation of SigI. The constitutive site-1 protease has not been identified for any RsgI homolog. Here, we report that RsgI's extracytoplasmic domain has structural and functional similarities to eukaryotic SEA domains that undergo autoproteolysis and have been implicated in mechanotransduction. We show that site-1 proteolysis in B. subtilis and Clostridial RsgI family members is mediated by enzyme-independent autoproteolysis of these SEA-like domains. Importantly, the site of proteolysis enables retention of the ectodomain through an undisrupted β-sheet that spans the two cleavage products. Autoproteolysis can be abrogated by relief of conformational strain in the scissile loop, in a mechanism analogous to eukaryotic SEA domains. Collectively, our data support the model that RsgI-SigI signaling is mediated by mechanotransduction in a manner that has striking parallels with eukaryotic mechanotransducive signaling pathways.