Genomic SELEX Screening of Regulatory Targets of Transcription Factors.

The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of transcriptional targets is determined via interaction with one of seven species of the sigma subunit and a total of approximately 300 species of transcription factor (TFs). For comprehensive identification of the regulatory targets of these two groups of regulatory proteins on the genome, we developed an in vitro approach, "Genomic SELEX" (gSELEX) screening. Here we describe a detailed protocol of the gSELEX screening system, which uses purified regulatory proteins and fragments of genomic DNA from E. coli. Moreover, we describe methods and examples of results using cell-free synthetic proteins.

Integration of molecular docking and molecular dynamics simulations with subtractive proteomics approach to identify the novel drug targets and their inhibitors in Streptococcus gallolyticus.

Streptococcus gallolyticus (Sg) is a non-motile, gram-positive bacterium that causes infective endocarditis (inflammation of the heart lining). Because Sg has gained resistance to existing antibiotics and there is currently no drug available, developing effective anti-Sg drugs is critical. This study combined core proteomics with a subtractive proteomics technique to identify potential therapeutic targets for Sg. Several bioinformatics approaches were used to eliminate non-essential and human-specific homologous sequences from the bacterial proteome. Then, virulence, druggability, subcellular localization, and functional analyses were carried out to specify the participation of significant bacterial proteins in various cellular processes. The pathogen's genome contained three druggable proteins, glucosamine-1phosphate N-acetyltransferase (GlmU), RNA polymerase sigma factor (RpoD), and pantetheine-phosphate adenylyltransferase (PPAT) which could serve as effective targets for developing novel drugs. 3D structures of target protein were modeled through Swiss Model. A natural product library containing 10,000 molecules from the LOTUS database was docked against therapeutic target proteins. Following an evaluation of the docking results using the glide gscore, the top 10 compounds docked against each protein receptor were chosen. LTS001632, LTS0243441, and LTS0236112 were the compounds that exhibited the highest binding affinities against GlmU, PPAT, and RpoD, respectively, among the compounds that were chosen. To augment the docking data, molecular dynamics simulations and MM-GBSA binding free energy were also utilized. More in-vitro research is necessary to transform these possible inhibitors into therapeutic drugs, though computer validations were employed in this study. This combination of computational techniques paves the way for targeted antibiotic development, which addresses the critical need for new therapeutic strategies against S. gallolyticus infections.

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.

WhiB-like proteins: Diversity of structure, function and mechanism.

The WhiB-Like (Wbl) proteins are a large family of iron-sulfur (Fe-S) cluster-containing transcription factors exclusively found in the phylum Actinobacteria, including the notable genera like Mycobacteria, Streptomycetes and Corynebacteria. These proteins play pivotal roles in diverse biological processes, such as cell development, redox stress response and antibiotic resistance. Members of the Wbl family exhibit remarkable diversity in their sequences, structures and functions, attracting great attention since their first discovery. This review highlights the most recent breakthroughs in understanding the structural and mechanistic aspects of Wbl-dependent transcriptional regulation.

A simple and unified protocol to purify all seven Escherichia coli RNA polymerase sigma factors.

RNA polymerase sigma factors are indispensable in the process of bacterial transcription. They are responsible for a given gene's promoter region recognition on template DNA and hence determine specificity of RNA polymerase and play a significant role in gene expression regulation. Here, we present a simple and unified protocol for purification of all seven Escherichia coli RNA polymerase sigma factors. In our approach, we took advantage of the His8-SUMO tag, known to increase protein solubilization. Sigma factors were first purified in N-terminal fusions with this tag, which was followed by tag removal with Ulp1 protease. This allowed to obtain proteins in their native form. In addition, the procedure is simple and requires only one resin type. With the general protocol we employed, we were able to successfully purify σD, σE, σS, and σN. Final step modification was required for σF, while for σH and σFecI, denaturing conditions had to be applied. All seven sigma factors were fully functional in forming an active holoenzyme with core RNA polymerase which we demonstrated with EMSA studies.

Structure of the SigF1-dependent pilA1 gene promoter and characterization of the light-activated response in the cyanobacterium Synechococcus elongatus PCC 7942.

In cyanobacteria that perform oxygenic photosynthesis, alternative sigma factors can play critical roles in environmental acclimation at the transcriptional initiation step. Here, we found in Synechococcus elongatus PCC 7942 that transcription of the pilA1 gene, encoding the type IV pilin, is dependent on one of the group 3 sigma factors, SigF1. We analyzed the promoter sequence determinants and proposed herein that the -10 and -35 boxes upstream of the transcriptional start site are critical for transcription. Interestingly, while the pilA1 promoter is activated by illumination, RNA polymerase containing SigF1 is already located on the promoter region under dark conditions, prior to illumination. This strongly suggests that promoter activation by light follows the recruitment of RNA polymerase during transcriptional initiation.

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/ß/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.

LIC_12757 from the pathogenic spirochaete Leptospira interrogans encodes an autoregulated ECF σE-type factor.

ECF (extracytoplasmic function) σ factors, members of the σ70-family, are the largest class of alternative σ factors which are stimulated in the presence of specific signals and direct RNA polymerase to transcribe a defined subset of genes. Thanks to them, bacterial pathogens can effectively reprogram their gene expression and, consequently, survive in the host and establish infection in a relatively short time. The number of ECF σ factors encoded within bacterial genomes is different depending on a given species and it reflects the likelihood that these bacteria will encounter harsh environmental conditions. The genome of L. interrogans, a zoonotic pathogen responsible for leptospirosis, is predicted to encode 11 ECF σE-type factors, but none of them have been characterized biochemically to date and their functions are still unknown. Here, we focused on one of the leptospiral ECF σ factors, namely LIC_12757, which was previously found to be up-regulated at elevated temperatures and may be related to the expression of clpB encoding an important L. interrogans virulence factor. We report cloning of the coding sequence of the LIC_12757 gene, its expression with the pET system and biochemical characterization of LIC_12757. By performing EMSA and in vitro transcription assays, we provide strong evidence that LIC_12757 indeed functions as a transcriptional factor that enables RNA polymerase to bind to the specific σE-type promoter and to initiate transcription. Interestingly, we demonstrate that LIC_12757 is autoregulated at the transcriptional level. Our study is a first step towards determining key aspects of LIC_12757 function in pathogenic Leptospira.

Multiple SigB homologues govern the transcription of the ssgBp promoter in the sporulation-specific ssgB gene in Streptomyces coelicolor A3(2).

Unlike Bacillus subtilis, Streptomyces coelicolor contains nine SigB homologues of the stress-response sigma factor SigB. By using a two-plasmid system, we previously identified promoters recognized by these sigma factors. Almost all promoters were recognized by several SigB homologues. However, no specific sequences of these promoters were found. One of these promoters, ssgBp, was selected to examine this cross-recognition in the native host. It controls the expression of the sporulation-specific gene ssgB. Using a luciferase reporter, the activity of this promoter in S. coelicolor and nine mutant strains lacking individual sigB homologous genes showed that sgBp is dependent on three sigma factors, SigH, SigN, and SigI. To determine which nucleotides in the-10 region are responsible for the selection of a specific SigB homologue, promoters mutated at the last three nucleotide positions were tested in the two-plasmid system. Some mutant promoters were specifically recognized by a distinct set of SigB homologues. Analysis of these mutant promoters in the native host showed the role of these nucleotides. A conserved nucleotide A at position 5 was essential for promoter activity, and two variable nucleotides at positions 4 and 6 were responsible for the partial selectivity of promoter recognition by SigB homologues.

Transcriptome fine-mapping in Fusobacterium nucleatum reveals FoxJ, a new σE-dependent small RNA with unusual mRNA activation activity.

The oral commensal Fusobacterium nucleatum can spread to extra-oral sites, where it is associated with diverse pathologies, including pre-term birth and cancer. Due to the evolutionary distance of F. nucleatum to other model bacteria, we lack a deeper understanding of the RNA regulatory networks that allow this bacterium to adapt to its various niches. As a first step in that direction, we recently showed that F. nucleatum harbors a global stress response governed by the extracytoplasmic function sigma factor, σE, which displays a striking functional conservation with Proteobacteria and includes a noncoding arm in the form of a regulatory small RNA (sRNA), FoxI. To search for putative additional σE-dependent sRNAs, we comprehensively mapped the 5' and 3' ends of transcripts in the model strain ATCC 23726. This enabled the discovery of FoxJ, a ~156-nucleotide sRNA previously misannotated as the 5' untranslated region (UTR) of ylmH. FoxJ is tightly controlled by σE and activated by the same stress conditions as is FoxI. Both sRNAs act as mRNA repressors of the abundant porin FomA, but FoxJ also regulates genes that are distinct from the target suite of FoxI. Moreover, FoxJ differs from other σE-dependent sRNAs in that it also positively regulates genes at the post-transcriptional level. We provide preliminary evidence for a new mode of sRNA-mediated mRNA activation, which involves the targeting of intra-operonic terminators. Overall, our study provides an important resource through the comprehensive annotation of 5' and 3' UTRs in F. nucleatum and expands our understanding of the σE response in this evolutionarily distant bacterium.IMPORTANCEThe oral microbe Fusobacterium nucleatum can colonize secondary sites, including cancer tissue, and likely deploys complex regulatory systems to adapt to these new environments. These systems are largely unknown, partly due to the phylogenetic distance of F. nucleatum to other model organisms. Previously, we identified a global stress response mediated by σE that displays functional conservation with the envelope stress response in Proteobacteria, comprising a coding and noncoding regulatory arm. Through global identification of transcriptional start and stop sites, we uncovered the small RNA (sRNA) FoxJ as a novel component of the noncoding arm of the σE response in F. nucleatum. Together with its companion sRNA FoxI, FoxJ post-transcriptionally modulates the synthesis of envelope proteins, revealing a conserved function for σE-dependent sRNAs between Fusobacteriota and Proteobacteria. Moreover, FoxJ activates the gene expression for several targets, which is a mode of regulation previously unseen in the noncoding arm of the σE response.

New layers of regulation of the general stress response sigma factor RpoS.

The general stress response (GSR) sigma factor RpoS from Escherichia coli has emerged as one of the key paradigms for study of how numerous signal inputs are accepted at multiple levels into a single pathway for regulation of gene expression output. While many studies have elucidated the key pathways controlling the production and activity of this sigma factor, recent discoveries have uncovered still more regulatory mechanisms which feed into the network. Moreover, while the regulon of this sigma factor comprises a large proportion of the E. coli genome, the downstream expression levels of all the RpoS target genes are not identically affected by RpoS upregulation but respond heterogeneously, both within and between cells. This minireview highlights the most recent developments in our understanding of RpoS regulation and expression, in particular those which influence the regulatory network at different levels from previously well-studied pathways.

An Improved Method for Eliminating or Creating Intragenic Bacterial Promoters.

Recent advances in genomic refactoring have been hindered by the ever-present complication of internal or cryptic transcriptional regulation. Typical approaches to these features have been to randomize or perform mass alterations to the gene sequences thought to contain the regulatory motifs; however, this approach can cause problems by altering translational speeds, introducing long distance DNA-DNA interaction effects, and inducing RNA toxicity. Previously, we developed a rational design approach named COdon Restrained Promoter SilEncing (CORPSE) which takes externally identified promoter sequences and uses position-specific scoring matrices as proxy promoter strengths to make minimal changes to promoter sequences to disable their activity. Additionally, through inverting our system we were also able to modify weak internal promoters to increase their activity. In this chapter, we augment our previous process with the biophysical model Promoter Calculator v1.0 developed by LaFleur et al. to combine promoter identification and activity prediction, with our algorithm to silently modify promoter sequences, to provide more robust promoter elimination and creation.

The RsfSR two-component system regulates SigF function by monitoring the state of the respiratory electron transport chain in Mycobacterium smegmatis.

In Mycobacterium smegmatis, the transcriptional activity of the alternative sigma factor SigF is posttranslationally regulated by the partner switching system consisting of SigF, the anti-SigF RsbW1, and three anti-SigF antagonists (RsfA, RsfB, and RsbW3). We previously demonstrated that expression of the SigF regulon is strongly induced in the Δaa3 mutant of M. smegmatis lacking the aa3 cytochrome c oxidase, the major terminal oxidase in the respiratory electron transport chain. Here, we identified and characterized the RsfSR two-component system involved in regulating the phosphorylation state of the major anti-SigF antagonist RsfB. RsfS (MSMEG_6130) is a histidine kinase with the cyclase/histidine kinase-associated sensing extracellular 3 domain at its N terminus, and RsfR (MSMEG_6131) is a receiver domain-containing protein phosphatase 2C-type phosphatase that can dephosphorylate phosphorylated RsfB. We demonstrated that phosphorylation of RsfR on Asp74 by RsfS reduces the phosphatase activity of RsfR toward phosphorylated RsfB and that the cellular abundance of the active unphosphorylated RsfB is increased in the Δaa3 mutant relative to the WT strain. We also demonstrated that the RsfSR two-component system is required for induction of the SigF regulon under respiration-inhibitory conditions such as inactivation of the cytochrome bcc1 complex and aa3 cytochrome c oxidase, as well as hypoxia, electron donor-limiting, high ionic strength, and low pH conditions. Collectively, our results reveal a key regulatory element involved in regulating the SigF signaling system by monitoring the state of the respiratory electron transport chain.

Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

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.

Spermidine constitutes a key determinant of motility and attachment of Salmonella Typhimurium through a novel regulatory mechanism.

Spermidine is a poly-cationic molecule belonging to the family of polyamines and is ubiquitously present in all organisms. Salmonella synthesizes, and harbours specialized transporters to import spermidine. A group of polyamines have been shown to assist in Salmonella Typhimurium's virulence and regulation of Salmonella pathogenicity Inslad 1 (SPI-1) genes and stress resistance; however, the mechanism remains elusive. The virulence trait of Salmonella depends on its ability to employ multiple surface structures to attach and adhere to the surface of the target cells before invasion and colonization of the host niche. Our study discovers the mechanism by which spermidine assists in the early stages of Salmonella pathogenesis. For the first time, we report that Salmonella Typhimurium regulates spermidine transport and biosynthesis processes in a mutually inclusive manner. Using a mouse model, we show that spermidine is critical for invasion into the murine Peyer's patches, which further validated our in vitro cell line observation. We show that spermidine controls the mRNA expression of fimbrial (fimA) and non-fimbrial adhesins (siiE, pagN) in Salmonella and thereby assists in attachment to host cell surfaces. Spermidine also regulated the motility through the expression of flagellin genes by enhancing the translation of sigma-28, which features an unusual start codon and a poor Shine-Dalgarno sequence. Besides regulating the formation of the adhesive structures, spermidine tunes the expression of the two-component system BarA/SirA to regulate SPI-1 encoded genes. Thus, our study unravels a novel regulatory mechanism by which spermidine exerts critical functions during Salmonella Typhimurium pathogenesis.

Artificial activation of both σH and Spo0A in Bacillus subtilis enforced initiation of spore development at the vegetatively growing phase.

When Bacillus subtilis cells face environmental deterioration, such as exhaustion of nutrients and an increase in cell density, they form spores. It is known that phosphorylation of Spo0A and activation of σH are key events at the initiation of sporulation. However, the initiation of sporulation is an extremely complicated process, and the relationship between these two events remains to be elucidated. To determine the minimum requirements for triggering sporulation initiation, we attempted to induce cell sporulation at the log phase, regardless of nutrients and cell density. In rich media such as Luria-Bertani (LB) medium, the cells of B. subtilis do not sporulate efficiently, possibly because of excess nutrition. When the amount of xylose in the LB medium was limited, σH -dependent transcription of the strain, in which sigA was under the control of the xylose-inducible promoter, was induced, and the frequency of sporulation was elevated according to the decreased level of σA. We also employed a fusion of sad67, which codes for an active form of Spo0A, and the IPTG-inducible promoter. The combination of lowered σA expression and activated Spo0A allowed the cells in the log phase to stop growing and rush into spore development. This observation of enforced initiation of sporulation in the mutant strain was detected even in the presence of the wild-type strain, suggesting that only intracellular events initiate and fulfill spore development regardless of extracellular conditions. Under natural sporulation conditions, the amount of σA did not change drastically throughout growth. Mechanisms that sequester σA from the core RNA polymerase and help σH to become active exist, but this has not yet been elucidated.

Structure and molecular mechanism of bacterial transcription activation.

Transcription activation is an important checkpoint of regulation of gene expression which occurs in response to different intracellular and extracellular signals. The key elements in this signal transduction process are transcription activators, which determine when and how gene expression is activated. Recent structural studies on a considerable number of new transcription activation complexes (TACs) revealed the remarkable mechanistic diversity of transcription activation mediated by different factors, necessitating a review and re-evaluation of the transcription activation mechanisms. In this review, we present a comprehensive summary of transcription activation mechanisms and propose a new, elaborate, and systematic classification of transcription activation mechanisms, primarily based on the structural features of diverse TAC components.

Sulfonamidyl derivatives of sigmacidin: Protein-protein interaction inhibitors targeting bacterial RNA polymerase and sigma factor interaction exhibiting antimicrobial activity against antibiotic-resistant bacteria.

RNA polymerase is an essential enzyme involved in bacterial transcription, playing a crucial role in RNA synthesis. However, it requires the association with sigma factors to initiate this process. In our previous work, we utilized a structure-based drug discovery approach to create benzoyl and benzyl benzoic acid compounds. These compounds were designed based on the amino acid residues within the key binding site of sigma factors, which are crucial for their interaction with RNA polymerase. By inhibiting bacterial transcription, these compounds exhibited notable antimicrobial activity, and we coined them as sigmacidins to highlight their resemblance to sigma factors and the benzoic acid structure. In this study, we further modified the compound scaffolds and developed a series of sulfonamidyl benzoic acid derivatives. These derivatives displayed potent antimicrobial activity, with minimum inhibitory concentrations (MICs) as low as 1 µg/mL, demonstrating their efficacy against bacteria. Furthermore, these compounds demonstrated low cytotoxicity, indicating their potential as safe antimicrobial agents. To ascertain their mechanism of action in interfering with bacterial transcription, we conducted biochemical and cellular assays. Overall, this study showcases the effectiveness of sulfonamidyl benzoic acid derivatives as antimicrobial agents by targeting protein-protein interactions involving RNA polymerase and sigma factors. Their strong antimicrobial activity and low cytotoxicity implicate their potential in combating antibiotic-resistant bacteria.

RpoS-Regulated Genes and Phenotypes in the Phytopathogenic Bacterium Pectobacterium atrosepticum.

The alternative sigma factor RpoS is considered to be one of the major regulators providing stress resistance and cross-protection in bacteria. In phytopathogenic bacteria, the effects of RpoS have not been analyzed with regard to cross-protection, and genes whose expression is directly or indirectly controlled by RpoS have not been determined at the whole-transcriptome level. Our study aimed to determine RpoS-regulated genes and phenotypes in the phytopathogenic bacterium Pectobacterium atrosepticum. Knockout of the rpoS gene in P. atrosepticum affected the long-term starvation response, cross-protection, and virulence toward plants with enhanced immune status. The whole-transcriptome profiles of the wild-type P. atrosepticum strain and its ΔrpoS mutant were compared under different experimental conditions, and functional gene groups whose expression was affected by RpoS were determined. The RpoS promoter motif was inferred within the promoter regions of the genes affected by rpoS deletion, and the P. atrosepticum RpoS regulon was predicted. Based on RpoS-controlled phenotypes, transcriptome profiles, and RpoS regulon composition, the regulatory role of RpoS in P. atrosepticum is discussed.