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Fast growth phenotypes are achieved through optimal transcriptomic allocation, in which cells must balance tradeoffs in resource allocation between diverse functions. One such balance between stress readiness and unbridled growth in E. coli has been termed the fear versus greed (f/g) tradeoff. Two specific RNA polymerase (RNAP) mutations observed in adaptation to fast growth have been previously shown to affect the f/g tradeoff, suggesting that genetic adaptations may be primed to control f/g resource allocation. Here, we conduct a greatly expanded study of the genetic control of the f/g tradeoff across diverse conditions. We introduced 12 RNA polymerase (RNAP) mutations commonly acquired during adaptive laboratory evolution (ALE) and obtained expression profiles of each. We found that these single RNAP mutation strains resulted in large shifts in the f/g tradeoff primarily in the RpoS regulon and ribosomal genes, likely through modifying RNAP-DNA interactions. Two of these mutations additionally caused condition-specific transcriptional adaptations. While this tradeoff was previously characterized by the RpoS regulon and ribosomal expression, we find that the GAD regulon plays an important role in stress readiness and ppGpp in translation activity, expanding the scope of the tradeoff. A phylogenetic analysis found the greed-related genes of the tradeoff present in numerous bacterial species. The results suggest that the f/g tradeoff represents a general principle of transcriptome allocation in bacteria where small genetic changes can result in large phenotypic adaptations to growth conditions.IMPORTANCETo increase growth, E. coli must raise ribosomal content at the expense of non-growth functions. Previous studies have linked RNAP mutations to this transcriptional shift and increased growth but were focused on only two mutations found in the proteins central region. RNAP mutations, however, commonly occur over a large structural range. To explore RNAP mutations impact, we have introduced 12 RNAP mutations found in laboratory evolution experiments and obtained expression profiles of each. The mutations nearly universally increased growth rates by adjusting said tradeoff away from non-growth functions. In addition to this shift, a few caused condition-specific adaptations. We explored the prevalence of this tradeoff across phylogeny and found it to be a widespread and conserved trend among bacteria.

Hallmarks of the developmental cycle of the obligate intracellular pathogenic bacterium Chlamydia are the primary differentiation of the infectious elementary body (EB) into the proliferative reticulate body (RB) and the secondary differentiation of RBs back into EBs. The mechanisms regulating these transitions remain unclear. In this report, we developed an effective novel strategy termed dependence on plasmid-mediated expression (DOPE) that allows for the knockdown of essential genes in Chlamydia. We demonstrate that GrgA, a Chlamydia-specific transcription factor, is essential for the secondary differentiation and optimal growth of RBs. We also show that GrgA, a chromosome-encoded regulatory protein, controls the maintenance of the chlamydial virulence plasmid. Transcriptomic analysis further indicates that GrgA functions as a critical regulator of all three sigma factors that recognize different promoter sets at developmental stages. The DOPE strategy outlined here should provide a valuable tool for future studies examining chlamydial growth, development, and pathogenicity.

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. [ABSTRACT FROM AUTHOR]

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 antisigma 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. [ABSTRACT FROM AUTHOR]

Solventogenic Clostridium species are important for establishing the sustainable industrial bioproduction of fuels and important chemicals such as acetone and butanol. The inherent versatility of these species in substrate utilization and the range of solvents produced during acetone butanol–ethanol (ABE) fermentation make solventogenic Clostridium an attractive choice for biotechnological applications such as the production of fuels and chemicals. The functional qualities of these microbes have thus been identified to be related to complex regulatory networks that play essential roles in modulating the metabolism of this group of bacteria. Yet, solventogenic Clostridium species still struggle to consistently achieve butanol concentrations exceeding 20 g/L in batch fermentation, primarily due to the toxic effects of butanol on the culture. Genomes of solventogenic Clostridium species have a relatively greater prevalence of genes that are intricately controlled by various regulatory molecules than most other species. Consequently, the use of genetic or metabolic engineering strategies that do not consider the underlying regulatory mechanisms will not be effective. Several regulatory factors involved in substrate uptake/utilization, sporulation, solvent production, and stress responses (Carbon Catabolite Protein A, Spo0A, AbrB, Rex, CsrA) have been identified and characterized. In this review, the focus is on newly identified regulatory factors in solventogenic Clostridium species, the interaction of these factors with previously identified molecules, and potential implications for substrate utilization, solvent production, and resistance/tolerance to lignocellulose-derived microbial inhibitory compounds. Taken together, this review is anticipated to highlight the challenges impeding the re-industrialization of ABE fermentation, and inspire researchers to generate innovative strategies for overcoming these obstacles. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: The membrane lipid phosphatidylcholine (PC) is crucial for the bacterium Sinorhizobium meliloti when generating nitrogen‐fixing nodules on the roots of their host plant. Although PC‐deficient mutants of S. meliloti cannot form any nodules on their host plant, suppressor mutants were isolated which partially recover the ability to trigger nodule formation. Among these suppressor mutants, we detected a reduced function mutant of FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids thereby probably affecting lipid packing. [ABSTRACT FROM AUTHOR]

Our new structure-based classification provides a more comprehensive/systematic understanding of bacterial transcriptional activation utilizing categorization based on the transcription factor (TF)/DNA/RNA polymerase (RNAP) interactions involved. The majority of transcription factors (TFs) bind to the upstream sites of the –35 element but differ in interaction with RNAP/DNA. Such differences often play a crucial role and could define important aspects of the molecular mechanism of transcription activation. Certain TFs bind to the promoter spacer region to enhance promoter recognition and activate transcription via DNA topology modification or adaptation of RNAP with remodeling or bridging interactions. Notably, numerous TFs interact only with RNAP rather than with the promoter DNA to activate transcription. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective To investigate whether the expression regulation of regulatory gene Sigma factors (sigA-sigM) is related to isoniazid resistance phenotype in isoniazid-resistant Mycobacterium tuberculosis (MTB) caused by katG mutations, and to provide reference for the study of the molecular mechanism of isoniazid resistance. Methods A total of 90 strains were collected from the patients undergoing first-line treatment at the Tianjin Tuberculosis Control Center during drug resistance testing from 2020 to 2022, of which 30 strains were sensitive strains without katG mutation, and 65 strains were isoniazidresistant strains caused by katG mutation, including 11 strains resistant only to isoniazid, 24 strains resistant to both isoniazid and streptomycin, and 30 strains multidrug-resistant to isoniazid, streptomycin, and rifampicin. After the strains were collected on the isoniazid drug-containing medium, the RNA of the strains was extracted, and the relative expression levels of Sigma factors were detected by reverse transcription-polymerase chain reaction(RT-PCR). The expression differences of Sigma factors in different isoniazid drug-resistant phenotypes were analyzed by the Mann-Whitney U test. Results The gene expression levels of sigA, sigC, sigF, sigG, sigH, sigI, sigJ, sigK, sigL in isoniazid mono-resistant group were significantly higher than those in pan-isoniazid-sensitive group (Z=4.368, 5.701, 6.865, 4.021, 5.126, 2.670, 5.983, 4.701, 5.490, P all<0.001). The gene expression levels of sigA, sigC, sigF, sigG, sigH, sigI, sigJ, sigK, sigL in poly-resistance group were significantly higher than those in pan-sensitivity group (Z=-5.017, -4.670, -4.667, -5.456, -4.083, -5.393, -4.712, -6.971, -8.206, -5.211, P all< 0.001). In multidrug-resistant (MDR) group, the gene expression levels of sigC, sigD, sigE, sigF, sigG, sigH, sigI, sigJ, sigK, sigL were significantly higher than those in pan-isoniazid-sensitive group (Z=-5.537, -4.003, -5.216, -7.328, -7.730, - 5.658, -4.440, -6.036, -4.862, -4.312, P all<0.001). The expression levels of sigB, sigF, sigG showed statistically significant differences in gene expression between the isoniazid mono-resistant, isoniazid poly-resistant, and isoniazid multidrug-resistant groups (Z=10.139, 7.735, 14.532, P all<0.001). The expression rates of sigF, sigG, sigI, sigJ, and sigL in the isoniazid-resistant group were significantly higher than those in isoniazid sensitive group (χ²=17.410. 45.673. 57.661. 42.896. 26.363, P all< 0.001). Conclusions sigF, sigG, sigI, sigJ, and sigL are associated with isoniazid resistance due to katG mutations. [ABSTRACT FROM AUTHOR]

The intracellular pathogen Chlamydia temporally regulates the expression of its genes, but the upstream signals that control transcription are not known. The best-studied regulatory pathway is a partner-switching mechanism that involves an anti-sigma factor, RsbW, which inhibits transcription by binding and sequestering the sigma subunit of RNA polymerase. RsbW is itself regulated by an anti-anti-sigma factor, RsbV, whose phosphorylation state is controlled by the phosphatase RsbU. In this study, we showed that Chlamydia trachomatis RsbU requires manganese or magnesium as a cofactor and dephosphorylates RsbV1 and RsbV2, which are the two chlamydial paralogs of RsbV. The gene for RsbU is adjacent to the enolase gene in a number of Chlamydia genomes, and we showed that eno and rsbU are cotranscribed from the same operon. In other bacteria, there is no known functional connection between the Rsb pathway and enolase, which is an enzyme in the glycolytic pathway. We found, however, that Chlamydia RsbU phosphatase activity was inhibited by phosphoenolpyruvate (PEP), the product of the enolase reaction, but not by 2-phosphoglycerate (2PGA), which is the substrate. These findings suggest that the enolase reaction and, more generally, glucose metabolism, may provide an upstream signal that regulates transcription in Chlamydia through the RsbW pathway. IMPORTANCE The RsbW pathway is a phosphorelay that regulates gene expression in Chlamydia, but its upstream signal has not been identified. We showed that RsbU, a phosphatase in this pathway, is inhibited by phosphoenolpyruvate, which is the product of the enolase reaction. As enolase is an enzyme in the glycolytic pathway, these results reveal an unrecognized link between glucose metabolism and gene regulation in chlamydiae. Moreover, as these intracellular bacteria acquire glucose from the infected host cell, our findings suggest that glucose availability may be an external signal that controls chlamydial gene expression.

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 2.0 Å 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.

Cyanobacteria safeguard their photosynthetic machinery from oxidative damage caused by adverse environmental factors such as high-intensity light. Together with many photoprotective compounds, they contain myxoxanthophylls, a rare group of glycosidic carotenoids containing a high number of conjugated double bonds. These carotenoids have been shown to: have strong photoprotective effects, contribute to the integrity of the thylakoid membrane, and upregulate in cyanobacteria under a variety of stress conditions. However, their metabolic potential has not been fully utilized in the stress biology of cyanobacteria and the pharmaceutical industry due to a lack of mechanistic understanding and their insufficient biosynthesis. This review summarizes current knowledge on: biological function, genetic regulation, biotechnological production, and pharmaceutical potential of myxoxanthophyll, with a focus on strain engineering and parameter optimization strategies for increasing their cellular content. The summarized knowledge can be utilized in cyanobacterial metabolic engineering to improve the stress tolerance of useful strains and enhance the commercial-scale synthesis of myxoxanthophyll for pharmaceutical uses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction–modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed. [ABSTRACT FROM AUTHOR]

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.

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.

Bacteria belonging to Streptomyces have the ability to produce a wide range of secondary metabolites through a shift from primary to secondary metabolism regulated by complex networks activated after vegetative growth terminates. Despite considerable effort to understand the regulatory elements governing gene expression related to primary and secondary metabolism in Streptomyces, system-level information remains limited. In this study, we integrated four multi-omics datasets from Streptomyces griseus NBRC 13350: RNA-seq, ribosome profiling, dRNA-seq, and Term-Seq, to analyze the regulatory elements of transcription and translation of differentially expressed genes during cell growth. With the functional enrichment of gene expression in different growth phases, one sigma factor regulon and four transcription factor regulons governing differential gene transcription patterns were found. In addition, the regulatory elements of transcription termination and post-transcriptional processing at transcript 3'-end positions were elucidated, including their conserved motifs, stem-loop RNA structures, and non-terminal locations within the polycistronic operons, and the potential regulatory elements of translation initiation and elongation such as 5'-UTR length, RNA structures at ribosome-bound sites, and codon usage were investigated. This comprehensive genetic information provides a foundational genetic resource for strain engineering to enhance secondary metabolite production in Streptomyces.

Light initiates chloroplast biogenesis in Arabidopsis by eliminating PHYTOCHROME-INTERACTING transcription FACTORs (PIFs), which in turn de-represses nuclear photosynthesis genes, and synchronously, generates a nucleus-to-plastid (anterograde) signal that activates the plastid-encoded bacterial-type RNA polymerase (PEP) to transcribe plastid photosynthesis genes. However, the identity of the anterograde signal remains frustratingly elusive. The main challenge has been the difficulty to distinguish regulators from the plethora of necessary components for plastid transcription and other essential chloroplast functions, such as photosynthesis. Here, we show that the genome-wide induction of nuclear photosynthesis genes is insufficient to activate the PEP. PEP inhibition is imposed redundantly by multiple PIFs and requires PIF3's activator activity. Among the nuclear-encoded components of the PEP holoenzyme, we identify four light-inducible, PIF-repressed sigma factors as anterograde signals. Together, our results elucidate that light-dependent inhibition of PIFs activates plastid photosynthesis genes via sigma factors as anterograde signals in parallel with the induction of nuclear photosynthesis genes.

Transcriptional regulation is a critical process to ensure expression of genes necessary for growth and survival in diverse environments. Transcription is mediated by multiple transcription factors including activators, repressors and sigma factors. Accurate computational prediction of the regulon of target genes for transcription factors is difficult and experimental identification is laborious and not scalable. Here, we demonstrate regulon identification by in vitro transcription-sequencing (RIViT-seq) that enables systematic identification of regulons of transcription factors by combining an in vitro transcription assay and RNA-sequencing. Using this technology, target genes of 11 sigma factors were identified in Streptomyces coelicolor A3(2). The RIViT-seq data expands the transcriptional regulatory network in this bacterium, discovering regulatory cascades and crosstalk between sigma factors. Implementation of RIViT-seq with other transcription factors and in other organisms will improve our understanding of transcriptional regulatory networks across biology.

Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.

The aim of this review is to summarize the current knowledge on the role of σ factors in a highly invasive spirochaete Leptospira interrogans responsible for leptospirosis that affects many mammals, including humans. This disease has a significant impact on public health and the economy worldwide. In bacteria, σ factors are the key regulators of gene expression at the transcriptional level and therefore play an important role in bacterial adaptative response to different environmental stimuli. These factors form a holoenzyme with the RNA polymerase core enzyme and then direct it to specific promoters, which results in turning on selected genes. Most bacteria possess several different σ factors that enable them to maintain basal gene expression, as well as to regulate gene expression in response to specific environmental signals. Recent comparative genomics and in silico genome‐wide analyses have revealed that the L. interrogans genome, consisting of two circular chromosomes, encodes a total of 14 σ factors. Among them, there is one putative housekeeping σ70‐like factor, and three types of alternative σ factors, i.e., one σ54, one σ28 and 11 putative ECF (extracytoplasmic function) σE‐type factors. Here, characteristics of these putative σ factors and their possible role in the L. interrogans gene regulation (especially in this pathogen's adaptive response to various environmental conditions, an important determinant of leptospiral virulence), are presented. [ABSTRACT FROM AUTHOR]

ABSTRACT Chlamydia trachomatis is a developmentally regulated, obligate intracellular bacterium that encodes three sigma factors: σ66, σ54, and σ28. σ66 is the major sigma factor controlling most transcription initiation during early- and mid-cycle development as the infectious elementary body (EB) transitions to the non-infectious reticulate body (RB) that replicates within an inclusion inside the cell. The roles of the minor sigma factors, σ54 and σ28, have not been well characterized to date; however, there are data to suggest each functions in late-stage development and secondary differentiation as RBs transition to EBs. As the process of secondary differentiation itself is poorly characterized, clarifying the function of these alternative sigma factors by identifying the genes regulated by them will further our understanding of chlamydial differentiation. We hypothesize that σ54 and σ28 have non-redundant and essential functions for initiating late gene transcription thus mediating secondary differentiation in Chlamydia. Here, we demonstrate the necessity of each minor sigma factor in successfully completing the developmental cycle. We have implemented and validated multiplexed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference techniques, novel to the chlamydial field to examine the effects of knocking down each alternative sigma factor individually and simultaneously. In parallel, we also overexpressed each sigma factor. Altering transcript levels for either or both alternative sigma factors resulted in a severe defect in EB production as compared to controls. Furthermore, RNA sequencing identified differentially expressed genes during alternative sigma factor dysregulation, indicating the putative regulons of each. These data demonstrate that the levels of alternative sigma factors must be carefully regulated to facilitate chlamydial growth and differentiation. IMPORTANCE Chlamydia trachomatis is a significant human pathogen in both developed and developing nations. Due to the organism's unique developmental cycle and intracellular niche, basic research has been slow and arduous. However, recent advances in chlamydial genetics have allowed the field to make significant progress in experimentally interrogating the basic physiology of Chlamydia. Broadly speaking, the driving factors of chlamydial development are poorly understood, particularly regarding how the later stages of development are regulated. Here, we employ a novel genetic tool for use in Chlamydia while investigating the effects of dysregulating the two alternative sigma factors in the organism that help control transcription initiation. We provide further evidence for both sigma factors' essential roles in late-stage development and their potential regulons, laying the foundation for deeper experimentation to uncover the molecular pathways involved in chlamydial differentiation.

ABSTRACT The functional significance of rpoN genes that encode two sigma factors in the Bradyrhizobium sp. strain DOA9 has been reported to affect colony formation, root nodulation characteristics, and symbiotic interactions with Aeschynomene americana. rpoN mutant strains are defective in cellular surface polysaccharide (CSP) production compared with the wild-type (WT) strain, and they accordingly exhibit smaller colonies and diminished symbiotic effectiveness. To gain deeper insights into the changes in CSP composition and the nodules of rpoN mutants, we employed synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy. FTIR analysis of the CSP revealed the absence of specific components in the rpoN mutants, including lipids, carboxylic groups, polysaccharide-pyranose rings, and β-galactopyranosyl residues. Nodules formed by DOA9WT exhibited a uniform distribution of lipids, proteins, and carbohydrates; mutant strains, particularly DOA9∆rpoNp:ΩrpoNc, exhibited decreased distribution uniformity and a lower concentration of C=O groups. Furthermore, Fe K-edge X-ray absorption near-edge structure and extended X-ray absorption fine structure analyses revealed deficiencies in the nitrogenase enzyme in the nodules of DOA9∆rpoNc and DOA9∆rpoNp:ΩrpoNc mutants; nodules from DOA9WT and DOA9∆rpoNp exhibited both leghemoglobin and the nitrogenase enzyme. IMPORTANCE This work provides valuable insights into how two rpoN genes affect the composition of cellular surface polysaccharides (CSPs) in Bradyrhizobium sp., which subsequently dictates root nodule chemical characteristics and nitrogenase production. We used advanced synchrotron methods, including synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy and X-ray absorption spectroscopy (XAS), for the first time in this field to analyze CSP components and reveal the biochemical changes occurring within nodules. These cutting-edge techniques confer significant advantages by providing detailed molecular information, enabling the identification of specific functional groups, chemical bonds, and biomolecule changes. This research not only contributes to our understanding of plant-microbe interactions but also establishes a foundation for future investigations and potential applications in this field. The combined use of the synchrotron-based FTIR and XAS techniques represents a significant advancement in facilitating a comprehensive exploration of bacterial CSPs and their implications in plant-microbe interactions.

Pseudomonas aeruginosa is a pathogen capable of colonizing virtually every human tissue. The host colonization competence and versatility of this pathogen are powered by a wide array of virulence factors necessary in different steps of the infection process. This includes factors involved in bacterial motility and attachment, biofilm formation, the production and secretion of extracellular invasive enzymes and exotoxins, the production of toxic secondary metabolites, and the acquisition of iron. Expression of these virulence factors during infection is tightly regulated, which allows their production only when they are needed. This process optimizes host colonization and virulence. In this work, we review the intricate network of transcriptional regulators that control the expression of virulence factors in P. aeruginosa, including one- and two-component systems and σ factors. Because inhibition of virulence holds promise as a target for new antimicrobials, blocking the regulators that trigger the production of virulence determinants in P. aeruginosa is a promising strategy to fight this clinically relevant pathogen. [ABSTRACT FROM AUTHOR]

The global characterization of transcriptional regulatory networks almost exclusively uses in vivo conditions, thereby providing a snapshot on multiple regulatory interactions at the same time. To complement these approaches, we developed and applied a method for characterizing bacterial promoters genome-wide by in vitro transcription coupled to transcriptome sequencing specific for native 5′-ends of transcripts. This method, called ROSE (run-off transcription/RNA-sequencing), only requires chromosomal DNA, ribonucleotides, RNA polymerase (RNAP) core enzyme, and a specific sigma factor, recognizing the corresponding promoters, which have to be analyzed. ROSE was performed on E. coli K-12 MG1655 genomic DNA using Escherichia coli RNAP holoenzyme (including σ70) and yielded 3226 transcription start sites, 2167 of which were also identified in in vivo studies, and 598 were new. Many new promoters not yet identified by in vivo experiments might be repressed under the tested conditions. Complementary in vivo experiments with E. coli K-12 strain BW25113 and isogenic transcription factor gene knockout mutants of fis, fur, and hns were used to test this hypothesis. Comparative transcriptome analysis demonstrated that ROSE could identify bona fide promoters that were apparently repressed in vivo. In this sense, ROSE is well-suited as a bottom-up approach for characterizing transcriptional networks in bacteria and ideally complementary to top-down in vivo transcriptome studies. [ABSTRACT FROM AUTHOR]

The ability of Staphylococcus aureus to infect many different tissue sites is enabled, in part, by its transcriptional regulatory network (TRN) that coordinates its gene expression to respond to different environments. We elucidated the organization and activity of this TRN by applying independent component analysis to a compendium of 108 RNA-sequencing expression profiles from two S. aureus clinical strains (TCH1516 and LAC). ICA decomposed the S. aureus transcriptome into 29 independently modulated sets of genes (i-modulons) that revealed: 1) High confidence associations between 21 i-modulons and known regulators; 2) an association between an i-modulon and σS, whose regulatory role was previously undefined; 3) the regulatory organization of 65 virulence factors in the form of three i-modulons associated with AgrR, SaeR, and Vim-3; 4) the roles of three key transcription factors (CodY, Fur, and CcpA) in coordinating the metabolic and regulatory networks; and 5) a low-dimensional representation, involving the function of few transcription factors of changes in gene expression between two laboratory media (RPMI, cation adjust Mueller Hinton broth) and two physiological media (blood and serum). This representation of the TRN covers 842 genes representing 76% of the variance in gene expression that provides a quantitative reconstruction of transcriptional modules in S. aureus, and a platform enabling its full elucidation.

Genome-scale studies of the bacterial regulatory network have been leveraged by declining sequencing cost and advances in ChIP (chromatin immunoprecipitation) methods. Of which, ChIP-exo has proven competent with its near-single base-pair resolution. While several algorithms and programs have been developed for different analytical steps in ChIP-exo data processing, there is a lack of effort in incorporating them into a convenient bioinformatics pipeline that is intuitive and publicly available. In this paper, we developed ChIP-exo Analysis Pipeline (ChEAP) that executes the one-step process, starting from trimming and aligning raw sequencing reads to visualization of ChIP-exo results. The pipeline was implemented on the interactive web-based Python development environment – Jupyter Notebook, which is compatible with the Google Colab cloud platform to facilitate the sharing of codes and collaboration among researchers. Additionally, users could exploit the free GPU and CPU resources allocated by Colab to carry out computing tasks regardless of the performance of their local machines. The utility of ChEAP was demonstrated with the ChIP-exo datasets of RpoN sigma factor in E. coli K-12 MG1655. To analyze two raw data files, ChEAP runtime was 2 min and 25 s. Subsequent analyses identified 113 RpoN binding sites showing a conserved RpoN binding pattern in the motif search. ChEAP application in ChIP-exo data analysis is extensive and flexible for the parallel processing of data from various organisms.

Bacteria use an array of sigma factors to regulate gene expression during different stages of their life cycles. Full-length, atomic-level structures of sigma factors have been challenging to obtain experimentally as a result of their many regions of intrinsic disorder. AlphaFold has now supplied plausible full-length models for most sigma factors. Here we discuss the current understanding of the structures and functions of sigma factors in the model organism, Bacillus subtilis, and present an X-ray crystal structure of a region of B. subtilis SigE, a sigma factor that plays a critical role in the developmental process of spore formation. [ABSTRACT FROM AUTHOR]

L. interrogans belongs to highly invasive spirochaetes causing leptospirosis in mammals, including humans. During infection, this pathogen is exposed to various stressors, and therefore, it must reprogram its gene expression to survive in the host and establish infection in a short duration of time. Host adaptation is possible thanks to molecular responses where appropriate regulators and signal transduction systems participate. Among the bacterial regulators, there are σ factors, including ECF (extracytoplasmic function) σ factors. The L. interrogans genome encodes 11 putative ECF σE-type factors. Currently, none of them has been characterized biochemically, and their functions are still unknown. One of them, LIC_10559, is the most likely to be active during infection because it is only found in the highly pathogenic Leptospira. The aim of this study was to achieve LIC_10559 overexpression to answer the question whether it may be a target of the humoral immune response during leptospiral infections. The immunoreactivity of the recombinant LIC_10559 was evaluated by SDS-PAGE, ECL Western blotting and ELISA assay using sera collected from Leptospira-infected animals and uninfected healthy controls. We found that LIC_10559 was recognized by IgG antibodies from the sera of infected animals and is, therefore, able to induce the host's immune response to pathogenic Leptospira. This result suggests the involvement of LIC_10559 in the pathogenesis of leptospirosis. [ABSTRACT FROM AUTHOR]

The Extracellular function (ECF) sigma factor SigE is one of the best characterized out of the 13 sigma factors encoded in the Mycobacterium tuberculosis chromosome. SigE is required for blocking phagosome maturation and full virulence in both mice and guinea pigs. Moreover, it is involved in the response to several environmental stresses as surface stress, oxidative stress, acidic pH, and phosphate starvation. Underscoring its importance in M. tuberculosis physiology, SigE is subjected to a very complex regulatory system: depending on the environmental conditions, its expression is regulated by three different sigma factors (SigA, SigE, and SigH) and a two-component system (MprAB). SigE is also regulated at the post-translational level by an anti-sigma factor (RseA) which is regulated by the intracellular redox potential and by proteolysis following phosphorylation from PknB upon surface stress. The set of genes under its direct control includes other regulators, as SigB, ClgR, and MprAB, and genes involved in surface remodeling and stabilization. Recently SigE has been shown to interact with PhoP to activate a subset of genes in conditions of acidic pH. The complex structure of its regulatory network has been suggested to result in a bistable switch leading to the development of heterogeneous bacterial populations. This hypothesis has been recently reinforced by the finding of its involvement in the development of persister cells able to survive to the killing activity of several drugs. [ABSTRACT FROM AUTHOR]

ABSTRACT Outer membrane protein A (OmpA) is the most abundant porin in bacterial outer membranes. KJΔOmpA299–356, an ompA C-terminal in-frame deletion mutant of Stenotrophomonas maltophilia KJ, exhibits pleiotropic defects, including decreased tolerance to menadione (MD)-mediated oxidative stress. Here, we elucidated the underlying mechanism of the decreased MD tolerance mediated by ΔompA299–356. The transcriptomes of wild-type S. maltophilia and the KJΔOmpA299–356 mutant strain were compared, focusing on 27 genes known to be associated with oxidative stress alleviation; however, no significant differences were identified. OmpO was the most downregulated gene in KJΔOmpA299–356. KJΔOmpA299–356 complementation with the chromosomally integrated ompO gene restored MD tolerance to the wild-type level, indicating the role of OmpO in MD tolerance. To further clarify the possible regulatory circuit involved in ompA defects and ompO downregulation, σ factor expression levels were examined based on the transcriptome results. The expression levels of three σ factors were significantly different (downregulated levels of rpoN and upregulated levels of rpoP and rpoE) in KJΔOmpA299–356. Next, the involvement of the three σ factors in the ΔompA299–356-mediated decrease in MD tolerance was evaluated using mutant strains and complementation assays. rpoN downregulation and rpoE upregulation contributed to the ΔompA299–356-mediated decrease in MD tolerance. OmpA C-terminal domain loss induced an envelope stress response. Activated σE decreased rpoN and ompO expression levels, in turn decreasing swimming motility and oxidative stress tolerance. Finally, we revealed both the ΔompA299–356-rpoE-ompO regulatory circuit and rpoE-rpoN cross regulation. IMPORTANCE The cell envelope is a morphological hallmark of Gram-negative bacteria. It consists of an inner membrane, a peptidoglycan layer, and an outer membrane. OmpA, an outer membrane protein, is characterized by an N-terminal β-barrel domain that is embedded in the outer membrane and a C-terminal globular domain that is suspended in the periplasmic space and connected to the peptidoglycan layer. OmpA is crucial for the maintenance of envelope integrity. Stress resulting from the destruction of envelope integrity is sensed by extracytoplasmic function (ECF) σ factors, which induce responses to various stressors. In this study, we revealed that loss of the OmpA-peptidoglycan (PG) interaction causes peptidoglycan and envelope stress while simultaneously upregulating σP and σE expression levels. The outcomes of σP and σE activation are different and are linked to β-lactam and oxidative stress tolerance, respectively. These findings establish that outer membrane proteins (OMPs) play a critical role in envelope integrity and stress tolerance.

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.

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction–modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed.

Multiple cell types sense fluid flow as an environmental cue. Flow can exert shear force (or stress) on cells, and the prevailing model is that biological flow sensing involves the measurement of shear force1,2. Here, we provide evidence for force-independent flow sensing in the bacterium Pseudomonas aeruginosa. A microfluidic-based transcriptomic approach enabled us to discover an operon of P. aeruginosa that is rapidly and robustly upregulated in response to flow. Using a single-cell reporter of this operon, which we name the flow-regulated operon (fro), we establish that P. aeruginosa dynamically tunes gene expression to flow intensity through a process we call rheosensing (as rheo- is Greek for flow). We further show that rheosensing occurs in multicellular biofilms, involves signalling through the alternative sigma factor FroR, and does not require known surface sensors. To directly test whether rheosensing measures force, we independently altered the two parameters that contribute to shear stress: shear rate and solution viscosity. Surprisingly, we discovered that rheosensing is sensitive to shear rate but not viscosity, indicating that rheosensing is a kinematic (force-independent) form of mechanosensing. Thus, our findings challenge the dominant belief that biological mechanosensing requires the measurement of forces.

Determining transcriptional and translational regulatory elements in GC-rich Streptomyces genomes is essential to elucidating the complex regulatory networks that govern secondary metabolite biosynthetic gene cluster (BGC) expression. However, information about such regulatory elements has been limited for Streptomyces genomes. To address this limitation, a high-quality genome sequence of β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27 064 is completed, which contains 7163 newly annotated genes. This provides a fundamental reference genome sequence to integrate multiple genome-scale data types, including dRNA-Seq, RNA-Seq and ribosome profiling. Data integration results in the precise determination of 2659 transcription start sites which reveal transcriptional and translational regulatory elements, including -10 and -35 promoter components specific to sigma (σ) factors, and 5'-untranslated region as a determinant for translation efficiency regulation. Particularly, sequence analysis of a wide diversity of the -35 components enables us to predict potential σ-factor regulons, along with various spacer lengths between the -10 and -35 elements. At last, the primary transcriptome landscape of the β-lactam biosynthetic pathway is analyzed, suggesting temporal changes in metabolism for the synthesis of secondary metabolites driven by transcriptional regulation. This comprehensive genetic information provides a versatile genetic resource for rational engineering of secondary metabolite BGCs in Streptomyces.

To colonize a host, bacteria depend on an ensemble of signaling systems to convert information about the various environments encountered within the host into specific cellular activities. How these signaling systems coordinate transitions between cellular states in vivo remains poorly understood. To address this knowledge gap, we investigated how the bacterial symbiont Vibrio fischeri initially colonizes the light organ of the Hawaiian bobtail squid Euprymna scolopes. Previous work has shown that the small RNA Qrr1, which is a regulatory component of the quorum-sensing system in V. fischeri, promotes host colonization. Here, we report that transcriptional activation of Qrr1 is inhibited by the sensor kinase BinK, which suppresses cellular aggregation by V. fischeri prior to light organ entry. We show that Qrr1 expression depends on the alternative sigma factor σ54 and the transcription factors LuxO and SypG, which function similar to an OR logic gate, thereby ensuring Qrr1 is expressed during colonization. Finally, we provide evidence that this regulatory mechanism is widespread throughout the Vibrionaceae family. Together, our work reveals how coordination between the signaling pathways underlying aggregation and quorum-sensing promotes host colonization, which provides insight into how integration among signaling systems facilitates complex processes in bacteria.

The Extracellular function (ECF) sigma factor SigE is one of the best characterized out of the 13 sigma factors encoded in the Mycobacterium tuberculosis chromosome. SigE is required for blocking phagosome maturation and full virulence in both mice and guinea pigs. Moreover, it is involved in the response to several environmental stresses as surface stress, oxidative stress, acidic pH, and phosphate starvation. Underscoring its importance in M. tuberculosis physiology, SigE is subjected to a very complex regulatory system: depending on the environmental conditions, its expression is regulated by three different sigma factors (SigA, SigE, and SigH) and a two-component system (MprAB). SigE is also regulated at the post-translational level by an anti-sigma factor (RseA) which is regulated by the intracellular redox potential and by proteolysis following phosphorylation from PknB upon surface stress. The set of genes under its direct control includes other regulators, as SigB, ClgR, and MprAB, and genes involved in surface remodeling and stabilization. Recently SigE has been shown to interact with PhoP to activate a subset of genes in conditions of acidic pH. The complex structure of its regulatory network has been suggested to result in a bistable switch leading to the development of heterogeneous bacterial populations. This hypothesis has been recently reinforced by the finding of its involvement in the development of persister cells able to survive to the killing activity of several drugs.

Erwinia amylovora causes a devastating fire blight disease in apples and pears. One of the main virulence determinants in E. amylovora is the hypersensitive response (HR) and pathogenicity (hrp)-type III secretion system (T3SS), which is activated by the RpoN-HrpL sigma factor cascade. However, the RpoN regulon in E. amylovora has not been investigated. In this study, we determined the RpoN regulon in E. amylovora by combining RNA-seq transcriptomic analysis with in silico binding site analysis. RNA-seq revealed that 262 genes, approximately 7.5% genes in the genome of E. amylovora, were differentially transcribed in the rpoN mutant as compared with the wild type. Specifically, genes associated with virulence, motility, nitrogen assimilation, the PspF system, stress response, and arginine biosynthesis are positively regulated by RpoN, whereas genes associated with biosynthesis of amino acids and sorbitol transport are negatively regulated by RpoN. In silico binding site analysis identified 46 potential target genes with a putative RpoN binding site, and the upstream sequences of six, three, and three genes also contain putative GlnG, PspF, and YfhA binding sites, respectively. Overall, RpoN directly regulates genes associated with virulence, nitrogen assimilation, the PspF system, motility and the YfhA/YfhK two-component regulatory system. [ABSTRACT FROM AUTHOR]

The bacterial heat-shock response is regulated by the alternative sigma factor, σ32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoters for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose-inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH (σ32) regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden change in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurred without arabinose induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased five-fold within 15 min of arabinose addition. Transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many genes essential for P. aeruginosa growth are induced by RpoH. Other stress response genes induced by RpoH are involved in damaged nucleic acid repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. Phenotypic analysis of P. aeruginosa ∆rpoH (PBAD-rpoH) showed that it is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also had increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside sensitivity of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes, such as aminoglycoside acetyl transferases, that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient-limiting conditions. [ABSTRACT FROM AUTHOR]

The sigma H (σΗ) and sigma E (σE) subunits of Corynebacterium glutamicum RNA polymerase belong to Group 4 of sigma factors, also called extracytoplasmic function (ECF) sigma factors. Genes of the C. glutamicum σΗ regulon that are involved in heat and oxidative stress response have already been defined, whereas the genes of the σE regulon, which is involved in cell surface stress response, have not been explored until now. Using the C. glutamicum RES167 strain and its derivative C. glutamicum ΔcseE with a deletion in the anti-σΕ gene, differential gene expression was analyzed by RNA sequencing. We found 296 upregulated and 398 downregulated genes in C. glutamicum ΔcseE compared to C. glutamicum RES167. To confirm the functional link between σΕ and the corresponding promoters, we tested selected promoters using the in vivo two-plasmid system with gfpuv as a reporter gene and by in vitro transcription. Analyses with RNAP+σΗ and RNAP+σΕ, which were previously shown to recognize similar promoters, proved that the σΗ and σE regulons significantly overlap. The σE-controlled genes were found to be involved for example in protein quality control (dnaK, dnaJ2, clpB, and clpC), the regulation of Clp proteases (clgR), and membrane integrity maintenance. The single-promoter analyses with σΗ and σΕ revealed that there are two groups of promoters: those which are exclusively σΗ-specific, and the other group of promoters, which are σΗ/σE-dependent. No exclusively σE-dependent promoter was detected. We defined the consensus sequences of exclusively σΗ-regulated promotors to be −35 GGAAt and − 10 GTT and σΗ/σE-regulated promoters to be −35 GGAAC and − 10 cGTT. Fifteen genes were found to belong to the σΗ/σΕ regulon. Homology modeling showed that there is a specific interaction between Met170 in σΗ and the nucleotides −31 and − 30 within the non-coding strand (AT or CT) of the σΗ-dependent promoters. In σE, Arg185 was found to interact with the nucleotides GA at the same positions in the σE-dependent promoters.

Experimental evolution of Escherichia coli K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1 knockout of phoB (encoding the positive regulator of the phosphate regulon). A phoB::kanR knockout increased growth at high pH. phoB mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in pcnB [poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions of torI, an inhibitor of TorR, which activates expression of torCAD (trimethylamine N-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay, rpoS deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded by yobG (mgrB) (PhoPQ regulator), rpoN (nitrogen starvation sigma factor), malI, and purR, as well as envelope proteins, such as those encoded by ompT and yahO Overall, E. coli evolution at high pH selects for mutations in key transcriptional regulators, including phoB and the stationary-phase sigma factor RpoS.IMPORTANCEEscherichia coli in its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.

Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation-specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell- and developmental stage-specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σH and σA , during sporulation. The results suggest that σH is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σA plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth.

A nuclear-encoded sigma (σ) factor is essential for the transcriptional regulation of plant chloroplast-encoded genes. Five putative maize σ factors have been identified by database searches, but their functions are unknown. We report a maize leaf color mutant etiolated/albino leaf 1 (eal1) that was derived from space mutation breeding. The eal1 mutant displays etiolated or albino leaves that then gradually turn to normal green at the seedling stage. The changes in eal1 leaf color are associated with changes in photosynthetic pigment content and chloroplast development. Map-based cloning revealed that a single amino-acid deletion changing Val480-Val481-Val482 to Val480-Val481, in the C-terminal domain σ4 of the putative σ factor ZmSig2A, is responsible for the eal1 mutation. In comparison with the expression level of the wild-type (WT) allele ZmSig2A+ in WT plants, much higher expression of the mutant allele ZmSig2AΔV in eal1 plants was detected before the eal1 plants turned to normal green. ZmSig2A shows the highest similarity to rice OsSig2A and Arabidopsis SIG2. Ectopic expression of ZmSig2A+ or ZmSig2AΔV driven by the cauliflower mosaic virus 35S promoter rescued the pale green leaf of the sig2 mutant, but ectopic expression of ZmSig2AΔV driven by the SIG2 promoter did not. We propose that the Val deletion generated a new weak allele of ZmSig2A that cannot completely abolish the ZmSig2A function. Some genes involved in chloroplast development and photosynthesis-associated nuclear genes showed significant expression differences between eal1 and WT plants. We conclude that ZmSig2A encoding a σ factor is essential for maize chloroplast development. The eal1 mutant with a weak allele of ZmSig2A represents a valuable genetic resource for investigating the regulation of ZmSig2A-mediated chloroplast development in maize. The eal1 mutation may be useful as a marker for early identification and elimination of false hybrids or transgene transmission in the application of genetic male sterility to commercial hybrid seed production.

ABSTRACT OmpA, the most abundant porin in Stenotrophomonas maltophilia KJ, exists as a two-domain structure with an N-terminal domain of β-barrel structure embedded in the outer membrane and a C-terminal domain collocated in the periplasm. KJΔOmpA299-356, an ompA mutant of S. maltophilia KJ with a truncated OmpA devoid of 299 to 356 amino acids (aa), was able to stably embed in the outer membrane. KJΔOmpA299-356 was more susceptible to β-lactams than wild-type KJ. We aimed to elucidate the mechanism underlying the ΔompA299-356-mediated increase in β-lactam susceptibility (abbreviated as “ΔOmpA299-356 phenotype”). KJΔOmpA299-356 displayed a lower ceftazidime (CAZ)-induced β-lactamase activity than KJ. Furthermore, KJ2, a L1/L2 β-lactamases-null mutant, and KJ2ΔOmpA299-356, a KJ2 mutant with truncated OmpA devoid of299 to 356 aa, had comparable β-lactam susceptibility. Both lines of evidence indicate that decreased β-lactamase activity contributes to the ΔOmpA299-356 phenotype. We analyzed the transcriptome results of KJ and KJΔOmpA299-356, focusing on PG homeostasis-associated genes. Among the 36 genes analyzed, the nagA gene was upregulated 4.65-fold in KJΔOmpA299-356. Deletion of the nagA gene from the chromosome of KJΔOmpA299-356 restored β-lactam susceptibility and CAZ-induced β-lactamase activity to wild-type levels, verifying that nagA-upregulation in KJΔOmpA299-356 contributes to the ΔOmpA299-356 phenotype. Furthermore, transcriptome analysis revealed that rpoE (Smlt3555) and rpoP (Smlt3514) were significantly upregulated in KJΔOmpA299-356. The deletion mutant construction, β-lactam susceptibility, and β-lactamase activity analysis demonstrated that σP, but not σE, was involved in the ΔOmpA299-356 phenotype. A real-time quantitative (qRT-PCR) assay confirmed that nagA is a member of the σP regulon. The involvement of the σP-NagA-L1/L2 regulatory circuit in the ΔOmpA299-356 phenotype was manifested. IMPORTANCE Porins of Gram-negative bacteria generally act as channels that allow the entry or extrusion of molecules. Moreover, the structural role of porins in stabilizing the outer membrane by interacting with peptidoglycan (PG) and the outer membrane has been proposed. The linkage between porin deficiency and antibiotic resistance increase has been reported widely, with a rationale for blocking antibiotic influx. In this study, a link between porin defects and β-lactam susceptibility increase was demonstrated. The underlying mechanism revealed that a novel σP-NagA-L1/L2 regulatory circuit is triggered due to the loss of the OmpA-PG interaction. This study extends the understanding on the porin defect and antibiotic susceptibility. Porin defects may cause opposite impacts on antibiotic susceptibility, which is dependent on the involvement of the defect. Blocking the porin channel role can increase antibiotic resistance; in contrast, the loss of porin structure role may increase antibiotic susceptibility.

Type 6 secretion systems (T6SSs) are specialized multiprotein complexes that inject protein effectors into prokaryotic and/or eukaryotic cells. We previously described the role of the T6SS of the phytopathogen Xanthomonas citri pv. citri as an anti‐eukaryotic nanoweapon that confers resistance to predation by the amoeba Dictyostelium discoideum. Transcription of the X. citri T6SS genes is induced by a signaling cascade involving the Ser/Thr kinase PknS and the extracytoplasmic function sigma factor EcfK. Here, we used a strain overexpressing a phosphomimetic constitutively active version of EcfK (EcfKT51E) to identify the EcfK regulon, which includes a previously uncharacterized transcription factor of the AraC‐family (TagK), in addition to T6SS genes and genes encoding protein homeostasis factors. Functional studies demonstrated that TagK acts downstream of EcfK, binding directly to T6SS gene promoters and inducing T6SS expression in response to contact with amoeba cells. TagK controls a small regulon, consisting of the complete T6SS, its accessory genes and additional genes encoded within the T6SS cluster. We conclude that a singular regulatory circuit consisting of a transmembrane kinase (PknS), an alternative sigma factor (EcfK) and an AraC‐type transcriptional regulator (TagK) promotes expression of the X. citri T6SS in response to a protozoan predator. [ABSTRACT FROM AUTHOR]

Using a mutant of Mycobacterium smegmatis lacking the major aa3 cytochrome c oxidase of the electron transport chain (Δaa3), we demonstrated that inhibition of the respiratory electron transport chain led to an increase in antibiotic resistance of M. smegmatis to isoniazid, rifampicin, ethambutol, and tetracycline. The alternative sigma factors SigB and SigE were shown to be involved in an increase in rifampicin resistance of M. smegmatis induced under respiration-inhibitory conditions. As in Mycobacterium tuberculosis, SigE and SigB form a hierarchical regulatory pathway in M. smegmatis through SigE-dependent transcription of sigB. Expression of sigB and sigE was demonstrated to increase in the Δaa3 mutant, leading to upregulation of the SigB-dependent genes in the mutant. The pho U2 (MSMEG_1605) gene implicated in a phosphate-signaling pathway and the MSMEG_1097 gene encoding a putative glycosyltransferase were identified to be involved in the SigB-dependent enhancement of rifampicin resistance observed for the Δaa3 mutant of M. smegmatis. The significance of this study is that the direct link between the functionality of the respiratory electron transport chain and antibiotic resistance in mycobacteria was demonstrated for the first time using an electron transport chain mutant rather than inhibitors of electron transport chain. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a leading cause of severe pneumonia. Our recent proteomic investigations into S. aureus invasion of human lung epithelial cells revealed three key adaptive responses: activation of the SigB and CodY regulons and upregulation of the hibernation-promoting factor SaHPF. Therefore, our present study aimed at a functional and proteomic dissection of the contributions of CodY, SigB and SaHPF to host invasion using transposon mutants of the methicillin-resistant S. aureus USA300. Interestingly, disruption of codY resulted in a "small colony variant" phenotype and redirected the bacteria from (phago)lysosomes into the host cell cytoplasm. Furthermore, we show that CodY, SigB and SaHPF contribute differentially to host cell adhesion, invasion, intracellular survival and cytotoxicity. CodY- or SigB-deficient bacteria experienced faster intracellular clearance than the parental strain, underscoring the importance of these regulators for intracellular persistence. We also show an unprecedented role of SaHPF in host cell adhesion and invasion. Proteomic analysis of the different mutants focuses attention on the CodY-perceived metabolic state of the bacteria and the SigB-perceived environmental cues in bacterial decision-making prior and during infection. Additionally, it underscores the impact of the nutritional status and bacterial stress on the initiation and progression of staphylococcal lung infections.