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σ 54 serves as an unconventional sigma factor with a distinct mechanism of transcription initiation, which depends on the involvement of a transcription activator. This unique sigma factor σ 54 is indispensable for orchestrating the transcription of genes crucial to nitrogen regulation, flagella biosynthesis, motility, chemotaxis and various other essential cellular processes. Currently, no comprehensive tools are available to determine σ 54 promoters and regulon in bacterial genomes. Here, we report a σ 54 promoter prediction method ProPr54, based on a convolutional neural network trained on a set of 446 validated σ 54 binding sites derived from 33 bacterial species. Model performance was tested and compared with respect to bacterial intergenic regions, demonstrating robust applicability. ProPr54 exhibits high performance when tested on various bacterial species, highly surpassing other available σ 54 regulon identification methods. Furthermore, analysis on bacterial genomes, which have no experimentally validated σ 54 binding sites, demonstrates the generalization of the model. ProPr54 is the first reliable in silico method for predicting σ 54 binding sites, making it a valuable tool to support experimental studies on σ 54 . In conclusion, ProPr54 offers a reliable, broadly applicable tool for predicting σ 54 promoters and regulon genes in bacterial genome sequences. A web server is freely accessible at http://propr54.molgenrug.nl., (© The Author(s) 2025. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

The σ54-σS sigma factor cascade plays a central role in regulating differential gene expression during the enzootic cycle of Borreliella burgdorferi, the Lyme disease pathogen. In this pathway, the primary transcription of rpoS (which encodes σS) is under the control of σ54 which is activated by a bacterial enhancer-binding protein (EBP), Rrp2. The σ54-dependent activation in B. burgdorferi has long been thought to be unique, requiring an additional factor, BosR, a homologue of classical Fur/PerR repressor/activator. However, how BosR is involved in this σ54-dependent activation remains unclear and perplexing. In this study, we demonstrate that BosR does not function as a regulator for rpoS transcriptional activation. Instead, it functions as a novel RNA-binding protein that governs the turnover rate of rpoS mRNA. We further show that BosR directly binds to the 5' untranslated region (UTR) of rpoS mRNA, and the binding region overlaps with a region required for rpoS mRNA degradation. Mutations within this 5'UTR region result in BosR-independent RpoS production. Collectively, these results uncover a novel role of Fur/PerR family regulators as RNA-binding proteins and redefine the paradigm of the σ54-σS pathway in B. burgdorferi., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The sigma (σ) factor of RNA holoenzymes is essential for identifying and binding to promoter regions during gene transcription in prokaryotes. σ54 promoters carried out various ancillary methods and environmentally responsive procedures; therefore, it is crucial to accurately identify σ54 promoter sequences to comprehend the underlying process of gene regulation. Herein, we come up with a convolutional neural network (CNN) based prediction tool named "iProm-Sigma54" for the prediction of σ54 promoters. The CNN consists of two one-dimensional convolutional layers, which are followed by max pooling layers and dropout layers. A one-hot encoding scheme was used to extract the input matrix. To determine the prediction performance of iProm-Sigma54, we employed four assessment metrics and five-fold cross-validation; performance was measured using a benchmark and test dataset. According to the findings of this comparison, iProm-Sigma54 outperformed existing methodologies for identifying σ54 promoters. Additionally, a publicly accessible web server was constructed.

Gene transcription is carried out by RNA polymerase (RNAP) and requires the conversion of the initial closed promoter complex, where DNA is double stranded, to a transcription-competent open promoter complex, where DNA is opened up. In bacteria, RNAP relies on σ factors for its promoter specificities. Using a special form of sigma factor (σ 54 ), which forms a stable closed complex and requires its activator that belongs to the AAA+ ATPases (ATPases associated with diverse cellular activities), we obtained cryo-electron microscopy structures of transcription initiation complexes that reveal a previously unidentified process of DNA melting opening. The σ 54 amino terminus threads through the locally opened up DNA and then becomes enclosed by the AAA+ hexameric ring in the activator-bound intermediate complex. Our structures suggest how ATP hydrolysis by the AAA+ activator could remove the σ 54 inhibition while helping to open up DNA, using σ 54 amino-terminal peptide as a pry bar.

Background: RpoN, also known as σ 54 , first reported in Escherichia coli, is a subunit of RNA polymerase that strictly controls the expression of different genes by identifying specific promoter elements. RpoN has an important regulatory function in carbon and nitrogen metabolism and participates in the regulation of flagellar synthesis, bacterial motility and virulence. However, little is known about the effect of RpoN in Plesiomonas shigelloides., Results: To identify pathways controlled by RpoN, RNA sequencing (RNA-Seq) of the WT and the rpoN deletion strain was carried out for comparison. The RNA-seq results showed that RpoN regulates ~ 13.2% of the P. shigelloides transcriptome, involves amino acid transport and metabolism, glycerophospholipid metabolism, pantothenate and CoA biosynthesis, ribosome biosynthesis, flagellar assembly and bacterial secretion system. Furthermore, we verified the results of RNA-seq using quantitative real-time reverse transcription PCR, which indicated that the absence of rpoN caused downregulation of more than half of the polar and lateral flagella genes in P. shigelloides, and the ΔrpoN mutant was also non-motile and lacked flagella. In the present study, the ability of the ΔrpoN mutant to kill E. coli MG1655 was reduced by 54.6% compared with that of the WT, which was consistent with results in RNA-seq, which showed that the type II secretion system (T2SS-2) genes and the type VI secretion system (T6SS) genes were repressed. By contrast, the expression of type III secretion system genes was largely unchanged in the ΔrpoN mutant transcriptome and the ability of the ΔrpoN mutant to infect Caco-2 cells was also not significantly different compared with the WT., Conclusions: We showed that RpoN is required for the motility and contributes to the killing ability of P. shigelloides and positively regulates the T6SS and T2SS-2 genes., (© 2022. The Author(s).)

Bacterial cells alter gene expression in response to changes in population density in a process called quorum sensing (QS). In Vibrio harveyi, LuxO, a low-cell-density activator of sigma factor-54 (RpoN), is required for transcription of five noncoding regulatory small RNAs (sRNAs), Qrr1 to Qrr5, which each repress translation of the master QS regulator, LuxR. Vibrio parahaemolyticus, the leading cause of bacterial seafoodborne gastroenteritis, also contains five Qrr sRNAs that control OpaR (the LuxR homolog), controlling capsule polysaccharide (CPS), motility, and metabolism. We show that in a Δ luxO deletion mutant, opaR was derepressed and CPS and biofilm were produced. However, in a Δ rpoN mutant, opaR was repressed, no CPS was produced, and less biofilm production was observed than in the wild type. To determine why opaR was repressed, expression analysis in Δ luxO showed that all five qrr genes were repressed, while in Δ rpoN the qrr2 gene was significantly derepressed. Reporter assays and mutant analysis showed that Qrr2 sRNA can act alone to control OpaR. Bioinformatics analysis identified a sigma-70 (RpoD) -35 -10 promoter overlapping the canonical sigma-54 (RpoN) -24 -12 promoter in the qrr2 regulatory region. The qrr2 sigma-70 promoter element was also present in additional Vibrio species, indicating that it is widespread. Mutagenesis of the sigma-70 -10 promoter site in the Δ rpoN mutant background resulted in repression of qrr2. Analysis of qrr quadruple deletion mutants, in which only a single qrr gene is present, showed that only Qrr2 sRNA can act independently to regulate opaR . Mutant and expression data also demonstrated that RpoN and the global regulator, Fis, act additively to repress qrr2 . Our data have uncovered a new mechanism of qrr expression and show that Qrr2 sRNA is sufficient for OpaR regulation. IMPORTANCE The quorum sensing noncoding small RNAs (sRNAs) are present in all Vibrio species but vary in number and regulatory roles among species. In the Harveyi clade, all species contain five qrr genes, and in Vibrio harveyi these are transcribed by sigma-54 and are additive in function. In the Cholerae clade, four qrr genes are present, and in Vibrio cholerae the qrr genes are redundant in function. In Vibrio parahaemolyticus, qrr2 is controlled by two overlapping promoters. In an rpoN mutant, qrr2 is transcribed from a sigma-70 promoter that is present in all V. parahaemolyticus strains and in other species of the Harveyi clade, suggesting a conserved mechanism of regulation. Qrr2 sRNA can function as the sole Qrr sRNA to control OpaR.

Pseudomonas aeruginosa uses three type six secretion systems (H1-, H2- and H3-T6SS) to manipulate its environment, subvert host cells and for microbial competition. These T6SS machines are loaded with a variety of effectors/toxins, many being associated with a specific VgrG. How P. aeruginosa transcriptionally coordinates the main T6SS clusters and the multiple vgrG islands spread through the genome is unknown. Here we show an unprecedented level of control with RsmA repressing most known T6SS-related genes. Moreover, each of the H2- and H3-T6SS clusters encodes a sigma factor activator (SFA) protein called, Sfa2 and Sfa3, respectively. SFA proteins are enhancer binding proteins necessary for the sigma factor RpoN. Using a combination of RNA-seq, ChIP-seq and molecular biology approaches, we demonstrate that RpoN coordinates the T6SSs of P. aeruginosa by activating the H2-T6SS but repressing the H1- and H3-T6SS. Furthermore, RpoN and Sfa2 control the expression of the H2-T6SS-linked VgrGs and their effector arsenal to enable very effective interbacterial killing. Sfa2 is specific as Sfa3 from the H3-T6SS cannot complement loss of Sfa2. Our study further delineates the regulatory mechanisms that modulate the deployment of an arsenal of T6SS effectors likely enabling P. aeruginosa to adapt to a range of environmental conditions., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

σ 54 factor (RpoN), a type of transcriptional regulatory factor, is widely found in pathogenic bacteria. It binds to core RNA polymerase (RNAP) and regulates the transcription of many functional genes in an enhancer-binding protein (EBP)-dependent manner. σ 54 has two conserved functional domains: the activator-interacting domain located at the N-terminal and the DNA-binding domain located at the C-terminal. RpoN directly binds to the highly conserved sequence, GGN 10 GC, at the -24/-12 position relative to the transcription start site of target genes. In general, bacteria contain one or two RpoNs but multiple EBPs. A single RpoN can bind to different EBPs in order to regulate various biological functions. Thus, the overlapping and unique regulatory pathways of two RpoNs and multiple EBP-dependent regulatory pathways form a complex regulatory network in bacteria. However, the regulatory role of RpoN and EBPs is still poorly understood in phytopathogenic bacteria, which cause economically important crop diseases and pose a serious threat to world food security. In this review, we summarize the current knowledge on the regulatory function of RpoN, including swimming motility, flagella synthesis, bacterial growth, type IV pilus (T4Ps), twitching motility, type III secretion system (T3SS), and virulence-associated phenotypes in phytopathogenic bacteria. These findings and knowledge prove the key regulatory role of RpoN in bacterial growth and pathogenesis, as well as lay the groundwork for further elucidation of the complex regulatory network of RpoN in bacteria.

RpoN (δ54) as a global regulator controls crucialvirulence-associated phenotype, which can regulate flagellum and exopolysaccharides (EPS) during pathogenic biofilm formation. However, the knowledge of the roles of rpoN in biofilm formation of V. alginolyticus is limited, especially at different cell densities. Herein, deletion mutant strain ΔrpoN, complementary strain ΔrpoN-C and negative control strain ΔrpoN-Z were constructed to investigate the effects of rpoN on biofilm formation of V. alginolyticus HN08155 based on flagellum and EPS at different cell density conditions. The results showed that all of strains can form biofilm, and biofilms of strains with rpoN were formed at low cell density (LCD) and detached at high cell density (HCD), while those of ΔrpoN and ΔrpoN-Z were absent at LCD and accumulated excessively with a spotty pellicle at HCD without detaching. The EPS contents of strains with rpoN was greater than that of ΔrpoN and ΔrpoN-Z at LCD, while the opposite trends were observed at HCD. The expression levels of rpoN were quantified, which were consistent with the trend of biofilm formation. It's worth noting that absence of rpoN resulted in the failure of biofilm detachment, lacking of flagellum and decreasing motility, indicating that rpoN was not necessary for biofilm formation, but it was essential for biofilm detachment., (Copyright © 2021. Published by Elsevier GmbH.)

Background: Glutamate and aspartate are preferred nutrients for a variety of microorganisms. In the case for many Pseudomonas spp., utilization of these amino acids is believed to be dependent on a transporter complex comprised of a periplasmic-solute binding protein (AatJ), two permease domains (AatQM) and an ATP-binding component (AatP). Notably, expression of this transporter complex is hypothesized to be regulated at the transcriptional level by the enhancer-binding protein AauR and the alternative sigma factor RpoN. The purpose of the current study was to determine the biological significance of the putative aatJ-aatQMP operon and its regulatory aauR and rpoN genes in the utilization of L-glutamate, L-glutamine, L-aspartate and L-asparagine in Pseudomonas aeruginosa PAO1., Results: Deletion of the aatJ-aatQMP, aauR or rpoN genes did not affect the growth of P. aeruginosa PAO1 on L-glutamate, L-glutamine, L-aspartate and L-asparagine equally. Instead, only growth on L-glutamate as the sole carbon source was abolished with the deletion of any one of these genes. Interestingly, growth of the aauR mutant on L-glutamate was readily restored via plasmid-based expression of the aatQMP genes, suggesting that it is the function of AatQMP (and not AatJ) that is limiting in the absence of the aauR gene. Subsequent analysis of beta-galactosidase reporters revealed that both aatJ and aatQ were induced in response to L-glutamate, L-glutamine, L-aspartate or L-asparagine in a manner dependent on the aauR and rpoN genes. In addition, both aatJ and aatQ were expressed at reduced levels in the absence of the inducing-amino acids and the regulatory aauR and rpoN genes. The expression of the aatJ-aatQMP genes is, therefore, multifaceted. Lastly, the expression levels of aatJ were significantly higher (> 5 fold) than that of aatQ under all tested conditions., Conclusions: The primary function of AauR in P. aeruginosa PAO1 is to activate expression of the aatJ-aatQMP genes in response to exogenous acidic amino acids and their amide derivatives. Importantly, it is the AauR-RpoN mediated induction of the aatQMP genes that is the pivotal factor enabling P. aeruginosa PAO1 to effectively utilize or consume L-glutamate as a sole or preferred nutrient.

Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ 54 system, which includes σ 54 -loaded RNA polymerase and transcriptional activators called enhancer binding proteins (EBPs), might be a common regulator of natural product genes. Here, we explored this idea by analyzing a selected group of putative σ 54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ 54 -RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ 54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ 54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.

Chlamydia bacteria are obligate intracellular organisms with a phylum-defining biphasic developmental cycle that is intrinsically linked to its ability to cause disease. The progression of the chlamydial developmental cycle is regulated by the temporal expression of genes predominantly controlled by RNA polymerase sigma (σ) factors. Sigma 54 (σ 54 ) is one of three sigma factors encoded by Chlamydia for which the role and regulon are unknown. CtcC is part of a two-component signal transduction system that is requisite for σ 54 transcriptional activation. CtcC activation of σ 54 requires phosphorylation, which relieves inhibition by the CtcC regulatory domain and enables ATP hydrolysis by the ATPase domain. Prior studies with CtcC homologs in other organisms have shown that expression of the ATPase domain alone can activate σ 54 transcription. Biochemical analysis of CtcC ATPase domain supported the idea of ATP hydrolysis occurring in the absence of the regulatory domain, as well as the presence of an active-site residue essential for ATPase activity (E242). Using recently developed genetic approaches in Chlamydia to induce expression of the CtcC ATPase domain, a transcriptional profile was determined that is expected to reflect the σ 54 regulon. Computational evaluation revealed that the majority of the differentially expressed genes were preceded by highly conserved σ 54 promoter elements. Reporter gene analyses using these putative σ 54 promoters reinforced the accuracy of the model of the proposed regulon. Investigation of the gene products included in this regulon supports the idea that σ 54 controls expression of genes that are critical for conversion of Chlamydia from replicative reticulate bodies into infectious elementary bodies. IMPORTANCE The factors that control the growth and infectious processes for Chlamydia are still poorly understood. This study used recently developed genetic tools to determine the regulon for one of the key transcription factors encoded by Chlamydia , sigma 54. Surrogate and computational analyses provide additional support for the hypothesis that sigma 54 plays a key role in controlling the expression of many components critical to converting and enabling the infectious capability of Chlamydia These components include those that remodel the membrane for the extracellular environment and incorporation of an arsenal of type III secretion effectors in preparation for infecting new cells., (Copyright © 2020 Soules et al.)

Homologous regulatory factors are widely present in bacteria, but whether homologous regulators synergistically or differentially regulate different biological functions remains mostly unknown. Here, we report that the homologous regulators RpoN1 and RpoN2 of the plant pathogen Xanthomonas campestris pv. campestris (Xcc) play different regulatory roles with respect to virulence traits, flagellar biosynthesis, and basal metabolism. RpoN2 directly regulated Xcc fliC and fliQ to modulate flagellar synthesis in X. campestris, thus affecting the swimming motility of X. campestris. Mutation of rpoN2 resulted in reduced production of biofilms and extracellular polysaccharides in Xcc. These defects may together cause reduced virulence of the rpoN2 mutant against the host plant. Moreover, we demonstrated that RpoN1 could regulate branched-chain fatty acid production and modulate the synthesis of diffusible signal factor family quorum sensing signals. Although RpoN1 and RpoN2 are homologues, the regulatory roles and biological functions of these proteins were not interchangeable. Overall, our report provides new insights into the two different molecular roles that form the basis for the transcriptional specialization of RpoN homologues., (© 2020 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Bacteria synthesize inorganic polyphosphate (polyP) in response to a variety of different stress conditions. polyP protects bacteria by acting as a protein-stabilizing chaperone, metal chelator, or regulator of protein function, among other mechanisms. However, little is known about how stress signals are transmitted in the cell to lead to increased polyP accumulation. Previous work in the model enterobacterium Escherichia coli has indicated that the RNA polymerase-binding regulatory protein DksA is required for polyP synthesis in response to nutrient limitation stress. In this work, I set out to characterize the role of DksA in polyP regulation in more detail. I found that overexpression of DksA increases cellular polyP content (explaining the long-mysterious phenotype of dksA overexpression rescuing growth of a dnaK mutant at high temperatures) and characterized the roles of known functional residues of DksA in this process, finding that binding to RNA polymerase is required but that none of the other functions of DksA appear to be necessary. Transcriptomics revealed genome-wide transcriptional changes upon nutrient limitation, many of which were affected by DksA, and follow-up experiments identified complex interactions between DksA and the stress-sensing alternative sigma factors FliA, RpoN, and RpoE that impact polyP production, indicating that regulation of polyP synthesis is deeply entwined in the multifactorial stress response network of E. coli IMPORTANCE Inorganic polyphosphate (polyP) is an evolutionarily ancient, widely conserved biopolymer required for stress resistance and pathogenesis in diverse bacteria, but we do not understand how its synthesis is regulated. In this work, I gained new insights into this process by characterizing the role of the transcriptional regulator DksA in polyP regulation in Escherichia coli and identifying previously unknown links between polyP synthesis and the stress-responsive alternative sigma factors FliA, RpoN, and RpoE., (Copyright © 2020 American Society for Microbiology.)

Bacterial enhancer-binding proteins (bEBPs) are specialised transcriptional activators. bEBPs are hexameric AAA + ATPases and use ATPase activities to remodel RNA polymerase (RNAP) complexes that contain the major variant sigma factor, σ 54 to convert the initial closed complex to the transcription competent open complex. Earlier crystal structures of AAA + domains alone have led to proposals of how nucleotide-bound states are sensed and propagated to substrate interactions. Recently, the structure of the AAA + domain of a bEBP bound to RNAP-σ 54 -promoter DNA was revealed. Together with structures of the closed complex, an intermediate state where DNA is partially loaded into the RNAP cleft and the open promoter complex, a mechanistic understanding of how bEBPs use ATP to activate transcription can now be proposed. This review summarises current structural models and the emerging understanding of how this special class of AAA + proteins utilises ATPase activities to allow σ 54 -dependent transcription initiation., Competing Interests: The authors declare no conflict of interest.

Cellular RNA polymerase is a multi-subunit macromolecular assembly responsible for gene transcription, a highly regulated process conserved from bacteria to humans. In bacteria, sigma factors are employed to mediate gene-specific expression in response to a variety of environmental conditions. The major variant σ factor, σ 54 , has a specific role in stress responses. Unlike σ 70 -dependent transcription, which often can spontaneously proceed to initiation, σ 54 -dependent transcription requires an additional ATPase protein for activation. As a result, structures of a number of distinct functional states during the dynamic process of transcription initiation have been captured using the σ 54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ 54 -dependent transcription initiation and DNA opening. Comparisons with σ 70 and eukaryotic polymerases reveal unique and common features during transcription initiation., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Transcriptional regulation by nuclease-deficient CRISPR/Cas is a popular and valuable tool for routine control of gene expression. CRISPR interference in bacteria can be reliably achieved with high efficiencies. Yet, options for CRISPR activation (CRISPRa) remained limited in flexibility and activity because they relied on σ 70 promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ 54 -dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ 54 -dependent promoters with biotechnology relevance in non-model bacteria. It also supports orthogonal gene regulation on multiple levels. Combining our CRISPRa with dxCas9 further expands flexibility in DNA targeting, and boosts dynamic ranges into regimes that enable construction of cascaded CRISPRa circuits. Application-wise, we construct a reusable scanning platform for readily optimizing metabolic pathways without library reconstructions. This eukaryote-like CRISPRa system is therefore a powerful and versatile synthetic biology tool for diverse research and industrial applications.

Sigma factor, as a unit of RNA polymerase holoenzyme, is a critical factor in the process of gene transcriptional regulation. It recognizes the specific DNA sites and brings the core enzyme of RNA polymerase to the upstream regions of target genes. Therefore, the prediction of the promoters for a particular sigma factor is essential for interpreting functional genomic data and observation. This paper develops a new method to predict sigma-54 promoters in bacterial genomes. The new method organically integrates motif finding and machine learning strategies to capture the intrinsic features of sigma-54 promoters. The experiments on E. coli benchmark test set show that our method has good capability to distinguish sigma-54 promoters from surrounding or randomly selected DNA sequences. The applications of the other three bacterial genomes indicate the potential robustness and applicative power of our method on a large number of bacterial genomes. The source code of our method can be freely downloaded at https://github.com/maqin2001/PromotePredictor.

Multidrug-resistant organisms are increasing in healthcare settings, and there are few antimicrobials available to treat infections from these bacteria. Pseudomonas aeruginosa is an opportunistic pathogen in burn patients and individuals with cystic fibrosis (CF), and a leading cause of nosocomial infections. P. aeruginosa is inherently resistant to many antibiotics and can develop resistance to others, limiting treatment options. P. aeruginosa has multiple sigma factors to regulate transcription. The alternative sigma factor, RpoN (σ 54 ), regulates many virulence genes and is linked to antibiotic resistance. Recently, we described a cis-acting peptide, RpoN*, which is a "molecular roadblock", binding consensus promoters at the -24 site, blocking transcription. RpoN* reduces virulence of P. aeruginosa laboratory strains, but its effects in clinical isolates was unknown. We investigated the effects of RpoN* on phenotypically varied P. aeruginosa strains isolated from CF patients. RpoN* expression reduced motility, biofilm formation, and pathogenesis in a P. aeruginosa-C. elegans infection model. Furthermore, we investigated RpoN* effects on antibiotic susceptibility in a laboratory strain. RpoN* expression increased susceptibility to several beta-lactam-based antibiotics in strain P. aeruginosa PA19660 Xen5. We show that using a cis-acting peptide to block RpoN consensus promoters has potential clinical implications in reducing virulence and improving antibiotic susceptibility.

Labrenzia aggregata LZB033 ( Rhodobacteraceae ), which produces dimethylsulfoniopropionate (DMSP) and reduces nitrate to nitrogen, was isolated from seawater of the East China Sea. Its genome encodes a large number of transcriptional regulators which may be important for its adaptation to diverse marine environments. The alternative σ 54 factor (RpoN) is a central regulator of many bacteria, regulating the transcription of multiple genes and controlling important cellular functions. However, the exact role of RpoN in Labrenzia spp. is unknown. In this study, an in-frame rpoN deletion mutant was constructed in LZB033, and the function of RpoN was determined. To systematically identify RpoN-controlled genes, we performed a detailed analysis of gene expression differences between the wild-type strain and the Δ rpoN mutant using RNA sequencing. The expression of 175 genes was shown to be controlled by RpoN. Subsequent phenotypic assays showed that the Δ rpoN mutant was attenuated in flagellar biosynthesis and swimming motility, utilized up to 13 carbon substrates differently, lacked the ability to assimilate malic acid, and displayed markedly decreased biofilm formation. In addition, stress response assays showed that the Δ rpoN mutant was impaired in the ability to survive under different challenge conditions, including osmotic stress, oxidative stress, temperature changes, and acid stress. Moreover, both the DMSP synthesis and catabolism rates of LZB033 decreased after rpoN was knocked out. Our work provides essential insight into the regulatory function of RpoN, revealing that RpoN is a key determinant for LZB033 flagellar formation, motility, biofilm formation, and environmental fitness, as well as DMSP production and degradation. IMPORTANCE This study established an in-frame gene deletion method in the alphaproteobacterium Labrenzia aggregata LZB033 and generated an rpoN gene mutant. A comparison of the transcriptomes and phenotypic characteristics between the mutant and wild-type strains confirmed the role of RpoN in L. aggregata LZB033 flagellar formation, motility, biofilm formation, and carbon usage. Most importantly, RpoN is a key factor for survival under different environmental challenge conditions. Furthermore, the ability to synthesize and metabolize dimethylsulfoniopropionate (DMSP) was related to RpoN. These features revealed RpoN to be an important regulator of stress resistance and survival for L. aggregata LZB033 in marine environments., (Copyright © 2019 American Society for Microbiology.)

The σ 54 regulon in Salmonella enterica serovar Typhimurium includes a predicted RNA repair operon encoding homologs of the metazoan Ro60 protein (Rsr), Y RNAs (YrlBA), RNA ligase (RtcB), and RNA 3'-phosphate cyclase (RtcA). Transcription from σ 54 -dependent promoters requires that a cognate bacterial enhancer binding protein (bEBP) be activated by a specific environmental or cellular signal; the cognate bEBP for the σ 54 -dependent promoter of the rsr-yrlBA-rtcBA operon is RtcR. To identify conditions that generate the signal for RtcR activation in S Typhimurium, transcription of the RNA repair operon was assayed under multiple stress conditions that result in nucleic acid damage. RtcR-dependent transcription was highly induced by the nucleic acid cross-linking agents mitomycin C (MMC) and cisplatin, and this activation was dependent on RecA. Deletion of rtcR or rtcB resulted in decreased cell viability relative to that of the wild type following treatment with MMC. Oxidative stress from peroxide exposure also induced RtcR-dependent transcription of the operon. Nitrogen limitation resulted in RtcR-independent increased expression of the operon; the effect of nitrogen limitation required NtrC. The adjacent toxin-antitoxin module, dinJ-yafQ , was cotranscribed with the RNA repair operon but was not required for RtcR activation, although YafQ endoribonuclease activated RtcR-dependent transcription. Stress conditions shown to induce expression the RNA repair operon of Escherichia coli ( rtcBA ) did not stimulate expression of the S Typhimurium RNA repair operon. Similarly, MMC did not induce expression of the E. coli rtcBA operon, although when expressed in S Typhimurium, E. coli RtcR responds effectively to the unknown signal(s) generated there by MMC exposure. IMPORTANCE Homologs of the metazoan RNA repair enzymes RtcB and RtcA occur widely in eubacteria, suggesting a selective advantage. Although the enzymatic activities of the eubacterial RtcB and RtcA have been well characterized, the physiological roles remain largely unresolved. Here we report stress responses that activate expression of the σ 54 -dependent RNA repair operon ( rsr-yrlBA-rtcBA ) of S Typhimurium and demonstrate that expression of the operon impacts cell survival under MMC-induced stress. Characterization of the requirements for activation of this tightly regulated operon provides clues to the possible functions of operon components in vivo , enhancing our understanding of how this human pathogen copes with environmental stressors., (Copyright © 2018 American Society for Microbiology.)

The rhizobacterium Pseudomonas protegens H78 biosynthesizes a number of antibiotic compounds, including pyoluteorin, 2,4-diacetylphloroglucinol, and pyrrolnitrin. Here, we investigated the global regulatory function of the nitrogen metabolism-related sigma factor RpoN in P. protegens H78 through RNA-seq and phenotypic analysis. During the mid- to late-log growth phase, transcriptomic profiling revealed that 562 genes were significantly upregulated, and 502 genes were downregulated by at least twofold at the RNA level in the rpoN deletion mutant in comparison with the wild-type strain H78. With respect to antibiotics, Plt biosynthesis and the expression of its operon were positively regulated, while Prn biosynthesis and the expression of its operon were negatively regulated by RpoN. RpoN is responsible for the global activation of operons involved in flagellar biogenesis and assembly, biofilm formation, and bacterial mobility. In contrast, RpoN was shown to negatively control a number of secretion system operons including one type VI secretion system operon (H1-T6SS), two pilus biogenesis operons (Flp/Tad-T4b pili and Csu-T1 pili), and one polysaccharide biosynthetic operon (psl). In addition, two operons that are involved in mannitol and inositol utilization are under the positive regulation of RpoN. Consistent with this result, the ability of H78 to utilize mannitol or inositol as a sole carbon source is positively influenced by RpoN. Taken together, the RpoN-mediated global regulation is mainly involved in flagellar biogenesis and assembly, bacterial mobility, biofilm formation, antibiotic biosynthesis, secretion systems, and carbon utilization in P. protegens H78.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen of humans, particularly those with cystic fibrosis. As a global regulator, RpoN controls a group of virulence-related factors and quorum-sensing (QS) genes in P. aeruginosa To gain further insights into the direct targets of RpoN in vivo , the present study focused on identifying the direct targets of RpoN regulation in QS and the type VI secretion system (T6SS). We performed chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) that identified 1,068 binding sites of RpoN, mostly including metabolic genes, a group of genes in QS ( lasI , rhlI , and pqsR ) and the T6SS ( hcpA and hcpB ). The direct targets of RpoN have been verified by electrophoretic mobility shifts assays (EMSA), lux reporter assay, reverse transcription-quantitative PCR, and phenotypic detection. The ΔrpoN ::Tc mutant resulted in the reduced production of pyocyanin, motility, and proteolytic activity. However, the production of rhamnolipids and biofilm formation were higher in the ΔrpoN ::Tc mutant than in the wild type. In summary, the results indicated that RpoN had direct and profound effects on QS and the T6SS. IMPORTANCE As a global regulator, RpoN controls a wide range of biological pathways, including virulence in P. aeruginosa PAO1. This work shows that RpoN plays critical and global roles in the regulation of bacterial pathogenicity and fitness. ChIP-seq provided a useful database to characterize additional functions and targets of RpoN in the future. The functional characterization of RpoN-mediated regulation will improve the current understanding of the regulatory network of quorum sensing and virulence in P. aeruginosa and other bacteria., (Copyright © 2018 American Society for Microbiology.)

Lysobacter is a Gram-negative genus comprising a group of environmental bacteria with abilities to produce abundant novel antibiotics, as well as adopting a unique type IV pilus (T4P)-mediated twitching motility (TM) that remains poorly understood. Here, we employ L. enzymogenes OH11 exhibiting significant antifungal activity as a working model to address this issue. Via mutating the 28 potential sigma factors in strain OH11, we have identified one protein RpoN OH11 (sigma 54) that is indispensable for T4P formation and TM. We further showed that RpoN OH11 not only regulates the transcription of pilA, but also another crucial gene chpA that encodes a hybrid two-component transduction system. The L. enzymogenes RpoN OH11 was found to directly bind to the promoter of chpA to control its transcription, which is found to be essential for the T4P-mediated TM. To our knowledge, such a transcriptional regulation performed by RpoN in control of bacterial TM has never been reported. Finally, we showed that L. enzymogenes OH11 could also produce biofilm that is likely employed by this strain to infect fungal pathogens. Mutation of rpoN OH11 , pilA and chpA all led to a significant decrease in biofilm formation, suggesting that the dual transcriptional regulation of pilA and chpA by RpoN OH11 plays a key role for RpoN OH11 to modulate the biofilm formation in L. enzymogenes. Overall, this study identified chpA as a new target of RpoN for controlling the T4P-mediated twitching motility and biofilm formation in L. enzymogenes OH11.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that infects immunocompromised and cystic fibrosis patients. Treatment is difficult due to antibiotic resistance, and new antimicrobials are needed to treat infections. The alternative sigma factor 54 (σ 54 , RpoN), regulates many virulence-associated genes. Thus, we evaluated inhibition of virulence in P. aeruginosa by a designed peptide (RpoN molecular roadblock, RpoN*) which binds specifically to RpoN consensus promoters. We expected that RpoN* binding to its consensus promoter sites would repress gene expression and thus virulence by blocking RpoN and/or other transcription factors. RpoN* reduced transcription of approximately 700 genes as determined by microarray analysis, including genes related to virulence. RpoN* expression significantly reduced motility, protease secretion, pyocyanin and pyoverdine production, rhamnolipid production, and biofilm formation. Given the effectiveness of RpoN* in vitro, we explored its effects in a Caenorhabditis elegans-P. aeruginosa infection model. Expression of RpoN* protected C. elegans in a paralytic killing assay, whereas worms succumbed to paralysis and death in its absence. In a slow killing assay, which mimics establishment and proliferation of an infection, C. elegans survival was prolonged when RpoN* was expressed. Thus, blocking RpoN consensus promoter sites is an effective strategy for abrogation of P. aeruginosa virulence.

Background: Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important crop diseases in the world. More insights into the mechanistic regulation of bacterial pathogenesis will help us identify novel molecular targets for developing effective disease control strategies. A large flagellar gene cluster is regulated under a three-tiered hierarchy by σ 54 factor RpoN2 and its activator FleQ, and σ 28 factor FliA. A hypothetical protein gene fliTX is located upstream of rpoN2, however, how it is regulated and how it is related to bacterial behaviors remain to be elucidated., Results: Sequence alignment analysis indicated that FliTX in Xoo is less well conserved compared with FliT proteins in Escherichia coli, Salmonella typhimurium, and Pseudomonas fluorescens. Co-transcription of fliTX with a cytosolic chaperone gene fliS and an atypical PilZ-domain gene flgZ in an operon was up-regulated by RpoN2/FleQ and FliA. Significantly shorter filament length and impaired swimming motility were observed in ∆fliTX compared with those in the wildtype strain. ∆fliTX also demonstrated reduced disease lesion length and in planta growth in rice, attenuated ability of induction of hypersensitive response (HR) in nonhost tobacco, and down-regulation of type III secretion system (T3SS)-related genes. In trans expression of fliTX gene in ∆fliTX restored these phenotypes to near wild-type levels., Conclusions: This study demonstrates that RpoN2- and FliA-regulated fliTX is indispensible for flagellar motility and virulence and provides more insights into mechanistic regulation of T3SS expression in Xoo.

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ 54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ 54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ 54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Dimethylarginine dimethylaminohydrolases (DDAHs) catalyze the hydrolysis of methylarginines to yield l-citrulline and methylamines as products. DDAHs and their central roles in methylarginine metabolism have been characterized for eukaryotic cells. While DDAHs are known to exist in some bacteria, including Streptomyces coelicolor and Pseudomonas aeruginosa , the physiological importance and genetic regulation of bacterial DDAHs remain poorly understood. To provide some insight into bacterial methylarginine metabolism, this study focused on identifying the key elements or factors regulating DDAH expression in P. aeruginosa PAO1. First, results revealed that P. aeruginosa can utilize N G , N G -dimethyl-l-arginine (ADMA) as a sole source of nitrogen but not carbon. Second, expression of the ddaH gene was observed to be induced in the presence of methylarginines, including N G -monomethyl-l-arginine (l-NMMA) and ADMA. Third, induction of the ddaH gene was shown to be achieved through a mechanism consisting of the putative enhancer-binding protein PA1196 and the alternative sigma factor RpoN. Both PA1196 and RpoN were essential for the expression of the ddaH gene in response to methylarginines. On the basis of the results of this study, PA1196 was given the name DdaR, for d imethylarginine d imethyl a minohydrolase r egulator. Interestingly, DdaR and its target ddaH gene are conserved only among P. aeruginosa strains, suggesting that this particular Pseudomonas species has evolved to utilize methylarginines from its environment. IMPORTANCE Methylated arginine residues are common constituents of eukaryotic proteins. During proteolysis, methylarginines are released in their free forms and become accessible nutrients for bacteria to utilize as growth substrates. In order to have a clearer and better understanding of this process, we explored methylarginine utilization in the metabolically versatile bacterium Pseudomonas aeruginosa PAO1. Our results show that the transcriptional regulator DdaR (PA1196) and the sigma factor RpoN positively regulate expression of dimethylarginine dimethylaminohydrolases (DDAHs) in response to exogenous methylarginines. DDAH is the central enzyme of methylarginine degradation, and its transcriptional regulation by DdaR-RpoN is expected to be conserved among P. aeruginosa strains., (Copyright © 2017 American Society for Microbiology.)

The sulfur oxidization (Sox) system is the central sulfur oxidization pathway of phototrophic and chemotrophic sulfur-oxidizing bacteria. Regulation and function of the Sox system in the chemotrophic Paracoccus pantotrophus has been elucidated; however, to date, no information is available on the regulation of this system in the chemolithotrophic Acidithiobacillus caldus, which is widely utilized in bioleaching. We described the novel tspSR-sox-like clusters in A. caldus and other chemolithotrophic sulfur-oxidizing bacteria containing Sox systems. The highly homologous σ 54 -dependent two-component signaling system (TspS/R), upstream of the sox operons in these novel clusters, was identified by phylogenetic analyses. A typical σ 54 -dependent promoter, P 1 , was identified upstream of soxX-I in the sox-I cluster of A. caldus MTH-04. The transcriptional start site (G) and the -12/-24 regions (GC/GG) of P 1 were determined by rapid amplification of cDNA ends (5'RACE), and the upstream activator sequences (UASs; TGTCCCAAATGGGACA) were confirmed by electrophoretic mobility shift assays (EMSAs) in vitro and by UAS-probe-plasmids assays in vivo. Sequence analysis of promoter regions in tspSR-sox-like clusters revealed that there were similar σ 54 -dependent promoters upstream of the soxX genes. Based on our results, we proposed a TspSR-mediated signal transduction and transcriptional regulation pathway for the Sox system in A. caldus. The regulation of σ 54 -dependent two-component systems (TCSs) for Sox pathways were explained for the first time in A. caldus, A. thiooxidans, T. tepidarius, and T. denitrificans, indicating the significance of modulating the sulfur oxidization in these chemolithotrophic sulfur oxidizers.

Rrp2 encodes a putative bacterial enhancer binding protein (bEBP) in Borrelia burgdorferi. Point mutation (G239C) of Rrp2 abolishes the transcriptional activation of σ 54 -dependent rpoS. In contrast to canonical bEBPs that are dispensable for bacterial growth, Rrp2 is essential for borrelial growth in BSK medium. It has been believed that Rrp2's ATPase activity is not required for cell growth, but experimental evidence supporting this notion has been lacking. In particular, it has remained unclear whether the residue G239 is involved in Rrp2's presumptive ATPase activity. To address these information gaps, we examined the roles of Rrp2's potential strategic signatures including the G239 residue and the putative Walker A and Walker B motifs. Herein it was showed that Rrp2 has ATP binding and hydrolysis activities engendered by the Walker A and B motifs respectively. However, these activities were not significantly impaired by a G239C mutation. Further mutagenesis analyses indicated that Rrp2's Walker A and B motifs are required for borrelial growth; mutations of key residues in these two motifs were lethal to B. burgdorferi. The combined data suggest that the Walker A and Walker B motifs of Rrp2 are involved in the control of another unknown RpoS-independent gene product(s) associated with borrelial replication., (© 2016 John Wiley & Sons Ltd.)

Bacterial sigma factors are subunits of RNA polymerase that direct the holoenzyme to specific sets of promoters in the genome and are a central element of regulating transcription. Most polymerase holoenzymes open the promoter and initiate transcription rapidly after binding. However, polymerase containing the members of the σ 54 family must be acted on by a transcriptional activator before DNA opening and initiation occur. A key domain in these transcriptional activators forms a hexameric AAA+ ATPase that acts through conformational changes brought on by ATP hydrolysis. Contacts between the transcriptional activator and σ 54 are primarily made through an N-terminal σ 54 activator interacting domain (AID). To better understand this mechanism of bacterial transcription initiation, we characterized the σ 54 AID by NMR spectroscopy and other biophysical methods and show that it is an intrinsically disordered domain in σ 54 alone. We identified a minimal construct of the Aquifex aeolicus σ 54 AID that consists of two predicted helices and retains native-like binding affinity for the transcriptional activator NtrC1. Using the NtrC1 ATPase domain, bound with the non-hydrolyzable ATP analog ADP-beryllium fluoride, we studied the NtrC1-σ 54 AID complex using NMR spectroscopy. We show that the σ 54 AID becomes structured after associating with the core loops of the transcriptional activators in their ATP state and that the primary site of the interaction is the first predicted helix. Understanding this complex, formed as the first step toward initiation, will help unravel the mechanism of σ 54 bacterial transcription initiation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Transcription initiation is highly regulated in bacterial cells, allowing adaptive gene regulation in response to environment cues. One class of promoter specificity factor called sigma54 enables such adaptive gene expression through its ability to lock the RNA polymerase down into a state unable to melt out promoter DNA for transcription initiation. Promoter DNA opening then occurs through the action of specialized transcription control proteins called bacterial enhancer-binding proteins (bEBPs) that remodel the sigma54 factor within the closed promoter complexes. The remodelling of sigma54 occurs through an ATP-binding and hydrolysis reaction carried out by the bEBPs. The regulation of bEBP self-assembly into typically homomeric hexamers allows regulated gene expression since the self-assembly is required for bEBP ATPase activity and its direct engagement with the sigma54 factor during the remodelling reaction. Crystallographic studies have now established that in the closed promoter complex, the sigma54 factor occupies the bacterial RNA polymerase in ways that will physically impede promoter DNA opening and the loading of melted out promoter DNA into the DNA-binding clefts of the RNA polymerase. Large-scale structural re-organizations of sigma54 require contact of the bEBP with an amino-terminal glutamine and leucine-rich sequence of sigma54, and lead to domain movements within the core RNA polymerase necessary for making open promoter complexes and synthesizing the nascent RNA transcript., (© 2016 The Author(s).)

The XylR/Pu regulatory node of the m-xylene biodegradation pathway of Pseudomonas putida mt-2 is one of the most intricate cases of processing internal and external cues into a single controlling element. Despite this complexity, the performance of the regulatory system is determined in vivo only by the occupation of Pu by m-xylene-activated XylR and σ(54)-RNAP. The stoichiometry between these three elements defines natural system boundaries that outline a specific functional space. This space can be expanded artificially following different strategies that involve either the increase of XylR or σ(54) or both elements at the same time (each using a different inducer). In this work we have designed a new regulatory architecture that drives the system to reach a maximum performance in response to one single input. To this end, we first explored using a simple mathematical model whether the output of the XylR/Pu node could be amended by simultaneously increasing σ(54) and XylR in response to only natural inducers. The exacerbation of Pu activity in vivo was tested in strains bearing synthetic transposons encoding xylR and rpoN (the σ(54) coding gene) controlled also by Pu, thereby generating a P. putida strain with the XylR/Pu output controlled by two intertwined feed forward loops (FFLs). The lack of a negative feedback loop in the expression node enables Pu activity to reach its physiological maximum in response to a single input. Only competition for cell resources might ultimately check the upper activity limit of such a rewired m-xylene sensing device.

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domain (PRD)-containing enhancer binding proteins (EBPs) are an important class of σ(54) -interacting transcriptional activators. Although PRD-containing EBPs are present in many Firmicutes, most of their regulatory functions remain unclear. In this study, the transcriptional regulons of about 50 PRD-containing EBPs in diverse Firmicutes species are reconstructed by using a comparative genomic approach, which contain the genes associated with utilization of β-glucosides, fructose/levan, mannose/glucose, pentitols, and glucosamine/fructosamine. We then present experimental evidence that the cel operon involved in cellobiose utilization is directly regulated by CelR and σ(54) (SigL) in Clostridium acetobutylicum. The predicted three CelR-binding sites and σ(54) promoter elements upstream of the cel operon are verified by in vitro binding assays. We show that CelR has an ATPase activity, which is strongly stimulated by the presence of DNA containing the CelR-binding sites. Moreover, mutations in any one of the three CelR-binding sites significantly decreased the cel promoter activity probably due to the need for all three DNA sites for maximal ATPase activity of CelR. It is suggested that CelR is regulated by PTS-mediated phosphorylation at His-551 and His-829, which exerts a positive effect and an inhibitory effect, respectively, on the CelR activity., (© 2015 John Wiley & Sons Ltd.)

Transcription regulation in a temporal and conditional manner underpins the lifecycle of enterobacterial pathogens. Upon exposure to a wide array of environmental cues, these pathogens modulate their gene expression via the RNA polymerase and associated sigma factors. Different sigma factors, either involved in general 'house-keeping' or specific responses, guide the RNA polymerase to their cognate promoter DNAs. The major alternative sigma54 factor when activated helps pathogens manage stresses and proliferate in their ecological niches. In this chapter, we review the function and regulation of the sigma54-dependent Phage shock protein (Psp) system-a major stress response when Gram-negative pathogens encounter damages to their inner membranes. We discuss the recent development on mechanisms of gene regulation, signal transduction and stress mitigation in light of different biophysical and biochemical approaches.

The alternative sigma factor RpoN is a unique regulator found among bacteria. It controls numerous processes that range from basic metabolism to more complex functions such as motility and nitrogen fixation. Our current understanding of RpoN function is largely derived from studies on prototypical bacteria such as Escherichia coli. Bacillus subtilis and Pseudomonas putida. Although the extent and necessity of RpoN-dependent functions differ radically between these model organisms, each bacterium depends on a single chromosomal rpoN gene to meet the cellular demands of RpoN regulation. The bacterium Ralstonia solanacearum is often recognized for being the causative agent of wilt disease in crops, including banana, peanut and potato. However, this plant pathogen is also one of the few bacterial species whose genome possesses dual rpoN genes. To determine if the rpoN genes in this bacterium are genetically redundant and interchangeable, we constructed and characterized ΔrpoN1, ΔrpoN2 and ΔrpoN1 ΔrpoN2 mutants of R. solanacearum GMI1000. It was found that growth on a small range of metabolites, including dicarboxylates, ethanol, nitrate, ornithine, proline and xanthine, were dependent on only the rpoN1 gene. Furthermore, the rpoN1 gene was required for wilt disease on tomato whereas rpoN2 had no observable role in virulence or metabolism in R. solanacearum GMI1000. Interestingly, plasmid-based expression of rpoN2 did not fully rescue the metabolic deficiencies of the ΔrpoN1 mutants; full recovery was specific to rpoN1. In comparison, only rpoN2 was able to genetically complement a ΔrpoN E. coli mutant. These results demonstrate that the RpoN1 and RpoN2 proteins are not functionally equivalent or interchangeable in R. solanacearum GMI1000.

In bacterial RNA polymerase (RNAP), the bridge helix and switch regions form an intricate network with the catalytic active centre and the main channel. These interactions are important for catalysis, hydrolysis and clamp domain movement. By targeting conserved residues in Escherichia coli RNAP, we are able to show that functions of these regions are differentially required during σ(70)-dependent and the contrasting σ(54)-dependent transcription activations and thus potentially underlie the key mechanistic differences between the two transcription paradigms. We further demonstrate that the transcription factor DksA directly regulates σ(54)-dependent activation both positively and negatively. This finding is consistent with the observed impacts of DksA on σ(70)-dependent promoters. DksA does not seem to significantly affect RNAP binding to a pre-melted promoter DNA but affects extensively activity at the stage of initial RNA synthesis on σ(54)-regulated promoters. Strikingly, removal of the σ(54) Region I is sufficient to invert the action of DksA (from stimulation to inhibition or vice versa) at two test promoters. The RNAP mutants we generated also show a strong propensity to backtrack. These mutants increase the rate of transcript-hydrolysis cleavage to a level comparable to that seen in the Thermus aquaticus RNAP even in the absence of a non-complementary nucleotide. These novel phenotypes imply an important function of the bridge helix and switch regions as an anti-backtracking ratchet and an RNA hydrolysis regulator., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bacterial RNA polymerases must associate with a σ factor to bind promoter DNA and initiate transcription. There are two families of σ factor: the σ70 family and the σ54 family. Members of the σ54 family are distinct in their ability to bind promoter DNA sequences, in the context of RNA polymerase holoenzyme, in a transcriptionally inactive state. Here, we map the genome-wide association of Escherichia coli σ54, the archetypal member of the σ54 family. Thus, we vastly expand the list of known σ54 binding sites to 135. Moreover, we estimate that there are more than 250 σ54 sites in total. Strikingly, the majority of σ54 binding sites are located inside genes. The location and orientation of intragenic σ54 binding sites is non-random, and many intragenic σ54 binding sites are conserved. We conclude that many intragenic σ54 binding sites are likely to be functional. Consistent with this assertion, we identify three conserved, intragenic σ54 promoters that drive transcription of mRNAs with unusually long 5' UTRs.

Enhancer-dependent transcription involving the promoter specificity factor σ(54) is widely distributed amongst bacteria and commonly associated with cell envelope function. For transcription initiation, σ(54)-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ(54) promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ(54) were evident, indicating loss of rpoN (encoding σ(54)) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ(54)-dependence in vivo not readily correlated with conventional σ(54) binding-sites., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Unlabelled: Salmonella enteric serovar Typhimurium, a major cause of food-borne illness, is capable of using a variety of carbon and nitrogen sources. Fructoselysine and glucoselysine are Maillard reaction products formed by the reaction of glucose or fructose, respectively, with the ε-amine group of lysine. We report here that S. Typhimurium utilizes fructoselysine and glucoselysine as carbon and nitrogen sources via a mannose family phosphotransferase (PTS) encoded by gfrABCD (glucoselysine/fructoselysine PTS components EIIA, EIIB, EIIC, and EIID; locus numbers STM14_5449 to STM14_5454 in S. Typhimurium 14028s). Genes coding for two predicted deglycases within the gfr operon, gfrE and gfrF, were required for growth with glucoselysine and fructoselysine, respectively. GfrF demonstrated fructoselysine-6-phosphate deglycase activity in a coupled enzyme assay. The biochemical and genetic analyses were consistent with a pathway in which fructoselysine and glucoselysine are phosphorylated at the C-6 position of the sugar by the GfrABCD PTS as they are transported across the membrane. The resulting fructoselysine-6-phosphate and glucoselysine-6-phosphate subsequently are cleaved by GfrF and GfrE to form lysine and glucose-6-phosphate or fructose-6-phosphate. Interestingly, although S. Typhimurium can use lysine derived from fructoselysine or glucoselysine as a sole nitrogen source, it cannot use exogenous lysine as a nitrogen source to support growth. Expression of gfrABCDEF was dependent on the alternative sigma factor RpoN (σ(54)) and an RpoN-dependent LevR-like activator, which we designated GfrR., Importance: Salmonella physiology has been studied intensively, but there is much we do not know regarding the repertoire of nutrients these bacteria are able to use for growth. This study shows that a previously uncharacterized PTS and associated enzymes function together to transport and catabolize fructoselysine and glucoselysine. Knowledge of the range of nutrients that Salmonella utilizes is important, as it could lead to the development of new strategies for reducing the load of Salmonella in food animals, thereby mitigating its entry into the human food supply., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ(54)) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate-dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ(54) holoenzyme at 3.8 angstroms reveals molecular details of σ(54) and its interactions with RNAP. The structure explains how σ(54) targets different regions in RNAP to exert its inhibitory function. Although σ(54) and the major σ factor, σ(70), have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription., (Copyright © 2015, American Association for the Advancement of Science.)

Sigma 54 is a transcriptional regulator predicted to play a role in physical interaction of bacteria with their environment, including virulence and biofilm formation. In order to study the role of Sigma 54 in Bacillus cereus, a comparative transcriptome and phenotypic study was performed using B. cereus ATCC 14579 WT, a markerless rpoN deletion mutant, and its complemented strain. The mutant was impaired in many different cellular functions including low temperature and anaerobic growth, carbohydrate metabolism, sporulation and toxin production. Additionally, the mutant showed lack of motility and biofilm formation at air-liquid interphase, and this correlated with absence of flagella, as flagella staining showed only WT and complemented strain to be highly flagellated. Comparative transcriptome analysis of cells harvested at selected time points during growth in aerated and static conditions in BHI revealed large differences in gene expression associated with loss of phenotypes, including significant down regulation of genes in the mutant encoding enzymes involved in degradation of branched chain amino acids, carbohydrate transport and metabolism, flagella synthesis and virulence factors. Our study provides evidence for a pleiotropic role of Sigma 54 in B. cereus supporting its adaptive response and survival in a range of conditions and environments.

Members of the genus Burkholderia are versatile bacteria capable of colonizing highly diverse environmental niches. In this study, we investigated the global response of the opportunistic pathogen Burkholderia cenocepacia H111 to nitrogen limitation at the transcript and protein expression levels. In addition to a classical response to nitrogen starvation, including the activation of glutamine synthetase, PII proteins, and the two-component regulatory system NtrBC, B. cenocepacia H111 also upregulated polyhydroxybutyrate (PHB) accumulation and exopolysaccharide (EPS) production in response to nitrogen shortage. A search for consensus sequences in promoter regions of nitrogen-responsive genes identified a σ(54) consensus sequence. The mapping of the σ(54) regulon as well as the characterization of a σ(54) mutant suggests an important role of σ(54) not only in control of nitrogen metabolism but also in the virulence of this organism., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Flagellar biogenesis in Helicobacter pylori is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ(80)), RpoN (σ(54)), and FliA (σ(28)), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaB and flgE) and FliA-dependent (flaA) genes. The MS ring (encoded by fliF) is one of the earliest flagellar structures assembled. Deletion of fliF resulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease in flaA transcript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions of fliH and genes encoding C ring components (fliM and fliY) decreased transcript levels of flaB and flgE but had little or no effect on transcript levels of flaA. Transcript levels of flaB and flgE were elevated in mutants where genes encoding rod proteins (fliE and flgBC) were deleted, while transcript levels of flaA was reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure., Importance: The mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of the H. pylori flagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of the H. pylori RpoN regulon, a hypothesis that can be tested by examining protein-protein interactions in vitro., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

A new approach at the transcriptional level was applied to lactate-based polyester production. Four σ factor disruptants, ΔrpoN, ΔrpoS, ΔfliA and ΔfecI, of Escherichia coli were used as hosts for poly(lactate-co-3-hydroxybutyrate) production from glucose. Among them, ΔrpoN caused dual positive effects of polymer production, enhanced cellular content and lactate fraction., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

σ(54)-dependent transcription controls a wide range of stress-related genes in bacteria and is tightly regulated. In contrast to σ(70), the σ(54)-RNA polymerase holoenzyme forms a stable closed complex at the promoter site that rarely isomerises into transcriptionally competent open complexes. The conversion into open complexes requires the ATPase activity of activator proteins that bind remotely upstream of the transcriptional start site. These activators belong to the large AAA protein family and the majority of them consist of an N-terminal regulatory domain, a central AAA domain and a C-terminal DNA binding domain. Here we use a functional variant of the NorR activator, a dedicated NO sensor, to provide the first structural and functional characterisation of a full length AAA activator in complex with its enhancer DNA. Our data suggest an inter-dependent and synergistic relationship of all three functional domains and provide an explanation for the dependence of NorR on enhancer DNA. Our results show that NorR readily assembles into higher order oligomers upon enhancer binding, independent of activating signals. Upon inducing signals, the N-terminal regulatory domain relocates to the periphery of the AAA ring. Together our data provide an assembly and activation mechanism for NorR., (© 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Vibrio parahaemolyticus is the leading cause of bacterial seafood-borne gastroenteritis worldwide, yet little is known about how this pathogen colonizes the human intestine. The alternative sigma factor RpoN/sigma-54 is a global regulator that controls flagellar synthesis, as well as a wide range of nonflagellar genes. We constructed an in-frame deletion mutation in rpoN (VP2670) in V. parahaemolyticus RIMD2210633, a clinical serogroup O3:K6 isolate, and examined the effects in vivo using a streptomycin-treated mouse model of colonization. We confirmed that deletion of rpoN rendered V. parahaemolyticus nonmotile, and it caused reduced biofilm formation and an apparent defect in glutamine synthetase production. In in vivo competition assays between the rpoN mutant and a wild-type RIMD2210633 strain marked with the β-galactosidase gene lacZ (WBWlacZ), the mutant colonized significantly more proficiently. Intestinal persistence competition assays also demonstrated that the rpoN mutant had enhanced fitness and outcompeted WBWlacZ. Mutants defective in the polar flagellum biosynthesis FliAP sigma factor also outcompeted WBWlacZ but not to the same level as the rpoN mutant, which suggested that lack of motility is not the sole cause of the fitness effect. In an in vitro growth competition assay in mouse intestinal mucus, the rpoN mutant also outcompeted the wild type and exhibited faster doubling times when grown in mucus and on individual components of mucus. Genes in the pathways for the catabolism of mucus sugars also had significantly higher expression levels in a ΔrpoN mutant than in the wild type. These data suggest that in V. parahaemolyticus, RpoN plays an important role in carbon utilization regulation, which may significantly affect host colonization.

RpoS (σ(38)) is required for cell survival under stress conditions, but it can inhibit growth if produced inappropriately and, consequently, its production and activity are elaborately regulated. Crl, a transcriptional activator that does not bind DNA, enhances RpoS activity by stimulating the interaction between RpoS and the core polymerase. The crl gene has two overlapping promoters, a housekeeping, RpoD- (σ(70)) dependent promoter, and an RpoN (σ(54)) promoter that is strongly up-regulated under nitrogen limitation. However, transcription from the RpoN promoter prevents transcription from the RpoD promoter, and the RpoN-dependent transcript lacks a ribosome-binding site. Thus, activation of the RpoN promoter produces a long noncoding RNA that silences crl gene expression simply by being made. This elegant and economical mechanism, which allows a near-instantaneous reduction in Crl synthesis without the need for transacting regulatory factors, restrains the activity of RpoS to allow faster growth under nitrogen-limiting conditions., Competing Interests: The authors declare no conflict of interest.

The expression of σ(54)-dependent Pseudomonas putida Pu promoter is activated by XylR activator when cells are exposed to a variety of aromatic inducers. In this study, the transcriptional activation of the P. putida Pu promoter was recreated in the heterologous host Escherichia coli. Here we show that the cAMP receptor protein (CRP), a well-known carbon utilization regulator, had an inhibitory effect on the expression of Pu promoter in a cAMP-dependent manner. The inhibitory effect was not activator specific. In vivo KMnO4 and DMS footprinting analysis indicated that CRP-cAMP poised the RNA polymerase at Pu promoter, inhibiting the isomerization step of the transcription initiation even in the presence of an activator. Therefore, the presence of PTS-sugar, which eliminates cAMP, could activate the poised RNA polymerase at Pu promoter to transcribe. Moreover, the activation region 1 (AR1) of CRP, which interacts directly with the αCTD (C-terminal domain of α-subunit) of RNA polymerase, was found essential for the CRP-mediated inhibition at Pu promoter. A model for the above observations is discussed.

One way that bacteria regulate the transcription of specific genes to adapt to environmental challenges is to use different σ factors that direct the RNA polymerase holoenzyme to distinct promoters. Unlike σ(70) RNA polymerase (RNAP), σ(54) RNAP is unable to initiate transcription without an activator: enhancer-binding protein (EBP). All EBPs contain one ATPase domain that belongs to the family of ATPases associated with various cellular activities (AAA+ ATPases). AAA+ ATPases use the energy of ATP hydrolysis to remodel different target macromolecules to perform distinct functions. These mechanochemical enzymes are known to form ring-shaped oligomers whose conformations strongly depend upon nucleotide status. Here, the crystallization of the AAA+ ATPase domain of an EBP from Aquifex aeolicus, NtrC1, in the presence of the non-hydrolyzable ATP analog ADP-BeFx is reported. X-ray diffraction data were collected from two crystals from two different protein fractions of the NtrC1 ATPase domain. Previously, this domain was co-crystallized with ADP and ATP, but the latter crystals were grown from the Walker B substitution variant E239A. Therefore, the new data sets are the first for a wild-type EBP ATPase domain co-crystallized with an ATP analog and they reveal a new crystal form. The resulting structure(s) will shed light on the mechanism of EBP-type transcription activators.