RpoN (sigma factor 54) contributes to bacterial fitness during tracheal colonization of Bordetella bronchiseptica.

The Gram-negative pathogenic bacterium Bordetella bronchiseptica is a respiratory pathogen closely related to Bordetella pertussis, the causative agent of whooping cough. Despite sharing homologous virulence factors, B. bronchiseptica infects a broad range of mammalian hosts, including some experimental animals, whereas B. pertussis is strictly adapted to humans. Therefore, B. bronchiseptica is often used as a representative model to explore the pathogenicity of Bordetella in infection experiments with laboratory animals. Although Bordetella virulence factors, including toxins and adhesins have been studied well, our recent study implied that unknown virulence factors are involved in tracheal colonization and infection. Here, we investigated bacterial genes contributing to tracheal colonization by high-throughput transposon sequencing (Tn-seq). After the screening, we picked up 151 candidate genes of various functions and found that a rpoN-deficient mutant strain was defective in tracheal colonization when co-inoculated with the wild-type strain. rpoN encodes σ54 , a sigma factor that regulates the transcription of various genes, implying its contribution to various bacterial activities. In fact, we found RpoN of B. bronchiseptica is involved in bacterial motility and initial biofilm formation. From these results, we propose that RpoN supports bacterial colonization by regulating various bacteriological functions.

iProm-Sigma54: A CNN Base Prediction Tool for σ54 Promoters.

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.

A general mechanism for transcription bubble nucleation in bacteria.

Bacterial transcription initiation requires σ factors for nucleation of the transcription bubble. The canonical housekeeping σ factor, σ70, nucleates DNA melting via recognition of conserved bases of the promoter -10 motif, which are unstacked and captured in pockets of σ70. By contrast, the mechanism of transcription bubble nucleation and formation during the unrelated σN-mediated transcription initiation is poorly understood. Herein, we combine structural and biochemical approaches to establish that σN, like σ70, captures a flipped, unstacked base in a pocket formed between its N-terminal region I (RI) and extra-long helix features. Strikingly, RI inserts into the nascent bubble to stabilize the nucleated bubble prior to engagement of the obligate ATPase activator. Our data suggest a general paradigm of transcription initiation that requires σ factors to nucleate an early melted intermediate prior to productive RNA synthesis.

RpoN is required for the motility and contributes to the killing ability of Plesiomonas shigelloides.

BACKGROUND: RpoN, also known as σ54, 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.

Mechanisms of DNA opening revealed in AAA+ transcription complex structures.

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 (σ54), 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 σ54 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 σ54 inhibition while helping to open up DNA, using σ54 amino-terminal peptide as a pry bar.

RpoN/Sfa2-dependent activation of the Pseudomonas aeruginosa H2-T6SS and its cognate arsenal of antibacterial toxins.

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.

Regulatory Small RNA Qrr2 Is Expressed Independently of Sigma Factor-54 and Can Function as the Sole Qrr Small RNA To Control Quorum Sensing in Vibrio parahaemolyticus.

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 ΔluxO deletion mutant, opaR was derepressed and CPS and biofilm were produced. However, in a ΔrpoN 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 ΔluxO showed that all five qrr genes were repressed, while in ΔrpoN 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 ΔrpoN 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.

The Regulatory Functions of σ54 Factor in Phytopathogenic Bacteria.

σ54 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. σ54 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, GGN10GC, 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.

Whole set of constitutive promoters for RpoN sigma factor and the regulatory role of its enhancer protein NtrC in Escherichia coli K-12.

The promoter selectivity of Escherichia coli RNA polymerase (RNAP) is determined by its promoter-recognition sigma subunit. The model prokaryote E. coli K-12 contains seven species of the sigma subunit, each recognizing a specific set of promoters. Using genomic SELEX (gSELEX) screening in vitro, we identified the whole set of 'constitutive' promoters recognized by the reconstituted RNAP holoenzyme alone, containing RpoD (σ70), RpoS (σ38), RpoH (σ32), RpoF (σ28) or RpoE (σ24), in the absence of other supporting regulatory factors. In contrast, RpoN sigma (σ54), involved in expression of nitrogen-related genes and also other cellular functions, requires an enhancer (or activator) protein, such as NtrC, for transcription initiation. In this study, a series of gSELEX screenings were performed to search for promoters recognized by the RpoN RNAP holoenzyme in the presence and absence of the major nitrogen response enhancer NtrC, the best-characterized enhancer. Based on the RpoN holoenzyme-binding sites, a total of 44 to 61 putative promoters were identified, which were recognized by the RpoN holoenzyme alone. In the presence of the enhancer NtrC, the recognition target increased to 61-81 promoters. Consensus sequences of promoters recognized by RpoN holoenzyme in the absence and presence of NtrC were determined. The promoter activity of a set of NtrC-dependent and -independent RpoN promoters was verified in vivo under nitrogen starvation, in the presence and absence of RpoN and/or NtrC. The promoter activity of some RpoN-recognized promoters increased in the absence of RpoN or NtrC, supporting the concept that the promoter-bound NtrC-enhanced RpoN holoenzyme functions as a repressor against RpoD holoenzyme. Based on our findings, we propose a model in which the RpoN holoenzyme fulfils the dual role of repressor and transcriptase for the same set of genes. We also propose that the promoter recognized by RpoN holoenzyme in the absence of enhancers is the 'repressive' promoter. The presence of high-level RpoN sigma in growing E. coli K-12 in rich medium may be related to the repression role of a set of genes needed for the utilization of ammonia as a nitrogen source in poor media. The list of newly identified regulatory targets of RpoN provides insight into E. coli survival under nitrogen-depleted conditions in nature.

Roles of rpoN in biofilm formation of Vibrio alginolyticus HN08155 at different cell densities.

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.

Utilization of L-glutamate as a preferred or sole nutrient in Pseudomonas aeruginosa PAO1 depends on genes encoding for the enhancer-binding protein AauR, the sigma factor RpoN and the transporter complex AatJQMP.

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.

The σ54 system directly regulates bacterial natural product genes.

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 σ54 system, which includes σ54-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 σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-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 σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.

Sigma 54-Regulated Transcription Is Associated with Membrane Reorganization and Type III Secretion Effectors during Conversion to Infectious Forms of Chlamydia trachomatis.

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 (σ54) 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 σ54 transcriptional activation. CtcC activation of σ54 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 σ54 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 σ54 regulon. Computational evaluation revealed that the majority of the differentially expressed genes were preceded by highly conserved σ54 promoter elements. Reporter gene analyses using these putative σ54 promoters reinforced the accuracy of the model of the proposed regulon. Investigation of the gene products included in this regulon supports the idea that σ54 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.

The route to transcription initiation determines the mode of transcriptional bursting in E. coli.

Transcription is fundamentally noisy, leading to significant heterogeneity across bacterial populations. Noise is often attributed to burstiness, but the underlying mechanisms and their dependence on the mode of promotor regulation remain unclear. Here, we measure E. coli single cell mRNA levels for two stress responses that depend on bacterial sigma factors with different mode of transcription initiation (σ70 and σ54). By fitting a stochastic model to the observed mRNA distributions, we show that the transition from low to high expression of the σ70-controlled stress response is regulated via the burst size, while that of the σ54-controlled stress response is regulated via the burst frequency. Therefore, transcription initiation involving σ54 differs from other bacterial systems, and yields bursting kinetics characteristic of eukaryotic systems.

Interactions between DksA and Stress-Responsive Alternative Sigma Factors Control Inorganic Polyphosphate Accumulation in Escherichia coli.

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. coliIMPORTANCE 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.

RpoN1 and RpoN2 play different regulatory roles in virulence traits, flagellar biosynthesis, and basal metabolism in Xanthomonas campestris.

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.

Bacterial Enhancer Binding Proteins-AAA+ Proteins in Transcription Activation.

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, σ54 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-σ54-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 σ54-dependent transcription initiation.

Engineered CRISPRa enables programmable eukaryote-like gene activation in bacteria.

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 σ70 promoters. Here we report a eukaryote-like bacterial CRISPRa system based on σ54-dependent promoters, which supports long distance, and hence multi-input regulation with high dynamic ranges. Our CRISPRa device can activate σ54-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.

Blocking RpoN reduces virulence of Pseudomonas aeruginosa isolated from cystic fibrosis patients and increases antibiotic sensitivity in a laboratory strain.

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 (σ54), 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.

Mechanisms of σ54-Dependent Transcription Initiation and Regulation.

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, σ54, has a specific role in stress responses. Unlike σ70-dependent transcription, which often can spontaneously proceed to initiation, σ54-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 σ54 system with both x-ray crystallography and cryo electron microscopy, furthering our understanding of σ54-dependent transcription initiation and DNA opening. Comparisons with σ70 and eukaryotic polymerases reveal unique and common features during transcription initiation.