Sigma Factor Research Articles

Identification of a comprehensive set of transcriptional regulators involved in the long-term survivability of Escherichia coli in soil.

Bacteria that typically do not thrive in soil can survive therein for long periods. While much research has been conducted on the external environmental factors affecting the long-term survival of bacteria in soil, their inherent factors are poorly understood. To adapt to environmental changes, bacteria alter their gene expression patterns using transcriptional regulators such as sigma factors. Using Escherichia coli as a model bacterium, we examined the effects of each transcriptional regulator on the long-term survivability of E. coli in soil. The survivability of 294 E. coli strains deficient in transcriptional regulators in soil was measured over 6weeks. The results showed that ten strains deficient in transcription factors significantly reduced survivability, whereas four deficient strains increased it. The functions common to several of these transcriptional regulators included carbon and nitrogen metabolism, stationary phase adaptation, and osmotic stress adaptation. These transcription factors are often global regulators and conserved among other pathogenic bacterial species. Taken together, we successfully identified a comprehensive set of transcription factors involved in the long-term survival of E. coli in soil. These findings will be useful for understanding the mechanisms underlying the adaptation of microorganisms to soil environments.

Roles of the rpoEc-chrR-chrA operon in superoxide tolerance and β-lactam susceptibility of Stenotrophomonas maltophilia

IntroductionThe rpoE-chrR pair is a regulatory system used by photosynthetic microorganisms to overcome singlet oxygen stress. rpoE and chrR encode the sigma factor σE and anti-sigma factor ChrR, respectively. Stenotrophomonas maltophilia, an opportunistic pathogen, is a multidrug-resistant gram-negative bacterium. Although it is not a photosynthetic microorganism, a rpoE-chrR homolog (smlt2377-smlt2378) was found in the S. maltophilia genome. In this study, we aimed to assess the significance of σEc-ChrR pair in oxidative stress alleviation and antibiotic susceptibility of S. maltophilia KJ.MethodsReverse transcription-polymerase chain reaction was used to validate the presence of operon. The contribution of rpoEc-chrR-chrA operon to oxidative stress alleviation and antibiotic susceptibility was evaluated using mutant constructs and stress-tolerance assays. RNA-seq transcriptome assay of wild-type KJ, KJΔChrR (chrR mutant), and KJΔChrRΔRpoEc (chrR/rpoEc double mutant) was performed to reveal the σEc regulon.ResultsThe rpoEc-chrR pair and downstream chrA formed an operon. Inactivation of chrR upregulated the expression of rpoEc-chrR-chrA operon in an σEc- and ChrA-dependent manner. σEc activation contributed to superoxide tolerance and increased β-lactam susceptibility but did not affect the tolerance to singlet oxygen and hydrogen peroxide. Transcriptome analysis revealed that expression of the nine-gene cluster, smlt2375-smlt2367, was significantly upregulated in KJΔChrR and reverted to the wild-type level in KJΔChrRΔRpoEc. smlt2375-smlt2367 cluster was located upstream of the rpoEc-chrR-chrA operon and divergently transcribed, seeming to be involved in membrane lipid modification. Deletion of smlt2375-smlt2367 cluster from the chromosome of KJΔChrR reverted the superoxide tolerance and β-lactam susceptibility to the wild-type level.DiscussionThe rpoEc-chrR pair of S. maltophilia was involved in superoxide tolerance and β-lactam susceptibility. Notably, a novel regulatory circuit involving rpoEc-chrR-chrA operon and smlt2375-smlt2367 cluster was revealed.

Regulatory role of PA3299.1 small RNA in Pseudomonas aeruginosa biofilm formation via modulation of algU and mucA expression.

Small RNAs (sRNAs) have emerged as key regulators of transcriptional factors and components within regulatory networks that govern bacterial biofilm formation. This study aimed to explore the regulatory role of the PA3299.1 sRNA in controlling biofilm formation in P. aeruginosa. Results showed that PA3299.1 expression was significantly elevated in both substratum-attached and colony biofilms compared to planktonic growth. Further investigation revealed that strains overexpressing PA3299.1 exhibited enhanced biofilm formation, while its deletion resulted in a substantial reduction in biofilm development. PA3299.1 was found to regulate the expression of AlgU and MucA, the sigma and anti-sigma factors, integral to the biofilm developmental network. In summary, this research identifies PA3299.1 as a critical regulator of biofilm formation and potentially a contributor to the pathogenicity of P. aeruginosa, that could help to develop new therapeutic strategies to manage biofilm-associated infections.

Optimizing petrophysical property prediction in fluvial-deltaic reservoirs: a multi-seismic attribute transformation and probabilistic neural network approach

Conventional techniques, which depend on geostatistical modeling, frequently fail to capture reservoir variability, especially when well data are sparse. To overcome this limitation, we develop a combined approach that integrates Multi-Seismic Attribute Transformation (MSAT) and Probabilistic Neural Network (PNN) techniques. By leveraging data from eight wells and post-stack seismic data from the Poseidon 3D area, gas-saturated deltaic-fluvial settings, we analyzed twelve seismic attributes, among which acoustic impedance low-frequency attribute, relative geological time, amplitude envelope, and amplitude-weighted frequency emerge as the most significant. Using the MSAT approach, we established strong connections between these attributes and porosity. Six attributes provide a correlation coefficient of 0.65 with the target log. To enhance precision, PNN and fine-tuning the sigma factor using well drop-out cross-validation analysis was utilized. This leads to a significant enhancement in model accuracy, reaching 76%. In addition, when the model training is concentrated within a 10-millisecond range around the Plover reservoir zone, the accuracy increases significantly to 89%. Our approach has proven to be highly effective, with a success rate of 73% as evidenced by validation through well drop-out analysis. This demonstrates that our method surpasses traditional methods. This innovative integration of seismic attribute-driven methodologies has indicated a major leap forward in identifying and characterizing reservoir heterogeneity. The results demonstrate that it enables more efficient future well-planning strategies and deepens our comprehension of deltaic-fluvial reservoir dynamics.

An extreme mutational hotspot in nlpD depends on transcriptional induction of rpoS.

Mutation rate varies within and between genomes. Within genomes, tracts of nucleotides, including short sequence repeats and palindromes, can cause localised elevation of mutation rate. Additional mechanisms remain poorly understood. Here we report an instance of extreme mutational bias in Pseudomonas fluorescens SBW25 associated with a single base-pair change in nlpD. These mutants frequently evolve in static microcosms, and have a cell-chaining (CC) phenotype. Analysis of 153 replicate populations revealed 137 independent instances of a C565T loss-of-function mutation at codon 189 (CAG to TAG (Q189*)). Fitness measures of alternative nlpD mutants did not explain the deterministic evolution of C565T mutants. Recognising that transcription can be mutagenic, and that codon 189 overlaps with a predicted promoter (rpoSp) for the adjacent stationary phase sigma factor, rpoS, transcription across this promoter region was measured. This confirmed rpoSp is induced in stationary phase and that C565T mutation caused significant elevation of transcription. The latter provided opportunity to determine the C565T mutation rate using a reporter-gene fused to rpoSp. Fluctuation assays estimate the C565T mutation rate to be ~5,000-fold higher than expected. In Pseudomonas, transcription of rpoS requires the positive activator PsrA, which we show also holds for SBW25. Fluctuation assays performed in a ∆psrA background showed a ~60-fold reduction in mutation rate confirming that the elevated rate of mutation at C565T mutation rate is dependent on induction of transcription. This hotspot suggests a generalisable phenomenon where the induction of transcription causes elevated mutation rates within defining regions of promoters.

Two new enzymes that liberate undecaprenyl-phosphate to replenish the carrier lipid pool during envelope stress.

The 55-carbon isoprenoid, undecaprenyl-phosphate (UndP), is a universal carrier lipid that ferries most glycans and glycopolymers across the cytoplasmic membrane in bacteria. In addition to peptidoglycan precursors, UndP transports O-antigen, capsule, wall teichoic acids, and sugar modifications. How this shared but limited lipid is distributed among competing pathways is just beginning to be elucidated. We recently reported that in the bacterium Bacillus subtilis, the stress-response sigma factor SigM and its cognate anti-sigma factor complex respond to changes in the free UndP pool. When levels are low, SigM activates genes that increase flux through the essential cell wall synthesis pathway, promote the recycling of the lipid carrier, and liberate the carrier from other polymer pathways. Here, we report that two additional enzymes under SigM control help maintain the free pool of UndP. One, UshA (YqjL), resembles alpha-beta hydrolases and liberates UndP from undecaprenyl-monophosphate-linked sugars. The other, UpsH (YpbG), resembles metallophosphoesterases and releases UndP from undecaprenyl-diphosphate-linked wall teichoic acids polymers but not lipid-linked peptidoglycan precursors. UshA becomes critical for growth when UndP-linked sugars are sequestered, and the carrier lipid pool is depleted. Similarly, UpsH becomes essential for viability when UndPP-linked intermediates accumulate. Mutations in the predicted catalytic residues of both putative hydrolases abrogate their function arguing that they act directly to release UndP. These findings define two new enzymes that liberate the carrier lipid from UndP- and UndPP-linked intermediates and bolster the model that the SigM stress-response pathway maintains the UndP pool and prioritizes its use for peptidoglycan synthesis.IMPORTANCEMotivated by the success of naturally occurring glycopeptide antibiotics like vancomycin, one arm of recent antibiotic discovery efforts has focused on compounds that bind lipid-linked precursors used to build extracytoplasmic polymers. Trapping these precursors depletes the universal carrier lipid undecaprenyl-phosphate, which is required for the synthesis of virtually all surface polymers, including peptidoglycan. Understanding how cells respond to this stress to restore the carrier lipid pool is critical to identifying effective drugs. Here, we report the identification of two new enzymes that are produced in response to the depletion of the carrier lipid pool. These enzymes recover the carrier lipid but cleave distinct lipid-linked precursors to do so.

Transcription Regulation of Flagellins: A Structural Perspective.

Bacterial flagella are complex molecular motors that are essential for locomotion and host colonization. They consist of 30 different proteins expressed in varying stoichiometries. Their assembly and function are governed by a hierarchical transcriptional regulatory network with multiple checkpoints primarily regulated by sigma factors. Expression of late flagellar genes requires the complete assembly of the flagellar basal body and hook. The extracellular segment of the flagellum, termed filament, is composed of self-assembling flagellin subunits encoded by the fliC gene and harbors potent antigenic epitopes. Structural studies have illuminated the molecular mechanisms underlying its assembly and its regulation at the transcription level. σ28, a key subunit of the RNA polymerase complex, binds to specific promoter sequences to initiate transcription of late flagellar genes, while its activity is controlled by the antisigma factor FlgM. This review summarizes current insights into the structural characterization of flagellins across various bacterial species, their transcription by σ28, and the structural mechanism controlling σ28 activity through FlgM. Additionally, we highlight the regulation of flagellin gene expression via transcription factors and their post-transcriptional regulation, providing a comprehensive overview of the intricate mechanisms that support bacterial motility and adaptation.

Positive feedback regulation between RpoS and BosR in the Lyme disease pathogen.

In Borrelia burgdorferi, the causative agent of Lyme disease, differential gene expression is primarily governed by the alternative sigma factor RpoS (σS). Understanding the regulation of RpoS is crucial for elucidating how B. burgdorferi is maintained throughout its enzootic cycle. Our recent studies have shown that the homolog of Fur/PerR repressor/activator BosR functions as an RNA-binding protein that controls the rpoS mRNA stability. However, the mechanisms regulating BosR, particularly in response to host signals and environmental cues, remain largely unclear. In this study, we uncovered a positive feedback loop between RpoS and BosR, wherein RpoS post-transcriptionally regulates BosR levels. Specifically, mutation or deletion of rpoS significantly reduced BosR levels, whereas artificial induction of rpoS resulted in a dose-dependent increase in BosR levels. Notably, RpoS does not affect bosR mRNA levels but instead modulates the turnover rate of the BosR protein. Moreover, we demonstrated that environmental cues do not directly influence bosR expression but instead induce rpoS transcription and RpoS production, thereby enhancing BosR protein levels. These findings reveal a new layer of complexity in the RpoN-RpoS regulatory pathway, challenging the existing paradigm and suggesting a need to re-evaluate the factors and signals previously implicated in regulating RpoS via BosR. This study provides new insights into the intricate regulatory networks underpinning B. burgdorferi's adaptation and survival in its enzootic cycle.IMPORTANCELyme disease is the most prevalent arthropod-borne infection in the United States. The etiological agent, Borreliella (or Borrelia) burgdorferi, is maintained in nature through an enzootic cycle involving a tick vector and a mammalian host. RpoS, the master regulator of differential gene expression, plays a crucial role in tick transmission and mammalian infection of B. burgdorferi. This study reveals a positive feedback loop between RpoS and a Fur/PerR homolog. Elucidating this regulatory network is essential for identifying potential therapeutic targets to disrupt B. burgdorferi's enzootic cycle. The findings also have broader implications for understanding the regulation of RpoS and Fur/PerR family in other bacteria.

LD-transpeptidase-mediated cell envelope remodeling enables developmental transitions and survival in Coxiella burnetii and Legionella pneumophila.

Coxiella burnetii and Legionella pneumophila are two phylogenetically related bacterial pathogens that exhibit extreme intrinsic resistance when they enter into a dormancy-like state. This enables both pathogens to survive extended periods in growth-limited environments. Survival is dependent upon their ability to undergo developmental transitions into two phenotypically distinct variants, one specialized for intracellular replication and another for prolonged survival in the environment and host. We currently lack an understanding of the mechanisms that mediate these developmental transitions. Here, we performed peptidoglycan (PG) glycoproteome analysis and showed significant enrichment of PG structures catalyzed by LD-transpeptidases (LDTs) in the survival variants of C. burnetii and L. pneumophila. This is supported by the upregulation of LDTs, resulting in susceptibility to carbapenem antibiotics. Furthermore, deletion of the most upregulated LDT, lpg1386, in L. pneumophila significantly changes PG architecture, survival, and susceptibility to antibiotics. Significantly regulated by RpoS, a stationary-phase sigma factor, LDT-dependent PG remodeling is differentially activated by the host intracellular growth environment compared to axenic culture. In addition, β-barrel tethering, a newly discovered mechanism of LDT-mediated cell envelope stabilization, seems not to be specific to the survival variants. Interestingly, an outer membrane (OM) long-chain fatty acid transporter (Lpg1810) is tethered to PG in L. pneumophila. Collectively, these findings show that LDT-mediated PG remodeling is a major determinant of developmental transitions and survival in C. burnetii and L. pneumophila. Understanding this mechanism might inform new therapeutic approaches for treating chronic infections caused by these pathogens, as well as suggest new methods to decontaminate environmental reservoirs during outbreaks.IMPORTANCECoxiella burnetii and L. pneumophila cause Q Fever and Legionnaire's disease in humans, respectively. There is a lack of effective treatments for fatal chronic infections caused by these pathogens, particularly chronic Q Fever. These bacteria survive long term in nutrient-limited environments by differentiating into phenotypically distinct survival variants. Our study revealed that LDTs, a group of PG remodeling enzymes, play a prominent role in the phenotypic differentiations of these bacteria. We show that LDT-targeting carbapenems are effective against the survival variants, thus demanding the exploration of carbapenems for treating chronic infections caused by these pathogens. We report the tethering of a unique OM fatty acid transporter to PG in L. pneumophila that could indicate a novel function of tethering, that is, the uptake of nutrient substrates. Homologs of this transporter are widely present in the Methylobacteriaceae family of bacteria known to survive in water systems like Legionella, thus suggesting a potentially conserved mechanism of bacterial survival in nutrient-limited environments.

ProPr54 web server: predicting σ54 promoters and regulon with a hybrid convolutional and recurrent deep neural network.

σ54 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 σ54 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 σ54 promoters and regulon in bacterial genomes. Here, we report a σ54 promoter prediction method ProPr54, based on a convolutional neural network trained on a set of 446 validated σ54 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 σ54 regulon identification methods. Furthermore, analysis on bacterial genomes, which have no experimentally validated σ54 binding sites, demonstrates the generalization of the model. ProPr54 is the first reliable in silico method for predicting σ54 binding sites, making it a valuable tool to support experimental studies on σ54. In conclusion, ProPr54 offers a reliable, broadly applicable tool for predicting σ54 promoters and regulon genes in bacterial genome sequences. A web server is freely accessible at http://propr54.molgenrug.nl.

Revival of the Escherichia coli heat shock response after two decades with a small Hsp in a critical but distinct act.

The heat stress response is an essential defense mechanism in all organisms. Heat shock proteins (Hsps) are produced in response to thermal stress, with their expression levels regulated by heat shock transcription factors. In Escherichia coli, the key transcription factor σ32 positively regulates Hsp expression. Studies from over two decades ago revealed that σ32 abundance is negatively controlled under normal conditions, mainly through degradation mechanisms involving DnaK, GroEL, and FtsH. Beyond this established mechanism, recent findings indicate that a small heat shock protein IbpA also plays a role in the translational regulation of σ32, adding a new layer to the established model. This review highlights the role of a new actor, IbpA, which strongly suppresses σ32 expression under non-stress conditions and markedly increases it during heat shock.

The SinR•SlrR heteromer attenuates transcription of a long operon of flagellar genes in Bacillus subtilis.

During growth, Bacillus subtilis differentiates into subpopulations of motile individuals and non-motile chains, associated with dispersal and biofilm formation respectively. The two cell types are dictated by the activity of the alternative sigma factor SigD encoded as the penultimate gene of the 27 kb long fla/che flagellar operon. The frequency of SigD-ON motile cells is increased by the heteromeric transcription factor SwrA•DegU that activates the fla/che promoter. Conversely, the frequency of motile cells is decreased by the heteromeric transcription factor SinR•SlrR, but the mechanism and location of inhibition is poorly understood. Here, using ChIP-Seq analysis, we determine the binding sites of the SinR•SlrR heteromer on the genome. We identified two sites within the fla/che operon that were both necessary and sufficient to attenuate transcript abundance by causing premature termination upstream of the gene that encodes SigD. Thus, cell motility and the transition to biofilm formation depend on the expression of a long operon governed by two opposing heteromeric transcription factors that operate at two different stages of the transcription cycle. More broadly, our study serves as a model for transcription factors that control transcriptional elongation and the regulation of long operons in bacteria.

Whole-Genome Sequencing of Resistance, Virulence and Regulation Genes in Extremely Resistant Strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a clinically significant opportunistic pathogen, renowned for its ability to acquire and develop diverse mechanisms of antibiotic resistance. This study examines the resistance, virulence, and regulatory mechanisms in extensively drug-resistant clinical strains of P. aeruginosa. Antibiotic susceptibility was assessed using the Minimum Inhibitory Concentration (MIC) method, and whole-genome sequencing (WGS) was performed on the Illumina NovaSeq platform. The analysis demonstrated a higher prevalence of virulence genes compared to resistance and regulatory genes. Key virulence factors identified included secretion systems, motility, adhesion, and biofilm formation. Resistance mechanisms observed comprised efflux pumps and beta-lactamases, while regulatory systems involved two-component systems, transcriptional regulators, and sigma factors. Additionally, phenotypic profiles were found to correlate with resistance genes identified through genotypic analysis. This study underscores the significant resistance and virulence of the clinical P. aeruginosa strains analyzed, highlighting the urgent need for alternative strategies to address infections caused by extensively drug-resistant bacteria.

Engineered Phage Enables Efficient Control of Gene Expression upon Infection of the Host Cell.

Recently, we developed a spatial phage-assisted continuous evolution (SPACE) system. This system utilizes chemotaxis coupled with the growth of motile bacteria during their spatial range expansion in soft agar to provide fresh host cells for iterative phage infection and selection pressure for preserving evolved genes of interest carried by phage mutants. Controllable mutagenesis activated only in a subpopulation of the migrating cells is essential in this system to efficiently generate mutated progeny phages from which desired individuals are selected during the directed evolution process. But, the widely adopted small molecule-dependent inducible system could hardly fulfill this purpose because it always affects all cells homogeneously. In this study, we developed a phage infection-induced gene expression system using modified Escherichia coli (E. coli) phage shock protein operon or sigma factors from Bacillus subtilis. Results showed that this system enabled efficient control of gene expression upon phage infection with dynamic output ranges from small to large using combinations of different engineered phages and corresponding promoters. This system was incorporated into SPACE to function as a phage infection-induced mutagenesis module and successfully facilitated the evolution of T7 RNA polymerase, which generated diverse mutants with altered promoter recognition specificity. We expect that phage infection-induced gene expression system could be further extended to more applications involving partial induction in a portion of a population and targeted induction in specific strains among a mixed bacterial community, which provides an important complement to small molecule-dependent inducible systems.

The SpxA1-TenA toxin-antitoxin system regulates epigenetic variations of Streptococcus pneumoniae by targeting protein synthesis.

Human pathogen Streptococcus pneumoniae forms multiple epigenetically and phenotypically distinct intra-populations by invertase PsrA-driven inversions of DNA methyltransferase hsdS genes in the colony opacity-determinant (cod) locus. As manifested by phase switch between opaque and transparent colonies, different genome methylation patterns or epigenomes confer pathogenesis-associated traits, but it is unknown how the pathogen controls the hsdS inversion orientations. Here, we report our finding of the SpxA1-TenA toxin-antitoxin (TA) system that regulates the orientations of hsdS inversions, and thereby bacterial epigenome and associated traits (e.g., colony opacity) by targeting pneumococcal protein synthesis. SpxA1 and TenA were found to constitute a highly conserved type II TA system in S. pneumoniae, primarily based on the observation that overexpressing toxin TenA led to growth arrest in E. coli and enhanced autolysis in S. pneumoniae, and the antitoxin SpxA1 repressed the transcription of the spxA1-tenA operon. When the transcription of tenA was de-repressed by a spontaneous AT di-nucleotide insertion/deletion in the promoter region of the spxA1-tenA operon, TenA bound to the ribosome maturation factor RimM, and thereby reduced the cellular level of alternative sigma factor ComX (known for the activation of natural transformation-associated genes). Attenuation of ComX expression in turn enhanced the transcription of the invertase gene psrA, which favored the formation of the transparent colony phase-associated hsdS allelic configurations in the cod locus. Phenotypically, moderate expression of TenA dramatically reshaped pneumococcal epigenome and colony opacity. Because spontaneous variations frequently occur during bacterial growth in the number of the AT di-nucleotides in the promoter region of the spxA1-tenA operon, this locus acts as a programmed genetic switch that generates pneumococcal subpopulations with epigenetic and phenotypic diversity.

Progression of ampC amplification during de novo amoxicillin resistance development in E. coli.

Beta-lactam antibiotics are the most applied antimicrobials in human and veterinarian health care. Hence, beta-lactam resistance is a major health problem. Gene amplification of AmpC beta-lactamase is a main contributor to de novo β-lactam resistance in Escherichia coli. However, the time course of amplification and the accompanying DNA mutations are unclear. Here, we study the progression of ampC amplification and ampC promoter mutations during the evolution of resistance induced by stepwise increasing amoxicillin concentrations. AmpC promoter mutations occurred by day 2, while the approximately eight-fold amplification occurred after more than 6 days of amoxicillin exposure. The combination of the amplification and the promoter mutations increased the ampC mRNA level by an average factor of 200 after 22 days. An IS1 insertion is identified in the amplification junction after resistance induction in the wild type (WT) and the ampC gene complementation strain (CompA), but not in ∆ampC, suggesting that the amplification depends on mobile genetic element transposition. In order to elucidate the correlation between gene mutations and ampC amplification, the DNA mutations acquired during resistance evolution by the WT, ∆ampC, and CompA were analyzed. Compared to evolved ∆ampC, several resistance-causing mutations are absent in evolved WT, while more mutations accumulated in stress response. The amoxicillin-resistant ∆ampC did not show amplification of the fragment around the original ampC position but exhibited a large duplication or triplication at another position, suggesting the essential role of the duplicated genes in resistance development.IMPORTANCEAmoxicillin is the most used antimicrobial against bacterial infections. DNA fragments containing ampC are amplified upon prolonged and stepwise increasing exposure to amoxicillin, causing resistance. These ampC-containing fragments have been identified in extended-spectrum beta-lactamase plasmids, which are considered the main cause of beta-lactam resistance. In this study, we document the time course of two important factors for ampC transcription enhancement, ampC amplification and ampC promoter mutations, during de novo amoxicillin resistance evolution. We propose that the transposon IS1 contributes to the amplification ampC region, that the sigma factor 70 regulates ampC overexpression, and that these combined form the backbone of a putative mechanism for ampC amplification.

Bb0689 contributes to the virulence of Borrelia burgdorferi in a murine model of Lyme disease.

Borrelia burgdorferi, the Lyme disease pathogen, continuously changes its gene expression profile in order to adapt to ticks and mammalian hosts. The alternative sigma factor RpoS plays a central role in borrelial host adaptation. Global transcriptome analyses suggested that more than 100 genes might be regulated by RpoS, but the main part of the regulon remains unexplored. Here, we showed that the expression of bb0689, a gene encoding an outer surface lipoprotein with unknown function, was activated by RpoS. By analyzing gene expression using luciferase reporter assays and quantitative reverse transcription PCR, we found that expression of bb0689 was induced by an elevated temperature, a reduced pH, and increased cell density during in vitro cultivation. The transcriptional start site and a functional promoter for gene expression were identified in the 5' regulatory region of bb0689. The promoter was responsive to environmental stimuli and influenced by RpoS. We also showed that bb0689 expression was expressed in B. burgdorferi during animal infection, suggesting the importance of this gene for infection. We further generated a bb0689 mutant and found that the infectivity of the mutant was severely attenuated in a murine infection model. Although bb0689-deficient spirochetes exhibited no defect during in vitro growth, they were defective in resistance to osmotic stress. Cis-complementation of the mutant with a wild-type copy of bb0689 fully rescued all phenotypes. Collectively, these results demonstrate that the RpoS-regulated gene bb0689 is a key contributor to the optimal infection of B. burgdorferi in animals.

The cyanobacterial circadian clock couples to pulsatile processes using pulse amplitude modulation.

Cellular processes are dynamic and often oscillatory, requiring precise coordination for optimal cell function.1,2,3,4,5,6,7 How distinct oscillatory processes can couple within a single cell remains an open question. Here, we use the cyanobacterial circadian clock8,9 as a model system to explore the coupling of oscillatory and pulsatile gene circuits. The cyanobacterial circadian clock generates 24-h oscillations in downstream targets10,11,12,13,14,15 to time processes across the day/night cycle.9,16,17,18,19,20,21,22 This timing is partly mediated by the clock's modulation of the activity of alternative sigma factors,14,23,24,25 which direct RNA polymerase to specific promoters.26 Using single-cell time-lapse microscopy and modeling, we find that the clock modulates the amplitude of expression pulses of the alternative sigma factor RpoD4, which occurs only at cell division. This pulse amplitude modulation (PAM), analogous to AM regulation in radio transmission,27 allows the clock to robustly generate a 24-h rhythm in rpoD4 expression despite rpoD4's pulsing frequency being non-circadian. By modulating cell division rates, we find that, as predicted by our model, PAM regulation generates the same 24-h period in rpoD4 pulse amplitude over a range of rpoD4 pulse frequencies. Furthermore, we identify a functional significance of rpoD4 expression levels: deletion of rpoD4 results in smaller cell sizes, whereas an increase in rpoD4 expression leads to larger cell sizes in a dose-dependent manner. Thus, our work reveals a link between the cell cycle, clock, and RpoD4 in cyanobacteria and suggests that PAM regulation can be a general mechanism for biological clocks to robustly modulate pulsatile downstream processes.

Promoter recognition specificity of Corynebacterium glutamicum stress response sigma factors σD and σH deciphered using computer modeling and point mutagenesis

This study aimed to reveal interactions of the stress response sigma subunits (factors) σD and σH of RNA polymerase and promoters in Gram-positive bacterium Corynebacterium glutamicum by combining wet-lab obtained data and in silico modeling. Computer modeling-guided point mutagenesis of C. glutamicum σH subunit led to the creation of a panel of σH variants. Their ability to initiate transcription from naturally occurring hybrid σD/σH-dependent promoter Pcg0441 and two control canonical promoters (σD-dependent PrsdA and σH-dependent PuvrD3) was measured and interpreted using molecular dynamics simulations of homology models of all complexes. The results led us to design the artificial hybrid promoter PD35H10 combining the −10 element of the PuvrD3 promoter and the −35 element of the PrsdA promoter. This artificial hybrid promoter PD35-rsdAH10-uvrD3 showed almost optimal properties needed for the bio-orthogonal transcription (not interfering with the native biological processes).

Structural insights into regulated intramembrane proteolysis by the positive alginate regulator MucP from Pseudomonas aeruginosa

Regulated intramembrane proteolysis (RIP) is a fundamentally conserved mechanism involving sequential cleavage by a membrane-bound Site-1 protease (S1P) and a transmembrane Site-2 protease (S2P). In the opportunistic pathogen Pseudomonas aeruginosa, the alternate sigma factor σ22 activates alginate production and in turn is regulated by the MucABCD system. The anti-sigma factor MucA, which inhibits σ22, is sequentially cleaved via RIP by AlgW (S1P) and MucP (S2P) respectively. In this study, we report high-resolution crystal structures of the MucP PDZ1 and PDZ2 domains. Structural and binding analysis confirms that MucP PDZ2 recognizes the carboxy-terminal Ala136 residue of MucA following Site-1 cleavage by AlgW, while the peptide binding groove of PDZ1 is obstructed by a short α-helix. A structure of MucP PDZ2 with bound MucA peptide shows how PDZ2 binds the newly exposed carboxyl terminus of MucA following AlgW cleavage. The ability of a ΔmucP strain of P. aeruginosa to form biofilms was reduced to a similar extent as a ΔalgW strain. This work paves the way for further studies of MucP and other PDZ-containing S2Ps in regulated intramembrane proteolysis.