Sigma Factor Research Articles

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.

EF-hand calcium sensor, EfhP, controls transcriptional regulation of iron uptake by calcium in Pseudomonas aeruginosa.

The human pathogen Pseudomonas aeruginosa (Pa) poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca2+ that are commonly present in CF nasal and lung fluids. In addition, a Ca2+-binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca2+-regulated virulence in P. aeruginosa. Here, we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca2+ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca2+ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ), and several virulence factors, such as the production of pyocins. The Ca2+ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca2+ and Fe, and this regulation required a Ca2+-dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca2+ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca2+ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to the host.IMPORTANCEPseudomonas aeruginosa (Pa) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca2+ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca2+ sensor, EfhP, is required for at least 1/3 of the Ca2+ response, including the majority of the iron uptake mechanisms and the production of pyocins. Transcription of efhP itself is regulated by Ca2+ and Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in Pa infections. The findings establish the regulatory interconnectedness between Ca2+ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca2+ and associated regulatory mechanisms will serve in the development of future therapeutics targeting Pa's dangerous infections.

Unveiling the hidden arsenal: new insights into Proteus mirabilis virulence in UTIs.

Proteus mirabilis is a Gram-negative bacterium commonly found in urinary tract infections (UTIs) and catheter-associated urinary tract infections (CAUTIs). The pathogenic mechanisms of Proteus mirabilis are complex and diverse, involving various virulence factors, including fimbriae, flagella, urease, polyphosphate kinase, lipopolysaccharides, cyclic AMP receptor protein, Sigma factor RpoE, and RNA chaperone protein Hfq. These factors play crucial roles in bacterial colonization, invasion, evasion of host immune responses, biofilm formation, and urinary stone formation. This paper is the first to comprehensively describe the hydrogenase system, autotransporter proteins, molybdate-binding protein ModA, and two-component systems as virulence factors in Proteus mirabilis, providing new insights into its pathogenic mechanisms in urinary tract infections. This review explores the mechanisms of biofilm formation by Proteus mirabilis and the various virulence factors involved in UTIs, revealing many newly discovered virulence factors from recent studies. These findings may offer new targets for clinical treatment of UTIs and vaccine development, highlighting the importance of understanding these virulence factors.

Novel function of single-target regulator NorR involved in swarming motility and biofilm formation revealed in Vibrio alginolyticus

NorR, as a single-target regulator, has been demonstrated to be involved in NO detoxification in bacteria under anaerobic conditions. Here, the norR gene was identified and deleted in the genome of Vibrio alginolyticus. The results showed that deletion of norR in Vibrio alginolyticus led to lower swarming motility and more biofilm formation on aerobic condition. Moreover, we proved that NorR from E. coli had a similar function in controlling motility. NorR overexpression led to increased resistance to oxidative stress and tetracycline. We also observed a reduced ability of the NorR-overexpressing strain to adapt to iron limitation condition. Transcriptome analysis showed that the genes responsible for bacterial motility and biofilm formation were affected by NorR. The expressions of several sigma factors (RpoS, RpoN, and RpoH) and response regulators (LuxR and MarR) were also controlled by NorR. Furthermore, Chip-qPCR showed that there is a direct binding between NorR and the promoter of rpoS. Based on these results, NorR appears to be a central regulator involved in biofilm formation and swarming motility in Vibrio alginolyticus.

MoaB2, a newly identified transcription factor, binds to σA in Mycobacterium smegmatis.

In mycobacteria, σA is the primary sigma factor. This essential protein binds to RNA polymerase (RNAP) and mediates transcription initiation of housekeeping genes. Our knowledge about this factor in mycobacteria is limited. Here, we performed an unbiased search for interacting partners of Mycobacterium smegmatis σA. The search revealed a number of proteins; prominent among them was MoaB2. The σA-MoaB2 interaction was validated and characterized by several approaches, revealing that it likely does not require RNAP and is specific, as alternative σ factors (e.g., closely related σB) do not interact with MoaB2. The structure of MoaB2 was solved by X-ray crystallography. By immunoprecipitation and nuclear magnetic resonance, the unique, unstructured N-terminal domain of σA was identified to play a role in the σA-MoaB2 interaction. Functional experiments then showed that MoaB2 inhibits σA-dependent (but not σB-dependent) transcription and may increase the stability of σA in the cell. We propose that MoaB2, by sequestering σA, has a potential to modulate gene expression. In summary, this study has uncovered a new binding partner of mycobacterial σA, paving the way for future investigation of this phenomenon.IMPORTANCEMycobacteria cause serious human diseases such as tuberculosis and leprosy. The mycobacterial transcription machinery is unique, containing transcription factors such as RbpA, CarD, and the RNA polymerase (RNAP) core-interacting small RNA Ms1. Here, we extend our knowledge of the mycobacterial transcription apparatus by identifying MoaB2 as an interacting partner of σA, the primary sigma factor, and characterize its effects on transcription and σA stability. This information expands our knowledge of interacting partners of subunits of mycobacterial RNAP, providing opportunities for future development of antimycobacterial compounds.

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.

Distinct impacts of each anti-anti-sigma factor ortholog of the chlamydial Rsb partner switching mechanism on development in Chlamydia trachomatis.

Partner switching mechanisms (PSMs) are signal transduction systems comprised of a sensor phosphatase (RsbU), an anti-sigma factor (RsbW, kinase), an anti-anti-sigma factor (RsbV, the RsbW substrate), and a target sigma factor. Chlamydia spp. are obligate intracellular bacterial pathogens of animals that undergo a developmental cycle transitioning between the infectious elementary body (EB) and replicative reticulate body (RB) within a host cell-derived vacuole (inclusion). Secondary differentiation events (RB to EB) are transcriptionally regulated, in part, by the housekeeping sigma factor (σ66) and two late-gene sigma factors (σ54 and σ28). Prior research supports that the PSM in Chlamydia trachomatis regulates availability of σ66. Pan-genome analysis revealed that PSM components are conserved across the phylum Chlamydiota, with Chlamydia spp. possessing an atypical arrangement of two anti-anti-sigma factors, RsbV1 and RsbV2. Bioinformatic analyses support RsbV2 as the homolog to the pan-genome-conserved RsbV with RsbV1 as an outlier. This, combined with in vitro data, indicates that RsbV1 and RsbV2 are structurally and biochemically distinct. Reduced levels or overexpression of RsbV1/RsbV2 did not significantly impact C. trachomatis growth or development. In contrast, overexpression of a non-phosphorylatable RsbV2 S55A mutant, but not overexpression of an RsbV1 S56A mutant, resulted in a 3 log reduction in infectious EB production without reduction in genomic DNA (total bacteria) or inclusion size, suggesting a block in secondary differentiation. The block was corroborated by reduced production of σ54/28-regulated late proteins and via transmission electron microscopy.IMPORTANCEChlamydia trachomatis is the leading cause of reportable bacterial sexually transmitted infections (STIs) and causes the eye infection trachoma, a neglected tropical disease. Broad-spectrum antibiotics used for treatment can lead to microbiome dysbiosis and increased antibiotic resistance development in other bacteria, and treatment failure for chlamydial STIs is a recognized clinical problem. Here, we show that disruption of a partner switching mechanism (PSM) significantly reduces infectious progeny production via blockage of reticulate body to elementary body differentiation. We also reveal a novel PSM expansion largely restricted to the species infecting animals, suggesting a role in pathogen evolution. Collectively, our results highlight the chlamydial PSM as a key regulator of development that could be a potential target for novel therapeutics.

Unveiling the novel regulatory roles of RpoD-family sigma factors in Salmonella Typhimurium heat shock response through systems biology approaches.

Three RpoD-family sigma factors, RpoD, RpoS, and RpoH, play critical roles in transcriptional regulation in Salmonella enterica serovar Typhimurium under heat shock conditions. However, the genome-wide regulatory mechanisms of these sigma factors in response to heat stress have remained elusive. In this study, we comprehensively identified 2,319, 2,226, and 213 genome-wide binding sites for RpoD, RpoS, and RpoH, respectively, under sublethal heat shock conditions (42°C). Machine learning-based transcriptome analysis was employed to infer the relative activity of iModulons, providing valuable insights into the transcriptional impact of heat shock. Integrative data analysis enabled the reconstruction of the transcriptional regulatory network of sigma factors, revealing how they modulate gene expression to adapt to heat stress, including responses to anaerobic and oxidative stresses. Notably, we observed a significant expansion of the RpoS sigmulon from 97 to 301 genes in response to heat shock, underscoring the crucial role of RpoS in regulating various metabolic processes. Moreover, we uncovered a competition mechanism between RpoD and RpoS within RpoS sigmulons, where RpoS significantly increases its binding within promoter regions shared with RpoD under heat shock conditions. These findings illuminate how three RpoD-family sigma factors coordinate multiple cellular processes to orchestrate the overall response of S. Typhimurium to heat stress.

Activation of the general stress response sigma factor SigB prevents competence development in Bacillus subtilis.

Bacillus subtilis exhibits a large number of different specific and general adaptation reactions, which need to be well balanced to sustain survival under largely unfavorable conditions. Under specific conditions, natural competence develops, which enables B. subtilis to actively take up exogenous DNA to integrate it into its own genome. In contrast to this specific adaptation, the general stress response is induced by a variety of exogenous stress and starvation stimuli, providing comprehensive protection and enabling survival of vegetative B. subtilis cells. In the present work, we reveal the molecular basis for the interconnection of these two important responses in the regulatory network. We describe that the master regulator of the general stress response SigB is activated by physiological stress stimuli, including daylight and ethanol stress, leading to the inactivation of the competence master regulator ComK by transcriptional anti-sense regulation, showing a strict hierarchy of adaptational responses under severe stress.

Light inducible gene expression system for Streptomyces

The LitR/CarH family comprises adenosyl B12-based photosensory transcriptional regulators that control light-inducible carotenoid production in nonphototrophic bacteria. In this study, we established a blue–green light-inducible hyperexpression system using LitR and its partner ECF-type sigma factor LitS in streptomycin-producing Streptomyces griseus NBRC 13350. The constructed multiple-copy number plasmid, pLit19, carried five genetic elements: pIJ101rep, the thiostrepton resistance gene, litR, litS, and σLitS-recognized light-inducible crtE promoter. Streptomyces griseus transformants harboring pLit19 exhibited a light-dependent hyper-production of intracellular reporter enzymes including catechol-2,3-dioxygenase and β-glucuronidase, extracellular secreted enzymes including laccase and transglutaminase, and secondary metabolites including melanin, flaviolin, and indigoidine. Cephamycin-producing Streptomyces sp. NBRC 13304, carrying an entire actinorhodin gene cluster, exhibited light-dependent actinorhodin production after the introduction of the pLit19 shuttle-type plasmid with the pathway-specific activator actII-ORF4. Insertion of sti fragment derived from Streptomyces phaeochromogenes pJV1 plasmid into pLit19 increased its light sensitivity, allowing gene expression under weak light irradiation. The two constructed Escherichia coli–Streptomyces shuttle-type pLit19 plasmids were found to have abilities similar to those of pLit19. We successfully established an optogenetically controlled hyperproduction system for S. griseus NBRC 13350 and Streptomyces sp. NBRC 13304.

Insertion sequence elements and unique symmetrical genomic regions mediate chromosomal inversions in Streptococcus pyogenes.

Chromosomal inversions are a phenomenon in many bacterial species, often across the axis of replication. Inversions have been shown to alter gene expression, changing persistence of colonisation and infection following environmental stresses. In Streptococcus pyogenes, inversions have been reported. However, frequency and molecular markers of inversions have not been systematically examined. Here, 249 complete S.pyogenes genomes were analysed using a pangenomic core gene synteny framework to identify sequences associated with inversions. 47% of genomes (118/249) contained at least one inversion, from 23 unique inversion locations. Chromosomal locations enabling inversions were usually associated with mobile elements (insertion sequences n=9 and prophages n=7). Two insertion sequences, IS1548 and IS1239, accounted for>80% of insertion sequences and were the only insertion sequences associated with inversions. The most observed inversion location (n=104 genomes, 88% of genomes with an inversion) occurs between two conserved regions encoding rRNAs, tRNAsand sigma factor genes. The regions are symmetrically placed around the origin of replication forming a unique chromosomal structure inS. pyogenes, relative to other streptococci. Cataloging of thechromosomal location and frequency of inversions can direct dissection of phenotypic changes following chromosomal inversions. The framework used here can be transferred to other bacterial species to characterise chromosomal inversions.

Investigation of the Sigma Approximation Technique for the Solution of the Time Spectral Equation System

Abstract The time spectral method (TSM) is commonly utilized to analyze periodic unsteady flows within turbomachines. However, the time spectral solutions in regions with large temporal and spatial gradients, such as wakes, shocks, and boundary layers, can be plagued with unphysical oscillations, known as the Gibbs phenomenon. This paper presents an investigation into the sigma approximation technique, which is capable of significantly attenuating the Gibbs phenomenon by dampening high-frequency nonlinear components in time spectral solutions. Central to this technique are three sigma parameters: the exponent, the cutoff number, and the number of harmonics, which collectively determine the damping effects for each frequency. However, the optimal combination of them remains to be ascertained considering the tradeoff between the accuracy of solutions and the reduction of unphysical oscillations. In this study, a two-row compressor configuration is first selected, and numerical simulations are performed to evaluate the efficacy of this technique under near-peak efficiency and near-stall operating conditions. It is found that Gibbs-type unphysical oscillations can be effectively mitigated without notably compromising solution accuracy through an optimal combination of sigma parameters. The NASA Stage 35 case study further verifies the effectiveness of the proposed optimal sigma parameter combination in preserving the accuracy of time spectral solutions based on the data from test rig. Moreover, the von Neumann analysis reveals that the sigma approximation technique can enhance the numerical stability of the time spectral equation system, thereby allowing the use of aggressive numerical parameters to accelerate convergence. The stability analysis results are verified by a two-dimensional bump under two different flow conditions and further assessed using the two-row compressor.

Environmental alkalization suppresses deployment of virulence strategies in Pseudomonas syringae pv. tomato DC3000.

Plant pathogenic bacteria encounter a drastic increase in apoplastic pH during the early stages of plant immunity. The effects of alkalization on pathogen-host interactions have not been comprehensively characterized. Here, we used a global transcriptomic approach to assess the impact of environmental alkalization on Pseudomonas syringae pv. tomato DC3000 in vitro. In addition to the Type 3 Secretion System, we found expression of genes encoding other virulence factors such as iron uptake and coronatine biosynthesis to be strongly affected by environmental alkalization. We also found that the activity of AlgU, an important regulator of virulence gene expression, was induced at pH 5.5 and suppressed at pH 7.8, which are pH levels that this pathogen would likely experience before and during pattern-triggered immunity, respectively. This pH-dependent control requires the presence of periplasmic proteases, AlgW and MucP, that function as part of the environmental sensing system that activates AlgU in specific conditions. This is the first example of pH-dependency of AlgU activity, suggesting a regulatory pathway model where pH affects the proteolysis-dependent activation of AlgU. These results contribute to deeper understanding of the role apoplastic pH has on host-pathogen interactions.IMPORTANCEPlant pathogenic bacteria, like Pseudomonas syringae, encounter many environmental changes including oxidative stress and alkalization during plant immunity, but the ecological effects of the individual responses are not well understood. In this study, we found that transcription of many previously characterized virulence factors in P. syringae pv. tomato DC3000 is downregulated by the level of environmental alkalization these bacteria encounter during the early stages of plant immune activation. We also report for the first time the sigma factor AlgU is post-translationally activated by low environmental pH through its natural activation pathway, which partially accounts for the expression Type 3 Secretion System virulence genes at acidic pH. The results of this study demonstrate the importance of extracellular pH on global regulation of virulence-related gene transcription in plant pathogenic bacteria.

Complex sporulation-specific expression of transcription termination factor Rho highlights its involvement in Bacillus subtilis cell differentiation

Termination factor Rho, responsible for the main factor-dependent pathway of transcription termination and the major inhibitor of antisense transcription, is an emerging regulator of various physiological processes in microorganisms. In Gram-positive bacterium Bacillus subtilis, Rho is involved in the control of cell adaptation to starvation and, in particular, in the control of sporulation, a complex differentiation program leading to formation of a highly resistant dormant spore. While the initiation of sporulation requires a decrease in Rho protein levels during the transition to stationary phase, the mechanisms regulating the expression of rho gene throughout the cell cycle remain largely unknown. Here we show that a drop in the activity of the vegetative SigA-dependent rho promoter causes the inhibition of rho expression in stationary phase. However, after the initiation of sporulation, rho gene is specifically reactivated in two compartments of the sporulating cell using distinct mechanisms. In the mother cell, rho expression occurs by read-through transcription initiated at the SigH-dependent promoter of the distal spo0F gene. In the forespore, rho gene is transcribed from the intrinsic promoter recognized by the alternative sigma factor SigF. These regulatory elements ensure the activity of Rho during sporulation, which appears important for the proper formation of spores. We provide experimental evidence that disruption of the spatiotemporal expression of rho during sporulation affects the resistance properties of spores, their morphology, and the ability to return to vegetative growth under favorable growth conditions.

Regulate the Risk Factors of Pseudomonas aeruginosa by some of Sigma Factor Genes

In this research, a study was conducted isolating and identification of Pseudomonas aeruginosa bacteria from clinical samples from various pathological conditions such as burns , otitis media , urinary tract infection , wounds, eye infection, blood infection, catheter infection, throat , vaginal infection and surgical. The present study includes 313 samples collected from patients who attend Alsader Medical City and Al- Hakim General Hospital during the period extended From September 2023 to December 2023 ( men and women ) with age groups between (1 -77) years. The identification of bacterial isolates were detected by classical and molecular technique (PCR), were the frequency among males 163 (52.07% ) was more than that in female 150 (47.92). The samples distribution is made according age group , it appears high for the following high (15.97) and (12.77) with group (16-30) years of male and female respectively . The results revealed that 57/313 specimens of the total number of samples are Pseudomonas aeruginosa by identified according to their morphological, physiological and biochemical properties. Also with molecular identification by using four primers wre the results showed 16S rRNA gene was detected in 57/57 (100%) P. aeruginosa isolates , the Las biofilm gene was detected in 43/57 (75.43%) , the algD gene was detected in 41/57 (71.92%) and 41/57 samples show positive results with the oprL gene. Six types of antibiotics were also tested to determine the resistance of the samples to them.

The plasmid-encoded members of paralogous gene family 52 are dispensable to the enzootic cycle of Borrelia burgdorferi.

Lyme disease, the leading vector-borne disease in the United States and Europe, develops after infection with Borrelia burgdorferi sensu lato bacteria. Transmission of the spirochete from the tick vector to a vertebrate host requires global changes in gene expression that are controlled, in part, by the Rrp2/RpoN/RpoS alternative sigma factor cascade. Transcriptional studies defining the B. burgdorferi RpoS regulon have suggested that RpoS activates the transcription of paralogous family 52 (PFam52) genes. In strain B31, PFam52 genes (bbi42, bbk53, and bbq03) encode a set of conserved hypothetical proteins with >89% amino acid identity that are predicted to be surface-localized. Extensive homology among members of paralogous families complicates studies of protein contributions to pathogenicity as the potential for functional redundancy will obfuscate findings. Using a sequential mutagenesis approach, we generated clones expressing a single PFam52 paralog, as well as a strain deficient in all three. The single paralog expressing strains were used to confirm BBI42, BBK53, and BBQ03 surface localization and RpoS regulation. Surprisingly, the PFam52-deficient strain was able to infect mice and complete the enzootic cycle similar to the wild-type parental strain. Indeed, the presence of numerous pseudogenes that contain frameshifts or internal stop codons among the PFam52 genes suggests that they may be subjected to gene loss in B. burgdorferi's reduced genome. Alternatively, the lack of phenotype might reflect the limitations of the experimental mouse infection model.

The role of two major nucleoid-associated proteins in Streptomyces, HupA and HupS, in stress survival and gene expression regulation

BackgroundStreptomyces are sporulating soil bacteria with enormous potential for secondary metabolites biosynthesis. Regulatory networks governing Streptomyces coelicolor differentiation and secondary metabolites production are complex and composed of numerous regulatory proteins ranging from specific transcriptional regulators to sigma factors. Nucleoid-associated proteins (NAPs) are also believed to contribute to regulation of gene expression. Upon DNA binding, these proteins impact DNA accessibility. Among NAPs, HU proteins are the most widespread and abundant. Unlike other bacteria, the Streptomyces genomes encode two HU homologs: HupA and HupS, which differ in structure and expression profile. However, it remained unclear whether the functions of both homologs overlap. Additionally, although both proteins have been shown to bind the chromosome, their rolesin transcriptional regulation have not been studied so far.ResultsIn this study, we explore whether HupA and HupS affect S. coelicolor growth under optimal and stressful conditions and how they control global gene expression. By testing both single and double mutants, we address the question of the complementarity of both HU homologs. We show that the lack of both hup genes led to growth and sporulation inhibition, as well as increased spore fragility. We also demonstrate that both HU homologs can be considered global transcriptional regulators, influencing expression of between 2% and 6% genes encoding among others proteins linked to global regulatory networks and secondary metabolite production.ConclusionsWe identify the independent HupA and HupS regulons, as well as genes under the control of both HupA and HupS proteins. Our data indicate a partial overlap between the functions of HupA and HupS during S. coelicolor growth. HupA and HupS play important roles in Streptomyces regulatory network and impact secondary metabolite clusters.

Listeriosis: Characteristics, Occurrence in Domestic Animals, Public Health Significance, Surveillance and Control.

Listeriosis is a dangerous zoonosis caused by bacteria of the genus Listeria, with Listeria monocytogenes (LM) being the most pathogenic species. Listeria monocytogenes has been detected in various animal species and in humans, and its ability to evolve from an environmental saprophyte to a powerful intracellular pathogen is driven by the invasion mechanisms and virulence factors that enable cell invasion, replication and cell-to-cell spread. Key regulatory systems, including positive regulatory factor A (PrfA) and the stress-responsive sigma factor σB, control the expression of virulence genes and facilitate invasion of host cells. Listeriosis poses a significant threat to cattle, sheep and goat herds, leading to abortions, septicemia and meningoencephalitis, and ruminants are important reservoirs for Listeria, facilitating transmission to humans. Other Listeria species such as Listeria ivanovii and Listeria innocua can also cause disease in ruminants. Resilience of LM in food processing environments makes it an important foodborne pathogen that is frequently transmitted through contaminated meat and dairy products, with contamination often occurring along the food production chain. In humans, listeriosis primarily affects immunocompromised individuals, pregnant women and the elderly and leads to severe conditions, such as meningitis, septicemia and spontaneous abortion. Possible treatment requires antibiotics that penetrate the blood-brain barrier. Despite the relatively low antimicrobial resistance, multidrug-resistant LM strains have been detected in animals, food and the environment. Controlling and monitoring the disease at the herd level, along with adopting a One Health approach, are crucial to protect human and animal health and to minimize the potential negative impacts on the environment.

Mycobacterial HelD connects RNA polymerase recycling with transcription initiation

Mycobacterial HelD is a transcription factor that recycles stalled RNAP by dissociating it from nucleic acids and, if present, from the antibiotic rifampicin. The rescued RNAP, however, must disengage from HelD to participate in subsequent rounds of transcription. The mechanism of release is unknown. We show that HelD from Mycobacterium smegmatis forms a complex with RNAP associated with the primary sigma factor σA and transcription factor RbpA but not CarD. We solve several structures of RNAP-σA-RbpA-HelD without and with promoter DNA. These snapshots capture HelD during transcription initiation, describing mechanistic aspects of HelD release from RNAP and its protective effect against rifampicin. Biochemical evidence supports these findings, defines the role of ATP binding and hydrolysis by HelD in the process, and confirms the rifampicin-protective effect of HelD. Collectively, these results show that when HelD is present during transcription initiation, the process is protected from rifampicin until the last possible moment.

RpoS activates formation of Salmonella Typhi biofilms and drives persistence in the gall bladder.

The development of strategies for targeting the asymptomatic carriage of Salmonella Typhi in chronic typhoid patients has suffered owing to our basic lack of understanding of the molecular mechanisms that enable the formation of S. Typhi biofilms. Traditionally, studies have relied on cholesterol-attached biofilms formed by a closely related serovar, Typhimurium, to mimic multicellular Typhi communities formed on human gallstones. In long-term infections, S. Typhi adopts the biofilm lifestyle to persist in vivo and survive in the carrier state, ultimately leading to the spread of infections via the fecal-oral route of transmission. In the present work, we studied S. Typhi biofilms directly, applied targeted as well as genome-wide genetic approaches to uncover unique biofilm components that do not conform to the CsgD-dependent pathway established in S. Typhimurium. We undertook a genome-wide Tn5 mutation screen in H58, a clinically relevant multidrug resistance strain of S. Typhi, in gallstone-mimicking conditions. We generated New Generation Sequencing libraries based on the ClickSeq technology to identify the key regulators, IraP and RpoS, and the matrix components Sth fimbriae, Vi capsule and lipopolysaccharide. We discovered that the starvation sigma factor, RpoS, was required for the transcriptional activation of matrix-encoding genes in vitro, and for S. Typhi colonization in persistent infections in vivo, using a heterologous fish larval model. An rpoS null mutant failed to colonize the gall bladder in chronic zebrafish infections. Overall, our work uncovered a novel RpoS-driven, CsgD-independent paradigm for the formation of cholesterol-attached Typhi biofilms, and emphasized the role(s) of stress signaling pathways for adaptation in chronic infections. Our identification of the biofilm regulators in S. Typhi paves the way for the development of drugs against typhoid carriage, which will ultimately control the increased incidence of gall bladder cancer in typhoid carriers.