A Ralstonia solanacearum effector targets TGA transcription factors to subvert salicylic acid signaling.

The bacterial pathogen Ralstonia solanacearum causes wilt disease on Arabidopsis thaliana and tomato (Solanum lycopersicum). This pathogen uses type III effectors to inhibit the plant immune system; however, how individual effectors interfere with plant immune responses, including transcriptional reprograming, remain elusive. Here, we show that the type III effector RipAB targets Arabidopsis TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors, the central regulators of plant immune gene regulation, via physical interaction in the nucleus to dampen immune responses. RipAB was required for R. solanacearum virulence on wild-type tomato and Arabidopsis but not Arabidopsis tga1 tga4 and tga2 tga5 tga6 mutants. Stable expression of RipAB in Arabidopsis suppressed the pathogen-associated molecular pattern-triggered reactive oxygen species (ROS) burst and immune gene induction as well as salicylic acid (SA) regulons including RBOHD and RBOHF, responsible for ROS production, all of which were phenocopied by the tga1 tga4 and tga2 tga5 tga6 mutants. We found that TGAs directly activate RBOHD and RBOHF expression and that RipAB inhibits this through interfering with the recruitment of RNA polymerase II. These results suggest that TGAs are the bona fide and major virulence targets of RipAB, which disrupts SA signaling by inhibiting TGA activity to achieve successful infection.

The associations between single nucleotide polymorphisms and diabetic retinopathy risk: an umbrella review.

This umbrella review was conducted aiming to assess the association between genetic variations and the development of diabetic retinopathy (DR) by collecting and evaluating available systematic reviews and meta-analysis results. We evaluated the methodological quality using the Measurement Tool to Assess Systematic Reviews (AMSTAR) 2.0, estimated the summary effect size by using the random effects model and calculated the 95% prediction intervals (PIs). Evidence from the included meta-analyses was graded according to established criteria as follows: convincing, highly suggestive, suggestive, weak, or not significant. This umbrella review included 32 meta-analyses of 52 candidate SNPs. The 12 selected meta-analyses were rated as "high," 2 studies were rated as "moderate," 11 studies were graded as "low," and the remaining 7 studies were graded as "critically low" in terms of methodological quality. Carriers of specific genotypes and alleles of the transcription Factor 7-like 2 C/T (TCF7L2 C/T) polymorphism (rs7903146, p < 0.001) might be more susceptible to the occurrence of DR in the homozygous and recessive models, and these associations were supported by "convincing" evidence. Significant associations were also found between interleukin-6 (IL-6) -174 G/C (rs1800795; p < 0.05) or vascular endothelial growth factor (VEGF) polymorphisms (rs2010963, rs699947, rs1570360, rs2010963, rs699947, rs2146323; all p values <0.05) and DR risk, but these associations were supported by "weak" evidence. The TCF7L2 C/T variant could be identified as a definitive genetic risk factor for the development and progression of DR. Data from additional in-depth studies are needed to establish robust evidence for the associations between polymorphisms of IL-6 or VEGF and DR.

CircHERC1 promotes non-small cell lung cancer cell progression by sequestering FOXO1 in the cytoplasm and regulating the miR-142-3p-HMGB1 axis.

BACKGROUND: Noncoding RNAs such as circular RNAs (circRNAs) are abundant in the human body and influence the occurrence and development of various diseases. Non-small cell lung cancer (NSCLC) is one of the most common malignant cancers. Information on the functions and mechanism of circRNAs in lung cancer is limited; thus, the topic needs more exploration. The purpose of this study was to identify aberrantly expressed circRNAs in lung cancer, unravel their roles in NSCLC progression, and provide new targets for lung cancer diagnosis and therapy. METHODS: High-throughput sequencing was used to analyze differential circRNA expression in patients with lung cancer. qRT‒PCR was used to determine the level of circHERC1 in lung cancer tissues and plasma samples. Gain- and loss-of-function experiments were implemented to observe the impacts of circHERC1 on the growth, invasion, and metastasis of lung cancer cells in vitro and in vivo. Mechanistically, dual luciferase reporter assays, fluorescence in situ hybridization (FISH), RNA immunoprecipitation (RIP) and RNA pull-down experiments were performed to confirm the underlying mechanisms of circHERC1. Nucleocytoplasmic localization of FOXO1 was determined by nucleocytoplasmic isolation and immunofluorescence. The interaction of circHERC1 with FOXO1 was verified by RNA pull-down, RNA immunoprecipitation (RIP) and western blot assays. The proliferation and migration of circHERC1 in vivo were verified by subcutaneous and tail vein injection in nude mice. RESULTS: CircHERC1 was significantly upregulated in lung cancer tissues and cells, ectopic expression of circHERC1 strikingly facilitated the proliferation, invasion and metastasis, and inhibited the apoptosis of lung cancer cells in vitro and in vivo. However, knockdown of circHERC1 exerted the opposite effects. CircHERC1 was mainly distributed in the cytoplasm. Further mechanistic research indicated that circHERC1 acted as a competing endogenous RNA of miR-142-3p to relieve the repressive effect of miR-142-3p on its target HMGB1, activating the MAPK/ERK and NF-κB pathways and promoting cell migration and invasion. More importantly, we found that circHERC1 could bind FOXO1 and sequester it in the cytoplasm, adjusting the feedback AKT pathway. The accumulation of FOXO1 in the cytosol and nuclear exclusion promoted cell proliferation and inhibited apoptosis. CircHERC1 is a new circRNA that promotes tumor function in NSCLC and may serve as a potential prognostic biomarker and therapeutic target for NSCLC. CONCLUSIONS: CircHERC1 is a new circRNA that promotes tumor function in NSCLC and may serve as a potential diagnosis biomarker and therapeutic target for NSCLC. Our findings indicate that circHERC1 facilitates the invasion and metastasis of NSCLC cells by regulating the miR-142-3p/HMGB1 axis and activating the MAPK/ERK and NF-κB pathways. In addition, circHERC1 can promote cell proliferation and inhibit apoptosis by sequestering FOXO1 in the cytoplasm to regulate AKT activity and BIM transcription.

Type III secretion by Yersinia pseudotuberculosis is reliant upon an authentic N-terminal YscX secretor domain.

YscX was discovered as an essential part of the Yersinia type III secretion system about 20 years ago. It is required for substrate secretion and is exported itself. Despite this central role, its precise function and mode of action remain unknown. In order to address this knowledge gap, this present study refocused attention on YscX to build on the recent advances in the understanding of YscX function. Our experiments identified an N-terminal secretion domain in YscX promoting its secretion, with the first five codons constituting a minimal signal capable of promoting secretion of the signal less ß-lactamase reporter. Replacing the extreme YscX N-terminus with known secretion signals of other Ysc-Yop substrates revealed that the YscX N-terminal segment contains non-redundant information needed for YscX function. Further, both in cis deletion of the YscX N-terminus in the virulence plasmid and ectopic expression of epitope-tagged YscX variants again lead to stable YscX production but not type III secretion of Yop effector proteins. Mislocalisation of the needle components, SctI and SctF, accompanied this general defect in Yops secretion. Hence, a coupling exists between YscX secretion permissiveness and the assembly of an operational secretion system.

Exploring symptom-level associations between anxiety and depression across developmental stages of adolescence: a network analysis approach.

BACKGROUND: Anxiety and depression often co-occur during adolescence, but the associations between symptoms of these two disorders in this developmental period are not yet fully understood. Network analysis provides a valuable approach to uncover meaningful associations among symptoms and offers insights for prevention and intervention strategies. This study aimed to investigate symptom-level associations between anxiety and depression using network analysis and to identify core symptoms, bridge symptoms, and differences in network structure across different stages of adolescence. METHODS: The cross-sectional study was conducted in March 2022 in Shenzhen, China. Participants completed the Generalized Anxiety Disorder Scale-7 and Patient Health Questionnaire Depression Scale, along with demographic questionnaires assessing age and gender. Chinese adolescents aged 10 to 17 who were in Grades 5 or 6 of elementary school, Grades 1 or 2 of middle school, or Grades 1 or 2 of high school, and who could comprehensively understand and read Chinese were recruited as participants. Students in Grade 3 of middle and high schools were excluded due to their upcoming high school or college entrance examinations. Based on age, participants were categorized into early, middle, and late developmental stages of adolescence. RESULTS: "Loss of control" was among the most central symptoms in the comorbidity network throughout all three developmental stages; "excessive worry" and "anhedonia" emerged as the core symptoms in early adolescence, and "restlessness" as the core symptom in late adolescence. "Anhedonia," "sad mood," and "fatigue" were identified as bridge symptoms between anxiety and depression across all three developmental stages of adolescence. The global strength of the network in middle adolescence was significantly higher compared to the other two stages. CONCLUSION: These findings highlight the core and bridge symptoms that require special attention and intervention at each stage of adolescence. Moreover, significantly higher network connectivity in middle adolescence suggests this is a critical period for intervention to prevent the development of comorbid mental disorders.

Birth spacing and risk of adverse pregnancy and birth outcomes: A systematic review and dose-response meta-analysis.

INTRODUCTION: The association between extreme birth spacing and adverse outcomes is controversial, and available evidence is fragmented into different classifications of birth spacing. MATERIAL AND METHODS: We conducted a systematic review of observational studies to evaluate the association between birth spacing (i.e., interpregnancy interval and interoutcome interval) and adverse outcomes (i.e., pregnancy complications, adverse birth outcomes). Pooled odds ratios (ORs) with 95% confidence intervals (CI) were calculated using a random-effects model, and the dose-response relationships were evaluated using generalized least squares trend estimation. RESULTS: A total of 129 studies involving 46 874 843 pregnancies were included. In the general population, compared with an interpregnancy interval of 18-23 months, extreme intervals (<6 months and ≥ 60 months) were associated with an increased risk of adverse outcomes, including preterm birth, small for gestational age, low birthweight, fetal death, birth defects, early neonatal death, and premature rupture of fetal membranes (pooled OR range: 1.08-1.56; p < 0.05). The dose-response analyses further confirmed these J-shaped relationships (pnon-linear < 0.001-0.009). Long interpregnancy interval was only associated with an increased risk of preeclampsia and gestational diabetes (pnon-linear < 0.005 and pnon-linear < 0.001, respectively). Similar associations were observed between interoutcome interval and risk of low birthweight and preterm birth (pnon-linear < 0.001). Moreover, interoutcome interval of ≥60 months was associated with an increased risk of cesarean delivery (pooled OR 1.72, 95% CI 1.04-2.83). For pregnancies following preterm births, an interpregnancy interval of 9 months was not associated with an increased risk of preterm birth, according to dose-response analyses (pnon-linear = 0.008). Based on limited evidence, we did not observe significant associations between interpregnancy interval or interoutcome interval after pregnancy losses and risk of small for gestational age, fetal death, miscarriage, or preeclampsia (pooled OR range: 0.76-1.21; p > 0.05). CONCLUSIONS: Extreme birth spacing has extensive adverse effects on maternal and infant health. In the general population, interpregnancy interval of 18-23 months may be associated with potential benefits for both mothers and infants. For women with previous preterm birth, the optimal birth spacing may be 9 months.

Novel co-crystal of 3-methylcinnamic acid with berberine (1:1): synthesis, characterization, and intestinal absorption property.

OBJECTIVE: To synthesis a novel 'Pharmaceutical Cocrystal' of berberine (BBR) with coformer 3-methylcinnamic acid (3MCA) for increasing its solubility and intestinal absorption property. SIGNIFICANCE: BBR-HCl has poor liposolubility, difficulty in penetrating the cell membrane and absorption in the gastrointestinal tract, low bioavailability, and limited clinical application. A new cocrystal is formed by the interaction between 3-MCA and BBR through molecular interaction, which improves the physicochemical properties, intestinal absorption property, and hygroscopicity. METHODS: The solvent evaporation method was used to synthesize BCR-3MCA cocrystal. The physicochemical properties of the crystals were confirmed by different spectral techniques, i.e. by X-ray diffraction (PXRD, SXRD), thermogravimetry and differential thermal analysis (DSC, TGA), and scanning electron microscopy (SEM). Hygroscopicity of the cocrystal was evaluated by dynamic water vapor sorption (DVS). The intestinal absorption property was evaluated by the Ussing chamber system. RESULTS: BBR and 3MCA can be directly self-assembled into uniform co-crystal by hydrogen bonds and π-π stacking interactions. Compared with BBR-HCl, the solubility of BBR-3MCA cocrystal in polar solvents of water, methanol, ethanol, and isopropanol increased by 13.9, 1.5, 4.7, and 15.8 times, respectively. The apparent absorption and the absorption rate constants were increased by 7.7 and 5.6 times, respectively. Surprisingly, BBR-3MCA co-crystal almost had no hygroscopicity. CONCLUSION: The absolute molecular structure of the co-crystal was further confirmed by single crystal X-ray diffraction. The hydrogen bonds drove the formation of X-like one-dimensional unit. Compared to the BBR-HCl, BBR-3MCA cocrystal displayed superior dissolution and solubility performance, improved physical-chemical properties and significantly improved intestinal absorption.

Genome-scale analyses of transcriptional start sites in Mycobacterium marinum under normoxic and hypoxic conditions.

BACKGROUND: Hypoxic stress plays a critical role in the persistence of Mycobacterium tuberculosis (Mtb) infection, but the mechanisms underlying this adaptive response remain ill defined. MATERIAL AND METHODS: In this study, using M. marinum as a surrogate, we analyzed hypoxic responses at the transcriptional level by Cappable-seq and regular RNA-seq analyses. RESULTS: A total of 6808 transcriptional start sites (TSSs) were identified under normoxic and hypoxic conditions. Among these TSSs, 1112 were upregulated and 1265 were downregulated in response to hypoxic stress. Using SigE-recognized consensus sequence, we identified 59 SigE-dependent promoters and all were upregulated under hypoxic stress, suggesting an important role for SigE in this process. We also compared the performance of Cappable-seq and regular RNA-seq using the same RNA samples collected from normoxic and hypoxic conditions, and confirmed that Cappable-seq is a valuable approach for global transcriptional regulation analyses. CONCLUSIONS: Our results provide insights and information for further characterization of responses to hypoxia in mycobacteria, and prove that Cappable-seq is a valuable approach for global transcriptional studies in mycobacteria.

CpxR regulates the Rcs phosphorelay system in controlling the Ysc-Yop type III secretion system in Yersinia pseudotuberculosis.

The CpxRA two-component regulatory system and the Rcs phosphorelay system are both employed by the Enterobacteriaceae family to preserve bacterial envelope integrity and function when growing under stress. Although both systems regulate several overlapping physiological processes, evidence demonstrating a molecular connection between Cpx and Rcs signalling outputs is scarce. Here, we show that CpxR negatively regulates the transcription of the rcsB gene in the Rcs phosphorelay system in Yersinia pseudotuberculosis. Interestingly, transcription of rcsB is under the control of three promoters, which were all repressed by CpxR. Critically, synthetic activation of Cpx signalling through mislocalization of the NlpE lipoprotein to the inner membrane resulted in an active form of CpxR that repressed activity of rcsB promoters. On the other hand, a site-directed mutation of the phosphorylation site at residue 51 in CpxR generated an inactive non-phosphorylated variant that was unable to regulate output from these rcsB promoters. Importantly, CpxR-mediated inhibition of rcsB transcription in turn restricted activation of the Ysc-Yop type III secretion system (T3SS). Moreover, active CpxR blocks zinc-mediated activation of Rcs signalling and the subsequent activation of lcrF transcription. Our results demonstrate a novel regulatory cascade linking CpxR-RcsB-LcrF to control production of the Ysc-Yop T3SS.

Targeting RNA helicase DHX33 blocks Ras-driven lung tumorigenesis in vivo.

Ras has been found to be mutated in 30% of non-small cell lung cancers, and its mutation has been regarded as a causal factor underlying tumorigenesis. However, no successful medicine has been developed so far to inhibit Ras for lung cancer treatment. We have previously identified DHX33 as a Ras downstream effector, promoting cell cycle progression and cell growth. In this study, with the K-Ras (G12D);DHX33 (lox/lox) mouse model, we discovered that genetic ablation of DHX33 inhibited tumor development. We further found that ablation of DHX33 altered the expression of nearly 2000 genes which are critical in cancer development such as cell cycle, apoptosis, glycolysis, Wnt signaling, and cell migration. Our study for the first time demonstrates the pivotal role of the DHX33 in Ras-driven lung cancer development in vivo and highlights that pharmacological targeting DHX33 can be a feasible option in treating Ras-mutant lung cancers.

Bilirubin Nanomedicines for the Treatment of Reactive Oxygen Species (ROS)-Mediated Diseases.

Reactive oxygen species (ROS) are chemically reactive species that are produced in cellular aerobic metabolism. They mainly include superoxide anion, hydrogen peroxide, hydroxyl radicals, singlet oxygen, ozone, and nitric oxide and are implicated in many physiological and pathological processes. Bilirubin, a cardinal pigment in the bile, has been increasingly investigated to treat cancer, diabetes, ischemia-reperfusion injury, asthma, and inflammatory bowel diseases (IBD). Indeed, bilirubin has been shown to eliminate ROS production, so it is now considered as a promising therapeutic agent for ROS-mediated diseases and can be used for the development of antioxidative nanomedicines. This review summarizes the current knowledge of the physiological mechanisms of ROS production and its role in pathological changes and focuses on discussing the antioxidative effects of bilirubin and its application in the experimental studies of nanomedicines. Previous studies have shown that bilirubin was mainly used as a responsive molecule in the microenvironment of ROS overproduction in neoplastic tissues for the development of anticancer nanodrugs; however, it could also exert powerful ROS scavenging activity in chronic inflammation and ischemia-reperfusion injury. Therefore, bilirubin, as an inartificial ROS scavenger, is expected to be used for the development of nanomedicines against more diseases due to the universality of ROS involvement in human pathological conditions.

How anatase TiO2 with {101} {001} and {100} surfaces affect the photooxidation process of roxithromycin.

TiO2 crystals are widely used in photocatalytic processes due to their low cost and fabulous catalytic performance. As described in our previous study, three types of TiO2 with the main surfaces of {101}, {001} and {100} were synthesized. In this study, the three types of TiO2 are used to investigate roxithromycin (ROX) photocatalytic degradation kinetics and the pH effect. For photocatalytic degradation, the obtained data have shown that the overall order of optimal degradation is shown as {101} > {001} > {100}. The photooxidation kinetics for {101} facet conforms to first-order kinetics at from pH 5 to pH 10, and most of the photooxidation kinetics for {001} and {100} facets are fitted well with the zero-order and second-order kinetics, respectively. The pH effects are varied to the three types of TiO2, of which {101} has the best degradation effect at pH values 4, 7 and 8, while {001} works best at pH 5 or pH 6, and {100} has a relatively obvious effect at pH 4 and pH 9. The relation between adsorption and oxidation has been tested and proved that the strong adsorption corresponds to the fast oxidation.

Yersinia pseudotuberculosis Exploits CD209 Receptors for Promoting Host Dissemination and Infection.

Yersinia pseudotuberculosis is a Gram-negative enteropathogen and causes gastrointestinal infections. It disseminates from gut to mesenteric lymph nodes (MLNs), spleen, and liver of infected humans and animals. Although the molecular mechanisms for dissemination and infection are unclear, many Gram-negative enteropathogens presumably invade the small intestine via Peyer's patches to initiate dissemination. In this study, we demonstrate that Y. pseudotuberculosis utilizes its lipopolysaccharide (LPS) core to interact with CD209 receptors, leading to invasion of human dendritic cells (DCs) and murine macrophages. These Y. pseudotuberculosis-CD209 interactions result in bacterial dissemination to MLNs, spleens, and livers of both wild-type and Peyer's patch-deficient mice. The blocking of the Y. pseudotuberculosis-CD209 interactions by expression of O-antigen and with oligosaccharides reduces infectivity. Based on the well-documented studies in which HIV-CD209 interaction leads to viral dissemination, we therefore propose an infection route for Y. pseudotuberculosis where this pathogen, after penetrating the intestinal mucosal membrane, hijacks the Y. pseudotuberculosis-CD209 interaction antigen-presenting cells to reach their target destinations, MLNs, spleens, and livers.

RbpA and σB association regulates polyphosphate levels to modulate mycobacterial isoniazid-tolerance.

To facilitate survival under drug stresses, a small population of Mycobacterium tuberculosis can tolerate bactericidal concentrations of drugs without genetic mutations. These drug-tolerant mycobacteria can be induced by environmental stresses and contribute to recalcitrant infections. However, mechanisms underlying the development of drug-tolerant mycobacteria remain obscure. Herein, we characterized a regulatory pathway which is important for the tolerance to isoniazid (INH) in Mycobacterium smegmatis. We found that the RNA polymerase binding protein RbpA associates with the stress response sigma factor σB , to activate the transcription of ppk1, the gene encoding polyphosphate kinase. Subsequently, intracellular levels of inorganic polyphosphate increase to promote INH-tolerant mycobacteria. Interestingly, σB and ppk1 expression varied proportionately in mycobacterial populations and positively correlated with tolerance to INH in individual mycobacteria. Moreover, sigB and ppk1 transcription are both induced upon nutrient depletion, a condition that stimulates the formation of INH-tolerant mycobacteria. Over-expression of ppk1 in rbpA knockdown or sigB deleted strains successfully restored the number of INH-tolerant mycobacteria under both normal growth and nutrient starved conditions. These data suggest that RbpA and σB regulate ppk1 expression to control drug tolerance both during the logarithmic growth phase and under the nutrition starved conditions.

PhoPR Positively Regulates whiB3 Expression in Response to Low pH in Pathogenic Mycobacteria.

During infection, Mycobacterium tuberculosis colonizes macrophages or necrotic granulomas, in which low pH is one of the major challenges. The PhoPR two-component regulatory system and the cytosolic redox sensor WhiB3 both play important roles in the response to low pH by M. tuberculosis However, whether close association exists between PhoPR and WhiB3 remains unclear. In this study, the positive regulation of whiB3 by PhoPR in mycobacteria was characterized. We observed that the expression patterns of the whiB3 gene under acidic conditions are different among mycobacterial species, suggesting that the regulation of whiB3 differs among mycobacteria. A sequence analysis of the whiB3 promoters (whiB3p) from M. tuberculosis and two closely related species, namely, M. marinum and M. smegmatis, showed that the whiB3p regions from M. tuberculosis and M. marinum contain a new type of PhoP box that is absent in the M. smegmatiswhiB3p Direct binding of PhoP to whiB3p from M. tuberculosis and M. marinum but not that from M. smegmatis was validated by in vitro protein-DNA binding assays. The direct activation of whiB3 by PhoPR under acidic conditions was further verified by reverse transcription-quantitative PCR (qRT-PCR) analysis in M. marinum Moreover, mutating the residues important for the phosphorylation pathway of PhoPR in M. marinum abolished the activation of whiB3 expression by PhoPR under acidic conditions, suggesting that low pH triggers the phosphorylation of PhoPR, which in turn activates the transcription of whiB3 Since the PhoP box was only identified in whiB3p of pathogenic mycobacteria, we suggest that the PhoPR-whiB3 regulatory pathway may have evolved to facilitate mycobacterial infection.IMPORTANCE The low pH in macrophages is an important barrier for infection by microbes. The PhoPR two-component regulatory system is required for the response to low pH and plays a role in redox homeostasis in Mycobacterium tuberculosis WhiB3, a cytosolic redox-sensing transcriptional regulator, is also involved in these processes. However, there is no direct evidence to demonstrate the regulation of WhiB3 by PhoPR. In this study, we found that PhoPR directly activates whiB3 expression in response to low pH. An atypical PhoP box in the whiB3 promoters has been identified and is only found in pathogenic mycobacteria, which suggests that the PhoPR-whiB3 regulatory pathway may facilitate mycobacterial infection. This study provides novel information for further characterization of the PhoPR regulon.

Association of ω with the C-Terminal Region of the ß' Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis.

The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the ß' subunit (ß'CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this ß'CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis Sequence alignment of the ω loop and the ß'CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly.IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α2ßß'ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis, and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

RgsA, an RpoS-dependent sRNA, negatively regulates rpoS expression in Pseudomonas aeruginosa.

As a master regulator, the alternative sigma factor RpoS coordinates the transcription of genes associated with protection against environmental stresses in bacteria. In Pseudomonas aeruginosa, RpoS is also involved in quorum sensing and virulence. The cellular RpoS level is regulated at multiple levels, whereas the post-transcriptional regulation of rpoS in P. aeruginosa remains unclear. To identify and characterize small regulatory RNAs (sRNAs) regulating RpoS in P. aeruginosa, an sRNA library expressing a total of 263 sRNAs was constructed to examine their regulatory roles on rpoS expression. Our results demonstrate that rpoS expression is repressed by the RpoS-dependent sRNA RgsA at the post-transcriptional level. Unlike OxyS, an sRNA previously known to repress rpoS expression under oxidative stress in Escherichia coli, RgsA represses rpoS expression during the exponential phase. This repression requires the RNA chaperone Hfq. Furthermore, the 71-77 conserved region of RgsA is necessary for full repression of rpoS expression, and the -25 to +27 region of rpoS mRNA is sufficient for RgsA-mediated rpoS repression. Together, our results not only add RgsA to the RpoS regulatory circuits but also highlight the complexity of interplay between sRNAs and transcriptional regulators in bacteria.

Characterization of a Minimal Type of Promoter Containing the -10 Element and a Guanine at the -14 or -13 Position in Mycobacteria.

Three key promoter elements, i.e., -10, -35, and T-15G-14N, are recognized by the σ subunit of RNA polymerase. Among them, promoters with the -10 element and either -35 or T-15G-14N are known to initiate transcription efficiently, but recent systematic analyses have identified a large group of promoters in Mycobacterium tuberculosis that contain only a -10 consensus. How these promoters initiate transcription remains poorly understood. Here, we show that promoters containing the -10 element and an upstream G located at the -14 or -13 position can successfully initiate transcription in mycobacteria. Importantly, this new type of promoter is active in the absence of other promoter consensuses, suggesting that it is a minimal promoter type. Mutation of the upstream G in promoters decreased the efficiencies of their binding with RNA polymerase and their abilities to initiate transcription in both in vitro and in vivo analyses. A glutamic acid in σ region 3.0 is essential for recognizing G-14 and G-13 and is conserved in both principal and principal-like σ factors in mycobacteria, indicating that recognition of this minimal type of promoter might be a common mechanism for transcription initiation. Consistently, more than 70% of the identified promoters in M. tuberculosis contained G-14 or G-13 upstream of the conserved -10 element, and thousands of promoters in representative mycobacterial species have been predicted using the -10 consensus and G-14 or G-13 Altogether, our study presents a universal mechanism for transcription initiation from a minimal promoter in mycobacteria, which might also be applicable to other bacteria.IMPORTANCE In contrast to the detailed information for recognizing classic promoters in the model organism Escherichia coli, very little is known about how transcription is initiated in the human pathogen Mycobacterium tuberculosis In this study, we characterized a new type of promoter in mycobacteria that requires only a -10 consensus and an upstream G-14 or G-13 Residues important for recognizing the -10 element and the upstream G are conserved in σA and σB from mycobacterial species. According to such features, thousands of promoters in mycobacteria can be predicted using the -10 consensus and G-14 or G-13, which suggests that transcription from this new type of promoter might be widespread. Our findings provide insightful information for characterizing promoters in mycobacteria.

RpoS-dependent sRNA RgsA regulates Fis and AcpP in Pseudomonas aeruginosa.

RgsA is a phylogenetically conserved small regulatory RNA (sRNA) in Pseudomonas species. This sRNA has been shown to be directly controlled by the major stationary phase and stress sigma factor σS (RpoS), and also indirectly regulated by the GacS/GacA two-component system. However, the role and the regulatory targets of this sRNA remain unclear. Here, two direct regulatory targets of RgsA, the mRNAs coding for the global transcriptional regulator Fis and the acyl carrier protein AcpP, were identified in P. aeruginosa. RgsA downregulates the synthesis of Fis and AcpP by base-pairing, and this regulation requires the RNA chaperone protein Hfq. Alignment of RgsA homologs in Pseudomonas revealed a conserved core region, which is strictly required for RgsA target recognition. Specifically, RgsA inhibits fis expression via an 11 + 11 bp RNA duplex, whereas this interaction region is not sufficient for repression and the 35 nt downstream region is also required. Interestingly, two functional start codons initiate fis mRNA translation and both are repressed by RgsA. Furthermore, deletion of rgsA significantly increased swarming motility in P. aeruginosa. Together, this study suggests a novel regulatory role of sRNA in which the versatile transcriptional regulator Fis and the stress regulator RpoS are connected by RgsA.

σ(E) -dependent activation of RbpA controls transcription of the furA-katG operon in response to oxidative stress in mycobacteria.

Mycobacterium tuberculosis adopts various strategies to cope with oxidative stress during infection. Transcriptional regulators, including σ factors, make important contributions to this stress response, but how these proteins cooperate with each other is largely unknown. In this study, the role of RbpA and its cooperation with σ factors in response to oxidative stress are investigated. Knock down expression of rbpA in Mycobacterium smegmatis attenuated bacterial survival in the presence of H2 O2 . Additionally, transcription of the rbpA gene was induced by H2 O2 in a σ(E) -dependent manner. After induction, RbpA interacts with the principal sigma factor, σ(A) , to control the transcription of furA-katG operon, which encodes an H2 O2 scavenging enzyme. Moreover, this regulation is responsible for the role of σ(E) in oxidative response because bacterial survival was attenuated and transcription of the furA-katG operon was down-regulated with H2 O2 treatment in sigE deletion mutant (ΔsigE), and over-expression of RbpA in ΔsigE strain restored all of these phenotypes. Taken together, our study first illustrated a mechanism for σ(E) in response to oxidative stress through regulation of rbpA transcription. This study was also the first to demonstrate that RbpA is required for the full response to oxidative stress by cooperating with the principal σ(A) .