Density of σ70 promoter-like sites in the intergenic regions dictates the redistribution of RNA polymerase during osmotic stress in Escherichia coli.

RNA polymerase (RNAP), the transcription machinery, shows dynamic binding across the genomic DNA under different growth conditions. The genomic features that selectively redistribute the limited RNAP molecules to dictate genome-wide transcription in response to environmental cues remain largely unknown. We chose the bacterial osmotic stress response model to determine genomic features that direct genome-wide redistribution of RNAP during the stress. Genomic mapping of RNAP and transcriptome profiles corresponding to the different temporal states after salt shock were determined. We found rapid redistribution of RNAP across the genome, primarily at σ70 promoters. Three subsets of genes exhibiting differential salt sensitivities were identified. Sequence analysis using an information-theory based σ70 model indicates that the intergenic regions of salt-responsive genes are enriched with a higher density of σ70 promoter-like sites than those of salt-sensitive genes. In addition, the density of promoter-like sites has a positive linear correlation with RNAP binding at different salt concentrations. The RNAP binding contributed by the non-initiating promoter-like sites is important for gene transcription at high salt concentration. Our study demonstrates that hyperdensity of σ70 promoter-like sites in the intergenic regions of salt-responsive genes drives the RNAP redistribution for reprograming the transcriptome to counter osmotic stress.

Nucleolus-like compartmentalization of the transcription machinery in fast-growing bacterial cells.

We have learned a great deal about RNA polymerase (RNA Pol), transcription factors, and the transcriptional regulation mechanisms in prokaryotes for specific genes, operons, or transcriptomes. However, we have only begun to understand how the transcription machinery is three-dimensionally (3D) organized into bacterial chromosome territories to orchestrate the transcription process and to maintain harmony with the replication machinery in the cell. Much progress has been made recently in our understanding of the spatial organization of the transcription machinery in fast-growing Escherichia coli cells using state-of-the-art superresolution imaging techniques. Co-imaging of RNA polymerase (RNA Pol) with DNA and transcription elongation factors involved in ribosomal RNA (rRNA) synthesis, and ribosome biogenesis has revealed similarities between bacteria and eukaryotes in the spatial organization of the transcription machinery for growth genes, most of which are rRNA genes. Evidence supports the notion that RNA Pol molecules are concentrated, forming foci at the clustering of rRNA operons resembling the eukaryotic nucleolus. RNA Pol foci are proposed to be active transcription factories for both rRNA genes expression and ribosome biogenesis to support maximal growth in optimal growing conditions. Thus, in fast-growing bacterial cells, RNA Pol foci mimic eukaryotic Pol I activity, and transcription factories resemble nucleolus-like compartmentation. In addition, the transcription and replication machineries are mostly segregated in space to avoid the conflict between the two major cellular functions in fast-growing cells.

Deacetylation of topoisomerase I is an important physiological function of E. coli CobB.

Escherichia coli topoisomerase I (TopA), a regulator of global and local DNA supercoiling, is modified by Nε-Lysine acetylation. The NAD+-dependent protein deacetylase CobB can reverse both enzymatic and non-enzymatic lysine acetylation modification in E. coli. Here, we show that the absence of CobB in a ΔcobB mutant reduces intracellular TopA catalytic activity and increases negative DNA supercoiling. TopA expression level is elevated as topA transcription responds to the increased negative supercoiling. The slow growth phenotype of the ΔcobB mutant can be partially compensated by further increase of intracellular TopA level via overexpression of recombinant TopA. The relaxation activity of purified TopA is decreased by in vitro non-enzymatic acetyl phosphate mediated lysine acetylation, and the presence of purified CobB protects TopA from inactivation by such non-enzymatic acetylation. The specific activity of TopA expressed from His-tagged fusion construct in the chromosome is inversely proportional to the degree of in vivo lysine acetylation during growth transition and growth arrest. These findings demonstrate that E. coli TopA catalytic activity can be modulated by lysine acetylation-deacetylation, and prevention of TopA inactivation from excess lysine acetylation and consequent increase in negative DNA supercoiling is an important physiological function of the CobB protein deacetylase.

Allosteric Activation of Bacterial Swi2/Snf2 (Switch/Sucrose Non-fermentable) Protein RapA by RNA Polymerase: BIOCHEMICAL AND STRUCTURAL STUDIES.

Members of the Swi2/Snf2 (switch/sucrose non-fermentable) family depend on their ATPase activity to mobilize nucleic acid-protein complexes for gene expression. In bacteria, RapA is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein that mediates RNAP recycling during transcription. It is known that the ATPase activity of RapA is stimulated by its interaction with RNAP. It is not known, however, how the RapA-RNAP interaction activates the enzyme. Previously, we determined the crystal structure of RapA. The structure revealed the dynamic nature of its N-terminal domain (Ntd), which prompted us to elucidate the solution structure and activity of both the full-length protein and its Ntd-truncated mutant (RapAΔN). Here, we report the ATPase activity of RapA and RapAΔN in the absence or presence of RNAP and the solution structures of RapA and RapAΔN either ligand-free or in complex with RNAP. Determined by small-angle x-ray scattering, the solution structures reveal a new conformation of RapA, define the binding mode and binding site of RapA on RNAP, and show that the binding sites of RapA and σ(70) on the surface of RNAP largely overlap. We conclude that the ATPase activity of RapA is inhibited by its Ntd but stimulated by RNAP in an allosteric fashion and that the conformational changes of RapA and its interaction with RNAP are essential for RNAP recycling. These and previous findings outline the functional cycle of RapA, which increases our understanding of the mechanism and regulation of Swi2/Snf2 proteins in general and of RapA in particular. The new structural information also leads to a hypothetical model of RapA in complex with RNAP immobilized during transcription.

Spatial organization of transcription machinery and its segregation from the replisome in fast-growing bacterial cells.

In a fast-growing Escherichia coli cell, most RNA polymerase (RNAP) is allocated to rRNA synthesis forming transcription foci at clusters of rrn operons or bacterial nucleolus, and each of the several nascent nucleoids contains multiple pairs of replication forks. The composition of transcription foci has not been determined. In addition, how the transcription machinery is three-dimensionally organized to promote cell growth in concord with replication machinery in the nucleoid remains essentially unknown. Here, we determine the spatial and functional landscapes of transcription and replication machineries in fast-growing E. coli cells using super-resolution-structured illumination microscopy. Co-images of RNAP and DNA reveal spatial compartmentation and duplication of the transcription foci at the surface of the bacterial chromosome, encompassing multiple nascent nucleoids. Transcription foci cluster with NusA and NusB, which are the rrn anti-termination system and are associated with nascent rRNAs. However, transcription foci tend to separate from SeqA and SSB foci, which track DNA replication forks and/or the replisomes, demonstrating that transcription machinery and replisome are mostly located in different chromosomal territories to maintain harmony between the two major cellular functions in fast-growing cells. Our study suggests that bacterial chromosomes are spatially and functionally organized, analogous to eukaryotes.

Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.

Transcription fidelity is critical for maintaining the accurate flow of genetic information. The study of transcription fidelity has been limited because the intrinsic error rate of transcription is obscured by the higher error rate of translation, making identification of phenotypes associated with transcription infidelity challenging. Slippage of elongating RNA polymerase (RNAP) on homopolymeric A/T tracts in DNA represents a special type of transcription error leading to disruption of open reading frames in Escherichia coli mRNA. However, the regions in RNAP involved in elongation slippage and its molecular mechanism are unknown. We constructed an A/T tract that is out of frame relative to a downstream lacZ gene on the chromosome to examine transcriptional slippage during elongation. Further, we developed a genetic system that enabled us for the first time to isolate and characterize E. coli RNAP mutants with altered transcriptional slippage in vivo. We identified several amino acid residues in the ß subunit of RNAP that affect slippage in vivo and in vitro. Interestingly, these highly clustered residues are located near the RNA strand of the RNA-DNA hybrid in the elongation complex. Our E. coli study complements an accompanying study of slippage by yeast RNAP II and provides the basis for future studies on the mechanism of transcription fidelity.

Structure and function of RapA: a bacterial Swi2/Snf2 protein required for RNA polymerase recycling in transcription.

One of the hallmarks of the Swi2/Snf2 family members is their ability to modify the interaction between DNA-binding protein and DNA in controlling gene expression. The studies of Swi2/Snf2 have been mostly focused on their roles in chromatin and/or nucleosome remodeling in eukaryotes. A bacterial Swi2/Snf2 protein named RapA from Escherichia coli is a unique addition to these studies. RapA is an RNA polymerase (RNAP)-associated protein and an ATPase. It binds nucleic acids including RNA and DNA. The ATPase activity of RapA is stimulated by its interaction with RNAP, but not with nucleic acids. RapA and the major sigma factor σ70 compete for binding to core RNAP. After one transcription cycle in vitro, RNAP is immobilized in an undefined posttranscription/posttermination complex (PTC), thus becoming unavailable for reuse. RapA stimulates RNAP recycling by ATPase-dependent remodeling of PTC, leading to the release of sequestered RNAP, which then becomes available for reuse in another cycle of transcription. Recently, the crystal structure of RapA that is also the first full-length structure for the entire Swi2/Snf2 family was determined. The structure provides a framework for future studies of the mechanism of RNAP recycling in transcription. This article is part of a Special Issue entitled: Snf2/Swi2 ATPase structure and function.

Polyphosphate binds to the principal sigma factor of RNA polymerase during starvation response in Helicobacter pylori.

Helicobacter pylori persists deep in the human gastric mucus layer in a harsh, nutrient-poor environment. Survival under these conditions depends on the ability of this human pathogen to invoke starvation/stress responses when needed. Unlike many bacteria, H. pylori lacks starvation/stress-responding alternative sigma factors, suggesting an additional mechanism might have evolved in this bacterium. Helicobacter pylori produces polyphosphate; however, the role and target of polyphosphate during starvation/stress have not been identified. Here we show that polyphosphate accumulated during nutrient starvation directly targets transcriptional machinery by binding to the principal sigma factor in H. pylori, uncovering a novel mechanism in microbial stress response. A positively charged Lys-rich region at the N-terminal domain of the major sigma factor is identified as the binding region for polyphosphate (region P) in vivo and in vitro, revealing a new element in sigma 70 family proteins. This interaction is biologically significant because mutant strains defective in the interaction undergo premature cell death during starvation. We suggested that polyphosphate is a second messenger employed by H. pylori to mediate gene expression during starvation/stress. The putative 'region P' is present in sigma factors of other human pathogens, suggesting that the uncovered interaction might be a general strategy employed by other pathogens.

Structure of RapA, a Swi2/Snf2 protein that recycles RNA polymerase during transcription.

RapA, as abundant as sigma70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. We report a structure of RapA that is also a full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and double-stranded DNA (dsDNA) suggests that RapA may bind to and translocate on dsDNA. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex for transcription reinitiation. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by sigma70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription.

[Nicotinamide-adenine dinucleotide phosphate oxidase p22phox expression in induced sputum cells for patients with obstructive sleep apnea hypopnea syndrome].

OBJECTIVE: the aim of the study was to observe the change of NADPH oxidase p22phox expression in the induced sputum cells for patients with obstructive sleep apnea hypopnea syndrome (OSAHS), and its relationship with the severity of OSAHS. METHODS: thirty OSAHS patients and 23 healthy controls were recruited in the study. Before sleep and in the next morning sputum induction was performed twice for each subject, cell numbers and differentials in induced sputum were counted. NADPH oxidase p22phox mRNA expression was measured in induced sputum cells by RT-PCR and the protein was measured by immunocytochemistry. RESULTS: the OSAHS group showed a significant higher percentage of neutrophils and a lower percentage of macrophages in the induced sputum compared to the control group (P < 0.05). Macrophages and neutrophils represented the main cell types expressing NADPH oxidase p22phox in induced sputum cells by immunocytochemistry. In the control group, level of NADPH oxidase p22phox mRNA and percentages of NADPH oxidase p22phox positive neutrophils and macrophages in sputum samples collected both in the morning and before sleep was significantly lower than in OSAHS group (P < 0.05). In OSAHS group, higher levels of NADPH oxidase p22phox mRNA and percentages of NADPH oxidase p22phox positive neutrophils and macrophages were found in the morning than at pre-sleep. A negative correlation was found between levels of NADPH oxidase p22phox mRNA and percentage of NADPH oxidase p22phox positive neutrophils and macrophages in the morning sputum were negatively associated with LspO(2), but positively associated with AHI. CONCLUSION: these findings suggest that there are changes of NADPH oxidase p22phox expression in induced sputum cells of OSAHS patients, and these changes are related to the severity of OSAHS.

Design of an adaptive stator for bundled piezo-walk motors.

Bundled piezoelectric motors combine several actuators to achieve high output power. However, mutual interference between the actuators leads to reduction in working efficiency. This work presents an adaptive stator that can reduce the mutual interference in bundled piezowalk motors. The stator consists of leaf springs for improving motor contact condition and proof masses that serve as an inertial body for maintaining high output force. Parameters of the proof masses and leaf springs are analyzed, and the working zone of the stator is discussed. A prototype of a linear motor with the designed adaptive stator is fabricated and tested. The maximum speed of a six-leg motor is 103 mm/s, and the stall force is approximately 1.2 N, driven with sinusoidal waveforms of 25Vpp at 30 kHz. Mutual interference between actuators is reduced remarkably with the adaptive stator. A comparison of six- and four-leg motors proves that motor performance has a linear relationship with the number of actuators, thereby indicating the potential of increasing output capability with additional actuators.

Regulation of cell growth during serum starvation and bacterial survival in macrophages by the bifunctional enzyme SpoT in Helicobacter pylori.

In Helicobacter pylori the stringent response is mediated solely by spoT. The spoT gene is known to encode (p)ppGpp synthetase activity and is required for H. pylori survival in the stationary phase. However, neither the hydrolase activity of the H. pylori SpoT protein nor the role of SpoT in the regulation of growth during serum starvation and intracellular survival of H. pylori in macrophages has been determined. In this study, we examined the effects of SpoT on these factors. Our results showed that the H. pylori spoT gene encodes a bifunctional enzyme with both a hydrolase activity and the previously described (p)ppGpp synthetase activity, as determined by introducing the gene into Escherichia coli relA and spoT defective strains. Also, we found that SpoT mediates a serum starvation response, which not only restricts the growth but also maintains the helical morphology of H. pylori. Strikingly, a spoT null mutant was able to grow to a higher density in serum-free medium than the wild-type strain, mimicking the "relaxed" growth phenotype of an E. coli relA mutant during amino acid starvation. Finally, SpoT was found to be important for intracellular survival in macrophages during phagocytosis. The unique role of (p)ppGpp in cell growth during serum starvation, in the stress response, and in the persistence of H. pylori is discussed.

[Effect of NADPH oxidase activity inhibitor apocynin on blood pressure in rats exposed to chronic intermittent hypoxia and the possible mechanisms].

OBJECTIVE: To investigate the effect of NADPH oxidase activity inhibitor apocynin on the blood pressure of rats exposed to chronic intermittent hypoxia (CIH). METHODS: Thirty healthy male SD rats were randomly divided into 3 groups of 10 each, a CIH group, an apocynin treatment group and a control group. The systolic blood pressure (SBP) was measured with tail-cuff method. RT-PCR and Western blot were used to examine mRNA and protein expression of NADPH oxidase subunit p22phox. The level of MDA, SOD, NO and .O(2)(-) were detected by colorimetric method. RESULTS: Compared to the normal group, the expression of p22phox mRNA and protein were significantly increased in CIH group (q = 3.202, 6.522, P < 0.05). There were no statistic difference of p22phox mRNA and protein expression between CIH group and apocynin treatment group (P > 0.05). Compared with those of CIH group, the levels of MDA, .O(2)(-) and the systolic blood pressure decreased significantly (P < 0.05), while SOD and NO activity increased significantly (q = 6.960, 4.385, P < 0.05) in apocynin treatment group. CONCLUSIONS: These results indicate that NADPH oxidase up-expression is closely associated with hypertension in OSAHS. Inhibition of NADPH oxidase activity may be hopefully served as a useful strategy for prevention and treatment of OSAHS-induced hypertension.

Extrachromosomal Nucleolus-Like Compartmentalization by a Plasmid-Borne Ribosomal RNA Operon and Its Role in Nucleoid Compaction.

In the fast-growing Escherichia coli cells, RNA polymerase (RNAP) molecules are concentrated and form foci at clusters of ribosomal RNA (rRNA) operons resembling eukaryotic nucleolus. The bacterial nucleolus-like organization, spatially compartmentalized at the surface of the compact bacterial chromosome (nucleoid), serves as transcription factories for rRNA synthesis and ribosome biogenesis, which influences the organization of the nucleoid. Unlike wild type that has seven rRNA operons in the genome in a mutant that has six (Δ6rrn) rRNA operons deleted in the genome, there are no apparent transcription foci and the nucleoid becomes uncompacted, indicating that formation of RNAP foci requires multiple copies of rRNA operons clustered in space and is critical for nucleoid compaction. It has not been determined, however, whether a multicopy plasmid-borne rRNA operon (prrnB) could substitute the multiple chromosomal rRNA operons for the organization of the bacterial nucleolus-like structure in the mutants of Δ6rrn and Δ7rrn that has all seven rRNA operons deleted in the genome. We hypothesized that extrachromosomal nucleolus-like structures are similarly organized and functional in trans from prrnB in these mutants. In this report, using multicolor images of three-dimensional superresolution Structured Illumination Microscopy (3D-SIM), we determined the distributions of both RNAP and NusB that are a transcription factor involved in rRNA synthesis and ribosome biogenesis, prrnB clustering, and nucleoid structure in these two mutants in response to environmental cues. Our results found that the extrachromosomal nucleolus-like organization tends to be spatially located at the poles of the mutant cells. In addition, formation of RNAP foci at the extrachromosomal nucleolus-like structure condenses the nucleoid, supporting the idea that active transcription at the nucleolus-like organization is a driving force in nucleoid compaction.

Growth rate regulation in Escherichia coli.

Growth rate regulation in bacteria has been an important issue in bacterial physiology for the past 50 years. This review, using Escherichia coli as a paradigm, summarizes the mechanisms for the regulation of rRNA synthesis in the context of systems biology, particularly, in the context of genome-wide competition for limited RNA polymerase (RNAP) in the cell under different growth conditions including nutrient starvation. The specific location of the seven rrn operons in the chromosome and the unique properties of the rrn promoters contribute to growth rate regulation. The length of the rrn transcripts, coupled with gene dosage effects, influence the distribution of RNAP on the chromosome in response to growth rate. Regulation of rRNA synthesis depends on multiple factors that affect the structure of the nucleoid and the allocation of RNAP for global gene expression. The magic spot ppGpp, which acts with DksA synergistically, is a key effector in both the growth rate regulation and the stringent response induced by nutrient starvation, mainly because the ppGpp level changes in response to environmental cues. It regulates rRNA synthesis via a cascade of events including both transcription initiation and elongation, and can be explained by an RNAP redistribution (allocation) model.

Relationship between reduced nicotinamide adenine dinucleotide phosphate oxidase subunit p22phox gene polymorphism and obstructive sleep apnea-hypopnea syndrome in the Chinese Han population.

BACKGROUND: Increased production of reactive oxygen species (ROS) is thought to play a major role in the pathogenesis of obstructive sleep apnea-hypopnea syndrome (OSAHS). The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is an important source of ROS. The p22phox subunit is polymorphic with a C242T variant that changes histidine-72 for a tyrosine in the potential heme binding site. This study aimed to investigate the relationship between NADPH oxidase subunit p22phox gene polymorphism and OSAHS. METHODS: The genotypes of p22phox polymorphism were determined by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) assay in 176 unrelated subjects of the Han population in southern region of China (including 107 OSAHS subjects and 69 non-OSAHS subjects), while the plasma concentration of superoxide dismutase (SOD) was detected in the two groups, and p22phox mRNA expression in peripheral blood mononuclear cell (PBMC) was determined with reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The phagocyte NADPH oxidase subunit p22phox mRNA expression was significantly increased in the OSAHS group than that in the non-OSAHS group (P < 0.01). Compared with the non-OSAHS control group ((85.31 +/- 9.23) U/ml), the levels of SOD were lower in patients with OSAHS ((59.65 +/- 11.61) U/ml (P < 0.01). There were significant differences in genotypes distribution in p22phox polymorphism between the two groups (P = 0.02). Compared with the non-OSAHS control group, the OSAHS group had a significantly higher T allele frequency in p22phox polymorphism (P = 0.03). There were independent effects of p22phox polymorphism on body mass index (BMI), neck circumference (NC), waist-to-hip ratio (WHR) in the OSAHS group, and the carriers of the T allele of p22phox polymorphism had greater NC, WHR, systolic blood pressure (SBP), diastolic blood pressure (DBP) and apnea-hypopnea index (AHI) (P < 0.05), but the carriers of the T allele had lower SOD (P < 0.01) and lowest SaO(2) (P = 0.04). There was no significant difference in p22phox mRNA expression between the OSAHS groups with or without T allele (P = 0.45). CONCLUSIONS: The NADPH oxidase subunit p22phox gene polymorphism may be associated with susceptibility to OSAHS, and it may be an important candidate gene for OSAHS.

Cross-resistance of Escherichia coli RNA polymerases conferring rifampin resistance to different antibiotics.

In this study we further defined the rifampin-binding sites in Escherichia coli RNA polymerase (RNAP) and determined the relationship between rifampin-binding sites and the binding sites of other antibiotics, including two rifamycin derivatives, rifabutin and rifapentine, and streptolydigin and sorangicin A, which are unrelated to rifampin, using a purified in vitro system. We found that there is almost a complete correlation between resistance to rifampin (Rif(r)) and reduced rifampin binding to 12 RNAPs purified from different rpoB Rif(r) mutants and a complete cross-resistance among the different rifamycin derivatives. Most Rif(r) RNAPs were sensitive to streptolydigin, although some exhibited weak resistance to this antibiotic. However, 5 out of the 12 Rif(r) RNAPs were partially resistant to sorangicin A, and one was completely cross-resistant to sorangicin A, indicating that the binding site(s) for these two antibiotics overlaps. Both rifampin and sorangicin A inhibited the transition step between transcription initiation and elongation; however, longer abortive initiation products were produced in the presence of the latter, indicating that the binding site for sorangicin A is within the rifampin-binding site. Competition experiments of different antibiotics with (3)H-labeled rifampin for binding to wild-type RNAP further confirmed that the binding sites for rifampin, rifabutin, rifapentine, and sorangicin A are shared, whereas the binding sites for rifampin and streptolydigin are distinct. Because Rif(r) mutations are highly conserved in eubacteria, our results indicate that this set of Rif(r) mutant RNAPs can be used to screen for new antibiotics that will inhibit the growth of Rif(r) pathogenic bacteria.