scholarly journals Dual Negative Control of spx Transcription Initiation from the P3 Promoter by Repressors PerR and YodB in Bacillus subtilis

2006 ◽  
Vol 189 (5) ◽  
pp. 1736-1744 ◽  
Author(s):  
Montira Leelakriangsak ◽  
Kazuo Kobayashi ◽  
Peter Zuber
Keyword(s):  
Oxidative Stress ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Point Mutations ◽  
Negative Control ◽  
Response To Treatment ◽  
Additive Increase

ABSTRACT The spx gene encodes an RNA polymerase-binding protein that exerts negative and positive transcriptional control in response to oxidative stress in Bacillus subtilis. It resides in the yjbC-spx operon and is transcribed from at least five promoters located in the yjbC regulatory region or in the yjbC-spx intergenic region. Induction of spx transcription in response to treatment with the thiol-specific oxidant diamide is the result of transcription initiation at the P3 promoter located upstream of the spx coding sequence. Previous studies conducted elsewhere and analyses of transcription factor mutants using transformation array technology have uncovered two transcriptional repressors, PerR and YodB, that target the cis-acting negative control elements of the P3 promoter. Expression of an spx-bgaB fusion carrying the P3 promoter is elevated in a yodB or perR mutant, and an additive increase in expression was observed in a yodB perR double mutant. Primer extension analysis of spx RNA shows the same additive increase in P3 transcript levels in yodB perR mutant cells. Purified YodB and PerR repress spx transcription in vitro when wild-type spx P3 promoter DNA was used as a template. Point mutations at positions within the P3 promoter relieved YodB-dependent repression, while a point mutation at position +24 reduced PerR repression. DNase I footprinting analysis showed that YodB protects a region that includes the P3 −10 and −35 regions, while PerR binds to a region downstream of the P3 transcriptional start site. The binding of both repressors is impaired by the treatment of footprinting reactions with diamide or hydrogen peroxide. The study has uncovered a mechanism of dual negative control that relates to the oxidative stress response of gram-positive bacteria.

scholarly journals Evidence that Oxidative Stress InducesspxA2Transcription in Bacillus anthracis Sterne through a Mechanism Requiring SpxA1 and Positive Autoregulation

2016 ◽  
Vol 198 (21) ◽  
pp. 2902-2913 ◽  
Author(s):  
Skye Barendt ◽  
Cierra Birch ◽  
Lea Mbengi ◽  
Peter Zuber
Keyword(s):  
Oxidative Stress ◽  
Bacillus Anthracis ◽  
Transcription Initiation ◽  
Bacterial Species ◽  
Negative Control ◽  
Mobility Shift ◽  
Content Type ◽  
Oxidative Protein Damage

ABSTRACTBacillus anthracispossesses two paralogs of the transcriptional regulator, Spx. SpxA1 and SpxA2 interact with RNA polymerase (RNAP) to activate the transcription of genes implicated in the prevention and alleviation of oxidative protein damage. ThespxA2gene is highly upregulated in infected macrophages, but how this is achieved is unknown. Previous studies have shown that thespxA2gene was under negative control by the Rrf2 family repressor protein, SaiR, whose activity is sensitive to oxidative stress. These studies also suggested thatspxA2was under positive autoregulation. In the present study, we show byin vivoandin vitroanalyses thatspxA2is under direct autoregulation but is also dependent on the SpxA1 paralogous protein. The deletion of eitherspxA1orspxA2reduced the diamide-inducible expression of anspxA2-lacZconstruct.In vitrotranscription reactions using purifiedB. anthracisRNAP showed that SpxA1 and SpxA2 protein stimulates transcription from a DNA fragment containing thespxA2promoter. Ectopically positionedspxA2-lacZfusion requires both SpxA1 and SpxA2 for expression, but the requirement for SpxA1 is partially overcome whensaiRis deleted. Electrophoretic mobility shift assays showed that SpxA1 and SpxA2 enhance the affinity of RNAP forspxA2promoter DNA and that this activity is sensitive to reductant. We hypothesize that the previously observed upregulation ofspxA2in the oxidative environment of the macrophage is at least partly due to SpxA1-mediated SaiR repressor inactivation and the positive autoregulation ofspxA2transcription.IMPORTANCERegulators of transcription initiation are known to govern the expression of genes required for virulence in pathogenic bacterial species. Members of the Spx family of transcription factors function in control of genes required for virulence and viability in low-GC Gram-positive bacteria. InBacillus anthracis, thespxA2gene is highly induced in infected macrophages, which suggests an important role in the control of virulence gene expression during the anthrax disease state. We provide evidence that elevated concentrations of oxidized, active SpxA2 result from an autoregulatory positive-feedback loop drivingspxA2transcription.

scholarly journals Bacillus subtilis ResD Induces Expression of the Potential Regulatory Genes yclJK upon Oxygen Limitation

2004 ◽  
Vol 186 (19) ◽  
pp. 6477-6484 ◽  
Author(s):  
Elisabeth Härtig ◽  
Hao Geng ◽  
Anja Hartmann ◽  
Angela Hubacek ◽  
Richard Münch ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Regulatory System ◽  
Oxygen Limitation ◽  
Anaerobic Growth ◽  
Nucleotide Substitutions ◽  
Anaerobic Induction ◽  
Two Component

ABSTRACT Transcription of the yclJK operon, which encodes a potential two-component regulatory system, is activated in response to oxygen limitation in Bacillus subtilis. Northern blot analysis and assays of yclJ-lacZ reporter gene fusion activity revealed that the anaerobic induction is dependent on another two-component signal transduction system encoded by resDE. ResDE was previously shown to be required for the induction of anaerobic energy metabolism. Electrophoretic mobility shift assays and DNase I footprinting experiments showed that the response regulator ResD binds specifically to the yclJK regulatory region upstream of the transcriptional start site. In vitro transcription experiments demonstrated that ResD is sufficient to activate yclJ transcription. The phosphorylation of ResD by its sensor kinase, ResE, highly stimulates its activity as a transcriptional activator. Multiple nucleotide substitutions in the ResD binding regions of the yclJ promoter abolished ResD binding in vitro and prevented the anaerobic induction of yclJK in vivo. A weight matrix for the ResD binding site was defined by a bioinformatic approach. The results obtained suggest the existence of a new branch of the complex regulatory system employed for the adaptation of B. subtilis to anaerobic growth conditions.

scholarly journals Transcription from the P3 Promoter of the Bacillus subtilis spx Gene Is Induced in Response to Disulfide Stress

2006 ◽  
Vol 189 (5) ◽  
pp. 1727-1735 ◽  
Author(s):  
Montira Leelakriangsak ◽  
Peter Zuber
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Intergenic Region ◽  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Promoter Sequence ◽  
Primer Extension ◽  
Control Element ◽  
Start Site

ABSTRACT The spx gene of Bacillus subtilis encodes a global regulator that controls transcription initiation in response to oxidative stress by interaction with RNA polymerase (RNAP). It is located in a dicistronic operon with the yjbC gene. The spx gene DNA complements an spx null mutation with respect to disulfide stress resistance, suggesting that spx is transcribed from a promoter located in the intergenic region of yjbC and spx. Transcription of the yjbC-spx operon has been reported to be driven by four promoters, three (P1, P2, and PB) residing upstream of yjbC and one (PM) located in the intergenic region between yjbC and spx. Primer extension analysis uncovered a second intergenic promoter, P3, from which transcription is elevated in cells treated with the thiol-specific oxidant diamide. P3 is utilized by the σA form of RNA polymerase in vitro without the involvement of a transcriptional activator. Transcriptional induction from P3 did not require an Spx-RNAP interaction and was observed in a deletion mutant lacking DNA upstream of position −40 of the P3 promoter start site. Deletion mutants with endpoints 3′ to the P3 transcriptional start site (positions +5, +15, and +30) showed near-constitutive transcription at the induced level, indicating the presence of a negative control element downstream of the P3 promoter sequence. Point mutations characterized by bgaB fusion expression and primer extension analyses uncovered evidence for a second cis-acting site in the P3 promoter sequence itself. The data indicate that spx transcription is under negative transcriptional control that is reversed when disulfide stress is encountered.

scholarly journals Investigation of the Wilson gene ATP7B transcriptional start site and the effect of core promoter alterations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Clemens Höflich ◽  
Angela Brieger ◽  
Stefan Zeuzem ◽  
Guido Plotz
Keyword(s):  
Wilson Disease ◽  
Transcription Initiation ◽  
Transcriptional Activity ◽  
Core Promoter ◽  
Transcriptional Start Site ◽  
Copper Metabolism ◽  
Biologically Relevant ◽  
The Core ◽  
Translational Start

AbstractPathogenic genetic variants in the ATP7B gene cause Wilson disease, a recessive disorder of copper metabolism showing a significant variability in clinical phenotype. Promoter mutations have been rarely reported, and controversial data exist on the site of transcription initiation (the core promoter). We quantitatively investigated transcription initiation and found it to be located in immediate proximity of the translational start. The effects human single-nucleotide alterations of conserved bases in the core promoter on transcriptional activity were moderate, explaining why clearly pathogenic mutations within the core promoter have not been reported. Furthermore, the core promoter contains two frequent polymorphisms (rs148013251 and rs2277448) that could contribute to phenotypical variability in Wilson disease patients with incompletely inactivating mutations. However, neither polymorphism significantly modulated ATP7B expression in vitro, nor were copper household parameters in healthy probands affected. In summary, the investigations allowed to determine the biologically relevant site of ATP7B transcription initiation and demonstrated that genetic variations in this site, although being the focus of transcriptional activity, do not contribute significantly to Wilson disease pathogenesis.

8. Evaluation of Snow Fungus Polysaccharides on benzo[a]pyrene-induced DNA Damage

2018 ◽  
Author(s):  
Daisy Liu
Keyword(s):  
Oxidative Stress ◽  
Dna Damage ◽  
Free Radical ◽  
Radical Scavenging ◽  
Chinese Hamster ◽  
Negative Control ◽  
Lung Fibroblasts ◽  
Protective Effects ◽  
Tremella Fuciformis

Snow fungus, Tremella fuciformis, has been demonstrated to have numerous health benefits including purported chemopreventive properties due to free radical-scavenging ability. Protective effects derived from snow fungus polysaccharides are evaluated on Chinese hamster lung fibroblasts (CCL-39) exposed to carcinogen benzo[a]pyrene known to cause free radical formation and oxidative stress to cells. In this experiment, it was hypothesized that the naturally occurring polysaccharides in snow fungus are able to protect against or reduce oxidative stress-induced DNA damage. Polysaccharides were isolated through an alkaline extraction and in-vitro digestion. DNA damage was measured using the single-cell gel electrophoresis comet assay after exposure to benzo[a]pyrene and polysaccharide extract to lung fibroblasts. Results were calculated using the mean and standard deviation data of tail length and area, respectively. Each damaged cell was measured and analyzed through ImageJ Editing Software. The results indicate a promising trend which depict snow fungus polysaccharides yielding lower levels of DNA damage compared to cells exposed to benzo[a]pyrene and compared to the negative control (phosphate buffered saline and Dulbecco’s cell medium). This study suggests polysaccharides from Tremella fuciformis could truly prevent cellular DNA damage by protecting against oxidative stress.

Molecular characterization of a developmentally regulated porcine skeletal myosin heavy chain gene and its 5′ regulatory region

1995 ◽  
Vol 108 (4) ◽  
pp. 1779-1789 ◽  
Author(s):  
K.C. Chang ◽  
K. Fernandes ◽  
M.J. Dauncey
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Chain Gene ◽  
Specific Expression ◽  
Slow Fibre ◽  
Skeletal Myosin

Members of the myosin heavy chain (MyHC) gene family show developmental stage- and spatial-specificity of expression. We report on the characterization and identification of a porcine skeletal fast MyHC gene, including its corresponding 5′ end cDNA and 5′ regulatory region. This MyHC isoform was found exclusively in skeletal muscles from about the last quarter of gestation through to adulthood. Expression of this isoform was higher postnatally and its spatial distribution resembled a rosette cluster; each with a ring of fast fibres surrounding a central slow fibre. This rosette pattern was absent in the adult diaphragm but about 20% of the fibres continued to express this MyHC isoform. Further in vivo expression studies, in a variety of morphologically and functionally diverse muscles, showed that this particular skeletal MyHC isoform was expressed in fast oxidative-glycolytic fibres, suggesting that it was the equivalent of the fast IIA isoform. Two domains in the upstream regulatory region were found to confer differentiation-specific expression on C2 myotubes (−1007 to -828 and -455 to -101), based on in vitro transient expression assays using the chloramphenicol acetyltransferase (CAT) reporter gene. Interestingly, for high levels of CAT expression to occur, a 3′ region, extending from the transcriptional start site to part. of intron 2, must be present in all the DNA constructs used.

scholarly journals Molecular Mechanisms Underlying the Positive Stringent Response of the Bacillus subtilis ilv-leu Operon, Involved in the Biosynthesis of Branched-Chain Amino Acids

2008 ◽  
Vol 190 (18) ◽  
pp. 6134-6147 ◽  
Author(s):  
Shigeo Tojo ◽  
Takenori Satomura ◽  
Kanako Kumamoto ◽  
Kazutake Hirooka ◽  
Yasutaro Fujita
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Transcription Initiation ◽  
Molecular Mechanisms ◽  
Stringent Response ◽  
Branched Chain Amino Acids ◽  
Base Substitution ◽  
Branched Chain

ABSTRACT Branched-chain amino acids are the most abundant amino acids in proteins. The Bacillus subtilis ilv-leu operon is involved in the biosynthesis of branched-chain amino acids. This operon exhibits a RelA-dependent positive stringent response to amino acid starvation. We investigated this positive stringent response upon lysine starvation as well as decoyinine treatment. Deletion analysis involving various lacZ fusions revealed two molecular mechanisms underlying the positive stringent response of ilv-leu, i.e., CodY-dependent and -independent mechanisms. The former is most likely triggered by the decrease in the in vivo concentration of GTP upon lysine starvation, GTP being a corepressor of the CodY protein. So, the GTP decrease derepressed ilv-leu expression through detachment of the CodY protein from its cis elements upstream of the ilv-leu promoter. By means of base substitution and in vitro transcription analyses, the latter (CodY-independent) mechanism was found to comprise the modulation of the transcription initiation frequency, which likely depends on fluctuation of the in vivo RNA polymerase substrate concentrations after stringent treatment, and to involve at least the base species of adenine at the 5′ end of the ilv-leu transcript. As discussed, this mechanism is presumably distinct from that for B. subtilis rrn operons, which involves changes in the in vivo concentration of the initiating GTP.

scholarly journals σIfromBacillus subtilis: Impact on Gene Expression and Characterization of σI-Dependent Transcription That Requires New Types of Promoters with Extended −35 and −10 Elements

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Olga Ramaniuk ◽  
Martin Převorovský ◽  
Jiří Pospíšil ◽  
Dragana Vítovská ◽  
Olga Kofroňová ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Iron Metabolism ◽  
Transcription Initiation ◽  
Iron Homeostasis ◽  
Model Organism ◽  
Content Type ◽  
Σ Factor

ABSTRACTThe σIsigma factor fromBacillus subtilisis a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σI-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σI. Further analysis revealed that the majority of these genes were affected indirectly by σI. The σIregulon, i.e., the genes directly regulated by σI, consists of 16 genes, of which eight (thedhbandykuoperons) are involved in iron metabolism. The involvement of σIin iron metabolism was confirmed phenotypically. Next, we set up anin vitrotranscription system and defined and experimentally validated the promoter sequence logo that, in addition to −35 and −10 regions, also contains extended −35 and −10 motifs. Thus, σI-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organismB. subtilis.IMPORTANCEIn bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σIregulon from the industrially important model Gram-positive bacteriumBacillus subtilis. We reveal that σIaffects expression of ∼130 genes, of which 16 are directly regulated by σI, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σI-dependent transcription. This study thus provides a comprehensive view on this underexplored component of theB. subtilistranscription machinery.

scholarly journals Phase Variation of Ag43 Is Independent of the Oxidation State of OxyR

2003 ◽  
Vol 185 (7) ◽  
pp. 2203-2209 ◽  
Author(s):  
Anu Wallecha ◽  
Jason Correnti ◽  
Vincent Munster ◽  
Marjan van der Woude
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Oxidation State ◽  
Regulatory Region ◽  
Phase Variation ◽  
In Vitro Transcription ◽  
Reduced Form ◽  
Target Sequences

ABSTRACT OxyR is a DNA binding protein that differentially regulates a cell's response to hydrogen peroxide-mediated oxidative stress. We previously reported that the reduced form of OxyR is sufficient for repression of transcription of agn43 from unmethylated template DNA, which is essential for deoxyadenosine methylase (Dam)- and OxyR-dependent phase variation of agn43. Here we provide evidence that the oxidized form of OxyR [OxyR(ox)] also represses agn43 transcription. In vivo, we found that exogenous addition of hydrogen peroxide, sufficient to oxidize OxyR, did not affect the expression of agn43. OxyR(ox) repressed in vitro transcription but only from an unmethylated agn43 template. The −10 sequence of the promoter and three Dam target sequences were protected in an in vitro DNase I footprint assay by OxyR(ox). Furthermore, OxyR(ox) bound to the agn43 regulatory region DNA with an affinity similar to that for the regulatory regions of katG and oxyS, which are activated by OxyR(ox), indicating that binding at agn43 can occur at biologically relevant concentrations. OxyR-dependent regulation of Ag43 expression is therefore unusual in firstly that OxyR binding at agn43 is dependent on the methylation state of Dam target sequences in its binding site and secondly that OxyR-dependent repression appears to be independent of hydrogen-peroxide mediated oxidative stress and the oxidation state of OxyR.

scholarly journals Improvement of α-Amylase Production by Modulation of Ribosomal Component Protein S12 in Bacillus subtilis 168

2006 ◽  
Vol 72 (1) ◽  
pp. 71-77 ◽  
Author(s):  
Kazuhiko Kurosawa ◽  
Takeshi Hosaka ◽  
Norimasa Tamehiro ◽  
Takashi Inaoka ◽  
Kozo Ochi
Keyword(s):  
Bacillus Subtilis ◽  
Antibiotic Production ◽  
Point Mutations ◽  
Protease Production ◽  
Gene Encoding ◽  
Translational Activity ◽  
Amylase Production ◽  
Ribosomal Protein S12

ABSTRACT The capacity of ribosomal modification to improve antibiotic production by Streptomyces spp. has already been demonstrated. Here we show that introduction of mutations that produce streptomycin resistance (str) also enhances α-amylase (and protease) production by a strain of Bacillus subtilis as estimated by measuring the enzyme activity. The str mutations are point mutations within rpsL, the gene encoding the ribosomal protein S12. In vivo as well as in vitro poly(U)-directed cell-free translation systems showed that among the various rpsL mutations K56R (which corresponds to position 42 in E. coli) was particularly effective at enhancing α-amylase production. Cells harboring the K56R mutant ribosome exhibited enhanced translational activity during the stationary phase of cell growth. In addition, the K56R mutant ribosome exhibited increased 70S complex stability in the presence of low Mg2+ concentrations. We therefore conclude that the observed increase in protein synthesis activity by the K56R mutant ribosome reflects increased stability of the 70S complex and is responsible for the increase in α-amylase production seen in the affected strain.

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