scholarly journals Genomic SELEX Search for Target Promoters under the Control of the PhoQP-RstBA Signal Relay Cascade

2007 ◽  
Vol 189 (13) ◽  
pp. 4791-4799 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Akiko Hasegawa ◽  
Emi Kanda ◽  
Takenori Miki ◽  
Kaneyoshi Yamamoto ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Consensus Sequence ◽  
Regulatory System ◽  
Regulatory Function ◽  
Mobility Shift ◽  
Promoter Assay ◽  
Signal Relay ◽  
Two Component ◽  
Genomic Selex

ABSTRACT RstBA, a two-component regulatory system of Escherichia coli with an unidentified regulatory function, is under the control of a Mg2+-sensing PhoQP two-component system. In order to identify the network of transcription regulation downstream of RstBA, we isolated a set of RstA-binding sequences from the E. coli genome by using the genomic SELEX system. A gel mobility shift assay indicated the binding of RstA to two SELEX DNA fragments, one including the promoter region of asr (acid shock RNA) and another including the promoter for csgD (a regulator of the curli operon). Using a DNase I footprinting assay, we determined the RstA-binding sites (RstA boxes) with the consensus sequence TACATNTNGTTACA. Transcription of the asr gene was induced 10- to 60-fold either in low-pH (pH 4.5) LB medium or in low-phosphate minimal medium as detected by promoter assay. The acid-induced in vivo transcription of asr was reduced after the deletion of rstA. In vivo transcription of the asr promoter was observed only in the presence of RstA. In agreement with the PhoQP-RstBA network, the addition of Mg2+ led to a severe reduction of the asr promoter activity, and the disruption of phoP also reduced the asr promoter activity, albeit to a lesser extent. These observations altogether indicate that RstA is an activator of asr transcription. In contrast, transcription of csgD was repressed by overexpression of RstA, indicating that RstA is a repressor for csgD. With these data taken together, we conclude that the expression of both asr and csgD is under the direct control of the PhoQP-RstBA signal relay cascade.

The nuclear orphan receptors COUP-TFII and Ear-2 act as silencers of the human oxytocin gene promoter

1997 ◽  
Vol 19 (2) ◽  
pp. 163-172 ◽  
Author(s):  
K Chu ◽  
HH Zingg
Keyword(s):  
Receptor Binding ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Gene Promoter ◽  
Fine Tuning ◽  
Orphan Receptor ◽  
Expression Vectors ◽  
Mobility Shift ◽  
Interaction Sites

We have previously shown that COUP-TFII and Ear-2, two members of the nuclear orphan receptor family, are able to repress oestrogen-stimulated transcriptional activity of the human oxytocin (OT) gene promoter by binding to a site that overlaps with the oestrogen response element (ERE) present in the 5' flanking region of the gene. Although most nuclear receptor-mediated transcriptional repression conforms with the paradigm of passive repression and involves competitive binding to an activator site, active repression, i.e. silencing of basal promoter activity, has been observed in a limited number of cases. Here we show by co-transfection experiments using COUP-TFII and Ear-2 expression vectors and reporter constructs containing OT gene promoter fragments linked to the chloramphenicol acetyltransferase gene that both COUP-TFII and Ear-2 are capable of silencing basal OT gene promoter activity by 54 and 75% respectively. 5' Deletion and footprint analyses revealed two areas of functionally important interaction sites: (1) a direct TGACC(T/C) repeat overlapping the ERE and (2) a more promoter-proximal area centred at - 90 containing three imperfect direct repeats (R1-R3) spaced by four nucleotides each. Mutagenesis of reporter constructs as well as electrophoretic mobility-shift assays demonstrated that each of the three proximal repeats R1-R3 contributed to orphan receptor binding and the silencing effect. Inasmuch as the orphan receptor-binding sites are not involved in mediating basal transcriptional activity of the OT gene promoter, the observed effects are best interpreted as active repression or promoter silencing. Moreover, since COUP-TFII and Ear-2 are both co-expressed in OT-expressing uterine epithelial cells, the novel transcriptional effects described here are likely to be of functional importance in the fine-tuning of uterine OT gene expression in vivo.

scholarly journals MisR/MisS Two-Component Regulon in Neisseria meningitidis

2007 ◽  
Vol 76 (2) ◽  
pp. 704-716 ◽  
Author(s):  
Yih-Ling Tzeng ◽  
Charlene M. Kahler ◽  
Xinjian Zhang ◽  
David S. Stephens
Keyword(s):  
Neisseria Meningitidis ◽  
Target Genes ◽  
Inner Core ◽  
Consensus Sequence ◽  
Meningococcal Infection ◽  
Type I ◽  
Mobility Shift ◽  
Iron Assimilation ◽  
Wide Range ◽  
Two Component

ABSTRACT Two-component regulatory systems are involved in processes important for bacterial pathogenesis. Inactivation of the misR/misS system in Neisseria meningitidis results in the loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. One hundred seventeen (78 up-regulated and 39 down-regulated) potential regulatory targets of the MisR/MisS (MisR/S) system were identified by transcriptional profiling of the NMBmisR mutant and the parental wild-type meningococcal strain NMB. The regulatory effect was further confirmed in a subset of target genes by quantitative real-time PCR and β-galactosidase transcriptional fusion reporter assays. The MisR regulon includes genes encoding proteins necessary for protein folding in the bacterial cytoplasm and periplasm, transcriptional regulation, metabolism, iron assimilation, and type I protein transport. Mutation in the MisR/S system caused increased sensitivity to oxidative stress and also resulted in decreased susceptibility to complement-mediated killing by normal human serum. To identify the direct targets of MisR regulation, electrophoretic mobility shift assays were carried out using purified MisR-His6 protein. Among 22 genes examined, misR directly interacted with 14 promoter regions. Six promoters were further investigated by DNase I protection assays, and a MisR-binding consensus sequence was proposed. Thus, the direct regulatory targets of MisR and the minimal regulon of the meningococcal MisR/S two-component signal transduction system were characterized. These data indicate that the MisR/S system influences a wide range of biological functions in N. meningitidis either directly or via intermediate regulators.

scholarly journals Novel Two-Component System MacRS Is a Pleiotropic Regulator That Controls Multiple Morphogenic Membrane Protein Genes inStreptomyces coelicolor

2018 ◽  
Vol 85 (4) ◽  
Author(s):  
Meng Liu ◽  
Peipei Zhang ◽  
Yanping Zhu ◽  
Ting Lu ◽  
Yemin Wang ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Consensus Sequence ◽  
Aerial Mycelium ◽  
Dnase I ◽  
Inverted Repeats ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Two Component

ABSTRACTAs with most annotated two-component systems (TCSs) ofStreptomyces coelicolor, the function of TCS SCO2120/2121 was unknown. Based on our findings, we have designated this TCS MacRS, formorphogenesis andactinorhodin regulator/sensor. Our study indicated that either single or double mutation of MacRS largely blocked production of actinorhodin but enhanced formation of aerial mycelium. Chromatin immunoprecipitation (ChIP) sequencing, using anS. coelicolorstrain expressing MacR-Flag fusion protein, identifiedin vivotargets of MacR, and DNase I footprinting of these targets revealed a consensus sequence for MacR binding, TGAGTACnnGTACTCA, containing two 7-bp inverted repeats. A genome-wide search revealed sites identical or highly similar to this consensus sequence upstream of six genes encoding putative membrane proteins or lipoproteins. These predicted sites were confirmed as MacR binding sites by DNase I footprinting and electrophoretic mobility shift assaysin vitroand by ChIP-quantitative PCRin vivo, and transcriptional analyses demonstrated that MacR significantly impacts expression of these target genes. Disruption of three of these genes,sco6728,sco4924, andsco4011, markedly accelerated aerial mycelium formation, indicating that their gene products are novel morphogenic factors. Two-hybrid assays indicated that these three proteins, which we have named morphogenic membrane protein A (MmpA; SCO6728), MmpB (SCO4924), and MmpC (SCO4011), interact with one another and with the putative membrane protein and MacR target SCO4225. Notably, SAV6081/82 and SVEN1780/81, homologs of MacRS TCS fromS. avermitilisandS. venezuelae, respectively, can substitute for MacRS, indicating functional conservation. Our findings reveal a role for MacRS in cellular morphogenesis and secondary metabolism inStreptomyces.IMPORTANCETCSs help bacteria adapt to environmental stresses by altering gene expression. However, the roles and corresponding regulatory mechanisms of most TCSs in theStreptomycesmodel strainS. coelicolorare unknown. We investigated the previously uncharacterized MacRS TCS and identified the core DNA recognition sequence, two seven-nucleotide inverted repeats, for the DNA-binding protein MacR. We further found that MacR directly controls a group of membrane proteins, including MmpA-C, which are novel morphogenic factors that delay formation of aerial mycelium. We also discovered that these membrane proteins interact with one another and that otherStreptomycesspecies have conserved MacRS homologs. Our findings suggest a conserved role for MacRS in morphogenesis and/or other membrane-associated activities. Additionally, our study showed that MacRS impacts, albeit indirectly, the production of the signature metabolite actinorhodin, further suggesting that MacRS and its homologs function as novel pleiotropic regulatory systems inStreptomyces.

In vivo regulation of the β-myosin heavy chain gene in hypertensive rodent heart

2001 ◽  
Vol 280 (5) ◽  
pp. C1262-C1276 ◽  
Author(s):  
Carola E. Wright ◽  
P. W. Bodell ◽  
F. Haddad ◽  
A. X. Qin ◽  
K. M. Baldwin
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Promoter Activity ◽  
Proximal Promoter ◽  
Future Research ◽  
Chain Gene ◽  
Direct Gene Transfer ◽  
Basal Region ◽  
Mobility Shift

The main goal of this study was to examine the transcriptional activity of different-length β-myosin heavy chain (β-MHC) promoters in the hypertensive rodent heart using the direct gene transfer approach. A hypertensive state was induced by abdominal aortic constriction (AbCon) sufficient to elevate mean arterial pressure by ∼45% relative to control. Results show that β-MHC promoter activity of all tested wild-type constructs, i.e., −3500, −408, −299, −215, −171, and −71 bp, was significantly increased in AbCon hearts. In the normal control hearts, expression of the −71-bp construct was comparable to that of the promoterless vector, but its induction by AbCon was comparable to that of the other constructs. Additional results, based on mutation analysis and DNA gel mobility shift assays targeting βe1, βe2, GATA, and βe3 elements, show that these previously defined cis-elements in the proximal promoter are indeed involved in maintaining basal promoter activity; however, none of these elements, either individually or collectively, appear to be major players in mediating the hypertension response of the β-MHC gene. Collectively, these results indicate that three separate regions on the β-MHC promoter are involved in the induction of the gene in response to hypertension: 1) a distal region between −408 and −3500 bp, 2) a proximal region between −299 and −215 bp, and 3) a basal region within −71 bp of the transcription start site. Future research needs to further characterize these responsive regions to more fully delineate β-MHC transcriptional regulation in response to pressure overload.

USF-1 and USF-2 trans-repress IL-1β-induced iNOS transcription in mesangial cells

2002 ◽  
Vol 283 (4) ◽  
pp. C1065-C1072 ◽  
Author(s):  
Ashish K. Gupta ◽  
Bruce C. Kone
Keyword(s):  
Dna Binding ◽  
Promoter Activity ◽  
Mesangial Cells ◽  
Luciferase Reporter ◽  
Mobility Shift ◽  
Inos Gene ◽  
E Box ◽  
Inos Promoter ◽  
Gel Mobility Shift

Transcriptional activation of the inducible nitric oxide synthase (iNOS) gene requires multiple interactions of cis elements and trans-acting factors. Previous in vivo footprinting studies (Goldring CE, Reveneau S, Algarte M, and Jeannin JF. Nucleic Acids Res 24: 1682–1687, 1996) of the murine iNOS gene demonstrated lipopolysaccharide-inducible protection of guanines in the region −904/−883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the −893/−888 E-box and correlating functional assays of the mutated iNOS promoter with upstream stimulatory factor (USF) DNA-binding activities, we demonstrate that the −893/−888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that USFs are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to interleukin-1β (IL-1β) in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs cannot bind to the −893/−888 E-box promoter region when the E-box is mutated. Cotransfection of USF-1 and USF-2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1β-simulated iNOS promoter activity. Cotransfection of dominant-negative USF-2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the −904/−883 region augmented IL-1β stimulation of iNOS promoter activity. Gel mobility shift assays showed that only USF-1 and USF-2 supershifted the USF protein-DNA complexes. These results demonstrated that USF binding to the E-box at −893/−888 serves to trans-repress basal expression and IL-1β induction of the iNOS promoter.

scholarly journals Modulated Binding of SATB1, a Matrix Attachment Region Protein, to the AT-Rich Sequence Flanking the Major Breakpoint Region of BCL2

2000 ◽  
Vol 20 (3) ◽  
pp. 868-877 ◽  
Author(s):  
Meera Ramakrishnan ◽  
Wen-Man Liu ◽  
Patricia A. DiCroce ◽  
Aleza Posner ◽  
Jian Zheng ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Interactions ◽  
Specific Binding ◽  
Chromosomal Translocation ◽  
Breakpoint Region ◽  
Matrix Attachment Region ◽  
Regulatory Function ◽  
Mobility Shift ◽  
Attachment Region

ABSTRACT The t(14,18) chromosomal translocation that occurs in human follicular lymphoma constitutively activates the BCL2 gene and disrupts control of apoptosis. Interestingly, 70% of the t(14,18) translocations are confined to three 15-bp clusters positioned within a 150-bp region (major breakpoint region or [MBR]) in the untranslated portion of terminal exon 3. We analyzed DNA-protein interactions in the MBR, as these may play some role in targeting the translocation to this region. An 87-bp segment (87MBR) immediately 3′ to breakpoint cluster 3 was essential for DNA-protein interaction monitored with mobility shift assays. We further delineated a core binding region within 87MBR: a 33-bp, very AT-rich sequence highly conserved between the human and mouse BCL2 gene (37MBR). We have purified and identified one of the core factors as the matrix attachment region (MAR) binding protein, SATB1, which is known to bind to AT-rich sequences with a high propensity to unwind. Additional factors in nuclear extracts, which we have not yet characterized further, increased SATB1 affinity for the 37MBR target four- to fivefold. Specific binding activity within 37MBR displayed cell cycle regulation in Jurkat T cells, while levels of SATB1 remained constant throughout the cell cycle. Finally, we demonstrated in vivo binding of SATB1 to the MBR, strongly suggesting the BCL2 major breakpoint region is a MAR. We discuss the potential consequences of our observations for both MBR fragility and regulatory function.

scholarly journals Differential transcriptional regulation of the human thrombin receptor gene by the Sp family of transcription factors in human endothelial cells

1998 ◽  
Vol 330 (3) ◽  
pp. 1469-1474 ◽  
Author(s):  
Yaxu WU ◽  
Johannes RUEF ◽  
N. Gadiparthi RAO ◽  
Cam PATTERSON ◽  
S. Marschall RUNGE
Keyword(s):  
Endothelial Cells ◽  
Promoter Activity ◽  
Receptor Gene ◽  
Thrombin Receptor ◽  
Wild Type ◽  
Mobility Shift ◽  
Human Thrombin ◽  
Gc Box

The mitogenic effects of thrombin are mediated by a G-protein-coupled receptor. Because the effects of thrombin are strongly influenced by the expression of its receptor, an understanding of its regulatory mechanisms is essential. To identify mechanisms of human thrombin receptor (HTR) gene regulation, a series of HTR-promoter-luciferase constructs were made and transfected into human microvascular endothelial cells for analysis. Deletion from bp -303 to -164 abolished reporter gene expression. Dimethyl sulphate treatment in vivo and DNase I footprinting in vitro demonstrated that a cluster of three GC box consensus sites was occupied, and electrophoretic mobility-shift assays established that Sp1 and Sp3 both bind to this 3ʹ GC box cluster. We mutated each of the three GC boxes individually and all three collectively within this 3ʹ cluster. Basal promoter activity was decreased to 46%, 78% and 29% of control for each of the GC boxes mutated individually, and to 6% when the three were mutated collectively. To test the individual abilities of Sp1 and Sp3 to activate or repress HTR transcription, we conducted co-transfection experiments with wild-type or mutated HTR-promoter-luciferase constructs. Co-transfection with Sp1 significantly augmented wild-type HTR promoter activity. Sp3 alone did not affect activity, and inhibited Sp1-mediated activation. Competition for shared binding sites by Sp1 and Sp3 might differentially regulate HTR expression in vascular endothelial cells.

scholarly journals The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

mBio ◽  
2011 ◽  
Vol 2 (1) ◽  
Author(s):  
Menglin Ma ◽  
Jorge Vidal ◽  
Juliann Saputo ◽  
Bruce A. McClane ◽  
Francisco Uzal
Keyword(s):  
Clostridium Perfringens ◽  
Regulatory System ◽  
Gas Gangrene ◽  
Necrotic Enteritis ◽  
Vegetative Cells ◽  
Content Type ◽  
Two Component ◽  
Type C ◽  
Beta Toxin

ABSTRACT Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells. IMPORTANCE Clostridium perfringens is an important human and veterinary pathogen. C. perfringens vegetative cells cause both histotoxic infections, e.g., traumatic gas gangrene, and infections originating when this bacterium grows in the intestines. The VirS/VirR two-component regulatory system has been shown to control the pathogenicity of C. perfringens type A strains in a mouse gas gangrene model, but there is no understanding of pathogenicity regulation when C. perfringens vegetative cells cause disease originating in the complex intestinal environment. The current study establishes that VirS/VirR controls vegetative cell pathogenicity when C. perfringens type C isolates cause hemorrhagic necrotic enteritis and lethal enterotoxemia (i.e., toxin absorption from the intestines into the circulation, allowing targeting of internal organs). This effect involves VirS/VirR-mediated regulation of beta toxin production in vivo. Therefore, VirS/VirR is the first identified global in vivo regulator controlling the ability of C. perfringens vegetative cells to cause gas gangrene and, at least some, intestinal infections.

scholarly journals ZraP is a periplasmic molecular chaperone and a repressor of the zinc-responsive two-component regulator ZraSR

2012 ◽  
Vol 442 (1) ◽  
pp. 85-93 ◽  
Author(s):  
Corinne Appia-Ayme ◽  
Andrea Hall ◽  
Elaine Patrick ◽  
Shiny Rajadurai ◽  
Thomas A. Clarke ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Regulatory System ◽  
Enteric Bacteria ◽  
Polymyxin B ◽  
Regulatory Function ◽  
Cationic Antimicrobial Peptide ◽  
Envelope Stress ◽  
Two Component ◽  
Periplasmic Proteins ◽  
Zinc Concentrations

The bacterial envelope is the interface with the surrounding environment and is consequently subjected to a barrage of noxious agents including a range of compounds with antimicrobial activity. The ESR (envelope stress response) pathways of enteric bacteria are critical for maintenance of the envelope against these antimicrobial agents. In the present study, we demonstrate that the periplasmic protein ZraP contributes to envelope homoeostasis and assign both chaperone and regulatory function to ZraP from Salmonella Typhimurium. The ZraP chaperone mechanism is catalytic and independent of ATP; the chaperone activity is dependent on the presence of zinc, which is shown to be responsible for the stabilization of an oligomeric ZraP complex. Furthermore, ZraP can act to repress the two-component regulatory system ZraSR, which itself is responsive to zinc concentrations. Through structural homology, ZraP is a member of the bacterial CpxP family of periplasmic proteins, which also consists of CpxP and Spy. We demonstrate environmental co-expression of the CpxP family and identify an important role for these proteins in Salmonella's defence against the cationic antimicrobial peptide polymyxin B.

scholarly journals Expression of Uptake Hydrogenase and Molybdenum Nitrogenase in Rhodobacter capsulatus Is Coregulated by the RegB-RegA Two-Component Regulatory System

2000 ◽  
Vol 182 (10) ◽  
pp. 2831-2837 ◽  
Author(s):  
Sylvie Elsen ◽  
Wanda Dischert ◽  
Annette Colbeau ◽  
Carl E. Bauer
Keyword(s):  
Photosynthetic Bacteria ◽  
Rhodobacter Capsulatus ◽  
Regulatory System ◽  
Photosynthetic Bacterium ◽  
Uptake Hydrogenase ◽  
Carbon And Nitrogen ◽  
Cellular Energy ◽  
Purple Photosynthetic Bacteria ◽  
Two Component

ABSTRACT Purple photosynthetic bacteria are capable of generating cellular energy from several sources, including photosynthesis, respiration, and H2 oxidation. Under nutrient-limiting conditions, cellular energy can be used to assimilate carbon and nitrogen. This study provides the first evidence of a molecular link for the coregulation of nitrogenase and hydrogenase biosynthesis in an anoxygenic photosynthetic bacterium. We demonstrated that molybdenum nitrogenase biosynthesis is under the control of the RegB-RegA two-component regulatory system in Rhodobacter capsulatus. Footprint analyses and in vivo transcription studies showed that RegA indirectly activates nitrogenase synthesis by binding to and activating the expression of nifA2, which encodes one of the two functional copies of the nif-specific transcriptional activator, NifA. Expression of nifA2 but notnifA1 is reduced in the reg mutants up to eightfold under derepressing conditions and is also reduced under repressing conditions. Thus, although NtrC is absolutely required fornifA2 expression, RegA acts as a coactivator ofnifA2. We also demonstrated that in regmutants, [NiFe]hydrogenase synthesis and activity are increased up to sixfold. RegA binds to the promoter of the hydrogenase gene operon and therefore directly represses its expression. Thus, the RegB-RegA system controls such diverse processes as energy-generating photosynthesis and H2 oxidation, as well as the energy-demanding processes of N2 fixation and CO2 assimilation.

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