CcpA Causes Repression of the phoPR Promoter through a Novel Transcription Start Site, PA6

ABSTRACT The Bacillus subtilis PhoPR two-component system is directly responsible for activation or repression of Pho regulon genes in response to phosphate deprivation. The response regulator, PhoP, and the histidine kinase, PhoR, are encoded in a single operon with a complex promoter region that contains five known transcription start sites, which respond to at least two regulatory proteins. We report here the identification of another direct regulator of phoPR transcription, carbon catabolite protein A, CcpA. This regulator functions in the presence of glucose or other readily metabolized carbon sources. The maximum derepression of phoPR expression in a ccpA mutant compared to a wild-type stain was observed under excess phosphate conditions with glucose either throughout growth in a high-phosphate defined medium or in a low-phosphate defined medium during exponential growth, a growth condition when phoPR transcription is low in a wild-type strain due to the absence of autoinduction. Either HPr or Crh were sufficient to cause CcpA dependent repression of the phoPR promoter in vivo. A ptsH1 (Hpr) crh double mutant completely relieves phoPR repression during phosphate starvation but not during phosphate replete growth. In vivo and in vitro studies showed that CcpA repressed phoPR transcription by binding directly to the cre consensus sequence present in the promoter. Primer extension and in vitro transcription studies revealed that the CcpA regulation of phoPR transcription was due to repression of PA6, a previously unidentified promoter positioned immediately upstream of the cre box. EσA was sufficient for transcription of PA6, which was repressed by CcpA in vitro. These studies showed direct repression by CcpA of a newly discovered EσA-responsive phoPR promoter that required either Hpr or Crh in vivo for direct binding to the putative consensus cre sequence located between PA6 and the five downstream promoters characterized previously.

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Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7232-7238.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7232-7238

Author(s):

W D Rapp ◽

D S Lupold ◽

S Mack ◽

D B Stern

Keyword(s):

Promoter Region ◽

Consensus Sequence ◽

Point Mutations ◽

In Vitro Transcription ◽

Wild Type ◽

Transcription Start ◽

Transcription System ◽

Mitochondrial Promoters ◽

Point Mutagenesis

Plant mitochondrial promoters are poorly conserved but generally share a loose consensus sequence spanning approximately 17 nucleotides. Using a homologous in vitro transcription system, we have previously shown that an 11-nucleotide sequence within this region comprises at least part of the maize mitochondrial atp1 promoter (W. Rapp and D. Stern, EMBO J. 11:1065-1073, 1992). We have extended this finding by using a series of linker-scanning and point mutations to define the atp1 promoter in detail. Our results show that mutations at positions -12 to +5, relative to the major transcription start site, can decrease initiation rates to between < 10 and 40% of wild-type levels. Some mutations, scattered throughout this region, have lesser effects or no effect. Taken together, our data suggest a model in which the atp1 promoter consists of a central domain extending from -7 to +5 and an upstream domain of 1 to 3 bp that is centered around -11 to -12. Because many mutations within this promoter region are tolerated in vitro, the maize atp1 promoter is distinct from the highly conserved yeast mitochondrial promoters.

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Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7232 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7232-7238 ◽

Cited By ~ 41

Author(s):

W D Rapp ◽

D S Lupold ◽

S Mack ◽

D B Stern

Keyword(s):

Promoter Region ◽

Consensus Sequence ◽

Point Mutations ◽

In Vitro Transcription ◽

Wild Type ◽

Transcription Start ◽

Transcription System ◽

Mitochondrial Promoters ◽

Point Mutagenesis

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Definition of the Binding Architecture to a Target Promoter of HP1043, the Essential Master Regulator of Helicobacter pylori

International Journal of Molecular Sciences ◽

10.3390/ijms22157848 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7848

Author(s):

Annamaria Zannoni ◽

Simone Pelliciari ◽

Francesco Musiani ◽

Federica Chiappori ◽

Davide Roncarati ◽

...

Keyword(s):

Helicobacter Pylori ◽

Response Regulator ◽

Consensus Sequence ◽

Binding Mode ◽

In Vitro Transcription ◽

Target Sequence ◽

Computational Alanine Scanning ◽

Definition Of ◽

Target Promoter

HP1043 is an essential orphan response regulator of Helicobacter pylori orchestrating multiple crucial cellular processes. Classified as a member of the OmpR/PhoB family of two-component systems, HP1043 exhibits a highly degenerate receiver domain and evolved to function independently of phosphorylation. Here, we investigated the HP1043 binding mode to a target sequence in the hp1227 promoter (Php1227). Scanning mutagenesis of HP1043 DNA-binding domain and consensus sequence led to the identification of residues relevant for the interaction of the protein with a target DNA. These determinants were used as restraints to guide a data-driven protein-DNA docking. Results suggested that, differently from most other response regulators of the same family, HP1043 binds in a head-to-head conformation to the Php1227 target promoter. HP1043 interacts with DNA largely through charged residues and contacts with both major and minor grooves of the DNA are required for a stable binding. Computational alanine scanning on molecular dynamics trajectory was performed to corroborate our findings. Additionally, in vitro transcription assays confirmed that HP1043 positively stimulates the activity of RNA polymerase.

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Abstract 229: TNF Receptor 1 Signaling Is Critically Involved in Mediating Angiotensin II-Induced Cardiac Fibrosis and Dysfunction

Circulation Research ◽

10.1161/res.111.suppl_1.a229 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Sandra B Haudek ◽

Jeff Crawford ◽

Erin Reineke ◽

Alberto A Allegre ◽

George E Taffet ◽

...

Keyword(s):

Angiotensin Ii ◽

Cardiac Fibrosis ◽

Smooth Muscle Actin ◽

Protein A ◽

Ang Ii ◽

Wild Type ◽

Monocyte Chemoattractant Protein 1 ◽

Reactive Fibrosis

Angiotensin-II (Ang-II) plays a key role in the development of cardiomyopathies, as it is associated with many conditions involving heart failure and pathologic hypertrophy. Using a murine model of Ang-II infusion, we found that Ang-II induced the synthesis of monocyte chemoattractant protein 1 (MCP-1) that mediated the uptake of CD34 + /CD45 + monocytic cells into the heart. These precursor cells differentiated into collagen-producing fibroblasts and were responsible for the Ang-II-induced development of reactive fibrosis. Preliminary in vitro data using our monocyte-to-fibroblast differentiation model, suggested that Ang-II required the presence of TNF to induce fibroblast maturation from monocytes. In vivo, they indicated that in mice deficient of both TNF receptors (TNFR1 and TNFR2), Ang-II-induced fibrosis was absent. We now assessed the hypothesis that specific TNFR1 signaling is necessary for Ang-II-mediated cardiac fibrosis. Mice deficient in either TNFR1 (TNFR1-KO) or TNFR2 (TNFR2-KO) were subjected to continuous infusion of Ang-II for 1 to 6 weeks (n=6-8/group). Compared to wild-type, we found that in TNFR1-KO, but not in TNFR2-KO mouse hearts, collagen deposition was attenuated, as was cardiac α-smooth muscle actin protein (a marker for activated fibroblasts). When we isolated viable cardiac fibroblasts and characterized them by flow cytometry, we found that Ang-II infusion in TNFR1-KO, but not in TNFR2-KO, resulted in a marked decrease of CD34 + /CD45 + cells. Quantitative RT-PCR demonstrated a striking reduction of type 1 and 3 collagen, as well of MCP-1 mRNA expression in TNFR1-KO mouse hearts. Further measurements of cardiovascular parameters indicated that TNFR1-KO animals developed lesser Ang-II-mediated LV remodeling, smaller changes in E-linear deceleration times/rates over time, and displayed a lower Tei index (a heart rate independent marker of cardiac function), indicating less stiffness in TNFR1-KO hearts compared to wild-type and TNFR2-KO hearts. The data suggest that Ang-II-dependent cardiac fibrosis requires TNF and its signaling through TNFR1 which enhances the induction of MCP-1 and uptake of monocytic fibroblast precursors that are associated with reactive fibrosis and cardiac remodeling and function.

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Joint Transcriptional Control of xpsR, the Unusual Signal Integrator of the Ralstonia solanacearum Virulence Gene Regulatory Network, by a Response Regulator and a LysR-Type Transcriptional Activator

Journal of Bacteriology ◽

10.1128/jb.180.10.2736-2743.1998 ◽

1998 ◽

Vol 180 (10) ◽

pp. 2736-2743 ◽

Cited By ~ 35

Author(s):

Jianzhong Huang ◽

Wandee Yindeeyoungyeon ◽

Ram P. Garg ◽

Timothy P. Denny ◽

Mark A. Schell

Keyword(s):

Binding Site ◽

Transcriptional Control ◽

Response Regulator ◽

Consensus Sequence ◽

Virulence Gene ◽

Transcriptional Activator ◽

Binding Assays ◽

Environmental Signals

ABSTRACT Ralstonia (Pseudomonas)solanacearum is a soil-borne phytopathogen that causes a wilting disease of many important crops. It makes large amounts of the exopolysaccharide EPS I, which it requires for efficient colonization, wilting, and killing of plants. Transcription of the epsoperon, encoding biosynthetic enzymes for EPS I, is controlled by a unique and complex sensory network that responds to multiple environmental signals. This network is comprised of the novel transcriptional activator XpsR, three distinct two-component regulatory systems (VsrAD, VsrBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that thexpsR promoter (P xpsR ) is simultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into theeps promoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of P xpsR to show the following. (i) PhcA primarily interacts with a typical 14-bp LysR-type consensus sequence around position −77, causing a sixfold activation of P xpsR ; a weaker, less-defined binding site between −183 and −239 likely enhances PhcA binding and activation via the −77 site another twofold. (ii) Full 70-fold activation of P xpsR requires the additional interaction of the VsrD response regulator (or its surrogate) with a 14-bp dyadic sequence centered around −315 where it enhances activation (and possibly binding) by PhcA; however, VsrD alone cannot activate P xpsR . (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect P xpsR activation or regulation.

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Molecular Characterization of the Mg2+-Responsive PhoP-PhoQ Regulon in Salmonella enterica

Journal of Bacteriology ◽

10.1128/jb.185.21.6287-6294.2003 ◽

2003 ◽

Vol 185 (21) ◽

pp. 6287-6294 ◽

Cited By ~ 84

Author(s):

Sergio Lejona ◽

Andrés Aguirre ◽

María Laura Cabeza ◽

Eleonora García Véscovi ◽

Fernando C. Soncini

Keyword(s):

Transcriptional Regulation ◽

Salmonella Enterica ◽

Response Regulator ◽

Consensus Sequence ◽

Direct Interaction ◽

Direct Repeat ◽

Infection Process ◽

Promoter Regions

ABSTRACT The PhoP/PhoQ two-component system controls the extracellular magnesium deprivation response in Salmonella enterica. In addition, several virulence-associated genes that are mainly required for intramacrophage survival during the infection process are under the control of its transcriptional regulation. Despite shared Mg2+ modulation of the expression of the PhoP-activated genes, no consensus sequence common to all of them could be detected in their promoter regions. We have investigated the transcriptional regulation and the interaction of the response regulator PhoP with the promoter regions of the PhoP-activated loci phoPQ, mgtA, slyB, pmrD, pcgL, phoN, pagC, and mgtCB. A direct repeat of the heptanucleotide sequence (G/T)GTTTA(A/T) was identified as the conserved motif recognized by PhoP to directly control the gene expression of the first five loci, among which the first four are ancestral to enterobacteria. On the other hand, no direct interaction of the response regulator with the promoter of phoN, pagC, or mgtCB was apparent by either in vitro or in vivo assays. These loci are Salmonella specific and were probably acquired by horizontal DNA transfer. Besides, sequence analysis of pag promoters revealed the presence of a conserved PhoP box in 6 out of the 12 genes analyzed. Our results strongly suggest that the expression of a set of Mg2+-controlled genes is driven by PhoP via unknown intermediate regulatory mechanisms that could also involve ancillary factors.

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Surfactant Protein D Increases Phagocytosis of Hypocapsular Cryptococcus neoformans by Murine Macrophages and Enhances Fungal Survival

Infection and Immunity ◽

10.1128/iai.00088-09 ◽

2009 ◽

Vol 77 (7) ◽

pp. 2783-2794 ◽

Cited By ~ 40

Author(s):

Scarlett Geunes-Boyer ◽

Timothy N. Oliver ◽

Guilhem Janbon ◽

Jennifer K. Lodge ◽

Joseph Heitman ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Defense Mechanisms ◽

Protein A ◽

Surfactant Protein ◽

Immune Defense ◽

Surfactant Protein D ◽

Wild Type ◽

Phagocytic Cells

ABSTRACT Cryptococcus neoformans is a facultative intracellular opportunistic pathogen and the leading cause of fungal meningitis in humans. In the absence of a protective cellular immune response, the inhalation of C. neoformans cells or spores results in pulmonary infection. C. neoformans cells produce a polysaccharide capsule composed predominantly of glucuronoxylomannan, which constitutes approximately 90% of the capsular material. In the lungs, surfactant protein A (SP-A) and SP-D contribute to immune defense by facilitating the aggregation, uptake, and killing of many microorganisms by phagocytic cells. We hypothesized that SP-D plays a role in C. neoformans pathogenesis by binding to and enhancing the phagocytosis of the yeast. Here, the abilities of SP-D to bind to and facilitate the phagocytosis and survival of the wild-type encapsulated strain H99 and the cap59Δ mutant hypocapsular strain are assessed. SP-D binding to cap59Δ mutant cells was approximately sixfold greater than binding to wild-type cells. SP-D enhanced the phagocytosis of cap59Δ cells by approximately fourfold in vitro. To investigate SP-D binding in vivo, SP-D−/− mice were intranasally inoculated with Alexa Fluor 488-labeled cap59Δ or H99 cells. By confocal microscopy, a greater number of phagocytosed C. neoformans cells in wild-type mice than in SP-D−/− mice was observed, consistent with in vitro data. Interestingly, SP-D protected C. neoformans cells against macrophage-mediated defense mechanisms in vitro, as demonstrated by an analysis of fungal viability using a CFU assay. These findings provide evidence that C. neoformans subverts host defense mechanisms involving surfactant, establishing a novel virulence paradigm that may be targeted for therapy.

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Phosphorylation of the Group A Streptococcal CovR Response Regulator Causes Dimerization and Promoter-Specific Recruitment by RNA Polymerase

Journal of Bacteriology ◽

10.1128/jb.00198-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4620-4626 ◽

Cited By ~ 45

Author(s):

Asiya A. Gusa ◽

Jinxin Gao ◽

Virginia Stringer ◽

Gordon Churchward ◽

June R. Scott

Keyword(s):

Rna Polymerase ◽

Virulence Factors ◽

Group A Streptococcus ◽

Response Regulator ◽

Regulatory System ◽

In Vitro Transcription ◽

Wild Type ◽

Group A ◽

The Difference

ABSTRACT The group A streptococcus (GAS), Streptococcus pyogenes, is an important human pathogen that causes infections ranging in severity from self-limiting pharyngitis to severe invasive diseases that are associated with significant morbidity and mortality. The pathogenic effects of GAS are mediated by the expression of virulence factors, one of which is the hyaluronic acid capsule (encoded by genes in the has operon). The expression of these virulence factors is controlled by the CovR/S (CsrR/S) two-component regulatory system of GAS which regulates, directly or indirectly, the expression of about 15% of the genome. CovR is a member of the OmpR/PhoB family of transcriptional regulators. Here we show that phosphorylation by acetyl phosphate results in dimerization of CovR. Dimerization was not observed using a D53A mutant of CovR, indicating that D53 is the site of phosphorylation in CovR. Phosphorylation stimulated binding of CovR to a DNA fragment containing the promoter of the has operon (Phas) approximately twofold. Binding of CovR D53A mutant protein to Phas was indistinguishable from the binding of wild-type unphosphorylated CovR. In vitro transcription, using purified GAS RNA polymerase, showed that wild-type CovR repressed transcription, and repression was stimulated more than sixfold by phosphorylation. In the presence of RNA polymerase, binding at Phas of phosphorylated, but not unphosphorylated, CovR was stimulated about fourfold, which accounts for the difference in the effect of phosphorylation on repression versus DNA binding. Thus, regulation of Phas by CovR is direct, and the degree of repression of Phas is controlled by the phosphorylation of CovR.

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An A257V Mutation in the Bacillus subtilis Response Regulator Spo0A Prevents Regulated Expression of Promoters with Low-Consensus Binding Sites

Journal of Bacteriology ◽

10.1128/jb.00590-09 ◽

2009 ◽

Vol 191 (17) ◽

pp. 5489-5498 ◽

Cited By ~ 3

Author(s):

Steve D. Seredick ◽

Barbara M. Seredick ◽

David Baker ◽

George B. Spiegelman

Keyword(s):

Dna Binding ◽

Binding Sites ◽

Transcription Initiation ◽

Molecular Basis ◽

Response Regulator ◽

Mutant Protein ◽

Wild Type ◽

Dna Binding Sites

ABSTRACT In Bacillus species, the master regulator of sporulation is Spo0A. Spo0A functions by both activating and repressing transcription initiation from target promoters that contain 0A boxes, the binding sites for Spo0A. Several classes of spo0A mutants have been isolated, and the molecular basis for their phenotypes has been determined. However, the molecular basis of the Spo0A(A257V) substitution, representative of an unusual phenotypic class, is not understood. Spo0A(A257V) is unusual in that it abolishes sporulation; in vivo, it fails to activate transcription from key stage II promoters yet retains the ability to repress the abrB promoter. To determine how Spo0A(A257V) retains the ability to repress but not stimulate transcription, we performed a series of in vitro and in vivo assays. We found unexpectedly that the mutant protein both stimulated transcription from the spoIIG promoter and repressed transcription from the abrB promoter, albeit twofold less than the wild type. A DNA binding analysis of Spo0A(A257V) showed that the mutant protein was less able to tolerate alterations in the sequence and arrangement of its DNA binding sites than the wild-type protein. In addition, we found that Spo0A(A257V) could stimulate transcription of a mutant spoIIG promoter in vivo in which low-consensus binding sites were replaced by high-consensus binding sites. We conclude that Spo0A(A257V) is able to bind to and regulate the expression of only genes whose promoters contain high-consensus binding sites and that this effect is sufficient to explain the observed sporulation defect.

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Development of an in vitro transcription system for Neurospora crassa mitochondrial DNA and identification of transcription initiation sites.

Molecular and Cellular Biology ◽

10.1128/mcb.9.9.3603 ◽

1989 ◽

Vol 9 (9) ◽

pp. 3603-3613 ◽

Cited By ~ 25

Author(s):

J C Kennell ◽

A M Lambowitz

Keyword(s):

Mitochondrial Dna ◽

Neurospora Crassa ◽

Transcription Initiation ◽

Consensus Sequence ◽

In Vitro Transcription ◽

Transcription System ◽

Group I ◽

Genes Encoding

We have developed an in vitro transcription system for Neurospora crassa mitochondrial DNA (mtDNA) and used it to identify transcription initiation sites at the 5' ends of the genes encoding the mitochondrial small and large rRNA and cytochrome b (cob). The in vitro transcription start sites correspond to previously mapped 5' ends of major in vivo transcripts of these genes. Sequences around the three transcription initiation sites define a 15-nucleotide consensus sequence, 5'-TTAGARA(T/G)G(T/G)ARTRR-3', all or part of which appears to be an element of an N. crassa mtDNA promoter. A somewhat looser 11-nucleotide consensus sequence, 5'-TTAGARR(T/G)R(T/G)A-3', was derived by including two additional promoters identified recently. Group I extranuclear mutants, such as [poky] and [SG-3], have a 4-base-pair (bp) deletion in the consensus sequence at the 5' end of the mitochondrial small rRNA and are grossly deficient in mitochondrial small rRNA (R. A. Akins and A. M. Lambowitz, Proc. Natl. Acad. Sci. USA 81:3791-3795, 1984). We show here that the 4-bp deletion in the consensus sequence decreases in vitro transcription from this site by more than 99%. N. crassa mtDNA is similar to Saccharomyces cerevisiae mtDNA in having multiple promoters, including separate promoters for the genes encoding the mitochondrial small and large rRNAs. Our results suggest that the primary effect of the 4-bp deletion in group I extranuclear mutants is to inhibit transcription of the mitochondrial small rRNA, leading to severe deficiency of mitochondrial small rRNA and small ribosomal subunits.

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