scholarly journals 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

1998 ◽  
Vol 180 (10) ◽  
pp. 2736-2743 ◽  
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.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

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

2003 ◽  
Vol 185 (21) ◽  
pp. 6287-6294 ◽  
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.

scholarly journals Alterations in the binding site of the cyclic AMP receptor protein at the Escherichia coli galactose operon regulatory region

1988 ◽  
Vol 253 (3) ◽  
pp. 809-818 ◽  
Author(s):  
K Gaston ◽  
B Chan ◽  
A Kolb ◽  
J Fox ◽  
S Busby
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Consensus Sequence ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Binding Assays ◽  
Cyclic Amp Receptor Protein ◽  
Stimulation Of

Gene manipulation techniques have been used to alter the binding site for the cyclic AMP-cyclic AMP receptor protein complex (cAMP-CRP) at the regulatory region of the Escherichia coli galactose (gal) operon. The effects of these changes on CRP-dependent stimulation of expression from the galP1 promoter in vivo have been measured, and gel binding assays have been used to measure the affinity of cAMP-CRP for the modified sites. Firstly we have deleted progressively longer sequences from upstream of the gal CRP site in order to locate the functional limit of the site. A deletion to -49, removing the first base that corresponds to the consensus sequence for a CRP binding site, is sufficient to reduce CRP binding and block CRP-dependent stimulation of P1. Secondly, we used synthetic oligonucleotides to invert the asymmetric nucleotide sequence at the gal CRP binding site or to make the sequence symmetric. Inversion of the site has little effect on CRP binding, the architecture of open complexes at P1 revealed by DNAase I footprinting, or the stimulation of transcription from P1. Making the site symmetric increases the affinity for CRP by over 50-fold and leads to increased transcription from P1, whilst hardly altering the DNAase I footprint of open complexes. Our results confirm that the strength of binding of CRP depends on the nature of the site and show that it is this that principally accounts for differences in CRP-dependent stimulation of transcription.

scholarly journals Identification of Novel Thermosensors in Gram-Positive Pathogens

2020 ◽  
Vol 7 ◽  
Author(s):  
Pilar Fernández ◽  
Alejandra Raquel Díaz ◽  
María Florencia Ré ◽  
Lucía Porrini ◽  
Diego de Mendoza ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Physiological Mechanism ◽  
Virulence Gene ◽  
Membrane Lipid ◽  
Gram Positive ◽  
Virulence Gene Expression ◽  
Cognate Response Regulator

Temperature is a crucial variable that every living organism, from bacteria to humans, need to sense and respond to in order to adapt and survive. In particular, pathogenic bacteria exploit host-temperature sensing as a cue for triggering virulence gene expression. Here, we have identified and characterized two integral membrane thermosensor histidine kinases (HKs) from Gram-positive pathogens that exhibit high similarity to DesK, the extensively characterized cold sensor histidine kinase from Bacillus subtilis. Through in vivo experiments, we demonstrate that SA1313 from Staphylococcus aureus and BA5598 from Bacillus anthracis, which likely control the expression of putative ATP binding cassette (ABC) transporters, are regulated by environmental temperature. We show here that these HKs can phosphorylate the non-cognate response regulator DesR, partner of DesK, both in vitro and in vivo, inducing in B. subtilis the expression of the des gene upon a cold shock. In addition, we report the characterization of another DesK homolog from B. subtilis, YvfT, also closely associated to an ABC transporter. Although YvfT phosphorylates DesR in vitro, this sensor kinase can only induce des expression in B. subtilis when overexpressed together with its cognate response regulator YvfU. This finding evidences a physiological mechanism to avoid cross talk with DesK after a temperature downshift. Finally, we present data suggesting that the HKs studied in this work appear to monitor different ranges of membrane lipid properties variations to mount adaptive responses upon cooling. Overall, our findings point out that bacteria have evolved sophisticated mechanisms to assure specificity in the response to environmental stimuli. These findings pave the way to understand thermosensing mediated by membrane proteins that could have important roles upon host invasion by bacterial pathogens.

scholarly journals Bordetella pertussis fim3gene regulation by BvgA: Phosphorylation controls the formation of inactive vs. active transcription complexes

2015 ◽  
Vol 112 (6) ◽  
pp. E526-E535 ◽  
Author(s):  
Alice Boulanger ◽  
Kyung Moon ◽  
Kimberly B. Decker ◽  
Qing Chen ◽  
Leslie Knipling ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Bordetella Pertussis ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Gene Activation ◽  
Virulence Gene ◽  
In Vitro Transcription ◽  
Transcription Complexes

Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and theBordetella pertussisRR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at Pfim3, the promoter for the virulence genefim3(fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at Pfim3. However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence offim3. We suggest that the presence of nonphosphorylated BvgA down-regulates Pfim3activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating Pfim3activation once the signal for BvgA phosphorylation is removed.

scholarly journals The Acid-Inducible asr Gene inEscherichia coli: Transcriptional Control by thephoBR Operon

1999 ◽  
Vol 181 (7) ◽  
pp. 2084-2093 ◽  
Author(s):  
Edita Sužiede˙liene˙ ◽  
Ke˛stutis Sužiede˙lis ◽  
Vaida Garbenčiute˙ ◽  
Staffan Normark
Keyword(s):  
Transcriptional Control ◽  
Signal Sequence ◽  
Consensus Sequence ◽  
Regulatory System ◽  
Chromosome Analysis ◽  
Luria Bertani ◽  
Computer Assisted ◽  
T7 Promoter

ABSTRACT Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, ∼450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. Theasr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encodedasr::lacZ fusion under a nativeasr promoter was reduced ∼15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of aphoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Δ(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoRregulatory system to sense an external acidity and regulate transcription of the asr gene.

scholarly journals The Paralogous MarR/DUF24-Family Repressors YodB and CatR Control Expression of the Catechol Dioxygenase CatE in Bacillus subtilis

2010 ◽  
Vol 192 (18) ◽  
pp. 4571-4581 ◽  
Author(s):  
Bui Khanh Chi ◽  
Kazuo Kobayashi ◽  
Dirk Albrecht ◽  
Michael Hecker ◽  
Haike Antelmann
Keyword(s):  
Bacillus Subtilis ◽  
Redox Regulation ◽  
Transcriptional Start Site ◽  
Consensus Sequence ◽  
Binding Assays ◽  
Catechol Dioxygenase ◽  
Disulfide Formation ◽  
Redox Sensing

ABSTRACT The redox-sensing MarR/DUF24-type repressor YodB controls expression of the azoreductase AzoR1 and the nitroreductase YodC that are involved in detoxification of quinones and diamide in Bacillus subtilis. In the present paper, we identified YodB and its paralog YvaP (CatR) as repressors of the yfiDE (catDE) operon encoding a catechol-2,3-dioxygenase that also contributes to quinone resistance. Inactivation of both CatR and YodB is required for full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis studies showed that CatR protects two inverted repeats with the consensus sequence TTAC-N5-GTAA overlapping the −35 promoter region (BS1) and the transcriptional start site (TSS) (BS2). The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue, which is required for redox sensing of CatR in vivo as shown by Cys-to-Ser mutagenesis. Our data suggest that CatR is modified by intermolecular disulfide formation in response to diamide and quinones in vitro and in vivo. Redox regulation of CatR occurs independently of YodB, and no protein interaction was detected between CatR and YodB in vivo using protein cross-linking and mass spectrometry.

Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

scholarly journals Hha Is a Negative Modulator of Transcription ofhilA, the Salmonella enterica Serovar Typhimurium Invasion Gene Transcriptional Activator

2001 ◽  
Vol 183 (22) ◽  
pp. 6620-6629 ◽  
Author(s):  
Thomas F. Fahlen ◽  
Rebecca L. Wilson ◽  
Jennifer D. Boddicker ◽  
Bradley D. Jones
Keyword(s):  
Salmonella Enterica ◽  
Transcriptional Activator ◽  
Negative Influence ◽  
Chromosomal Dna ◽  
Invasive Phenotype ◽  
Environmental Signals ◽  
Serovar Typhimurium ◽  
Suboptimal Condition

ABSTRACT An early step in the establishment of Salmonella enterica serovar Typhimurium murine infection is the penetration of the intestinal mucosa of the small intestine. The majority of the genes responsible for the Salmonellainvasive phenotype are encoded on Salmonellapathogenicity island 1, and their transcription is controlled by thehilA transcriptional activator. The expression ofhilA is regulated by environmental signals including oxygen, osmolarity, pH, and growth phase such that the presence of any one suboptimal condition results in repression of hilAexpression and the invasive phenotype. We have conducted a search for negative regulators of hilA by introduction of aSalmonella enterica serovar Typhimurium chromosomal DNA gene bank into a Salmonella enterica serovar TyphimuriumhilA::Tn5lacZY reporter strain. This screen has identified the hha gene as a regulator that exerts a negative influence on hilA expression. Plasmid-encoded hha significantly reduceshilA::Tn5lacZY chromosomal expression, as well as expression of the invasion genesinvF, prgH, and sipC. Anhha null mutation results in substantial derepression of both chromosomally encoded and plasmid-encodedhilA::Tn5lacZY expression. Introduction of plasmid-encoded hha into strain SL1344 results in attenuation of invasion using in vitro and in vivo assays. Importantly, purified Hha protein was found to bind to ahilA DNA promoter fragment, suggesting that the regulatory activity of the Hha protein occurs at thehilA promoter. These data add detail to the developing model of the regulation of Salmonella invasion genes.

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

2006 ◽  
Vol 188 (4) ◽  
pp. 1266-1278 ◽  
Author(s):  
Ankita Puri-Taneja ◽  
Salbi Paul ◽  
Yinghua Chen ◽  
F. Marion Hulett
Keyword(s):  
Response Regulator ◽  
Consensus Sequence ◽  
Protein A ◽  
Carbon Sources ◽  
In Vitro Transcription ◽  
Defined Medium ◽  
Wild Type ◽  
Transcription Start

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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