scholarly journals FleQ, the Major Flagellar Gene Regulator in Pseudomonas aeruginosa, Binds to Enhancer Sites Located Either Upstream or Atypically Downstream of the RpoN Binding Site

2002 ◽  
Vol 184 (19) ◽  
pp. 5251-5260 ◽  
Author(s):  
Jeevan Jyot ◽  
Nandini Dasgupta ◽  
Reuben Ramphal
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Binding Sites ◽  
Direct Interaction ◽  
Significant Loss ◽  
Transcription Start Sites ◽  
Dnase I Footprinting ◽  
Gel Shift Assay ◽  
Polymerase Binding

ABSTRACT In Pseudomonas aeruginosa, flagellar genes are regulated in a cascade headed by FleQ, an NtrC/NifA-type activator. FleQ and RpoN positively regulate expression of flhA, fliE, fliL, and fleSR genes, among others. Direct interaction of FleQ with flhA, fliE, fliL, and fleSR promoters was demonstrated by gel shift assay, along with experiments to conclusively determine the specificity of its binding. DNase I footprinting was performed to determine the FleQ binding sites on flhA, fliE, fliL, and fleSR promoters. No sequence conservation among these binding sites was observed. Primer extension analysis revealed the transcription start sites (TSSs) to be localized above the FleQ binding sites in flhA, fliE, and fliL promoters. Analysis of the above data revealed FleQ binding to be in the leader sequence of these promoters, whereas FleQ binding was 67 bp upstream of the TSS in the fleSR promoter. Mutagenesis of the FleQ binding site in the flhA promoter confirmed its functionality in vivo. Deletion of the flhA promoter upstream of the RNA polymerase binding site did not result in a significant loss of promoter activity. These results point to two modes of regulation by an NtrC-type regulator in the flagellar hierarchy in P. aeruginosa, the first being the typical model of activation from a distance via looping in the fleSR promoter and the second involving flhA, fliE, and fliL promoters, where FleQ binds in the downstream vicinity of the promoter and activates transcription without looping.

scholarly journals Transcriptional Repression Mediated by LysR-Type Regulator CatR Bound at Multiple Binding Sites

1998 ◽  
Vol 180 (9) ◽  
pp. 2367-2372 ◽  
Author(s):  
Sudha A. Chugani ◽  
Matthew R. Parsek ◽  
A. M. Chakrabarty
Keyword(s):  
Binding Site ◽  
Structural Gene ◽  
Binding Sites ◽  
Transcriptional Start Site ◽  
Dnase I ◽  
Site Directed Mutagenesis ◽  
Dnase I Footprinting ◽  
Gel Shift

ABSTRACT The catBCA operon of Pseudomonas putidaencodes enzymes involved in the catabolism of benzoate. Transcription of this operon requires the LysR-type transcriptional regulator CatR and an inducer molecule, cis,cis-muconate. Previous gel shift assays and DNase I footprinting have demonstrated that CatR occupies two adjacent sites proximal to thecatBCA promoter in the presence of the inducer. We report the presence of an additional binding site for CatR downstream of thecatBCA promoter within the catB structural gene. This site, called the internal binding site (IBS), extends from +162 to +193 with respect to the catB transcriptional start site and lies within the catB open reading frame. Gel shift analysis and DNase I footprinting determined that CatR binds to this site with low affinity. CatR binds cooperatively with higher affinity to the IBS in the presence of the two upstream binding sites. Parallel in vivo and in vitro studies were conducted to determine the role of the internal binding site. We measured β-galactosidase activity ofcatB-lacZ transcriptional fusions in vivo. Our results suggest a probable cis-acting repressor function for the internal binding site. Site-directed mutagenesis of the IBS verified this finding. The location of the IBS within the catBstructural gene, the cooperativity observed in footprinting studies, and phasing studies suggest that the IBS likely participates in the interaction of CatR with the upstream binding sites by looping out the intervening DNA.

scholarly journals Functional Analyses of the RsmY and RsmZ Small Noncoding Regulatory RNAs inPseudomonas aeruginosa

2018 ◽  
Vol 200 (11) ◽  
Author(s):  
Kayley H. Janssen ◽  
Manisha R. Diaz ◽  
Matthew Golden ◽  
Justin W. Graham ◽  
Wes Sanders ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Site ◽  
Binding Sites ◽  
Rna Binding ◽  
Opportunistic Pathogen ◽  
Binding Properties ◽  
Content Type ◽  
Preferential Binding ◽  
Regulatory Rnas

ABSTRACTPseudomonas aeruginosais a Gram-negative opportunistic pathogen with distinct acute and chronic virulence phenotypes. Whereas acute virulence is typically associated with expression of a type III secretion system (T3SS), chronic virulence is characterized by biofilm formation. Many of the phenotypes associated with acute and chronic virulence are inversely regulated by RsmA and RsmF. RsmA and RsmF are both members of the CsrA family of RNA-binding proteins and regulate protein synthesis at the posttranscriptional level. RsmA activity is controlled by two small noncoding regulatory RNAs (RsmY and RsmZ). Bioinformatic analyses suggest that RsmY and RsmZ each have 3 or 4 putative RsmA binding sites. Each predicted binding site contains a GGA sequence presented in the loop portion of a stem-loop structure. RsmY and RsmZ regulate RsmA, and possibly RsmF, by sequestering these proteins from target mRNAs. In this study, we used selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) chemistry to determine the secondary structures of RsmY and RsmZ and functional assays to characterize the contribution of each GGA site to RsmY/RsmZ activity. Our data indicate that RsmA has two preferential binding sites on RsmY and RsmZ, while RsmF has one preferential binding site on RsmY and two sites on RsmZ. Despite RsmF and RsmA sharing a common consensus site, RsmF binding properties are more restrictive than those of RsmA.IMPORTANCECsrA homologs are present in many bacteria. The opportunistic pathogenPseudomonas aeruginosauses RsmA and RsmF to inversely regulate factors associated with acute and chronic virulence phenotypes. RsmA has an affinity for RsmY and RsmZ higher than that of RsmF. The goal of this study was to understand the differential binding properties of RsmA and RsmF by using the RsmY and RsmZ regulatory small RNAs (sRNAs) as a model. Mutagenesis of the predicted RsmA/RsmF binding sites on RsmY and RsmZ revealed similarities in the sites required to control RsmA and RsmF activityin vivo. Whereas binding by RsmA was relatively tolerant of binding site mutations, RsmF was sensitive to disruption to all but two of the sites, further demonstrating that the requirements for RsmF binding activityin vivoandin vitroare more stringent than those for RsmA.

scholarly journals In Vivo and In Vitro Effects of Integration Host Factor at the DmpR-Regulated ς54-Dependent Po Promoter

2001 ◽  
Vol 183 (9) ◽  
pp. 2842-2851 ◽  
Author(s):  
Chun Chau Sze ◽  
Andrew D. Laurie ◽  
Victoria Shingler
Keyword(s):  
Rna Polymerase ◽  
Binding Site ◽  
Binding Sites ◽  
Integration Host Factor ◽  
Host Factor ◽  
Physical Interaction ◽  
Rich Region ◽  
Polymerase Binding

ABSTRACT Transcription from the Pseudomonas CF600-derived ς54-dependent promoter Po is controlled by the aromatic-responsive activator DmpR. Here we examine the mechanism(s) by which integration host factor (IHF) stimulates DmpR-activated transcriptional output of the Po promoter both in vivo and in vitro. In vivo, the Po promoter exhibits characteristics that typify many ς54-dependent promoters, namely, a phasing-dependent tolerance with respect to the distance from the regulator binding sites to the distally located RNA polymerase binding site, and a strong dependence on IHF for optimal promoter output. IHF is shown to affect transcription via structural repercussions mediated through binding to a single DNA signature located between the regulator and RNA polymerase binding sites. In vitro, using DNA templates that lack the regulator binding sites and thus bypass a role of IHF in facilitating physical interaction between the regulator and the transcriptional apparatus, IHF still mediates a DNA binding-dependent stimulation of Po transcription. This stimulatory effect is shown to be independent of previously described mechanisms for the effects of IHF at ς54 promoters such as aiding binding of the regulator or recruitment of ς54-RNA polymerase via UP element-like DNA. The effect of IHF could be traced to promotion and/or stabilization of open complexes within the nucleoprotein complex that may involve an A+T-rich region of the IHF binding site and promoter-upstream DNA. Mechanistic implications are discussed in the context of a model in which IHF binding results in transduction of DNA instability from an A+T-rich region to the melt region of the promoter.

scholarly journals Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

2008 ◽  
Vol 190 (7) ◽  
pp. 2496-2504 ◽  
Author(s):  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Jun-Rong Wei ◽  
Jwu-Ching Shu ◽  
Chia-Chen Lu ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Start Codon ◽  
Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

scholarly journals Participation of Ets transcription factors in the glucocorticoid response of the rat tyrosine aminotransferase gene.

1994 ◽  
Vol 14 (6) ◽  
pp. 4116-4125 ◽  
Author(s):  
M L Espinás ◽  
J Roux ◽  
J Ghysdael ◽  
R Pictet ◽  
T Grange
Keyword(s):  
Transcription Factors ◽  
Binding Site ◽  
Cell Line ◽  
Binding Sites ◽  
Tyrosine Aminotransferase ◽  
Hierarchical Level ◽  
Detectable Effect ◽  
Independent Manner ◽  
Glucocorticoid Response

We have previously shown that two remote glucocorticoid-responsive units (GRUs) of the rat tyrosine aminotransferase (TAT) gene contain multiple binding sites for several transcription factor families, including the glucocorticoid receptor (GR). We report here the identification of two novel binding sites for members of the Ets family of transcription factors in one of these GRUs. One of these binding sites overlaps the major GR-binding site (GRBS), whereas the other is located in its vicinity. Inactivation of the latter binding site leads to a twofold reduction of the glucocorticoid response, whereas inactivation of the site overlapping the GRBS has no detectable effect. In vivo footprinting analysis reveals that the active site is occupied in a glucocorticoid-independent manner, in a TAT-expressing cell line, even though it is located at a position where there is a glucocorticoid-dependent alteration of the nucleosomal structure. This same site is not occupied in a cell line that does not express TAT but expresses Ets-related DNA-binding activities, suggesting the existence of an inhibitory effect of chromatin structure at a hierarchical level above the nucleosome. The inactive Ets-binding site that overlaps the GRBS is not occupied even in TAT-expressing cells. However, this same overlapping site can confer Ets-dependent stimulation of both basal and glucocorticoid-induced levels when it is isolated from the GRU and duplicated. Ets-1 expression in COS cells mimics the activity of the Ets-related activities present in hepatoma cells. These Ets-binding sites could participate in the integration of the glucocorticoid response of the TAT gene with signal transduction pathways triggered by other nonsteroidal extracellular stimuli.

scholarly journals Could Azithromycin Be Part of Pseudomonas aeruginosa Acute Pneumonia Treatment?

2021 ◽  
Vol 12 ◽  
Author(s):  
Anne-Gaëlle Leroy ◽  
Jocelyne Caillon ◽  
Nathalie Caroff ◽  
Alexis Broquet ◽  
Stéphane Corvec ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Trials ◽  
Respiratory Infections ◽  
Direct Interaction ◽  
Membered Ring ◽  
Careful Examination ◽  
Major Interest ◽  
Acute Pneumonia

Azithromycin (AZM) is a 15-membered-ring macrolide that presents a broad-spectrum antimicrobial activity against Gram-positive bacteria and atypical microorganisms but suffers from a poor diffusion across the outer-membrane of Gram-negative bacilli, including Pseudomonas aeruginosa (PA). However, AZM has demonstrated clinical benefits in patients suffering from chronic PA respiratory infections, especially cystic fibrosis patients. Since the rise of multidrug-resistant PA has led to a growing need for new therapeutic options, this macrolide has been proposed as an adjunctive therapy. Clinical trials assessing AZM in PA acute pneumonia are scarce. However, a careful examination of the available literature provides good rationales for its use in that context. In fact, 14- and 15-membered-ring macrolides have demonstrated immunomodulatory and immunosuppressive effects that could be of major interest in the management of acute illness. Furthermore, growing evidence supports a downregulation of PA virulence dependent on direct interaction with the ribosomes, and based on the modulation of several key regulators from the Quorum Sensing network. First highlighted in vitro, these interesting properties of AZM have subsequently been confirmed in the animal models. In this review, we systematically analyzed the literature regarding AZM immunomodulatory and anti-PA effects. In vitro and in vivo studies, as well as clinical trials were reviewed, looking for rationales for AZM use in PA acute pneumonia.

scholarly journals CfaD-Dependent Expression of a Novel Extracytoplasmic Protein from Enterotoxigenic Escherichia coli

2007 ◽  
Vol 189 (14) ◽  
pp. 5060-5067 ◽  
Author(s):  
M. Carolina Pilonieta ◽  
Maria D. Bodero ◽  
George P. Munson
Keyword(s):  
Escherichia Coli ◽  
Binding Sites ◽  
Secretory Pathway ◽  
Dnase I ◽  
Enterotoxigenic Escherichia Coli ◽  
Watery Diarrhea ◽  
Dnase I Footprinting ◽  
Virulence Regulator

ABSTRACT H10407 is a strain of enterotoxigenic Escherichia coli (ETEC) that utilizes CFA/I pili to adhere to surfaces of the small intestine, where it elaborates toxins that cause profuse watery diarrhea in humans. Expression of the CFA/I pilus is positively regulated at the level of transcription by CfaD, a member of the AraC/XylS family. DNase I footprinting revealed that the activator has two binding sites upstream of the pilus promoter cfaAp. One site extends from positions −23 to −56, and the other extends from positions −73 to −103 (numbering relative to the transcription start site of cfaAp). Additional CfaD binding sites were predicted within the genome of H10407 by computational analysis. Two of these sites lie upstream of a previously uncharacterized gene, cexE. In vitro DNase I footprinting confirmed that both sites are genuine binding sites, and cexEp::lacZ reporters demonstrated that CfaD is required for the expression of cexE in vivo. The amino terminus of CexE contains a secretory signal peptide that is removed during translocation across the cytoplasmic membrane through the general secretory pathway. These studies suggest that CexE may be a novel ETEC virulence factor because its expression is controlled by the virulence regulator CfaD, and its distribution is restricted to ETEC.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (11) ◽  
pp. 5648-5659
Author(s):  
F J McNally ◽  
J Rine
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Binding Sites ◽  
Consensus Sequence ◽  
Protein A ◽  
Replication Initiation ◽  
Yeast Saccharomyces Cerevisiae ◽  
Autonomously Replicating Sequence ◽  
Functional Components

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.

scholarly journals Regulation of the Rhodobacter sphaeroides 2.4.1 hemA Gene by PrrA and FnrL

2008 ◽  
Vol 190 (20) ◽  
pp. 6769-6778 ◽  
Author(s):  
Britton Ranson-Olson ◽  
Jill H. Zeilstra-Ryalls
Keyword(s):  
Binding Site ◽  
Rhodobacter Sphaeroides ◽  
Binding Sites ◽  
Aminolevulinic Acid ◽  
Acid Synthesis ◽  
P2 Promoter ◽  
Indirect Action ◽  
Hema Gene

ABSTRACT Part of the oxygen responsiveness of Rhodobacter sphaeroides 2.4.1 tetrapyrrole production involves changes in transcription of the hemA gene, which codes for one of two isoenzymes catalyzing 5-aminolevulinic acid synthesis. Regulation of hemA transcription from its two promoters is mediated by the DNA binding proteins FnrL and PrrA. The two PrrA binding sites, binding sites I and II, which are located upstream of the more-5′ hemA promoter (P1), are equally important to transcription under aerobic conditions, while binding site II is more important under anaerobic conditions. By using phosphoprotein affinity chromatography and immunoblot analyses, we showed that the phosphorylated PrrA levels in the cell increase with decreasing oxygen tensions. Then, using both in vivo and in vitro methods, we demonstrated that the relative affinities of phosphorylated and unphosphorylated PrrA for the two binding sites differ and that phosphorylated PrrA has greater affinity for site II. We also showed that PrrA regulation is directed toward the P1 promoter. We propose that the PrrA component of anaerobic induction of P1 transcription is attributable to higher affinity of phosphorylated PrrA than of unphosphorylated PrrA for binding site II. Anaerobic activation of the more-3′ hemA promoter (P2) is thought to involve FnrL binding to an FNR consensuslike sequence located upstream of the P2 promoter, but the contribution of FnrL to P1 induction may be indirect since the P1 transcription start is within the putative FnrL binding site. We present evidence suggesting that the indirect action of FnrL works through PrrA and discuss possible mechanisms.

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