scholarly journals Dual Role of the PhoP∼P Response Regulator: Bacillus amyloliquefaciens FZB45 Phytase Gene Transcription Is Directed by Positive and Negative Interactions with the phyC Promoter

2006 ◽  
Vol 188 (19) ◽  
pp. 6953-6965 ◽  
Author(s):  
Oliwia Makarewicz ◽  
Sarah Dubrac ◽  
Tarek Msadek ◽  
Rainer Borriss
Keyword(s):  
Transcription Initiation ◽  
Bacillus Amyloliquefaciens ◽  
Promoter Region ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Consensus Sequence ◽  
Start Codon ◽  
In Vitro Assays ◽  
Unusual Structure ◽  
Phytase Gene

ABSTRACT Several Bacillus strains secrete phytase, an enzyme catalyzing dephosphorylation of myo-inositol hexakisphosphate (phytate). We identified the phyC (phytase) gene from environmental Bacillus amyloliquefaciens FZB45 as a member of the phosphate starvation-inducible PhoPR regulon. In vivo and in vitro assays revealed that PhoP∼P is essential for phyC transcription. The transcriptional start site was identified downstream of a σA-like promoter region located 27 bp upstream of the probable translation ATG start codon. Inspection of the phyC promoter sequence revealed an unusual structure. The− 35 and −10 regions are separated by a window of 21 bp. A pair of tandemly repeated PhoP TT(T/A/C)ACA binding boxes was located within and upstream of the −35 consensus promoter region. A single PhoP box was found within the −10 consensus promoter region. DNase I footprinting experiments performed with isolated PhoP confirmed that PhoP∼P binds at two sites overlapping with the phyC −35 and −10 consensus promoter region. While binding of dimeric PhoP∼P at −35 is essential for activation of the phyC promoter, binding of PhoP∼P at− 10 suppresses promoter activity. A sixfold enhancement of phyC gene expression was registered after T:G substitution of nucleotide −13 (mutant MUT13), which eliminates PhoP binding at the single PhoP box without impairing the −10 consensus sequence. Moreover, MUT13 also expressed phyC during phosphate-replete growth, suggesting that the repressing effect due to binding of PhoP∼P at −10 was abolished. A model is presented in which transcription initiation of phyC is positively and negatively affected by the actual concentration of the PhoP∼P response regulator.

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 Modulation of covR Expression in Streptococcus mutans UA159

2008 ◽  
Vol 190 (13) ◽  
pp. 4478-4488 ◽  
Author(s):  
Patrick Chong ◽  
Laura Drake ◽  
Indranil Biswas
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Growth Phase ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Mobility Shift

ABSTRACT The biofilm-forming Streptococcus mutans is a gram-positive bacterium that resides in the human oral cavity and is considered to be the primary etiological agent in the formation of dental caries. The global response regulator CovR, which lacks a cognate sensor kinase, is essential for the pathogenesis and biofilm formation of this bacterium, but it is not clear how covR expression is regulated in S. mutans. In this communication, we present the results of our studies examining various factors that regulate the expression of covR in S. mutans UA159. The results of Southern hybridization and PCR analysis indicated that CovR is an orphan response regulator in various isolates of S. mutans. The transcriptional start site for covR was found to be 221 base pairs upstream of the ATG start codon, and site-directed mutagenesis of the upstream TATAAT box confirmed our findings. The expression of covR is growth phase dependent, with maximal expression observed during exponential-growth phase. While changes to the growth temperature did not significantly affect the expression of covR, increasing the pH or the concentration of Mg2+ in the growth medium leads to an increase in covR expression. The results of semiquantitative reverse transcriptase PCR analysis and in vivo transcriptional-fusion reporter assays indicated that CovR autoregulates its own expression; this was verified by the results of electrophoretic mobility shift assays and DNase I protection assays, which demonstrated direct binding of CovR to the promoter region. Apparently, regulation by Mg2+ and the autoregulation of covR are not linked. A detailed analysis of the regulation of CovR may lead to a better understanding of the pathogenesis of S. mutans, as well as providing further insight into the prevention of dental caries.

scholarly journals Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region

2008 ◽  
Vol 190 (19) ◽  
pp. 6467-6474 ◽  
Author(s):  
Oliwia Makarewicz ◽  
Svetlana Neubauer ◽  
Corinna Preusse ◽  
Rainer Borriss
Keyword(s):  
Bacillus Amyloliquefaciens ◽  
Promoter Region ◽  
Response Regulator ◽  
Dependent Manner ◽  
Coding Region ◽  
Mobility Shift ◽  
Dnase I Footprinting ◽  
Regulator Protein ◽  
Transition State Regulator ◽  
Gel Mobility Shift

ABSTRACT We have previously identified the phyC gene of Bacillus amyloliquefaciens FZB45, encoding extracellular phytase, as a member of the PhoP regulon, which is expressed only during phosphate starvation. Its σA-dependent promoter is positively and negatively regulated by the phosphorylated PhoP response regulator in a phosphate-dependent manner (O. Makarewicz, S. Dubrac, T. Msadek, and R. Borriss, J. Bacteriol. 188:6953-6965, 2006). Here, we provide experimental evidence that the transcription of phyC underlies a second control mechanism exerted by the global transient-phase regulator protein, AbrB, which hinders its expression during exponential growth. Gel mobility shift and DNase I footprinting experiments demonstrated that AbrB binds to two different regions in the phyC promoter region that are separated by about 200 bp. One binding site is near the divergently orientated yodU gene, and the second site is located downstream of the phyC promoter and extends into the coding region of the phyC gene. Cooperative binding to the two distant binding regions is necessary for the AbrB-directed repression of phyC transcription. AbrB does not affect the transcription of the neighboring yodU gene.

scholarly journals Characterization of the Fructosyltransferase Gene of Actinomyces naeslundii WVU45

2000 ◽  
Vol 182 (13) ◽  
pp. 3649-3654 ◽  
Author(s):  
Lori J. Bergeron ◽  
Evangelia Morou-Bermudez ◽  
Robert A. Burne
Keyword(s):  
Transcription Initiation ◽  
Transcriptional Start Site ◽  
Initiation Site ◽  
Start Codon ◽  
Gram Positive ◽  
Regulation Of Expression ◽  
Significant Similarity ◽  
Gram Negative ◽  
Gram Positive Bacteria ◽  
Actinomyces Naeslundii

ABSTRACT Oral actinomycetes produce fructosyltransferase (FTF) enzymes which convert sucrose into polymers of d-fructose, known as levans, and these polymers are thought to contribute to the persistence and virulence of the organisms. A gene encoding FTF was isolated fromActinomyces naeslundii WVU45; the deduced amino acid sequence showed significant similarity to known levansucrases of gram-negative environmental isolates but was less similar to FTFs from gram-positive bacteria. A transcriptional start site was mapped by primer extension 70 bp 5′ from the putative start codon. Promoter fusions to a chloramphenicol acetyltransferase gene were used to confirm that there was a functional promoter driving ftfexpression and to show that sequences located 86 to 218 bp upstream of the transcription initiation site were required for optimalftf expression. Quantitative slot blot analysis against total RNA from cells grown on different sugars or from different growth phases revealed that ftf was constitutively transcribed. Thus, the A. naeslundii FTF is more similar in primary sequence and the regulation of expression to levansucrases of gram-negative bacteria than gram-positive bacteria.

Partial cloning and characterization of Slc12a2: the gene encoding the secretory Na+-K+-2Cl−cotransporter

1997 ◽  
Vol 273 (4) ◽  
pp. C1267-C1277 ◽  
Author(s):  
Jeffrey Randall ◽  
Tina Thorne ◽  
Eric Delpire
Keyword(s):  
Transcription Initiation ◽  
Collecting Duct ◽  
Consensus Sequence ◽  
Cell Volume Regulation ◽  
Start Codon ◽  
Luciferase Reporter ◽  
Nucleotide Sequence Analysis ◽  
Ribonuclease Protection Assay ◽  
Gene Encoding ◽  
Spliced Variant

The Slc12a2 gene encodes a widely expressed bumetanide-sensitive Na+-K+-2Cl−cotransporter that participates in various functions such as Cl− secretion and cell volume regulation. We isolated and characterized 75 kilobases of the murine gene encoding the cotransporter. The cotransport protein is encoded by 27 exons. Ribonuclease protection assay and primer extension demonstrated tissue-specific transcription initiation sites located within 270 base pairs upstream of the start codon. Nucleotide sequence analysis of the proximal 5′-flanking region revealed the presence of a weak TATA box, multiple Sp1/GC consensus sites, and the consensus sequence of a putative transcriptional initiator. Transfection of luciferase reporter gene constructs in mouse inner medullary collecting duct (mIMCD-3) cells confirmed the location of the minimal promoter within a 120-base pair fragment upstream of the cDNA. We also report the identification of an alternatively spliced variant of the cotransporter, expressed primarily in brain. This new spliced variant lacks exon 21, which encodes a 16-amino acid peptide located in the COOH-terminal tail of the protein. The absence of this exon causes the loss of the single protein kinase A consensus site of the cotransport protein.

scholarly journals Regulation of Alginate Biosynthesis inPseudomonas syringae pv. syringae

1999 ◽  
Vol 181 (11) ◽  
pp. 3478-3485 ◽  
Author(s):  
Mohamed K. Fakhr ◽  
Alejandro Peñaloza-Vázquez ◽  
Ananda M. Chakrabarty ◽  
Carol L. Bender
Keyword(s):  
Sigma Factor ◽  
Promoter Region ◽  
Biosynthetic Pathway ◽  
Response Regulator ◽  
Consensus Sequence ◽  
Gene Encoding ◽  
Environmental Signals ◽  
Alginate Production ◽  
Key Enzyme ◽  
Alginate Biosynthesis

ABSTRACT Both Pseudomonas aeruginosa and the phytopathogenP. syringae produce the exopolysaccharide alginate. However, the environmental signals that trigger alginate gene expression in P. syringae are different from those inP. aeruginosa with copper being a major signal in P. syringae. In P. aeruginosa, the alternate sigma factor encoded by algT (ς22) and the response regulator AlgR1 are required for transcription of algD, a gene which encodes a key enzyme in the alginate biosynthetic pathway. In the present study, we cloned and characterized the gene encoding AlgR1 from P. syringae. The deduced amino acid sequence of AlgR1 from P. syringae showed 86% identity to its P. aeruginosa counterpart. Sequence analysis of the region flankingalgR1 in P. syringae revealed the presence ofargH, algZ, and hemC in an arrangement virtually identical to that reported in P. aeruginosa. An algR1 mutant, P. syringaeFF5.32, was defective in alginate production but could be complemented when algR1 was expressed in trans. ThealgD promoter region in P. syringae(PsalgD) was also characterized and shown to diverge significantly from the algD promoter in P. aeruginosa. Unlike P. aeruginosa, algR1was not required for the transcription of algD in P. syringae, and PsalgD lacked the consensus sequence recognized by AlgR1. However, both the algD andalgR1 upstream regions in P. syringae contained the consensus sequence recognized by ς22, suggesting thatalgT is required for transcription of both genes.

scholarly journals Poly-3-Hydroxybutyrate Degradation in Rhizobium (Sinorhizobium) meliloti: Isolation and Characterization of a Gene Encoding 3-Hydroxybutyrate Dehydrogenase

1999 ◽  
Vol 181 (3) ◽  
pp. 849-857 ◽  
Author(s):  
P. Aneja ◽  
T. C. Charles
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Sinorhizobium Meliloti ◽  
Transcriptional Start Site ◽  
Consensus Sequence ◽  
Start Codon ◽  
Start Site ◽  
Gene Encoding ◽  
Reading Frame ◽  
Isolation And Characterization

ABSTRACT We have cloned and sequenced the 3-hydroxybutyrate dehydrogenase-encoding gene (bdhA) from Rhizobium (Sinorhizobium) meliloti. The gene has an open reading frame of 777 bp that encodes a polypeptide of 258 amino acid residues (molecular weight 27,177, pI 6.07). The R. meliloti Bdh protein exhibits features common to members of the short-chain alcohol dehydrogenase superfamily. bdhA is the first gene transcribed in an operon that also includes xdhA, encoding xanthine oxidase/dehydrogenase. Transcriptional start site analysis by primer extension identified two transcription starts. S1, a minor start site, was located 46 to 47 nucleotides upstream of the predicted ATG start codon, while S2, the major start site, was mapped 148 nucleotides from the start codon. Analysis of the sequence immediately upstream of either S1 or S2 failed to reveal the presence of any known consensus promoter sequences. Although a ς54 consensus sequence was identified in the region between S1 and S2, a corresponding transcript was not detected, and a rpoN mutant of R. meliloti was able to utilize 3-hydroxybutyrate as a sole carbon source. The R. meliloti bdhA gene is able to confer uponEscherichia coli the ability to utilize 3-hydroxybutyrate as a sole carbon source. An R. meliloti bdhA mutant accumulates poly-3-hydroxybutyrate to the same extent as the wild type and shows no symbiotic defects. Studies with a strain carrying alacZ transcriptional fusion to bdhAdemonstrated that gene expression is growth phase associated.

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

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.

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

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

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

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