scholarly journals Expression of the cpdA Gene, Encoding a 3′,5′-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex

2008 ◽  
Vol 191 (3) ◽  
pp. 922-930 ◽  
Author(s):  
Han-Suk Kim ◽  
Sung-Min Kim ◽  
Hyun-Jung Lee ◽  
Soon-Jung Park ◽  
Kyu-Ho Lee
Keyword(s):  
Regulatory Region ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Upstream Region ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Multicopy Plasmid ◽  
Camp Receptor Protein ◽  
Camp Phosphodiesterase ◽  
Camp Receptor

ABSTRACT The intracellular level of cyclic 3′,5′-AMP (cAMP), a signaling molecule that mediates a variety of cellular processes, is finely modulated by the regulation of its synthesis, excretion, and degradation. In this study, cAMP phosphodiesterase (CpdA), an enzyme that catalyzes the conversion of cAMP to AMP, was characterized in a pathogenic bacterium, Vibrio vulnificus. The cpdA gene exists in an operon composed of mutT, yqiB, cpdA, and yqiA, the transcription of which was initiated at position −22 upstream of mutT. A cpdA-null mutant of V. vulnificus contained significantly higher levels of cAMP than the wild type but showed no detectable cAMP when a multicopy plasmid of the cpdA gene was provided in trans, suggesting that CpdA is responsible for cAMP degradation. Cellular contents of the CpdA protein decreased dramatically in both cya and crp mutants. In addition, levels of expression of the cpdA::luxAB transcription fusion decreased in cya and crp mutants. The level of expression of cpdA::luxAB in the cya mutant increased in a concentration-dependent manner upon the exogenous addition of cAMP. The cAMP-cAMP receptor protein (CRP) complex bound directly to the upstream region of mutT, which includes a putative CRP-binding sequence centered at position −95.5 relative to the transcription start site. Site-directed mutagenesis or the deletion of this sequence in the cpdA::luxAB transcription fusion resulted in the loss of regulation by cAMP and CRP. Thus, this study demonstrates that CpdA plays a crucial role in determining the intracellular cAMP level and shows for the first time that the expression of cpdA is activated by the cAMP-CRP complex via direct binding to the regulatory region.

scholarly journals Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917

2015 ◽  
Vol 81 (22) ◽  
pp. 7687-7696 ◽  
Author(s):  
Huihui Yan ◽  
Feifei Bao ◽  
Liping Zhao ◽  
Yanying Yu ◽  
Jiaqin Tang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Carbon Sources ◽  
Coli Strain ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Upstream Region ◽  
Camp Receptor Protein ◽  
Content Type ◽  
Camp Receptor

ABSTRACTHeparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription.1H nuclear magnetic resonance (NMR) analysis was further performed to determine theEscherichia colistrain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains.

scholarly journals Positive and Negative Transcriptional Regulation of the Escherichia coli Gluconate Regulon Gene gntTby GntR and the Cyclic AMP (cAMP)-cAMP Receptor Protein Complex

1998 ◽  
Vol 180 (7) ◽  
pp. 1777-1785 ◽  
Author(s):  
Norbert Peekhaus ◽  
T. Conway
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Catabolite Repression ◽  
Transcriptional Start Site ◽  
Negative Regulator ◽  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Start Site ◽  
Camp Receptor Protein ◽  
Camp Receptor

ABSTRACT The gntT gene of Escherichia coli is specifically induced by gluconate and repressed via catabolite repression. Thus, gluconate is both an inducer and a repressor ofgntT expression since gluconate is a catabolite-repressing sugar. In a gntR deletion mutant, the expression of a chromosomal gntT::lacZ fusion is both high and constitutive, confirming that GntR is the negative regulator of gntT. Indeed, GntR binds to two consensus gnt operator sites; one overlaps the −10 region of the gntT promoter, and the other is centered at +120 with respect to the transcriptional start site. The binding of GntR to these sites was proven in vitro by gel redardation assays and in vivo by site-directed mutagenesis of the binding sites. Binding of GntR to the operators is eliminated by gluconate and also by 6-phosphogluconate at a 10-fold-higher concentration. Interestingly, when gntR deletion strains are grown in the presence of gluconate, there is a twofold decrease in gntTexpression which is independent of catabolite repression and binding of GntR to the operator sites. This novel response of gntRmutants to the inducer is termed ultrarepression. Transcription ofgntT is activated by binding of the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex to a CRP binding site positioned at −71 upstream of the gntT transcription start site.

scholarly journals Sycrp2 Is Essential for Twitching Motility in the CyanobacteriumSynechocystissp. Strain PCC 6803

2018 ◽  
Vol 200 (21) ◽  
Author(s):  
Wei-Yu Song ◽  
Sha-Sha Zang ◽  
Zheng-Ke Li ◽  
Guo-Zheng Dai ◽  
Ke Liu ◽  
...  
Keyword(s):  
Cell Motility ◽  
Receptor Protein ◽  
Upstream Region ◽  
Twitching Motility ◽  
Camp Receptor Protein ◽  
Mobility Shift ◽  
Content Type ◽  
Receptor Proteins ◽  
Camp Receptor ◽  
Pcc 6803

ABSTRACTTwo cAMP receptor proteins (CRPs), Sycrp1 (encoded bysll1371) and Sycrp2 (encoded bysll1924), exist in the cyanobacteriumSynechocystissp. strain PCC 6803. Previous studies have demonstrated that Sycrp1 has binding affinity for cAMP and is involved in motility by regulating the formation of pili. However, the function of Sycrp2 remains unknown. Here, we report thatsycrp2disruption results in the loss of motility ofSynechocystissp. PCC 6803, and that the phenotype can be recovered by reintroducing thesycrp2gene into the genome ofsycrp2-disrupted mutants. Electron microscopy showed that thesycrp2-disrupted mutant lost the pilus apparatus on the cell surface, resulting in a lack of cell motility. Furthermore, the transcript level of thepilA9-pilA11operon (essential for cell motility and regulated by the cAMP receptor protein Sycrp1) was markedly decreased insycrp2-disrupted mutants compared with the wild-type strain. Moreover, yeast two-hybrid analysis and a pulldown assay demonstrated that Sycrp2 interacted with Sycrp1 to form a heterodimer and that Sycrp1 and Sycrp2 interacted with themselves to form homodimers. Gel mobility shift assays revealed that Sycrp1 specifically binds to the upstream region ofpilA9. Together, these findings indicate that inSynechocystissp. PCC 6803, Sycrp2 regulates the formation of pili and cell motility by interacting with Sycrp1.IMPORTANCEcAMP receptor proteins (CRPs) are widely distributed in cyanobacteria and play important roles in regulating gene expression. Although many cyanobacterial species have two cAMP receptor-like proteins, the functional links between them are unknown. Here, we found that Sycrp2 in the cyanobacteriumSynechocystissp. strain PCC 6803 is essential for twitching motility and that it interacts with Sycrp1, a known cAMP receptor protein involved with twitching motility. Our findings indicate that the two cAMP receptor-like proteins in cyanobacteria do not have functional redundancy but rather work together.

scholarly journals Control of Transposon-mediated Activation of theglpFKOperon ofEscherichia coliby two DNA binding Proteins

2016 ◽  
Author(s):  
Zhongge Zhang ◽  
Milton H. Saier
Keyword(s):  
Insertion Sequence ◽  
Gene Activation ◽  
Regulatory Region ◽  
Receptor Protein ◽  
Upstream Region ◽  
Upstream Regulatory Region ◽  
Wild Type ◽  
Transposon Insertion ◽  
Glycerol Utilization ◽  
Camp Receptor

AbstractEscherichia colicells deleted for the cyclic AMP (cAMP) receptor protein (Crp) gene (Δcrp) cannot utilize glycerol because cAMP-Crp is a required positive activator of glycerol utilization operonglpFK. We have previously shown that a transposon, Insertion Sequence 5 (IS5), can reversibly insert into the upstream regulatory region of the operon so as to activateglpFKand enable glycerol utilization. GlpR, which repressesglpFKtranscription, binds to theglpFKupstream region near the site of IS5insertion, and prevents insertion. We here show that the cAMP-Crp complex, which also binds to theglpFKupstream regulatory region, also inhibits IS5hopping into the activating site. This finding allowed us to identify conditions under which wild type cells can acquireglpFK-activating IS5insertions. Maximal rates of IS5insertion into the activating site require the presence of glycerol as well as a non-metabolizable sugar analogue that lowers cytoplasmic cAMP concentrations. Under these conditions, IS5insertional mutants accumulate and outcompete the wild type cells. Because of the widespread distribution of glucose analogues in nature, this mechanism of gene activation could have evolved by natural selection.

scholarly journals cAMP receptor protein (CRP) positively regulates the yihU–yshA operon in Salmonella enterica serovar Typhi

Microbiology ◽  
2011 ◽  
Vol 157 (3) ◽  
pp. 636-647 ◽  
Author(s):  
J. M. Villarreal ◽  
I. Hernández-Lucas ◽  
F. Gil ◽  
I. L. Calderón ◽  
E. Calva ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Salmonella Enterica ◽  
Regulatory Region ◽  
Enteric Bacteria ◽  
Transcriptional Unit ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Salmonella Enterica Serovar Typhi ◽  
Genetic Components ◽  
Camp Receptor

Salmonella enterica serovar Typhi (S. Typhi) is the aetiological agent of typhoid fever in humans. This bacterium is also able to persist in its host, causing a chronic disease by colonizing the spleen, liver and gallbladder, in the last of which the pathogen forms biofilms in order to survive the bile. Several genetic components, including the yihU–yshA genes, have been suggested to be involved in the survival of Salmonella in the gallbladder. In this work we describe how the yihU–yshA gene cluster forms a transcriptional unit regulated positively by the cAMP receptor global regulator CRP (cAMP receptor protein). The results obtained show that two CRP-binding sites on the regulatory region of the yihU–yshA operon are required to promote transcriptional activation. In this work we also demonstrate that the yihU–yshA transcriptional unit is carbon catabolite-repressed in Salmonella, indicating that it forms part of the CRP regulon in enteric bacteria.

scholarly journals DdrI, a cAMP Receptor Protein Family Member, Acts as a Major Regulator for Adaptation ofDeinococcus radioduransto Various Stresses

2018 ◽  
Vol 200 (13) ◽  
Author(s):  
Laura Meyer ◽  
Geneviève Coste ◽  
Suzanne Sommer ◽  
Jacques Oberto ◽  
Fabrice Confalonieri ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Division ◽  
Heat Shock ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Central Metabolism ◽  
Camp Receptor Protein ◽  
Content Type ◽  
Dna Damaging Agents ◽  
Camp Receptor

ABSTRACTTheDNAdamageresponseddrIgene encodes a transcription regulator belonging to the cAMP receptor protein (CRP) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects and sensitivity to DNA-damaging agents and to oxidative stress. Here, we show that the absence of the DdrI protein also confers sensitivity to heat shock treatment, and several genes involved in heat shock response were shown to be upregulated in a DdrI-dependent manner. Interestingly, expression of theEscherichia coliCRP partially compensates for the absence of the DdrI protein. Microscopic observations of ΔddrImutant cells revealed an increased proportion of two-tetrad and anucleated cells in the population compared to the wild-type strain, indicating that DdrI is crucial for the completion of cell division and/or chromosome segregation. We show that DdrI is also involved in the megaplasmid MP1 stability and in efficient plasmid transformation by facilitating the maintenance of the incoming plasmid in the cell. Thein silicoprediction of putative DdrI binding sites in theD. radioduransgenome suggests that hundreds of genes, belonging to several functional groups, may be regulated by DdrI. In addition, the DdrI protein absolutely requires cAMP forin vitrobinding to specific target sequences, and it acts as a dimer. All these data underline the major role of DdrI inD. radioduransphysiology under normal and stress conditions by regulating, both directly and indirectly, a cohort of genes involved in various cellular processes, including central metabolism and specific responses to diverse harmful environments.IMPORTANCEDeinococcus radioduranshas been extensively studied to elucidate the molecular mechanisms responsible for its exceptional ability to withstand lethal effects of various DNA-damaging agents. A complex network, including efficient DNA repair, protein protection against oxidation, and diverse metabolic pathways, plays a crucial role for its radioresistance. The regulatory networks orchestrating these various pathways are still missing. Our data provide new insights into the crucial contribution of the transcription factor DdrI for theD. radioduransability to withstand harmful conditions, including UV radiation, mitomycin C treatment, heat shock, and oxidative stress. Finally, we highlight that DdrI is also required for accurate cell division, for maintenance of plasmid replicons, and for central metabolism processes responsible for the overall cell physiology.

scholarly journals A cAMP-independent carbohydrate-driven mechanism inhibits tnaA expression and TnaA enzyme activity in Escherichia coli

Microbiology ◽  
2014 ◽  
Vol 160 (9) ◽  
pp. 2079-2088 ◽  
Author(s):  
Gang Li ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Receptor Protein ◽  
Intracellular Camp ◽  
Camp Receptor Protein ◽  
Additional Mechanism ◽  
E Coli ◽  
Tolc Protein ◽  
Camp Receptor ◽  
Camp Levels

When Escherichia coli is grown in a medium lacking glucose or another preferred carbohydrate, the concentration of cAMP–cAMP receptor protein (cAMP–CRP) increases, and this latter complex regulates the expression of more than 180 genes. To respond rapidly to changes in carbohydrate availability, E. coli must maintain a suitable intracellular concentration of cAMP by either exporting or degrading excess cAMP. Currently, cAMP export via the TolC protein is thought to be more efficient at reducing these levels than is CpdA-mediated degradation of cAMP. Here, we compared the contributions of TolC and CpdA by measuring the expression of cAMP-regulated genes that encode tryptophanase (TnaA) and β-galactosidase. In the presence of exogenous cAMP, a tolC mutant produced intermediate levels of these enzymes, suggesting that cAMP levels were held in check by CpdA. Conversely, a cpdA mutant produced much higher amounts of these enzymes, indicating that CpdA was more efficient than TolC at reducing cAMP levels. Surprisingly, expression of the tnaA gene halted rapidly when glucose was added to cells lacking both TolC and CpdA, even though under these conditions cAMP could not be removed by either pathway and tnaA expression should have remained high. This result suggests the existence of an additional mechanism that eliminates intracellular cAMP or terminates expression of some cAMP–CRP-regulated genes. In addition, adding glucose and other carbohydrates rapidly inhibited the function of pre-formed TnaA, indicating that TnaA is regulated by a previously unknown carbohydrate-dependent post-translational mechanism.

scholarly journals Environmental Signals Controlling Production of Hemagglutinin/Protease in Vibrio cholerae

2001 ◽  
Vol 69 (10) ◽  
pp. 6549-6553 ◽  
Author(s):  
Jorge A. Benitez ◽  
Anisia J. Silva ◽  
Richard A. Finkelstein
Keyword(s):  
Vibrio Cholerae ◽  
Deletion Mutant ◽  
Receptor Protein ◽  
Intracellular Camp ◽  
Wild Type ◽  
Camp Receptor Protein ◽  
Environmental Signals ◽  
Camp Receptor ◽  
Camp Levels

ABSTRACT Vibrio cholerae hemagglutinin/protease (Hap) was induced upon nutrient limitation and strongly repressed by glucose. Hap was not produced in a mutant defective in the cyclic AMP (cAMP) receptor protein, suggesting that intracellular cAMP levels mediate Hap expression. No difference was found in Hap production between anrpoS deletion mutant and its isogenic wild-type precursor, indicating that the alternate ςs factor is not essential for Hap expression. Based on these and previous results, we discuss the role of Hap in the pathogenesis of cholera.

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