Probing the Mechanism of CRP Activation by Site-Directed Mutagenesis: The Role of Serine 128 in the Allosteric Pathway of cAMP Receptor Protein Activation

Biochemistry ◽  
1995 ◽  
Vol 34 (34) ◽  
pp. 10816-10826 ◽  
Author(s):  
Xiaodong Cheng ◽  
Lubomir Kovac ◽  
J. Ching Lee
Keyword(s):  
Site Directed Mutagenesis ◽  
Receptor Protein ◽  
Directed Mutagenesis ◽  
Camp Receptor Protein ◽  
Protein Activation ◽  
Camp Receptor ◽  
Allosteric Pathway

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 Impacts of Global Transcriptional Regulators on Persister Metabolism

2015 ◽  
Vol 59 (5) ◽  
pp. 2713-2719 ◽  
Author(s):  
Wendy W. K. Mok ◽  
Mehmet A. Orman ◽  
Mark P. Brynildsen
Keyword(s):  
Cyclic Amp ◽  
Transcriptional Regulators ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Catabolic Activity ◽  
Camp Receptor

ABSTRACTBacterial persisters are phenotypic variants with an extraordinary capacity to tolerate antibiotics, and they are hypothesized to be a main cause of chronic and relapsing infections. Recent evidence has suggested that the metabolism of persisters can be targeted to develop therapeutic countermeasures; however, knowledge of persister metabolism remains limited due to difficulties associated with isolating these rare and transient phenotypic variants. By using a technique to measure persister catabolic activity, which is based on the ability of metabolites to enable aminoglycoside (AG) killing of persisters, we investigated the role of seven global transcriptional regulators (ArcA, Cra, cyclic AMP [cAMP] receptor protein [CRP], DksA, FNR, Lrp, and RpoS) on persister metabolism. We found that removal of CRP resulted in a loss of AG potentiation in persisters for all metabolites tested. These results highlight a central role for cAMP/CRP in persister metabolism, as its perturbation can significantly diminish the metabolic capabilities of persisters and effectively eliminate the ability of AGs to eradicate these troublesome bacteria.

Regulatory role of cAMP receptor protein over Escherichia coli fumarase genes

2012 ◽  
Vol 50 (3) ◽  
pp. 426-433 ◽  
Author(s):  
Yu-Pei Chen ◽  
Hsiao-Hsien Lin ◽  
Chi-Dung Yang ◽  
Shin-Hong Huang ◽  
Ching-Ping Tseng
Keyword(s):  
Escherichia Coli ◽  
Regulatory Role ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Camp Receptor

scholarly journals Positive Effect of Carbon Sources on Natural Transformation in Escherichia coli: Role of Low-Level Cyclic AMP (cAMP)-cAMP Receptor Protein in the Derepression ofrpoS

2015 ◽  
Vol 197 (20) ◽  
pp. 3317-3328 ◽  
Author(s):  
Mengyue Guo ◽  
Huanyu Wang ◽  
Nengbin Xie ◽  
Zhixiong Xie
Keyword(s):  
Escherichia Coli ◽  
Natural Transformation ◽  
Carbon Sources ◽  
Transformation Frequency ◽  
Receptor Protein ◽  
Camp Receptor Protein ◽  
Content Type ◽  
E Coli ◽  
Camp Receptor

ABSTRACTNatural plasmid transformation ofEscherichia coliis a complex process that occurs strictly on agar plates and requires the global stress response factor σS. Here, we showed that additional carbon sources could significantly enhance the transformability ofE. coli. Inactivation of phosphotransferase system genes (ptsH,ptsG, andcrr) caused an increase in the transformation frequency, and the addition of cyclic AMP (cAMP) neutralized the promotional effect of carbon sources. This implies a negative role of cAMP in natural transformation. Further study showed thatcrpandcyaAmutations conferred a higher transformation frequency, suggesting that the cAMP-cAMP receptor protein (CRP) complex has an inhibitory effect on transformation. Moreover, we observed thatrpoSis negatively regulated by cAMP-CRP in early log phase and that bothcrpandcyaAmutants show no transformation superiority whenrpoSis knocked out. Therefore, it can be concluded that both thecrpandcyaAmutations derepressrpoSexpression in early log phase, whereby they aid in the promotion of natural transformation ability. We also showed that the accumulation of RpoS during early log phase can account for the enhanced transformation aroused by additional carbon sources. Our results thus demonstrated that the presence of additional carbon sources promotes competence development and natural transformation by reducing cAMP-CRP and, thus, derepressingrpoSexpression during log phase. This finding could contribute to a better understanding of the relationship between nutrition state and competence, as well as the mechanism of natural plasmid transformation inE. coli.IMPORTANCEEscherichia coli, which is not usually considered to be naturally transformable, was found to spontaneously take up plasmid DNA on agar plates. Researching the mechanism of natural transformation is important for understanding the role of transformation in evolution, as well as in the transfer of pathogenicity and antibiotic resistance genes. In this work, we found that carbon sources significantly improve transformation by decreasing cAMP. Then, the low level of cAMP-CRP derepresses the general stress response regulator RpoS via a biphasic regulatory pattern, thereby contributing to transformation. Thus, we demonstrate the mechanism by which carbon sources affect natural transformation, which is important for revealing information about the interplay between nutrition state and competence development inE. coli.

scholarly journals Genomic mapping of cAMP receptor protein (CRPMt) inMycobacterium tuberculosis: relation to transcriptional start sites and the role of CRPMtas a transcription factor

2014 ◽  
Vol 42 (13) ◽  
pp. 8320-8329 ◽  
Author(s):  
Christina Kahramanoglou ◽  
Teresa Cortes ◽  
Nishad Matange ◽  
Debbie M. Hunt ◽  
Sandhya S. Visweswariah ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Receptor Protein ◽  
Genomic Mapping ◽  
Camp Receptor Protein ◽  
Camp Receptor ◽  
Transcriptional Start Sites

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.

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