scholarly journals Cyclic AMP Receptor Protein RegulatescspE, an Early Cold-Inducible Gene, in Escherichia coli

2011 ◽  
Vol 193 (22) ◽  
pp. 6142-6151 ◽  
Author(s):  
Sheetal Uppal ◽  
Svetlana R. Maurya ◽  
Ramesh S. Hire ◽  
Narendra Jawali
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Cyclic Amp ◽  
Target Site ◽  
Class I ◽  
Receptor Protein ◽  
Upstream Region ◽  
Core Motif ◽  
Content Type ◽  
Cyclic Amp Receptor Protein

cspE, a member of thecspAfamily of cold shock proteins inEscherichia coli, is an early cold-inducible protein. The nucleic acid melting ability and transcription antiterminator activity of CspE have been reported to be critical for growth at low temperature. Here, we show that the cyclic AMP receptor protein (CRP), a global regulator involved in sugar metabolism, upregulatescspEinE. coli. Sequence analysis of thecspEupstream region revealed a putative CRP target site centered at −61.5 relative to the transcription start. The binding of CRP to this target site was demonstrated using electrophoretic mobility shift assays. The presence of this site was shown to be essential for PcspEactivation by CRP. Mutational analysis of the binding site indicated that the presence of an intact second core motif is more important than the first core motif for CRP-PcspEinteraction. Based on the promoter architecture, we classified PcspEas a class I CRP-dependent promoter. This was further substantiated by our data demonstrating the involvement of the AR1 domain of CRP in PcspEtranscription. Furthermore, the substitutions in the key residues of the RNA polymerase α-subunit C-terminal domain (α-CTD), which are important for class I CRP-dependent transcription, showed the involvement of 265 and 287 determinants in PcspEtranscription. In addition, the deletion ofcrpled to a growth defect at low temperature, suggesting that CRP plays an important role in cold adaptation.

scholarly journals Cyclic AMP Receptor Protein-Dependent Activation of the Escherichia coli acsP2 Promoter by a Synergistic Class III Mechanism

2003 ◽  
Vol 185 (17) ◽  
pp. 5148-5157 ◽  
Author(s):  
Christine M. Beatty ◽  
Douglas F. Browning ◽  
Stephen J. W. Busby ◽  
Alan J. Wolfe
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class I ◽  
Receptor Protein ◽  
Class Iii ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Rna Polymerase Holoenzyme

ABSTRACT The cyclic AMP receptor protein (CRP) activates transcription of the Escherichia coli acs gene, which encodes an acetate-scavenging enzyme required for fitness during periods of carbon starvation. Two promoters direct transcription of acs, the distal acsP1 and the proximal acsP2. In this study, we demonstrated that acsP2 can function as the major promoter and showed by in vitro studies that CRP facilitates transcription by “focusing” RNA polymerase to acsP2. We proposed that CRP activates transcription from acsP2 by a synergistic class III mechanism. Consistent with this proposal, we showed that CRP binds two sites, CRP I and CRP II. Induction of acs expression absolutely required CRP I, while optimal expression required both CRP I and CRP II. The locations of these DNA sites for CRP (centered at positions −69.5 and −122.5, respectively) suggest that CRP interacts with RNA polymerase through class I interactions. In support of this hypothesis, we demonstrated that acs transcription requires the surfaces of CRP and the C-terminal domain of the α subunit of RNA polymerase holoenzyme (α-CTD), which is known to participate in class I interactions: activating region 1 of CRP and the 287, 265, and 261 determinants of the α-CTD. Other surface-exposed residues in the α-CTD contributed to acs transcription, suggesting that the α-CTD may interact with at least one protein other than CRP.

scholarly journals Determinants of the C-Terminal Domain of the Escherichia coli RNA Polymerase α Subunit Important for Transcription at Class I Cyclic AMP Receptor Protein-Dependent Promoters

2002 ◽  
Vol 184 (8) ◽  
pp. 2273-2280 ◽  
Author(s):  
Nigel J. Savery ◽  
Georgina S. Lloyd ◽  
Stephen J. W. Busby ◽  
Mark S. Thomas ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Rna Polymerase ◽  
Class Ii ◽  
Class I ◽  
Receptor Protein ◽  
Α Subunit ◽  
Cyclic Amp Receptor Protein ◽  
Terminal Domain

ABSTRACT Alanine scanning of the Escherichia coli RNA polymerase α subunit C-terminal domain (αCTD) was used to identify amino acid side chains important for class I cyclic AMP receptor protein (CRP)-dependent transcription. Key residues were investigated further in vivo and in vitro. Substitutions in three regions of αCTD affected class I CRP-dependent transcription from the CC(−61.5) promoter and/or the lacP1 promoter. These regions are (i) the 287 determinant, previously shown to contact CRP during class II CRP-dependent transcription; (ii) the 265 determinant, previously shown to be important for αCTD-DNA interactions, including those required for class II CRP-dependent transcription; and (iii) the 261 determinant. We conclude that CRP contacts the same target in αCTD, the 287 determinant, at class I and class II CRP-dependent promoters. We also conclude that the relative contributions of individual residues within the 265 determinant depend on promoter sequence, and we discuss explanations for effects of substitutions in the 261 determinant.

scholarly journals Repression by Cyclic AMP Receptor Protein at a Distance

mBio ◽  
2012 ◽  
Vol 3 (5) ◽  
Author(s):  
David J. Lee ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Transcription Factors ◽  
Cyclic Amp ◽  
Promoter Activity ◽  
Bacterial Genome ◽  
Receptor Protein ◽  
Transcription Start ◽  
Content Type ◽  
Cyclic Amp Receptor Protein ◽  
Α Subunits

ABSTRACT In a previous study of promoters dependent on the Escherichia coli cyclic AMP receptor protein (CRP), carrying tandem DNA sites for CRP, we found that the upstream-bound CRP could either enhance or repress transcription, depending on its location. Here, we have analyzed the interactions between CRP and the C-terminal domains of the RNA polymerase α subunits at some of these promoters. We report that the upstream-bound CRP interacts with these domains irrespective of whether it up- or downregulates promoter activity. Hence, disruption of this interaction can lead to either down- or upregulation, depending on its location. IMPORTANCE Many bacterial promoters carry multiple DNA sites for transcription factors. While most factors that downregulate promoter activity bind to targets that overlap or are downstream of the transcription start and −10 element, very few cases of repression from upstream locations have been reported. Since more Escherichia coli promoters are regulated by cyclic AMP receptor protein (CRP) than by any other transcription factor, and since multiple DNA sites for CRP are commonplace at promoters, our results suggest that promoter downregulation by transcription factors may be more prevalent than hitherto thought, and this will have implications for the annotation of promoters from new bacterial genome sequences.

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.

Mannose utilization in Escherichia coli requires cyclic AMP but not an exogenous inducer

1980 ◽  
Vol 26 (12) ◽  
pp. 1508-1511 ◽  
Author(s):  
Ann D. E. Fraser ◽  
Hiroshi Yamazaki
Keyword(s):  
Escherichia Coli ◽  
Carbon Source ◽  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Sole Carbon Source ◽  
Receptor Protein ◽  
Deficient Mutant ◽  
Phosphotransferase System ◽  
Cyclic Amp Receptor Protein ◽  
Cyclic Monophosphate

It has not been clarified whether the utilization of mannose by Escherichia coli requires adenosine 3′,5′-cyclic monophosphate (cyclic AMP). Using an adenylyl cyclase deficient mutant (CA8306B) and a cyclic AMP receptor protein (CRP) deficient mutant (5333B) we have shown that the utilization of mannose is dependent on the cyclic AMP–CRP complex. 2-Deoxyglucose (DG) is a nonmetabolizable glucose analog specific for the phosphotransferase system (PTS) which transports mannose (termed here PTSM). Growth of CA8306B on glycerol is unaffected by addition of the analog, whereas growth of the strain on glycerol plus cyclic AMP ceases im mediately upon addition of DG. These results suggest that the formation of PTSM is dependent on cyclic AMP. In addition, CA8306B grown on glycerol plus cyclic AMP can immediately utilize mannose when transferred to a medium containing mannose as a sole carbon source, whereas the same strain grown on glycerol without cyclic AMP cannot utilize mannose when so transferred. These results suggest that the formation of PTSM does not require an exogenous inducer.

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