Engineering global regulator cAMP receptor protein (CRP) of E. coli for improved biobutanol tolerance

Vfr, a global regulator of Pseudomonas aeruginosa virulence factors, is a homologue of the Escherichia coli cAMP receptor protein, CRP. Vfr is 91 % similar to CRP and maintains many residues important for CRP to bind cAMP, bind DNA, and interact with RNA polymerase at target promoters. While vfr can complement an E. coli crp mutant in β-galactosidase production, tryptophanase production and catabolite repression, crp can only complement a subset of Vfr-dependent phenotypes in P. aeruginosa. Using specific CRP binding site mutations, it is shown that Vfr requires the same nucleotides as CRP for optimal transcriptional activity from the E. coli lac promoter. In contrast, CRP did not bind Vfr target sequences in the promoters of the toxA and regA genes. Footprinting analysis revealed Vfr protected sequences upstream of toxA, regA, and the quorum sensing regulator lasR, that are similar to but significantly divergent from the CRP consensus binding sequence, and Vfr causes similar DNA bending to CRP in bound target sequences. Using a preliminary Vfr consensus binding sequence deduced from the Vfr-protected sites, Vfr target sequences were identified upstream of the virulence-associated genes plcN, plcHR, pbpG, prpL and algD, and in the vfr/orfX, argH/fimS, pilM/ponA intergenic regions. From these sequences the Vfr consensus binding sequence, 5′-ANWWTGNGAWNY : AGWTCACAT-3′, was formulated. This study suggests that Vfr shares many of the same functions as CRP, but has specialized functions, at least in terms of DNA target sequence binding, required for regulation of a subset of genes in its regulon.

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Determination of the rates of synthesis and degradation of adenosine 3′,5′-cyclic monophosphate in Escherichia coli CRP− and CRP+ strains

Canadian Journal of Biochemistry ◽

10.1139/o78-130 ◽

1978 ◽

Vol 56 (9) ◽

pp. 849-852 ◽

Cited By ~ 12

Author(s):

Ann D. E. Fraser ◽

Hiroshi Yamazaki

Keyword(s):

Escherichia Coli ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Balanced Growth ◽

E Coli ◽

Cyclic Monophosphate ◽

Camp Receptor ◽

Extracellular Camp ◽

Synthesis And Degradation

We have developed a method for estimating the rates of synthesis and degradation of adenosine 3′,5′-cyclic monophosphate (cAMP) in Escherichia coli during balanced growth. Applying this method, we have found that an E. coli CRP− mutant 5333 (deficient for cAMP receptor protein) synthesizes cAMP about 25 times faster than does its CRP+ parent 1100. This accounts for the abnormally high intracellular and extracellular cAMP accumulation in 5333.

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Regulation of ytfK by cAMP-CRP Contributes to SpoT-Dependent Accumulation of (p)ppGpp in Response to Carbon Starvation YtfK Responds to Glucose Exhaustion

Frontiers in Microbiology ◽

10.3389/fmicb.2021.775164 ◽

2021 ◽

Vol 12 ◽

Author(s):

Laura Meyer ◽

Elsa Germain ◽

Etienne Maisonneuve

Keyword(s):

Escherichia Coli ◽

Cyclic Amp ◽

Catabolite Repression ◽

Stringent Response ◽

Receptor Protein ◽

Glucose Starvation ◽

Camp Receptor Protein ◽

E Coli ◽

Camp Receptor ◽

Glucose Availability

Guanosine penta- or tetraphosphate (known as (p)ppGpp) serves as second messenger to respond to nutrient downshift and other environmental stresses, a phenomenon called stringent response. Accumulation of (p)ppGpp promotes the coordinated inhibition of macromolecule synthesis, as well as the activation of stress response pathways to cope and adapt to harmful conditions. In Escherichia coli, the (p)ppGpp level is tightly regulated by two enzymes, the (p)ppGpp synthetase RelA and the bifunctional synthetase/hydrolase SpoT. We recently identified the small protein YtfK as a key regulator of SpoT-mediated activation of stringent response in E. coli. Here, we further characterized the regulation of ytfK. We observed that ytfK is subjected to catabolite repression and is positively regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. Importantly, YtfK contributes to SpoT-dependent accumulation of (p)ppGpp and cell survival in response to glucose starvation. Therefore, regulation of ytfK by the cAMP-CRP appears important to adjust (p)ppGpp level and coordinate cellular metabolism in response to glucose availability.

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Faculty Opinions recommendation of Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029209.346568 ◽

2005 ◽

Author(s):

Ian Booth

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Receptor Protein ◽

Camp Receptor Protein ◽

E Coli ◽

Camp Receptor

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Faculty Opinions recommendation of Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029209.342598 ◽

2005 ◽

Author(s):

Stephen Spiro

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Receptor Protein ◽

Camp Receptor Protein ◽

E Coli ◽

Camp Receptor

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The E. coli cAMP receptor protein can be used in a eukaryotic system as a repressor activated by cAMP

Biochemical Society Transactions ◽

10.1042/bst025188s ◽

1997 ◽

Vol 25 (2) ◽

pp. 188S-188S

Author(s):

VOLPE FILIPPO

Keyword(s):

Receptor Protein ◽

Camp Receptor Protein ◽

E Coli ◽

Camp Receptor

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Point mutations in the DNA- and cNMP-binding domains of the homologue of the cAMP receptor protein (CRP) in Mycobacterium bovis BCG: implications for the inactivation of a global regulator and strain attenuation

Microbiology ◽

10.1099/mic.0.27444-0 ◽

2005 ◽

Vol 151 (2) ◽

pp. 547-556 ◽

Cited By ~ 35

Author(s):

Claire L. Spreadbury ◽

Mark J. Pallen ◽

Tim Overton ◽

Marcel A. Behr ◽

Serge Mostowy ◽

...

Keyword(s):

Dna Binding ◽

Mycobacterium Bovis ◽

Point Mutations ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Global Regulator ◽

Reporter System ◽

Binding Domains ◽

Camp Receptor

The genome of Mycobacterium tuberculosis H37Rv includes a homologue of the CRP/FNR (cAMP receptor protein/fumarate and nitrate reduction regulator) family of transcription regulators encoded by Rv3676. Sequencing of the orthologous gene from attenuated Mycobacterium bovis Bacille Calmette–Guérin (BCG) strains revealed point mutations that affect the putative DNA-binding and cNMP-binding domains of the encoded protein. These mutations are not present in the published sequences of the Rv3676 orthologues in M. bovis, M. tuberculosis or Mycobacterium leprae. An Escherichia coli lacZ reporter system was used to show that the M. tuberculosis Rv3676 protein binds to DNA sites for CRP, but this DNA binding was decreased or abolished with the Rv3676 protein counterparts from BCG strains. The DNA-binding ability of the M. tuberculosis Rv3676 protein was decreased by the introduction of base changes corresponding to the BCG point mutations. Conversely, the DNA binding of the BCG Rv3676 proteins from BCG strains was restored by removing the mutations. These data show that in this reporter system the point mutations present in the Rv3676 orthologue in BCG strains render its function defective (early strains) or abolished (late strains) and suggest that this protein might be naturally defective in M. bovis BCG strains. This raises the possibility that a contributing factor to the attenuation of BCG strains may be an inability of this global regulator to control the expression of genes required for in vivo survival and persistence.

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