A novel function of the cAMP-CRP complex in Escherichia coli: cAMP-CRP functions as an adaptor for the CytR repressor in the deo operon

Unlike classical bacterial repressors, the CytR repressor of Escherichia coli cannot independently regulate gene expression. Here we show that CytR binding to the deoP2 promoter relies on interaction with the master gene regulatory protein, CRP, and, furthermore, that cAMP-CRP and CytR bind co-operatively to deoP2. Using mutant promoters we show that tandem, properly spaced DNA-bound cAMP-CRP complexes are required for this co-operative binding. These data suggest that CytR forms a bridge between tandem cAMP-CRP complexes, and that cAMP-CRP functions as an adaptor for CytR. The implications of this new version of negative control in E. coli on bacterial gene expression and on combinatorial gene regulation in higher organisms are discussed.     

The cyclic AMP (cAMP)-cAMP receptor protein complex functions both as an activator and as a corepressor at the tsx-p2 promoter of Escherichia coli K-12.

The tsx-p2 promoter is one of at least seven Escherichia coli promoters that are activated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and negatively regulated by the CytR repressor. DNase I footprinting assays were used to study the interactions of these regulatory proteins with the tsx-p2 promoter region and to characterize tsx-p2 regulatory mutants exhibiting an altered response to CytR. We show that the cAMP-CRP activator complex recognizes two sites in tsx-p2 that are separated by 33 bp: a high-affinity site (CRP-1) overlaps the -35 region, and a low-affinity site (CRP-2) is centered around position -74 bp. The CytR repressor protects a DNA segment that is located between the two CRP sites and partially overlaps the CRP-1 target. In combination, the cAMP-CRP and CytR proteins bind cooperatively to tsx-p2, and the nucleoprotein complex formed covers a region of 78 bp extending from the CRP-2 site close to the -10 region. The inducer for the CytR repressor, cytidine, does not prevent in vitro DNA binding of CytR, but releases the repressor from the nucleoprotein complex and leaves the cAMP-CRP activator bound to its two DNA targets. Thus, cytidine interferes with the cooperative DNA binding of cAMP-CRP and CytR to tsx-p2. We characterized four tsx-p2 mutants exhibiting a reduced response to CytR; three carried mutations in the CRP-2 site, and one carried a mutation in the region between CRP-1 and the -10 sequence. Formation of the cAMP-CRP-CytR DNA nucleoprotein complex in vitro was perturbed in each mutant. These data indicate that the CytR repressor relies on the presence of the cAMP-CRP activator complex to regulate tsx-p2 promoter activity and that the formation of an active repression complex requires the combined interactions of cAMP-CRP and CytR at tsx-p2.     

Restored DNA-binding of the cAMP-CRP activator complex reestablishes negative regulation by the CytR repressor in the deoP2 promoter in Escherichia coli.

Summary We have investigated in vivo the coupling between CytR regulation of the deoP2 promoter in Escherichia coli and the DNA-binding specificity of the cAMP-CRP (cAMP receptor protein) complex in order to obtain a more detailed picture of the role played by cAMP-CRP in CytR regulation. By introducing CRP proteins that exhibit an altered DNA binding specificity into a strain containing a mutant deoP2 promoter in which cAMP-CRP activation was decreased and CytR regulation completely abolished, we show that CytR regulation of this promoter can be reestablished by restored the DNA binding of the cAMP-CRP complex. Hence, CytR regulation of deoP2 can be modulated by simply varying DNA binding of cAMP-CRP. These data confirm the crucial role played by the cAMP-CRP activator complex in CytR regulation of the deoP2 promoter.     

Tandem CRP binding sites in the deo operon of Escherichia coli K-12

The locations of DNA binding by the cyclic AMP receptor protein (CRP) in the deo operon of Escherichia coli have been determined by the DNase I footprinting procedure. Two high affinity sites were found around positions -35 and -90, preceding the second deo promoter. In vitro data on induction of gene fusions that join different parts of the deoP -2 regulatory region to the lac genes suggest that: (1) both CRP binding sites are needed for high expression from the deoP -2 region; and (2) negative regulation by the cytR repressor is accomplished by preventing the cAMP-CRP complex from binding to the second target.     

Interaction of negative (CytT) and positive (cAMP-CRP) regulation in the promoter region of the uridine phosphorylase (udp) gene in Escherichia coli K-12

Interaction of negative (CytR) and positive (cAMP-CRP) control in the promoter region of the uridine phosphorylase (udp) gene of Escherichia coli has been studied by using udp-lac operon fusions in which the structural lacZ gene is expressed from the wild type promoter udpP+ or from mutant promoters udpP1 and udpP18. The specific activity of beta-galactosidase was examined in these fusions in cytR+ and cytR- backgrounds after introduction of specific mutations in crp locus, crp* and crp(a) altering interaction of CRP protein with catabolite-sensitive promoters. The data obtained using crp* mutation confirm the proposed model of the udp gene regulation, according to which CytR repressor protein interferes with CRP binding site in the promoter-operator region of the udp gene and thereby prevents the positive action of cAMP-CRP complex on the udp expression. Additional data in favor of this model were obtained using crp(a) mutation which most probably alters the structure of CRP protein in such a way that it exhibits more high affinity to the udp promoter, as compared to the CytR repressor protein. Indeed, taken by itself, the crp(a) mutation did not lead to any increase in the expression of udpP+-lac fusion under the conditions of cAMP limitation (on glucose-grown cells), in spite of whether or not the CytR repressor was present. However, when combined with the ptsG mutation or when cells were grown on succinate medium, complete constitutive expression of udpP+-lac fusion is observed, even in the presence of the cytR gene product. The effect of the crp(a) mutation was virtually the same in strains harboring udpP1-lac fusion. These data are in accordance with suggestion that udpP1 is a mutation in the site of the promoter-operator region that responds to the cytR gene product, while the corresponding binding site for CRP protein is still unaltered in this mutant. On the other hand, the crp(a) mutation causes only slight alteration in the expression of udpP18-lac fusion, providing additional evidence that udpP18 mutation seems to comprise a modification of the promoter-operator region, where binding sites for CRP and CytR proteins overlap.     

Design of camp–CRP-activated promoters in Escherichia coli

We have studied the deoP2 promoter of Escherichia coli to define features that are required for optimal activation by the complex of adenosine 3',5' monophosphate (cAMP) and the cAMP receptor protein (CRP). Systematic mutagenesis of deoP2 shows that the distance between the CRP site and the -10 hexamer is the crucial factor in determining whether the promoter is activated by cAMP-CRP. Based on these observations, we propose that cAMP-CRP-activated promoters can be created by correctly aligning a CRP target and a -10 hexamer. This idea has been successfully tested by converting both a CRP-independent promoter and a sequence resembling the consensus -10 hexamer to strongly cAMP-CRP-activated promoters.