Environmentally regulated algD promoter is responsive to the cAMP receptor protein in Escherichia coli

The environmentally activated algD promoter of Pseudomonas aeruginosa has been shown to be influenced by DNA supercoiling. It is believed that protein-induced bending or looping is required for this activation. We studied the role of Escherichia coli cAMP-CRP on algD promoter activation in E. coli and show that a functional CRP is required for this activation. We also demonstrate that the algD promoter is sensitive to glucose repression both in E. coli and P. aeruginosa. Deletion of a putative consensus CRP binding sequence upstream of the algD promoter renders the promoter non-responsive to glucose repression. The involvement of cAMP-CRP complex in the activation of the algD promoter in E. coli has been demonstrated directly through binding of a 255 base pair DNA fragment containing the putative consensus CRP binding sequence. Other fragments, upstream or downstream but without any consensus CRP binding sequence, did not show any binding with CRP. A CRP-like analogue, similar to that in Xanthomonas campestris, but capable of activating genes without forming a complex with cAMP, is believed to allow glucose repression in P. aeruginosa.     

Differential control by IHF and cAMP of two oppositely oriented genes, hpt and gcd, in Escherichia coli: significance of their partially overlapping regulatory elements

The hpt gene, which encodes hypoxanthine phosphoribosyltransferase, is located next to, but transcribed in the opposite direction to, the gcd gene, which codes for a membrane-bound glucose dehydrogenase, at 3.1 min on the Escherichia coli genome. In their promoter-operator region, putative regulatory elements for integration host factor (IHF) and for the complex comprising 3', 5'-cyclic AMP (cAMP) and its receptor protein (CRP) are present, and they overlap the promoters for hpt and gcd, respectively. The involvement of IHF and cAMP-CRP, as well as the corresponding putative cis-acting elements, in the expression of the two genes was investigated by using lacZ operon fusions. In an adenylate cyclase-deficient strain, addition of cAMP increased the expression of hpt and reduced the expression of gcd. In agreement with this observation, the introduction of mutations into the putative binding element for the cAMP-CRP complex enhanced the expression of gcd. In contrast, mutations introduced into the putative IHF-binding elements increased the level of hpt expression. Similar results were obtained with IHF-defective strains. Thus, the expression of the two genes is regulated in a mutually exclusive manner. Additional experiments with mutations at the -10 sequence of the gcd promoter suggest that the binding of RNA polymerase to the hpt promoter interferes with the interaction of RNA polymerase with the gcd promoter, and vice versa.     

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.     

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.