Cyclic AMP and its receptor protein are required for expression of transfer genes of conjugative plasmid F in Escherichia coli

A number of cya and crp mutants of Escherichia coli HfrH were analyzed for several Tra functions of the F plasmid. The mutants were observed to be deficient in conjugal donor ability, absorption of phages MS2 and Q beta and surface exclusion. These defects were suppressed in cya mutants grown with cAMP supplementation. A cAMP concentration of 3 X 10(-4) M produced maximal suppression of donor ability defect in a cya strain. cAMP did not suppress the Tra- phenotype of crp mutants. Latent periods of MS2 were shorter in cya and crp bacteria. Phage T7 development appeared similar in wild type, cya, and crp cells. It is concluded that tra genes of F plasmid are expressed only to a small extent in cya and crp mutants and that cAMP and its receptor protein are required for the normal expression of tra genes.     

Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli.

Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta-galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53-62 and 141-148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter-domain region of the N-terminal cAMP-binding and C-terminal DNA-binding domains.     

Catabolite repression of Escherichia coli heat-stable enterotoxin activity.

The Escherichia coli heat-stable enterotoxin (ST) coded for by plasmid pYK007 (Apr ST+) showed a dependence for cyclic adenosine 3',5'-monophosphate (cAMP) to express ST activity in an adenyl cyclase (cya) deletion mutant; no ST activity was detected in the presence of cAMP in a cAMP receptor protein (crp) deletion mutant or in a double deletion mutant (delta cya delta crp). The cya-crp effect on ST activity could not be accounted for by a modification of the copy number of plasmid deoxyribonucleic acid per chromosome equivalent or by an alteration in the secretion of an active intracellular enterotoxin.     

UV resistance of E. coli K-12 deficient in cAMP/CRP regulation.

Deletion of genes for adenylate cyclase (delta cya) or cAMP receptor protein (delta crp) in E. coli K-12 confers a phenotype that includes resistance to UV radiation (254 nm). Such mutations lead to UV resistance of uvr+, uvrA, lexA and recA strains which could partly be abolished by the addition of cAMP to delta cya but not to delta crp strain culture medium. This effect was not related to either inducibility of major DNA repair genes or growth rate of the bacteria. Enhanced survival was also observed for UV-irradiated lambda bacteriophage indicating that a repair mechanism of UV lesions was involved in this phenomenon.     

Glucose lowers CRP* levels resulting in repression of the lac operon in cells lacking cAMP

CRP—cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene ( crp* ). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of β-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of β-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS—PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp * mRNA levels. The level of CRP* protein correlates with β-galactosidase activity. When the crp promoter is replaced with the bla promoter, the inhibitory effect of glucose on crp * expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya⁻ crp * cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.     

Mechanism of CRP-mediated cya suppression in Escherichia coli.

Escherichia coli strain NCR30 contains a cya lesion and a second-site cya suppressor mutation that lies in the crp gene. NCR30 shows a pleiotropic phenotypic reversion to the wild-type state in expressing many operons that require the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex for positive control. In vivo beta-galactosidase synthesis in NCR30 was sensitive to glucose-mediated repression, which was relieved not only by cAMP but also by cyclic GMP and cyclic CMP. The CRP isolated from NCR30 differed from the protein isolated from wild-type E. coli in many respects. The mutant protein bound cAMP with four to five times greater affinity than wild-type CRP. Protease digestion studies indicated that native NCR30 CRP exists in the cAMP-CRP complex-like conformation. The protein conferred a degree of cAMP independence on the in vitro synthesis of beta-galactosidase. In addition, the inherent positive control activity of the mutant protein in vitro was enhanced by those nucleotides that stimulate in vivo beta-galactosidase synthesis in NCR30. The results of this study supported the conclusion that the crp allele of NCR30 codes for a protein having altered effector specificity yet capable of promoting positive control over catabolite-sensitive operons in the absence of an effector molecule.     

Antipain lethality to Escherichia coli: dependence upon cyclic adenosine 3'',5''-monophosphate and its receptor protein.

Antipain kills Escherichia coli K-12 cells in an exponential manner beginning 1 h after its addition. Mutant strains, delta cya and crp, which are unable to synthesize cyclic adenosine 3',5'-monophosphate (cAMP) and the cAMP receptor protein, respectively, are not affected. Addition of cAMP (5 mM) to antipain-treated mutant strains causes killing of delta cya cells, but not crp cells. Thus the lethal effect of antipain is dependent upon cAMP and its receptor protein.     

Cyclic AMP-dependent osmoregulation of crp gene expression in Escherichia coli

We have found that the cyclic AMP (cAMP) receptor protein (CRP)-cAMP regulatory complex in Escherichia coli is subject to osmoregulation at the level of crp gene expression. This osmoregulation was lost in a cya mutant strain but could be restored by external addition of cAMP, suggesting that the intracellular level of cAMP is a key factor in the osmoregulation of CRP. The ability of the cell to maintain optimal CRP activity was essential for the growth and survival of the bacteria under low-osmolarity conditions as shown by studies with different crp mutant alleles. A suppressor mutant with a novel amino acid substitution (L124R) in CRP showed restored growth at low osmolarity. CRP(L124R) was not activated by cAMP and was shown to be dominant negative over the wild type. Our findings suggest that the fine-tuning of the CRP activity may be critical for bacterial viability and adaptability to changing osmotic conditions.