Generation of deletions in the 3'-flanking sequences of the Escherichia coli crp gene that induce cyclic AMP suppressor functions

Abstract

The crp structural gene and its 3'-flanking sequences were subcloned into M13mp8, and in vitro deletions were constructed in both the 5' and 3' ends of the gene by using Bal 31 nuclease. Deletions ranged in size from 24 to 250 base pairs at the 5' end of crp. Sixteen deletions generated at the 3' end of the gene ranged in size from 133 to 675 base pairs. The majority of deletions extended into the crp structural gene. Another class of deletions, i.e., Acrp-4, Acrp-17, and Acrp-2, had endpoints extending in the 3'-flahking sequences external to the crp structural gene. Deletions were subcloned into pBR322 and transformed into the Estherichia coli cya crp deletion strain NCR438. Transformants containing plasmid pBM4 with the Acrp4 mutation, a deletion of 133 base pairs, were cyclic AMP independent. Strain NCR440 harboring this plasmid expressed 1-galactosidase and threonine dehydratase activities and fermented lactose, ribose, arabinose, and xylose in the absence of exogenous cyclic AMP. The Acrp-4 mutation also caused strain NC1k440 to be hypersensitive to exogenous cyclic AMP. The cylic AMP receptor protein expressed in maxicells from pBM4 carrying the Acrp4 mutation comigrnted with the wild-type protein on eletrophoretic gels. The Acrp4 mutation demonstrates that sequences distal to the crp structural gene can mediate cyclic AMP suppressor functions.

Contributions of SpoT Hydrolase, SpoT Synthetase, and RelA Synthetase to Carbon Source Diauxic Growth Transitions in Escherichia coli

“…An inverse linear relationship is observed between the amount of cAMP added and the lag time, but the typical hierarchy again persists. Finally, the ability to utilize different sugars was estimated with EMB plates ( Barton and Melton, 1987 ) with the test set of strains comparing effects of crp or cyaA mutants ( Figure 2C ). The test set of strains are found to utilize all five sugars examined, while crp or cyaA mutants can use only glucose and partially utilize N -acetyl glucosamine.…”

“…Minimal medium M9 liquid and plates were prepared as described in Vinella et al (2012) and MOPS media for 32 P labeling was prepared as described in Neidhardt et al (1974) . EMB plates were prepared as described in Barton and Melton (1987) . When required, the following antibiotics were added at the indicated concentrations: 50 μg/ml ampicillin (Ap) and 20 μg/ml kanamycin (Km).…”

Salmonella typhimurium deletion mutants lacking adenylate cyclase and cyclic AMP receptor protein are avirulent and immunogenic

“…Deletion of the crp gene precludes recovery of crp* (23, 57) mutations that permit transcription of genes and operons requiring activation by the CRP in the absence of cAMP. Similarly, the absence of the crp gene also presumably precludes recovery of csm suppressor mutations (44) that are closely linked to the crp gene (6) and which can also obviate the need for cAMP in cya mutants. Even though the Acya revertant strains remained avirulent, we have not tested enough independent revertant isolates of cya::TnJO and Acya strains to conclude that'virulence could never be restored.…”

Cyclic AMP in prokaryotes.

“…A second class of CRP* mutant has been reported (23,124). Mutants of this class differ from those described above in that the mutation(s) that confers the cAMP-independent phenotype is in the stem structure of a putative rho-independent terminator located in the 3'-noncoding region of crp.…”