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., delta crp-4, delta crp-17, and delta crp-2, had endpoints extending in the 3'-flanking sequences external to the crp structural gene. Deletions were subcloned into pBR322 and transformed into the Escherichia coli cya crp deletion strain NCR438. Transformants containing plasmid pBM4 with the delta crp4 mutation, a deletion of 133 base pairs, were cyclic AMP independent. Strain NCR440 harboring this plasmid expressed beta-galactosidase and threonine dehydratase activities and fermented lactose, ribose, arabinose, and xylose in the absence of exogenous cyclic AMP. The delta crp-4 mutation also caused strain NCR440 to be hypersensitive to exogenous cyclic AMP. The cylic AMP receptor protein expressed in maxicells from pBM4 carrying the delta crp-4 mutation comigrated with the wild-type protein on electrophoretic gels. The delta crp-4 mutation demonstrates that sequences distal to the crp structural gene can mediate cyclic AMP suppressor functions.