Characterization and generation of Escherichia coli adenylate cyclase deletion mutants

Abstract

Escherichia coli Acya-283 is a 75-bp in-frame deletion overlapping the 5' end of Acya-854; Acya-201 is a 41-bp frameshift deletion overlapping the 3' end of Acya-854. Sequence repeats were found at the boundaries of Acya-283 and Acya-201, suggesting a mechanism for deletion formation. Recombinant DNA procedures were used to construct a strain in which the total cya structural gene in the chromosome was replaced by the kanamycin resistance gene.Numerous studies on the physiology of Escherichia coli have taken advantage of strains that are incapable of accumulating cyclic AMP (cAMP). Brickman et al. (3) described three spontaneous deletions in the adenylate cyclase gene (cya). One of these mutations (Acya-854) has become the most widely used Acya background. Recently, Glaser et al. (7) have characterized the Acya-854 mutation as a 200-bp frameshift deletion. This study was designed to characterize the remaining two cya deletions described by Brickman et al. and to design a cya total deletion strain.(A preliminary report of some of these findings has been presented as an abstract [18].)Characterization of adenylate cyclase deletions. Adenylate cyclase activity in toluene-treated cells (11) of the parent strain CA8000 (3) was compared with those of cells of the Acya-201 (strain CA8300) and Acya-283 (strain CA8303) deletion strains. Although there was easily detectable activity in the wild-type cells, there was no detectable activity in either of the deletion strains. The lack of adenylate cyclase activity in the deletion strains might be due either to the absence of the protein, resulting from a premature translation termination or instability of the protein product, or, alternatively, to the accumulation of an inactive protein. The analyses described below were performed to evaluate these possibilities.Polymerase chain reaction (PCR) analysis of Acya-283 compared with that of the wild-type DNA indicates clearly that the deletion is located somewhere between bases 901 and 1320 (Fig. 1A, lanes 4 and 5) and that the size of the deletion is approximately 80 bp. Similarly, PCR analysis of Acya-201 compared with that of the wild-type DNA shows that the deletion is located in the same fragment (Fig. 1B, lanes 4 and 5) and that the deletion size is approximately 50 bp.A precise analysis of the locations of Acya-283 and Acya-201 was made by sequencing asymmetric PCR products (Fig. 2). Acya-283 was found to span the region from bases 1002 to 1076 (numbering as described in reference 1). This deletion is * Corresponding author. t Present address: Department of Neurology, School of Medicine, University of Maryland at Baltimore, Baltimore, MD 21201. therefore a 75-bp in-frame deletion and might be expected to produce a truncated gene product. Acya-201 was found to cover the region from bases 1219 to 1259; this corresponds to a 41-bp deletion. Since this deletion is out of frame, it is likely that premature translation termination would result. Inspection of the cya sequence (1) indicates that there is a stop codon (TGA) in the...

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