Mutations that affect transcription and cyclic AMP-CRP regulation of the adenylate cyclase gene (cya) of Salmonella typhimurium.
We studied the expression of the cya promoter(s) in cya-lac fusion strains of Salmonella typhimurium and demonstrated cAMP receptor protein (CRP)-dependent repression by cAMP. Expression of cya was reduced about fourfold in cultures grown in acetate minimal medium as compared to cultures grown in glucose-6-phosphate minimal medium. Expression of cya was also reduced about fourfold by addition of 5 mM cAMP to cultures grown in glucose minimal medium. We constructed in vitro deletion and insertion mutations altering a major cya promoter (P2) and a putative CRP binding site overlapping P2. These mutations were recombined into the chromosome by allele replacement with M13mp::cya recombinant phages and the regulation of the mutant promoters was analyzed. A 4-bp deletion of the CRP binding site and a 4-bp insertion in this site nearly eliminated repression by cAMP. A mutant with the P2 promoter and the CRP binding site both deleted exhibited an 80% reduction in cya expression; the 20% residual expression was insensitive to cAMP repression. This mutant retained a Cya+ phenotype. Taken together, the results establish that the cya gene is transcribed from multiple promoters one of which, P2, is negatively regulated by the cAMP-CRP complex. Correction for the contribution to transcription by the cAMP-CRP nonregulated cya promoters indicates that the P2 promoter is repressed at least eightfold by cAMP-CRP.
Fandl
J P
JP
Department of Microbiology, University of California, Davis 95616.
Thorner
L K
LK
Artz
S W
SW
D000085:Acetates / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000235:genetics* / Q000378:metabolism; D000483:Alleles; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005958:Glucosephosphates / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011953:Receptors, Cyclic AMP / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014158:Transcription, Genetic
D000085:Acetates; D003470:Culture Media; D005958:Glucosephosphates; D011953:Receptors, Cyclic AMP; D000242:Cyclic AMP; D000262:Adenylyl Cyclases