The role of the lac promotor locus in the regulation of beta-galactosidase synthesis by cyclic 3',5'-adenosine monophosphate.

We have recently reported that cyclic 3',5'-adenosine monophosphate (cyclic 3',5'-AMP) stimulates the production of j8-galactosidase and tryptophanase in Escherichia coli.1 Investigation of the effect of cyclic 3',5'-AMP on 13-galactosidase synthesis revealed that the nucleotide acts at the level of transcription to increase the synthesis offl-galactosidase messenger RNA (mRNA). 2 This effect of cyclic 3',5'-AMP is distinct from the well-characterized repressoroperator control of the lac operon,' since cyclic 3',5'-AMP is active in mutants of both the repressor and the operator loci. Further, cyclic 3',5'-AMP overcomes the transient repression of fi-galactosidase synthesis produced by the addition of glucose to glycerol-grown E. coli.2 We have now studied the regulation of f3galactosidase synthesis in mutants of the lac promotor region. These mutants have recently been isolated and described by Scaife and Beckwith.4 They respond to inducers of the lac operon, but when fully induced make only about 5 per cent as much of the lac enzymes as does their parent strain, E. coli K12 3000. We find that cyclic 3',5'-AMP does not stimulate ,3-galactosidase synthesis in one of these promotor mutants, strain L8. In addition, while glucose produces a normal permanent repression of fl-galactosidase synthesis in this mutant, it does not produce a transient repression of 8-galactosidase synthesis when added to glycerol-grown cells. The studies with strain L8, reported in this paper, suggest that the site at which cyclic 3',5'-AMP acts to stimulate fl-galactosidase synthesis is the lac promotor, and that transient repression of the lac operon is mediated by changes in the concentration of cyclic 3',5'-AM\P. Materials and Methods. -The following strains of E. coli K12 were used: 3000 (the parent strain, lac i+p+o+z+y+a+); L8 and L29, two promotor mutants that make about 5% as much j#-galactosidase as does 3000;4' 5 64, a revertant of L8 that makes normal amounts of j3-galactosidase and that is genetically indistinguishable from 3000;6 82 and N27, two other revertants of L8 that contain a second mutation within the promotor locus, and that make, respectively, 15-20%o and 50-70% as much #-galactosidase as does the parent strain;6 o' and o,0. , two operator-constitutive mutants;5 and i5 2, a constitutive i-gene mutant.5 Strain 3000 was a gift from Dr. E. Steers; all other strains were given to us by Dr. J. Beckwith. Bacteria were grown aerobically at 370C in medium A, a minimal medium,7 supplemented with thiamine. Glucose (0.5%) or glycerol (0.5%0) served as carbon sources. Growth was measured turbidimetrically at 560 nm in a Coleman Jr. spectrophotometer. All strains grew with doubling times of 50-60 min on glucose and 70-80 min on glycerol; cyclic 3',5'-AMP had no significant effect on their growth rates. j3-Galactosidase was induced by the addition of 10-3 M isopropyl-l-thio-,B-D-galactoside (IPTG) and was assayed in toluene-treated cells by the method of Pardee, Jacob, and Monod.8 Tryptophanase was induced by the addition of 2.5 X 10-4 M tryptophan and was assayed in toluene-treated cells by the method of Bilezikian et al.9 Other materials and methods have been described previously. ' 2