GLUCOSE LOWERS CRP-ASTERISK LEVELS RESULTING IN REPRESSION OF THE LAC OPERON IN CELLS LACKING CAMP

CRP-cAMP-dependent operons of Escherichia coli can be expressed in cells lacking functional adenylate cyclase when they carry a second-site mutation in the crp gene (crp*). It is known that the expression of these operons is repressed by glucose, but the molecular mechanism underlying this cAMP-independent catabolite repression has been a long-standing mystery. Here we address the question of how glucose inhibits the expression of beta-galactosidase in the absence of cAMP. We have isolated several mutations in the crp gene that confer a CRP* phenotype. The expression of beta-galactosidase is reduced by glucose in cells carrying these mutations. Using Western blotting and/or SDS-PAGE analysis, we demonstrate that glucose lowers the cellular concentration of CRP* through a reduction in crp* mRNA levels. The level of CRP* protein correlates with beta-galactosidase activity. When the crp promoter is replaced with the crp promoter, the inhibitory effect of glucose on crp* expression is virtually abolished. These data strongly suggest that the lowered level of CRP* caused by glucose mediates catabolite repression in cya(-) crp* cells and that the autoregulatory circuit of the crp gene is involved in the down-regulation of CRP* expression by glucose.