Multiple regulation of the activity of adenylate cyclase in Escherichia coli

Abstract

We have studied the correlation between the activities of adenylate cyclase (ATP pyrophosphatelyase-(cyclizing); EC 4.6.1.1) and in vivo rates of synthesis and intracellular concentrations of adenosine 3',5' cyclic monophosphate (cAMP) under various growth conditions in wild-type Escherichia coli and in mutants lacking or overproducing the cAMP receptor protein (CAP). We showed that when wild-type bacteria are grown in the presence of a variety of carbon sources the intracellular concentrations of cAMP are inversely related to the adenylate cyclase activities determined in permeabilized cells, suggesting that the carbon source-dependent modulation of cAMP levels is not directly related to the regulation of adenylate cyclase activity. In mutants lacking functional CAP (crp) the in vivo rates of cAMP synthesis are several hundred-fold higher than in the wild-type parent without a parallel increase of adenylate cyclase activities. In a strain carrying multiple copies of the crp gene and overproducing CAP the activity of adenylate cyclase is severely inhibited, although the in vivo rate of cAMP synthesis is similar to the parental strain. We interpret these results as indicating that CAP controls mainly the activity rather than the synthesis of adenylate cyclase.

The calmodulin‐sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col

“…Adenylate cyclase synthesis differed widely in the two plasmids (see below, Table 1 Table 1. Bacteria carrying both plasmids , PVUC-1 and pOIA4, 5 or 6, synthesized high cAMP levels, 20 to 100 times higher than wild-type cya + E. coli (Joseph et al, 1982). Although more than 95 % of the total cAMP is extracellular, the intracellular concentrations correlate roughly with the total concentrations (Joseph and Ullmann, unpublished coli, carrying pOlA7 or 8, was enhanced by about 50 times when calmodulin was added to an in vitro assay.…”

Mechanism responsible for glucose–lactose diauxie in Escherichia coli: challenge to the cAMP model

“…To determine the intracellular cAMP level, culture samples were taken and centrifuged at 12 000 g for 2 min at 4 8 C. The pellets were suspended in 20 L of H 2 O, and heated for 5 min at 100 8 C. Samples were centrifuged at 12 000 g for 2 min at 4 8 C, and supernatants were removed and mixed with 60 L ethanol, then centrifuged at 12 000 g for 10 min at 4 8 C. The supernatants were dried, and pellets were suspended in 10 L of H 2 O and used for the determination of intracellular cAMP levels. The intracellular concentration of cAMP was calculated on the assumption that an OD 600 of 1.4 corresponds to 10 9 cells/mL (Miller 1972) and the volume of the cells is 2 2 10 -12 mL (Joseph et al 1982).…”

Aspects of the Regulation of Adenylate Cyclase Synthesis in Escherichia coli K12

“…Experimental evidence has recently been accumulated which substantiates the latter hypothesis (Aiba, 1985;Kawamukai et al, 1985). However, the actual repression of cya transcription by cAMP has been found to be weak (two-to sixfold); thus it would hardly explain the large effects observed in uiuo (30-to 100-fold increase in crp strains) (Joseph et al, 1982). To elucidate the control of cAMP synthesis, it appeared necessary to investigate the details of cya expression.…”