Evelyne Joseph

Multiple regulation of the activity of adenylate cyclase in Escherichia coli

SummaryWe 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.

Two functional domains in adenylate cyclase of Escherichia coli.

To study the regulation of Escherichia coli adenylate cyclase, plasmids that carry part of the cya gene, as well as plasmids containing hybrid genes having part of cya fused to part of lacZ, were constructed. This allowed us to propose that the enzyme is made of at least two well-defined domains. The NH2 terminus carries the ATP leads to cAMP catalytic activity, whereas the COOH-terminal end corresponds to a regulatory polypeptide domain.

Role of 2-ketobutyrate as an alarmone in E. coli K12: Inhibition of adenylate cyclase activity mediated by the phosphoenolpyruvate: Glycose phosphotransferase transport system

Summary2-ketobutyrate and its analogues were found to inhibit strongly and transiently the rate of β-galactosidase synthesis in Escherichia coli K12. This effect was ascribed to a strong and transient inhibition of the adenylate cyclase activity. By using pts mutants, we showed, in agreement with our previous results (Daniel et al. 1983), that the likely target of 2-ketobutyrate and its analogues is the phosphoenolpyruvate: glycose phosphotransferase transport system (PTS). Furthermore, evidence for such a cascade effect caused by 2-ketobutyrate and its analogues allowed us to corroborate our previous proposal (Daniel et al. 1983) that 2-ketobutyrate, a precursor of isoleucine, acts as an E. coli alarmone monitoring the passage from anaerobic to aerobic growth conditions.

Transcription - translation coupling and polarity : A possible role of cyclic AMP

Using specific mutants exhibiting altered translational rates we found that when the rate of protein synthesis is reduced the polarity of the lactose and galactose operons is increased. This polarity is in part relieved by cyclic AMP. On the basis of in vitro hybridization experiments were propose that the cyclic AMP-CAP complex and the ribosomes display similar functions, namely destabilization of the RNA-DNA hybrid formed during transcription.

β-Galactosidase immunoassay for the measurement of cyclic AMP☆

Abstract A specific and sensitive β-galactosidase immunoassay is described. A cyclic AMP-β-galactosidase conjugate is used as the labeled cyclic AMP derivative in a competitive binding assay. By determining the β-galactosidase activity, the concentration of the antibody-bound conjugate is easily detected. Cyclic AMP levels and adenylate cyclase activities in Escherichia coli K12 can be determined by using this β-galactosidase immunoassay. Comparative studies performed between this enzyme immunoassay and the conventional radioimmunoassay show satisfactory correlation between the two methods.

crpX mutants of Escherichia coli K12: Specific regulatory effects of altered cyclic AMP receptor proteins

SummaryWe attempted to correlate structural modifications of the adenosine 3′,5′ cyclic monophosphate (cAMP) receptor protein (CAP), to changes in some of its in vivo regulatory functions such as (i) stimulation of the lactose operon expression and (ii) control of adenylate cyclase activity. A radioimmunological procedure was used to study the structure of CAP synthesized by three mutants (crpX) grown under various conditions, in the presence or absence of endogenous or exogenous cAMP. In one mutant CAP appears to be sensitive to thermal inactivation. In another mutant CAP is particularly sensitive to degradation in the absence of cAMP; this degradation is enhanced by high temperature and during stationary phase of grwoth, and prevented by the addition of glucose. Functional alterations of CAP were not found to follow structural changes strictly. In the crpX mutants and in strains carrying the crp+ or other crp allele, the stimulation of the lactose operon expression and the modulation of the in vivo rates of cAMP synthesis appear to vary in parallel, favoring an indirect mechanism of regulation of adenylate cyclase by CAP.

Modulation of the lactose operon mRNA turnover by inhibitors of dihydrofolate reductase.

Abstract The decay rate of lactose messenger RNA is strongly decreased when inhibitors of dihydrofolate reductase are added to the growth medium. The effect is not immediate: it appears after one generation of growth in the presence of the inhibitor. Additional genetic evidence suggests that the level of one carbon-tetrahydrofolate might be involved in the processing of mRNA.