Martine Crasnier

The catalytic domain of Escherichia coli K-12 adenylate cyclase as revealed by deletion analysis of the cya gene

In Escherichia coli, adenylate cyclase activity is regulated by phosphorylated EnzymeIIAGlc, a component of the phosphotransferase system for glucose transport. In strains deficient in EnzymeIIAGlc, CAMP levels are very low. Adenylate cyclase containing the D414N substitution produces a low level of cAMP and it has been proposed that D414 may be involved in the process leading to activation by EnzymeIIAGlc. In this work, spontaneous secondary mutants producing large amounts of cAMP in strains deficient in EnzymeIIAGlc were obtained. The secondary mutations were all deletions located in the cya gene around the D414N mutation, generating adenylate cyclases truncated at the carboxyl end. Among them, a 48 kDa protein (half the size of wild-type adenylate cyclase) was shown to produce ten times more cAMP than wild-type adenylate cyclase in strains deficient in EnzymeIIAGlc. In addition, this protein was not regulated in strains grown on glucose and diauxic growth was abolished. This allowed the definition of a catalytic domain that is not regulated by the phosphotransferase system and produces levels of cAMP similar to that of regulated wild-type adenylate cyclase in wild-type strains grown in the absence of glucose. Further analysis allowed the characterization of the COOH-terminal regulatory domain, which is proposed to be inhibitory to the activity of the catalytic domain.

Regulation of Escherichia coli adenylate cyclase activity during hexose phosphate transport

In Escherichia coli, cAMP levels vary with the carbon source used in the culture medium. These levels are dependent on the cellular concentration of phosphorylated EnzymeIIAglc, a component of the glucose-phosphotransferase system, which activates adenylate cyclase (AC). When cells are grown on glucose 6-phosphate (Glc6P), the cAMP level is particularly low. In this study, we investigated the mechanism leading to the low cAMP level when Glc6P is used as the carbon source, i.e. the mechanism preventing the activation of AC by phosphorylated EnzymeIIAglc. Glc6P is transported via the Uhp system which is inducible by extracellular Glc6P. The Uhp system comprises a permease UhpT and three proteins UhpA, UhpB and UhpC which are necessary for uhpT gene transcription. Controlled expression of UhpT in the absence of the regulatory proteins (UhpA, UhpB and UhpC) allowed us to demonstrate that (i) the Uhp regulatory proteins do not prevent the activation of AC by direct interaction with EnzymeIIAglc and (ii) an increase in the amount of UhpT synthesized (corresponding to an increase in the amount of Glc6P transported) correlates with a decrease in the cAMP level. We present data indicating that Glc6P per se or its degradation is unlikely to be responsible for the low cAMP level. It is concluded that the level of cAMP in the cell is determined by the flux of Glc6P through UhpT.

Characterization of Escherichia coli adenylate cyclase mutants with modified regulation.

In Escherichia coli there is a large increase of cAMP synthesis in crp strains, which are deficient in the catabolite gene activator protein. In this work it was shown that this increase in cAMP synthesis does not occur in crp crr strains, deficient in both the catabolite gene activator protein and enzymeIII-glucose, a component of the phosphotransferase system. It was also shown that the other components of the phosphotransferase system are required to obtain the increase of cAMP synthesis in a crp background. Adenylate cyclase mutants were obtained, by random mutagenesis, which had partial adenylate cyclase activity but which did not exhibit increased levels of cAMP in a crp background. For three mutants the mutation was identified as a single point mutation. This allowed the identification of residues arginine 188, aspartic acid 414 and glycine 463 which could be involved in the catabolite gene activator protein dependent activation process.

pH-Regulation of acid phosphatase of plant cell walls: An example of adaptation to the intracellular milieu

Plant cell walls contain several hydrolases [l-4]. Some of them play a central role in cell extension by promoting local hydrolysis of acidic cell wall polysaccharides. Others control hydrolysis and transport of extracellular metabolites in the cell. In sycamore (Acer pseudoplutunus) cells cultured in vitro, the most abundant of these enzymes is an acid phosphatase whose activity may be detected at the outer surface of unbroken cells [5]. Owing to the polyanionic nature of primary plant cell walls, the local pH within the cell-wall matrix may be quite different from the one prevailing in the bulk phase. This difference may be as large as several pH units (61. Moreover, the Donnan potential in the cell wall matrix, generated by negative fixed charges of polygalacturonates, is tightly controlled by ionic strength of the outer bulk phase; that is, the ionic strength of the solution outside the cell. Therefore depending on the experimental conditions, the local pH in the cell wall may vary by several pH units. tured in vitro in liquid medium under sterile conditions, as in [5]. Cell disruption was done in a French press under 1000 kg/cm2. Cell wall preparations free of cytoplasmic contaminations were obtained as in [5]. About 55% of cell-wall acid phosphatase activity was solubilized by raising the ionic strength of the cell wall fragment suspension. The enzyme was purified to homogeneity from this soluble extract as in [5]. Acid phosphatase on cell wall fragments does not result from an artefact created by cell disruption because the same enzyme may be obtained by raising the ionic strength of a suspension of intact unbroken cells. This enzyme is a monomeric glycoprotein of 100 000 Mr [5].

Elution of acid phosphatase from sycamore cell walls

Abstract In isolated cell wall fragments of cultured sycamore cells, acid phosphatase activity was preferentially located on one side. This side has been identified as the external side of the cell wall. About 60% of acid phosphatase activity could be solubilized from cell wall fragments. Electron micrographs showed that the majority of the solubilized enzyme came from the external area of the wall.

Electric Repulsion Effects and the Dynamic Behaviour of Enzymes Embedded in Biological Polyelectrolytes

The dynamic behaviour of an enzyme within an insoluble polyelectrolyte, such as a membrane or a cell wall, may be quite different from the one it should display if it were in free solution.

Detection of an adenylate cyclase gene in Pasteurella species

A Pasteurella multocida adenylate cyclase gene has been previously cloned in Escherichia coli and sequenced. A 1200 bp HpaI fragment from the coding region was used as a probe to analyse the presence of the gene in different Pasteurella species and subspecies, Actinobacillus ureae (formerly P. ureae) and group EF-4 bacteria. Thirty-seven strains were checked for the presence of the gene. It was shown that the adenylate cyclase gene was detected only in the species Pasteurella multocida.