Escherichia coli adenylate cyclase homepage

In 1965, when cAMP was first discovered in Escherichia coli by R. Makman and E. Sutherland, it was noticed that the inhibitory effect of glucose on cAMP synthesis was also observed with various substrates, even though glucose was more effective than the other substrates [JBC Online].

In 1975, it was established by W. Epstein and coworkers that carbon sources control the intracellular levels of cAMP by regulating its synthesis [PNAS].  The authors also showed that intracellular levels of cAMP were not regulated by variations in the efflux rate of cAMP, a result that was later confirmed by studying cAMP transport with membrane vesicles [JB].

The effect of the carbon source on cAMP levels has since been corroborated by several laboratories worldwide.  Escherichia coli cells grown in a minimal medium supplemented with any one of a variety of carbon sources exhibit different levels of cAMP.  For instance, the following carbon sources are arranged in order beginning with the one leading to the lowest up to the highest level of cAMP: (1) glucose-6-phosphate, (2) glucose, (3) mannitol, (4) gluconate, (5) fructose, (6) lactate and (7) glycerol.

The variations in the cAMP level can be monitored by measuring the activity of an enzyme.  The one widely chosen is β-galactosidase which hydrolyzes lactose to glucose and galactose.  The lacZ gene, encoding β-galactosidase, is part of the lac operon which is under positive control of the CAP-cAMP complex [JBC Online].  Consequently, under specific experimental conditions, there is an almost linear relationship between intracellular cAMP concentrations and β-galactosidase activities.

A partial explanation for the cAMP variations came with the study of the phosphotransferase system (PTS) which was discovered by W. Kundig and coworkers in 1964 [Medline] and further analyzed in 1990 [Medline].  In 1993, the PTS was the object of a thorough review by P. Postma and coworkers [MMBR].  In 2005, comparative genomic analyses of the PTS were published [MMBR].  The 1993 review was updated in 2006 with an emphasis on Gram-positive bacteria [MMBR] [Note: There is a gross error entailing negative consequences in the Introduction of the 2006 review, page 941.  Wild type E. coli does not exhibit diauxic growth in the presence of fructose and another less favorable sugar!  See Jacques Monod "Recherches sur la croissance des cultures bactériennes" 1942].  A 2007 issue of the "Journal of Molecular Microbiology and Biotechnology" brings a special focus on the PTS of Gram-positive bacteria [Table of Contents].

The PTS is a bacterial membrane transport system that allows the concomitant transport and phosphorylation of carbohydrates, essentially sugars (PTS-sugars).  Transport and phosphorylation occur, at the expense of phosphoenolpyruvate (PEP), through a phosphoryl cascade involving two cytosoluble proteins, Enzyme I and HPr, and the Enzyme II complexes that are specific for each sugar transported.  The phosphoryl transfer involves phosphorylation of histidine residues [BBA-Proteins & Proteomics].  The different enzyme II complexes are characterized by their domains (A, B, C and possibly D) present either on a single polypeptide chain or as several polypeptides [JB].

Formation of stable transition state complexes between the different PTS proteins may occur during transport [JBC Online].  The sub-cellular distribution of Enzyme I, the first protein of the PTS complex, varies with growth conditions [PNAS].  Kinetic studies have indicated that Enzyme I acts as a dimer and can phosphorylate HPr without dissociating to a monomer [Medline].  Effects of ligands on Enzyme I have been explained by a molecular model involving a "swivel" upon binding of PEP [JBC Online] [PNAS].  Motifs have been determined in HPr that are crucial, and highly specific, to the molecular interactions of HPr with its targeted Enzyme IIA domains [JB].

Additional kinetic studies of sugar binding and phosphotransfer reactions between Enzyme II domains are leading to model-based predictions of the kinetic behavior of the PTS, especially the glucose-PTS [JBC Online].

As a related issue, it is worth mentioning that the PTS has been described as a drug target system for the identification of novel and highly specific anti-microbials [Patent Storm].  Also, worth noting, a PTS permease can be necessarily required to allow for toxicity caused by secreted bactericidal compounds [JB].

Considering this rather complex transport system, it can be generalized that the rate of transport of any PTS-sugar depends on the concentration of the carrier proteins and the rate of phosphate transfer between these carrier proteins.  When two PTS-sugars are present in the culture medium, competition for uptake is likely to occur according to the same factors.  Interestingly however in Escherichia coli the PTS evolved for glucose to be taken up preferentially and the key factor in this preferential uptake is the glucose-specific IIA protein, Enzyme IIA^glc, the product of the crr gene.

Enzyme IIA^glc has been characterized by kinetic studies of different mutants including truncated forms [JBC Online].  Such studies supported the proposal that the N-terminal 18 residue domain attaches to the membrane thereby stabilizing the interaction between Enzyme IIA^glc and the IIB domain of Enzyme IICB^glc (the glucose permease) during glucose transport [JBC Online].

Visit the RCSB Protein Data Bank RCSB PDB [Nucleic Acids Research] for structural data (you may enter IIAGLC as keyword).

Discovery of PTS secondary regulatory functions involving Enzyme IIA^glc, i.e., regulation of adenylate cyclase and the phenomenon of inducer exclusion [JBC Online] provided an explanation for the preferential uptake of glucose over other sugars, particularly non-PTS-sugars, by Escherichia coli.