Escherichia coli adenylate cyclase homepage [Contents]

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].

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].  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 [Annual Review of Biochemistry].  In 1993 the PTS was the object of a thorough review by P. Postma and coworkers [MMBR].  The 1993 review was updated in 2006 with an emphasis on Gram-positive bacteria [MMBR][Author's Correction].  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].

Comparative genomic analyses of the PTS were reported by screening 202 sequenced genomes [MMBR].  By using both phylogenetic methods and analysis of genome context within 222 sequenced genomes HGT (Horizontal Gene Transfer) was implicated in the evolution of the PTS [BMC Evolutionary Biology].  The PTS has been found in archaea, for example Thermofilum pendens [LBNL].

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 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].  The phosphoryl transfer involves phosphorylation of histidine and cysteine residues [BBA-Proteins & Proteomics] [The Journal of Physical Chemistry B].

Formation of stable transition state complexes between the different PTS proteins may occur during transport [JBC].  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 [Biochemistry].  Effects of ligands on Enzyme I have been explained by a molecular model involving a "swivel" upon binding of PEP [JBC] [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].  Because binding between Enzyme I and HPr does not involve significant conformational changes HPr acts as a phospho-relay between Enzyme I and the Enzyme II complexes [JBC].

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].

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].  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].

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] provided an explanation for the preferential uptake of glucose over other sugars, particularly non-PTS-sugars, by Escherichia coli.