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Escherichia coli adenylate cyclase homepage: Chapter I

In vivo regulation of adenylate cyclase

In 1965, when cAMP was first discovered in Escherichia coli by Makman and 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 [J Biol Chem].

In 1975, it was established by Epstein, Rothman-Denes and Hesse that carbon sources control the intracellular levels of cAMP by regulating its synthesis [Proc Natl Acad Sci U S A].  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 [J Bacteriol].

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 [β-galactosidase at UniProtKB].  The lacZ gene, encoding β-galactosidase, is part of the lac operon which is under positive control of the CAP-cAMP complex [J Biol Chem].  Consequently, under specific experimental conditions, there is an almost linear relationship between intracellular cAMP concentrations and β-galactosidase activities.  Of interest, the lacZ gene, due to its well-studied regulation, has been used in numerous studies including the study of adaptive mutations in E. coli [PLoS Genet].

A partial explanation for the cAMP variations came with the study of the phosphotransferase system (PTS) which was discovered by Kundig, Ghosh and Roseman1 in 1964 [PubMed] and further analyzed in 1990 [Annu Rev Biochem].  In 1993 the PTS was the object of a thorough review by Postma, Lengeler and Jacobson [Microbiol Mol Biol Rev].  The 1993 review was updated in 2006 with an emphasis on Gram-positive bacteria [Microbiol Mol Biol Rev Author's Correction].  A 2007 issue of the 'Journal of Molecular Microbiology and Biotechnology' brought 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 [Microbiol Mol Biol Rev].  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 Evol Biol].  The PTS has been found in archaea, for example Thermofilum pendens [LBNL].  In 2013 a previously uncharacterized PTS was discovered in the pathogen Salmonella enterica [J Bacteriol].  

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 phosphoryl transfer between two cytosoluble proteins, Enzyme I (EI) and HPr, and the different Enzymes II which are specific for each PTS-substrate transported.  The Enzymes II are characterized by their domains (A, B, C and possibly D) present either as a single polypeptide chain or as several polypeptides [J Bacteriol].  The phosphoryl transfer involves phosphorylation of histidine and cysteine residues [Biochim Biophys Acta] [J Phys Chem B].

Phosphotransferase system
The typical PTS phosphorylation cascade from PEP to the PTS substrate

Formation of stable transition state complexes between the different PTS proteins may occur during transport [J Biol Chem].  The sub-cellular distribution of EI, the first phosphotransfer protein of the PTS complex, varies with growth conditions [Proc Natl Acad Sci U S A], an observation which later was not corroborated [EMBO J].  Kinetic studies have indicated that EI acts as a dimer and can phosphorylate HPr without dissociating to a monomer [Biochemistry].  The isolated C-terminal domain of EI is able, as a dimer, to hydrolyze PEP into pyruvate and inorganic phosphate although not as efficiently as full length EI.  Such domain provides a model system for studying conformational dynamics regulating EI activity [J Biol Chem].  Effects of ligands on EI have been explained by a molecular model involving a 'swivel' upon binding of PEP [J Biol Chem] [Proc Natl Acad Sci U S A].  The interactions between Enzyme I and its inhibitor α-ketoglutarate (which accumulates during nitrogen starvation) have been studied by NMR and enzymatic assays [ACS Chem Biol].

Binding between EI and HPr does not involve significant conformational changes, HPr acting as a phospho-relay between EI and the Enzyme II complexes [J Biol Chem].  Motifs have been determined in HPr that are crucial, and highly specific, to the molecular interactions of HPr with its targeted Enzyme IIA domains [J Bacteriol].  HPr also interacts with regulatory proteins, for example MtlR which regulates the expression of the mannitol operon [Sci Rep] [PubMed Commons].

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 [J Biol Chem].  Enzyme II mutants have been constructed to study phosphotranfer between domains, for example mannitol Enzyme II [J Biol Chem].  Structural studies highlight differences between the IIC domains for example IICMtl and IICChb [Biochim Biophys Acta] and IICGlc [J Struct Biol].

Color-coded version of the PTS

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.  Also, worth noting, a PTS permease can be necessarily required to allow for toxicity caused by secreted bactericidal compounds [J Bacteriol].

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 IIAGlc, the product of the crr (carbohydrate repression resistant) gene [IIAGlc at UniProtKB].

Enzyme IIAGlc has been characterized by kinetic studies of different mutants including truncated forms [J Biol Chem].  Such studies supported the proposal that the N-terminal 18 residue domain attaches to the membrane thereby stabilizing the interaction between Enzyme IIAGlc and the IIB domain of Enzyme IICBGlc (the glucose permease) during glucose transport [J Biol Chem].

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

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


1 Hexosamine metabolism, sialic acids, and the phosphotransferase system: Saul Roseman's contributions to glycobiology [J Biol Chem]; Saul Roseman: His many contributions to biochemistry over eight decades [Proc Natl Acad Sci USA]

To Chapter II: The glucose specific IIA protein   Chapter II