As originally proposed by Feucht and Saier, the phosphorylated form of Enzyme IIAGlc acts as an activator of adenylate cyclase [J Bacteriol]. When glucose is present in the culture medium, a decrease in the concentration of phosphorylated Enzyme IIAGlc is observed which correlates with a decrease in adenylate cyclase activity. Therefore, in the presence of glucose, the cAMP level is low as compared to other carbon sources. It is to be understood that any regulation affecting glucose transport will also affect cAMP synthesis.
Most PTS-sugar permeases require higher levels of cAMP for their synthesis, which is controlled by the CAP-cAMP complex. Thus, by producing a low cAMP level, glucose transport indirectly lowers the synthesis of most PTS-permeases. Consequently, when several PTS-sugars are present in the culture medium, glucose is likely to be taken up preferentially.
Synthesis of Enzyme IICBGlc, the glucose permease encoded by ptsG, is controlled by CAP-cAMP, and therefore should also be lowered by a low level of cAMP. However, synthesis of Enzyme IICBGlc is not solely regulated by CAP-cAMP. It is additionally regulated by the Mlc protein, a glucose inducible transcriptional regulator [Mol Microbiol] [J Bacteriol] previously characterized as the product of the mlc (making large colonies) gene [PubMed], and whose effect on ptsG transcription may be perceived as buffering the effect caused by the low level of cAMP.
During glucose transport, transcriptional repression of ptsG by Mlc is abolished due to binding of Mlc to the dephosphorylated form of Enzyme IICBGlc [EMBO J]. Sequestration of Mlc by Enzyme IIBGlc requires attachment of Enzyme IIBGlc to the cytoplasmic membrane [J Biol Chem]. Interestingly, membrane sequestration of Mlc was found to be a crucial factor in Mlc inactivation [Mol Microbiol] however such finding may have to be re-analyzed considering more recent data [J Bacteriol] [J Bacteriol]. Upon elucidation of Mlc crystal structure, it was proposed that binding of tetrameric Mlc to Enzyme IICBGlc triggers the release of dimeric Mlc from its operator sites [J Biol Chem]. Structural analyses of the Mlc-IIBGlc complex suggest that phosphorylation of Enzyme IIBGlc impedes complex formation [Proc Natl Acad Sci U S A] [IICBGlc at UniProtKB] [Mlc at UniProtKB].
Under glucose-limited conditions of growth, transcription of ptsG is repressed by RpoS, a global stress response regulator [Res Microbiol]. Under conditions of intracellular glucose-6-phosphate accumulation, ptsG mRNA is degraded due to transcriptional activation of sgrS (encoding the small RNA SgrS) by SgrR [Mol Microbiol] [Mol Microbiol]. The sugar efflux transporter A (SetA) contributes to the glucose-phosphate stress response [J Bacteriol]. The effect of SgrT on the transport of glucose, as reported [J Bacteriol], must be taken with caution [PubMed Commons].
In 2008 the complex regulation of ptsG expression in E. coli was elegantly reported [FEMS Microbiol Rev]. In 2010 the sophisticated transcription of ptsG became even more complex [J Mol Microbiol Biotechnol].
Another entry exists for glucose that also involves Enzyme IIAGlc, and a protein designated MalX, the product of the malX gene. This entry however is not effective in wild type strains but in mutant strains lacking both Enzyme IICBGlc and mannose-specific Enzyme IIC and IID (which can be used for glucose transport). The phosphorylation of MalX by Enzyme IIAGlc also correlates with a decrease in adenylate cyclase activity (Crasnier-Mednansky, unpublished results).
The role of phosphorylated Enzyme IIAGlc as an activator of adenylate cyclase is also exemplified by the so-called CAP-dependent activation process [Microbiology].
Finally, mutants of Enzyme IIAGlc that have lost the capability to be phosphorylated failed to activate adenylate cyclase [J Bacteriol].
Another regulatory function of the PTS, called inducer exclusion, also involves Enzyme IIAGlc, and provides a way for glucose to compete with other non-PTS carbon sources for entry in the cell. In fact, the unphosphorylated form of Enzyme IIAGlc can inhibit by direct interaction the permease of non-PTS sugars, for example the lactose permease (LacY) thereby blocking the entry of lactose and subsequent formation of allolactose, the inducer of the lactose operon. As a consequence β-galactosidase is not synthesized. Thus inducer exclusion can be observed on minimal medium plates supplemented with X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside), a colorimetric substrate for β-galactosidase. When β-galactosidase is present in the cell, X-gal is hydrolyzed and the colonies appear blue. However, when glucose is added to the plate unphosphorylated Enzyme IIAGlc prevents lactose entrance by interacting with the lactose permease. Therefore wild type strains grow and appear as white colonies.
In 2003, the three-dimensional structure of Escherichia coli lactose permease was solved by Abramson et al. [Science Magazine]. See the model from the cover of Science. For a global perspective, it is of interest to read "Membrane protein doppelgangers" from The Scientist, August 25, 2003 issue (a subscription is required).
Lactose permease belongs to the Major Facilitator Superfamily (MFS) of transport proteins. Its mode of action, as reported by Abramson et al., is astonishing for its simplicity; lactose permease acts as a "gate-keeper". The gate opens when lactose binds to the sugar binding pocket unless glucose is available to the cell, in which case the gate remains closed. Amino-acids essential to sugar recognition play a dynamic role in the opening of the gate [J Biol Chem]. Additionally measurement of interhelical distance changes during sugar binding supported the proposed mechanism involving access of the sugar binding site to either side of the membrane [Proc Natl Acad Sci U S A]. Thus the sugar binding site is alternately accessible to the periplasmic and cytoplasmic side of the membrane during the reciprocal opening and closing of the gate, as further demonstrated [Proc Natl Acad Sci U S A] and conclusively established [Proc Natl Acad Sci U S A]. Opening and closing of the gate involves opening and closing of a large periplasmic cavity [Proc Natl Acad Sci U S A] with the opening being the limiting step for sugar binding [Proc Natl Acad Sci U S A] [Proc Natl Acad Sci U S A]. The structure of LacY with a high-affinity lactose analog (an α-substituted galactoside) was reported allowing analysis of LacY specificity and affinity for sugar [Proc Natl Acad Sci U S A]. Electrogenic events linked to lactose permease activity have been investigated [Proc Natl Acad Sci U S A].
Solving the molecular mechanism underlying lactose transport has marvelously magnified the comprehension of the inducer exclusion process.
Unphosphorylated Enzyme IIAGlc inhibits uptake of several other non-PTS sugars by binding to their transport systems. It also inhibits glycerol kinase by direct binding thereby preventing inducer synthesis. Specific molecular interactions between unphosphorylated Enzyme IIAGlc and its target proteins have been identified, for examples the MalK subunits of the maltose transporter [J Biol Chem] and glycerol kinase [Science Magazine] [Note: Enzyme IIIGlc was renamed Enzyme IIAGlc]. In 2013 crystals of the maltose transporter (MalFGK2) bound with Enzyme IIAGlc were obtained and further characterized thus leading to a molecular understanding of inducer exclusion [Nature]1.
Regulation of adenylate cyclase and inducer exclusion are linked together because any change in the ratio of phosphorylated over unphosphorylated Enzyme IIAGlc will affect both phenomena.
An interaction between unphosphorylated Enzyme IIAGlc and the product of the yafA gene (renamed frsA for fermentation/respiration switch) has been reported [J Biol Chem]. Such interaction may possibly interfere with the regulatory functions of Enzyme IIAGlc. The yafA gene is adjacent to the gpt gene and transcribed from its own promoter [Gene]. The product of the yafA gene (or FrsA) was classified among databases as a putative hydrolase of the α/β super family. Later on, FrsA was reported to have a pyruvate decarboxylase activity [Nat Chem Biol].
In 2015, an interaction was demonstrated between unphosphorylated Enzyme IIAGlc and the EAL domain of CsrD, a regulatory protein involved in the degradation of sRNAs by housekeeping nucleases [Mol Microbiol] [PubMed Commons]. Also, CsrD-controlled CsrA bound specifically to crp mRNA with no major effect on CRP expression at 37 °C and possibly a slight effect at 22 °C [J Bacteriol].
1 The title of the Nature article Carbon catabolite repression of the maltose transporter revealed by X-ray crystallography is inaccurate and misleading. A better title would have been related to inducer exclusion, not carbon catabolite repression.