Regulation of the phosphotransferase system (PTS)-mediated sugar uptake in Corynebacterium glutamicum in response to perturbations of the central metabolism

Corynebacterium glutamicum is a Gram-positive bacterium used in the biotechnological production of amino acids. It co-metabolizes most substrates, such as glucose and sucrose. The uptake and concomitant phosphorylation of those two substrates is mediated by the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), consisting of the common proteins HPr and EI, and an array of substrate-specific EII permeases. The PTS plays a central role in the regulation of nutrient uptake and metabolism in bacteria. However, the regulatory functions of the PTS in C. glutamicum are not understood. As the availability of NADPH is a limiting factor for the biosynthesis of amino acids, the deletion of pgi, encoding the enzyme phosphoglucoisomerase, is a promising approach for strain improvement. It blocks the first step of glycolysis and directs the glucose-derived carbon flux towards the NADPH-producing pentose phosphate pathway. However, despite that C. glutamicum Δpgi grows well with sucrose as a sole carbon source, addition of glucose arrests growth by causing repression of ptsS, encoding the sucrose-specific EIIsuc, and a drastic sucrose uptake inhibition. The regulatory mechanism behind these phenomena was unknown and has been investigated here. It was shown that the glucose addition inhibits sucrose uptake in C. glutamicum Δpgi prior to ptsS-repression and this fast process is not prevented by transcriptional or translational inhibitors. Analysis of the phosphorylation state of HPr - the last common component of the PTS phosphorylation cascade - indicated that the uptake inhibition is caused by a rapid depletion of HPr~P. The addition of non-PTS substrates which generate carbon flux towards glucose-6-P like e.g. maltose or glucose-6-P, uptake of which was enabled by the heterologously expressed transporter UhpT, led to similar growth and sucrose uptake inhibition as the addition of glucose. Unlike glucose, those substrates do not consume PEP for their uptake, so that the HP~P depletion is not caused by a decrease of the PEP/pyruvate ratio but by a glucose-6-P stress response mechanism. Perception of the glucose-6-P stress and the following response initiation requires the glucose-specific EIIglc as in EIIglc-deficient pgi mutants sucrose uptake was not inhibited by glucose, glucose-6-P or maltose addition. Further, it was shown that the low ptsS-mRNA levels observed in C. glutamicum Δpgi after glucose addition are a consequence of transcriptional repression by the regulator SugR. EMSA studies indicated fructose-1-P and to a lesser extent fructose-6-P as inhibitors of the SugR binding to the ptsS-promoter region. Taken together, this work shows that EIIglc is part of a novel mechanism for the perception of sugar-P stress which leads to instantaneous inhibition of the PTS phosphorylation cascade and consequently PTS activity in C. glutamicum. Additionally, this rapid uptake inhibition leads to low fructose-1-P formation and thus by an inducer exclusion mechanism, to SugR-dependent reduction of ptsS-expression. A suppressor mutation in the gene cg0790 (lpdA) was found to improve significantly the growth, sucrose uptake and ptsS-expression of C. glutamicum Δpgi during cultivation in the presence of glucose. The role of the novel regulatory mechanism for PTS regulation in C. glutamicum is also discussed.