A third glucose uptake bypass in Corynebacterium glutamicum ATCC 31833

In Corynebacterium glutamicum, the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) has long been the only known glucose uptake system, but we recently found suppressor mutants emerging from a PTS-negative strain of C. glutamicum ATCC 31833 on glucose agar plates, and identified two alternative potential glucose uptake systems, the myo-inositol transporters encoded by iolT1 and iolT2. The expression of either gene renders the PTS-negative strain WTΔptsH capable of growing on glucose. In the present study, we found a suppressor strain that still grew on glucose even after the iolT1 and iolT2 genes were both disrupted under the PTS-negative background. Whole-genome sequencing of the suppressor strain SPH1 identified a G-to-T exchange at 134 bp upstream of the bglF gene encoding an EII component of the β-glucoside-PTS, which is found in limited wild-type strains of C. glutamicum. Introduction of the mutation into strain WTΔptsH allowed the PTS-negative strain to grow on glucose. Reverse transcription-quantitative PCR analysis revealed that the mutation upregulates the bglF gene by approximately 11-fold. Overexpression of bglF under the gapA promoter in strain WTΔptsH rendered the strain capable of growing on glucose, and deletion of bglF in strain SPH1 abolished the growth again, proving that bglF is responsible for glucose uptake in the suppressor strain. Simultaneous disruption of three glucokinase genes, glk (Cgl2185, NCgl2105), ppgK (Cgl1910, NCgl1835), and Cgl2647 (NCgl2558), in strain SPH1 resulted in no growth on glucose. Plasmid-mediated expression of any of the three genes in the triple-knockout mutant restored the growth on glucose. These results indicate that C. glutamicum ATCC 31833 has an additional non-PTS glucose uptake route consisting of the bglF-specified EII permease and native glucokinases.