A single V317A or V317M substitution in Enzyme II of a newly identified β-glucoside phosphotransferase and utilization system of Corynebacterium glutamicum R extends its specificity towards cellobiose

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Abstract
A catabolic system involved in the utilization ofβ-glucosides inCorynebacterium glutamicumR and its spontaneous mutant variants allowing uptake of cellobiose were investigated. The system comprises aβ-glucoside-specific Enzyme IIBCA component (genebglF) of the phosphotransferase system (PTS), a phospho-β-glucosidase (bglA) and an antiterminator protein (bglG) from the BglG/SacY family of transcription regulators. The results suggest that transcription antitermination is involved in control of induction and carbon catabolite repression ofbglgenes, which presumably form an operon. Functional analysis of thebglFandbglAproducts revealed that they are simultaneously required for uptake, phosphorylation and breakdown of methylβ-glucoside, salicin and arbutin. Although cellobiose is not normally a substrate for BglF permease and is not utilized byC. glutamicumR, cellobiose-utilizing mutants can be obtained. The mutation responsible was mapped to thebgllocus and sequenced, and point mutations were found in codon 317 ofbglF. These led to substitutions V317A and/or V317M near the putative PTS active-site H313 in the membrane-spanning IIC domain of BglF and allowed BglF to act on cellobiose. Such results strengthen the evidence that the IIC domains can be regarded as selectivity filters of the PTS.
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