Characterisation of phosphotransferase systems of Clostridium beijerinckii belonging to the mannose/fructose/sorbose family
Cairney, Camilla May
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After many years of decline, the production of acetone, butanol and ethanol via bacterial fermentation (ABE fermentation) is gathering renewed interest from those in the fuel industry. This is due to an increasing awareness of the damages fossil fuels have on our environment and also their limited supply. Species classified within the genus Clostridium, particularly Clostridium acetobutylicum and Clostridium beijerinckii, are long time favourites for use in ABE production. In order to re-establish the ABE fermentation process a better understanding of the physiology and metabolism of these strains is required. The PEP-dependent phosphotransferase system (PTS), which catalyses both uptake and phosphorylation of its substrates, is a major mechanism of sugar uptake in anaerobic bacteria. The genome of Clostridium beijerinckii 8052 encodes a total of 43 complete phosphotransferase systems, nine of which are categorised as belonging to the mannose/fructose/sorbose superfamily. Genes encoding four of these nine systems were amplified by PCR and transformed into Escherichia coli ZSC113, a strain incapable of phosphorylating mannose and glucose, in order to determine the transport function of each system via complementation of the fermentation phenotype on indicator agar. The PTSs encoded by cbei0712-0713, cbei3871-3874 and cbei0957-0958 all gave negative fermentation phenotypes for both mannose and glucose. PTS cbei0965-0966, however, gave a positive fermentation phenotype for mannose, but not for glucose. As the strain E. coli ZSC113 was isolated 40 years ago following chemical mutagenesis, the exact positions of mutations have been unknown. Therefore the manXYZ genes encoding the PTS, responsible for transporting mannose, glucose and other sugars, were amplified by PCR and sequenced to search for mutations. It was found that one mutation lay in manX, three in manY and one in manZ.