Studies on the glucose family phosphotransferases of Clostridium beijerinckii
Essalem, Mohemed Essalem Emhemed
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Revival of the ABE fermentation will be enhanced by the ability of bacterial strains to utilise cheap, renewable substrates containing a range of fermentable carbohydrates. Development of an effective process will, however, depend on a detailed understanding of the mechanisms of uptake and metabolism of the available sugars. The predominant mechanism for uptake of sugars and sugar derivatives in the clostridia is the phosphoenolpyruvate (PEP) - dependent phosphotransferase system (PTS), which not only catalyses the concurrent uptake and phosphorylation of its substrate but also plays a central role in regulation of carbohydrate metabolism. Complete characterization of the PTS in the solventogenic clostridia will therefore be instrumental in developing strategies for constructing effective fermentation strains. The Clostridium beijerinckii 8052 genome encodes 43 complete phosphotransferase systems, including sixteen belonging to the glucose-glucoside family. Three of the PTSs are members of the glucose subgroup in a phylogenetic branch, and might therefore transport glucose. Since glucose has been shown to repress utilization of other sugars by Clostridium beijerinckii, these systems could also potentially be involved in glucose sensing and carbon catabolite repression (CCR). The cbei 0751 gene encoding a IICBA PTS permease was amplified by PCR, and cloned into Escherichia coli ZSC113, a mutant which cannot take up and phosphorylate glucose and mannose. Transformants showed a positive fermentation phenotype for glucose and mannose. Extracts showed glucose PTS activity, and cbei 0751 was therefore shown to be a functional glucose PTS. The activity was inhibited by mannose confirming that the system also recognises mannose as a substrate. The expression of this gene appeared to be constitutive although quantitative expression was not performed. Similar experiments were used to investigate the function of a second system encoded by cbei 4983 (IICB) and cbei 4982 (IIA). Although these genes were successfully cloned, their function could not be identified. Since the cbei 4984 gene encodes a putative glycoside hydrolase, this suggests that the primary function of this PTS may be to transport and phosphorylate a disaccharide, but further experimental analysis is required to identify the substrate of this system. Attempts to inactivate the two phosphotransferases to examine the effect on the cells were not successful.