Cloning and sequencing of genes involved in glycolysis from Clostridium acetobutylicum
Belouski, Edward John
Master of Arts
Complementation studies were carried out with a Clostridium acetobutylicum ATCC 824 plasmid library and Escherichia coli glycolytic mutants. Degenerate primers were designed from homologous regions in known glycolysis genes, and used to screen the selected plasmids. The gene encoding phosphofructokinase and the surrounding DNA was sequenced. The gene encoding pyruvate kinase was discovered downstream of the phosphofructokinase gene. It appears that phosphofructokinase and pyruvate kinase form an operon. The gene encoding glyceraldehyde-3-phosphate dehydrogenase was also found in a cosmid library, but only partially sequenced. Phosphofructokinase from C. acetobutylicum has high homology to the phosphofructokinases from Bacillus stearothermophilus, Bacillus macquarensis, Escherichia coli and other microbes. Conserved residues involved in the binding of substrates, cofactors and allosteric effectors are found in the C. acetobutylicum homologue. The pyruvate kinase/phosphofructokinase operon may act as a "regulatory operon" for glycolysis in C. acetobutylicum.