CLONING, SEQUENCE, AND EXPRESSION OF THE PHOSPHOFRUCTOKINASE GENE OF CLOSTRIDIUM-ACETOBUTYLICUM ATCC-824 IN ESCHERICHIA-COLI

The pfk gene encoding phosphofructokinase (Pfk) from the anaerobic bac terium Clostridium acetobutylicum ATCC 824 was cloned and sequenced. T he gene was identified in a plasmid library by complementation of an E , coli pfk mutant and by the ability to amplify a fragment by PCR usin g primers based on homologous regions of Pfk from other microorganisms . Nucleotide sequence analysis revealed a coding region for a 319-aa p rotein homologous to Pfks from other organisms, Enzyme assay and abili ty to complement the growth defects of E, coli pfk mutants confirmed t he expression of the clostridial pfk gene. The pyruvate kinase (pyk) g ene was identified adjacent pfk. Such an arrangement for the genes enc oding key regulators of glycolytic flux had not yet been described in a strict anaerobe. This gene arrangement has been found in other Gram- positive organisms, but not in Gram-negative organisms.