Identification of a phosphofructokinase-encoding gene from Streptococcus thermophilus CNRZ1205 – a novel link between carbon metabolism and gene regulation?

Abstract.

In order to isolate genes encoding so-called Two-Component Regulatory Systems from the lactic acid bacterium Streptococcus thermophilus, a cloning strategy was employed based on suppression of the alkaline phosphatase-negative phenotype displayed by the Escherichia coli strain ANCC22. Several suppressing clones were obtained which were shown to produce alkaline phosphatase activity. Sequence analysis of four of these clones revealed the presence of overlapping DNA inserts representing two ORFs, designated pfkT and pykT, whose deduced protein products exhibit significant similarity to phosphofructokinases and pyruvate kinases, respectively, from a variety of bacteria. A plasmid bearing pfkT was shown to complement a phosphofructokinase-negative mutant of E. coli, showing that this gene indeed specifies phosphofructokinase activity. It was shown that suppression of the alkaline phosphatase-negative phenotype of E. coli ANCC22 due to the presence of pfkT is caused by modulation of the intracellular level of acetyl phosphate.