Effect of pfkA chromosomal interruption on growth, sporulation, and production of organic acids in Bacillus subtilis

Phosphofructolcinase (Pfk) plays a key role in the regulation of carbohydrate metabolism. Its activity can be used as an indicator of glycolytic flux in a microorganism. We have cloned and characterized the pfkA gene from Bacillus subtilis, which encodes the enzyme phosphofructokinase. This gene was insertionally inactivated at the chromosomal level in a wild type strain and in strains lacking the PEP: sugar phosphotranferase system (PTS). Although the pykA gene is immediately downstream of the pflcA gene, forming a constitutive operon in B. subtilis, the pyruvate kinase activity was not altered in the pfkA mutant. The inactivation of the pfkA gene had a strong impact on the growth of the B. subtilis wild type strain and PTS mutants in Spizizen's minimal media and Schaeffer's sporulation media. Pfk inactivation was also reflected by the timing and percentage of sporulation of the wild type and PTS mutants in sporulation media as well as in the production of organic by-products (pyruvate, lactate, and acetate).