Identification of a gene cluster encoding Krebs cycle oxidative enzymes linked to the pyruvate carboxylase gene in Lactococcus lactis ssp lactis C2

We identified a 14-kb pyruvate carboxylase gene-containing fragment from a lactococcal CB-lambda phage genomic library. Downstream of the pyruvate car boxylase gene-containing fragment, a gene cluster coding for open reading f rames displaying extensive homology to citrate synthase, aconitase, and a t runcated isocitrate dehydrogenase was identified. However, the truncation w as shown to have occurred during the cloning by two noncontiguous Sau3AI fr agments ligating together. The lactococcal citrate synthase gene consisted of 1323 bp and encoded a 441-amino acid citrate synthase protein. The lacto coccal aconitase gene was 2544 bp and encoded an 848-amino acid protein. Co rresponding to the complete citrate synthase gene, citrate synthase activit y was detected in Lactococcus lactis ssp. Lactis C2. Isocitrate dehydrogena se activity was found to be missing in Lactococcus Lactis C2, suggesting th at the gene may be incomplete or is not expressed, resulting in a requireme nt for glutamic acid in lactococci.