PURIFICATION OF LEUCONOSTOC-MESENTEROIDES CITRATE LYASE AND CLONING AND CHARACTERIZATION OF THE CITCDEFG GENE-CLUSTER

A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mes enteroides and was shown to contain three subunits, The first 42 amino acids of the beta subunit were identified, as well as an internal pep tide sequence spanning some 20 amino acids into the alpha subunit, Usi ng degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp, cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding cit rate lyase of L. mesenteroides is organized in three open reading fram es, citD, citE, and citF, encoding, respectively, the three citrate ly ase subunits gamma (acyl carrier protein [ACP]), beta (citryl-S-ACP ly ase; EC 4.1.3.34), and alpha (citrate:acetyl-ACP transferase; EC 2.8.3 .10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebs iella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found, The deduced protein is similar to CitG of the other bacteri a, and its function remains unknown. Expression of the citCDEFG gene c luster in Escherichia coli led to the detection of a citrate lyase act ivity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group, This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.