CLONING AND CHARACTERIZATION OF THE GENES ENCODING THE MALOLACTIC ENZYME AND THE MALATE PERMEASE OF LEUCONOSTOC-OENOS

Using degenerated primers from conserved regions of the protein sequen ces of malic enzymes, we amplified a 324-bp DNA fragment by PCR from L euconostoc oenos and used this fragment as a probe for screening a Leu conostoc oenos genomic bank, Of the 2,990 clones in the genomic bank e xamined, 7 with overlapping fragments were isolated by performing colo ny hybridization experiments, Sequencing 3,453 bp from overlapping fra gments revealed two open reading frames that were 1,623 and 942 nucleo tides long and were followed by a putative terminator structure, The f irst deduced protein (molecular weight, 59,118) is very similar (level of similarity, 66%) to the malolactic enzyme of Lactococcus lactis; a s in several malic enzymes, highly conserved protein regions are prese nt, The synthesis of a protein with an apparent molecular mass of 60 k Da was highlighted by the results of labelling experiments performed w ith Escherichia coli minicells, The gene was expressed in E. coli and Saccharomyces cerevisiae and conferred ''malolactic activity'' to thes e species, The second open reading frame encodes a putative 34,190-Da protein which has the characteristics of a carrier protein and mag hav e 10 membrane-spanning segments organized around a central hydrophilic core, Energy-dependent L-[C-14]malate transport was observed with E. coli dicarboxylic acid transport-deficient mutants carrying the malate permease-expressing vector, Our results suggest that in Leuconostoc o enos the genes that encode the malolactic enzyme and a malate carrier protein are organized in a cluster.