MOLECULAR CHARACTERIZATION OF GENES INVOLVED IN THE PRODUCTION OF THEBACTERIOCIN LEUCOCIN-A FROM LEUCONOSTOC GELIDUM

Leucocin A is a small heat-stable bacteriocin produced by Leuconostoc gelidum UAL187. A 2,9-kb fragment of plasmid DNA that contains the leu cocin structural gene and a second open reading frame (ORF) in an oper on was previously cloned (J, W. Hastings, M. Sailer, It. Johnson, K. L . Boy, J. C. Vederas, and M. E. Stiles, J. Bacteriol. 173:7498-7500, 1 991), When a 1-kb Draf-HpaI fragment containing this operon was introd uced into a bacteriocin-negative variant (UAL187-13), immunity but no leucocin production was detected. Leucocin production was observed whe n an 8-kb SacI-HindIII fragment of the leucocin plasmid was introduced into L. gelidum UAL187-13 and Lactococcus lactis IL1403. Nucleotide s equence analysis of this 8-kb fragment revealed the presence of three ORFs in an operon upstream of and on the strand opposite from the leuc ocin structural gene, The first ORF (lcaE) encodes a putative protein of 149 amino acids with no apparent function in leucocin A production. The second ORF (lcaC) contains 717 codons that encode a protein homol ogous to members of the HlyB family of ATP-binding cassette transporte rs. The third ORF (lcaD) contains 457 codons that encode a protein wit h marked similarity to LcnD, a protein essential for the expression of the lactococcal bacteriocin lactococcin A. Deletion mutations in lcaC and lcaD resulted in loss of leucocin production, indicating that Lca C and LcaD are involved in production and translocation of leucocin A, The secretion apparatus for lactococcin A did not complement mutation s in the lcaCD genes to express leucocin A in L. lactis, However, lact ococcin A production was observed when the structural and immunity gen es for this bacteriocin were introduced into a Leucocin producer of L. gelidum UAL187, indicating that lactococcin A could be exported by th e leucocin A secretion machinery.