Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum.
Leucocin A-UAL 187 is a bacteriocin produced by Leuconostoc gelidum UAL 187, a lactic acid bacterium isolated from vacuum-packaged meat. The bacteriocin was purified by ammonium sulfate or acid (pH 2.5) precipitation, hydrophobic interaction chromatography, gel filtration, and reversed-phase high-performance liquid chromatography with a yield of 58% of the original activity. Leucocin A is stable at low pH and heat resistant, and the activity of the pure form is enhanced by the addition of bovine serum albumin. It is inactivated by a range of proteolytic enzymes. The molecular weight was determined by mass spectrometry to be 3,930.3 +/- 0.4. Leucocin A-UAL 187 contains 37 amino acids with a calculated molecular weight of 3,932.3. A mixed oligonucleotide (24-mer) homologous to the sequence of the already known N terminus of the bacteriocin hybridized to a 2.9-kb HpaII fragment of a 7.6-MDa plasmid from the producer strain. The fragment was cloned into pUC118 and then subcloned into a lactococcal shuttle vector, pNZ19. DNA sequencing revealed an operon consisting of a putative upstream promoter, a downstream terminator, and two open reading frames flanked by a putative upstream promoter and a downstream terminator. The first open reading frame downstream of the promoter contains 61 amino acids and is identified as the leucocin structural gene, consisting of a 37-amino-acid bacteriocin and a 24-residue N-terminal extension. No phenotypic expression of the bacteriocin was evident in several lactic acid bacteria that were electrotransformed with pNZ19 containing the 2.9-kb cloned fragment of the leucocin A plasmid.
Hastings
J W
JW
Department of Food Science, University of Alberta, Edmonton, Canada.
Sailer
M
M
Johnson
K
K
Roy
K L
KL
Vederas
J C
JC
Stiles
M E
ME
D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000096:biosynthesis* / Q000378:metabolism*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007934:Leuconostoc / Q000235:genetics*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D015183:Restriction Mapping; D014357:Trypsin
D001430:Bacteriocins; D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; C071726:leucocin A-UAL 187; D014357:Trypsin