Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp.
Lactacin F is a heat-stable bacteriocin produced by Lactobacillus acidophilus 11088. A 63-mer oligonucleotide probe deduced from the N-terminal lactacin F amino acid sequence was used to clone the putative laf structural gene from plasmid DNA of a lactacin F-producing transconjugant, L. acidophilus T143. One clone, NCK360, harbored a recombinant plasmid, pTRK160, which contained a 2.2-kb EcoRI fragment of the size expected from hybridization experiments. An Escherichia coli-L. acidophilus shuttle vector was constructed, and a subclone (pTRK162) containing the 2.2-kb EcoRI fragment was introduced by electroporation into two lactacin F-negative strains, L. acidophilus 89 and 88-C. Lactobacillus transformants containing pTRK162 expressed lactacin F activity and immunity. Bacteriocin produced by the transformants exhibited an inhibitory spectrum and heat stability identical to those of the wild-type bacteriocin. An 873-bp region of the 2.2-kb fragment was sequenced by using a 20-mer degenerate lactacin F-specific primer to initiate sequencing from within the lactacin F structural gene. Analysis of the resulting sequence identified an open reading frame which could encode a protein of 75 amino acids. The 25 N-terminal amino acids for lactacin F were identified within the open reading frame along with an N-terminal extension, possibly a signal sequence. The lactacin F N-terminal sequence, through the remainder of the open reading frame (57 amino acids; 6.3 kDa), correlated extremely well with composition analyses of purified lactacin F which also predicted a size of 51 to 56 amino acid residues. Molecular characterization of lactacin F identified a small hydrophobic peptide that may be representative of a common bacteriocin class in lactic acid bacteria.
Muriana
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PM
Department of Food Science, North Carolina State University, Raleigh 27695-7624.
Klaenhammer
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D000595:Amino Acid Sequence; D001430:Bacteriocins / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000235:genetics*; D007779:Lactobacillus acidophilus / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010641:Phenotype; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping
D001430:Bacteriocins; D004269:DNA, Bacterial; C067573:lactacin F