Isolation and characterization of acidocin A and cloning of the bacteriocin gene from Lactobacillus acidophilus

• Published in: Applied and Environmental Microbiology Vol. 61; no. 3; pp. 1061 - 1067
• Main Authors: Kanatani, K. (Tamon Sake Brewing Co., Ltd., Nishinomiya, Hyogo, Japan), Oshimura, M, Sano, K
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.03.1995
• Subjects: Amino Acid Sequence; Amino Acids - analysis; Bacteria; Bacteria - drug effects; BACTERIOCINAS; BACTERIOCINE; Bacteriocins - chemistry; Bacteriocins - genetics; Bacteriocins - isolation & purification; Bacteriocins - pharmacology; Bacteriology; Base Sequence; Biochemistry; Biological and medical sciences; BIOSINTESIS; BIOSYNTHESE; Chromatography, Liquid; Cloning, Molecular - methods; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; CULTIVOS INICIADORES; CULTURE STARTER; Food industries; Food microbiology; Fundamental and applied biological sciences. Psychology; GENE; Gene Expression Regulation, Bacterial; GENES; Genes, Bacterial - genetics; LACTOBACILLUS ACIDOPHILUS; Lactobacillus acidophilus - genetics; LAIT FERMENTE; LECHE FERMENTADA; Microbiology; Molecular Sequence Data; Open Reading Frames - genetics; Operon - genetics; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Protein Precursors - genetics; PURIFICACION; PURIFICATION; Restriction Mapping; SECUENCIA NUCLEICA; Sequence Analysis; Sequence Analysis, DNA; SEQUENCE NUCLEIQUE; Transformation, Bacterial; Lactic acid bacteria; Nucleotide sequence; Bacteriocin; Sequence alignment; Listeria monocytogenes; Homology; Lactobacillus acidophilus; Open reading frame; Food industry; Bacteria; Lactobacillaceae; Chemical composition; Antibacterial agent; Application; Comparative study; Aminoacid sequence; Structural analysis
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.61.3.1061-1067.1995

Acidocin A, a bacteriocin produced by Lactobacillus acidophilus TK9201, is active against closely related lactic acid bacteria and food-borne pathogens including Listeria monocytogenes. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential ion-exchange and reversed-phase chromatographies. The molecular mass was determined by high-performance liquid chromatography gel filtration to be 6,500 Da. The sequence of the first 16 amino acids of the N terminus was determined, and oligonucleotide probes based on this sequence were constructed to detect the acidocin A structural gene acdA. The probes hybridized to the 4.5-kb EcoRI fragment of a 45-kb plasmid, pLA9201, present in L. acidophilus TK9201, and the hybridizing region was further localized to the 0.9-kb KpnI-XbaI fragment. Analysis of the nucleotide sequence of this fragment revealed that acidocin A was synthesized as an 81-amino-acid precursor including a 23-amino-acid N-terminal extension. An additional open reading frame (ORF2) encoding a 55-amino-acid polypeptide was found downstream of and in the same operon as acdA. Transformants containing this ORF2 became resistant to acidocin A, suggesting that ORF2 encodes an immunity function for acidocin A. The 7.2-kb SacI-XbaI fragment containing the upstream region of acdA of pLA9201 was necessary for acidocin A expression in the acidocin A-deficient mutant, L. acidophilus TK9201-1, and other Lactobacillus strains