Molecular characterization of lacticins FS56 and FS92, two broad-spectrum bacteriocins produced by Lactococcus lactis strains

Ying Mao, Purdue University

Abstract

Lactococcus lactis FS56 and FS92, isolated from retail food products, produce broad-spectrum antimicrobial substances active against foodborne pathogens, including Listeria monocytogenes . Lacticin FS56 and FS92, bacteriocins produced by Lc. lactis strains FS56 and FS92, respectively, were purified to homogeneity by ammonium sulfate precipitation, solid-phase extraction, and reverse phase HPLC. Comparison of stained and indicator-overlayed Tricine-SDS-PAGE gels indicated that both are peptides of less than 3.5 kDa. MALDI-Mass spectrometric analysis of three peaks collected from HPLC containing lacticin FS56 showed that they all contained a 5.2 kDa peptide. Further characterization of lacticin FS92 indicated that it is heat stable and resistant to trypsin but sensitive to various other proteolytic enzymes. This bacteriocin has a bactericidal mode of action against the sensitive strain Lactobacillus delbrueckii 4797. Amino acid compositional analysis confirmed the hydrophobic nature of lacticin FS92 and estimated that lacticin FS92 contained at least 64 amino acids. Database comparisons based on composition did not result in any matches. MALDI mass spectrometric analysis of HPLC-purified lacticin FS92 demonstrated a major peak with a mass of 6.32 kDa. However, its N-terminal amino acids could not be identified, indicating that the N-terminus of purified lacticin FS92 is blocked. To study the genes associated with lacticin FS92 production, a method for generation of a transposon Tn917 insertional mutant library in Lc. lactis FS92 was developed. By transforming the temperature sensitive plasmid pTV1-OK harboring Tn917, a mutant library was generated. With temperature up-shifting, transformants were selected for antibiotic resistance of the transposon and then screened for loss of the plasmid pTV1-OK. Southern hybridization indicated the random insertion of Tn9l7 into the chromosome of Lc. lactis FS92. By screening the Tn9l7 mutant library for bacteriocin deficient phenotypes, one lacticin FS92 negative strain, M85, was isolated. The mutant M85 was examined for its growth under different pHs, salt concentrations, and temperatures. API carbohydrate spectra, SDS-PAGE cell surface protein profiles, and plasmid profiles also were analyzed for the mutant to compare with its parental strain, Lc. lactis FS92. The results indicated no significant difference between the wild type and the mutant cells. Southern hybridization analysis of single restriction enzyme digests of chromosomal DNA from M85 revealed that only one copy of the Tn9l7 inserted into the mutant. This suggested that the transposon Tn9l7 insertional mutation occurs within the gene potentially involving the bacteriocin production. The bacteriocin deficient mutant M85 was recovered by shot-gun cloning of the insert DNA adjacent to Tn9l7. The protein deduced from the sequence analysis showed a strong similarity to the transmembrane protein of several other bacteria and to proteins of the ATP-binding-cassette (ABC) transporter. These results indicated that the gene is responsible for the export of the bacteriocin from the cell.

Degree

Ph.D.

Advisors

Muriana, Purdue University.

Subject Area

Microbiology|Molecular biology

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