Purification and transpositional inactivation of Lacticin FS92, a broad-spectrum bacteriocin produced by Lactococcus lactis FS92

Lactococcus lactis FS92, a raw pork isolate produces a broad-spectrum bacteriocin (lacticin FS92) with activity against Bacillus spp., Clostridium spp., and Listeria monocytogenes. Lacticin FS92 was purified to homogeneity by ammonium sulfate flocculation, solid-phase extraction and reversed phased HPLC. Tricine-SDS-PAGE analysis indicated a mass of <3.5 kDa whereas MALDI-Mass spectrometry gave two peaks with masses of 3.16 and 6.32 kDa. Further characterization of lacticin FS92 indicated that it is heat-stable, has a bactericidal mode of action, and is resistant to trypsin but sensitive to other proteolytic enzymes. Amino acid composition analysis estimated that it contains approximately 32 amino acids. Failure to obtain N-terminal amino acid sequence using automated Edman degradation sequence analysis suggests that lacticin FS92 is N-blocked. The temperature-sensitive plasmid, pTV1-OK, containing the transposon, Tn917 was used in temperature-upshift experiments to generate chromosomal insertions in the L. lactis FS92 chromosome. One transposant, L. lactis FS92-M85 contained a single-copy chromosomal insertion of Tn917 that inactivated lacticin FS92 phenotypic expression (Bac(-)) but maintained lacticin FS92 immunity (Bac(R)). A flanking 389-bp sequence surrounding the Tn917 insertion was cloned and sequenced, but had little homology (< 50%) to known sequences. These data provide characterization of an anti-listerial bacteriocin that may have potential application as a food preservative. (C) 2001 Academic Press.