Functional analysis of the gene encoding immunity to lactacin F, lafI, and its use as a Lactobacillus-specific, food-grade genetic marker

Abstract

Lactacin F is a two-component class II bacteriocin produced by Lactobacillus johnsonii VPI 11088. The laf operon is composed of the bacteriocin structural genes, lafA and lafX, and a third open reading frame, ORFZ. Two strategies were employed to study the function of ORFZ. This gene was disrupted in the chromosome of NCK64, a lafA729 lafX ORFZ derivative of VPI 11088. A disruption cassette consisting of ORFZ interrupted with a cat gene was cloned into pSA3 and introduced into NCK64. Manipulation of growth temperatures and antibiotic selection resulted in homologous recombination which disrupted the chromosomal copy of ORFZ with the cat gene. This ORFZ mutation resulted in loss of immunity to lactacin F but had little effect on production of LafX, which is not bactericidal without LafA. Expression of ORFZ in this ORFZ- background rescued the immune phenotype. Expression of ORFZ in a bacteriocin-sensitive derivative of VPI 11088 also reestablished immunity. These data indicate that ORFZ, renamed lafI, encodes the immunity factor for the lactacin F system. The sensitivity of various Lactobacillus strains to lactacin F was further evaluated. Lactacin F inhibited 11 strains including several members of the A1, A2, A3, A4, B1, and B2 L. acidophilus homology groups. Expression of lafI in bacteriocin-sensitive strains L. acidophilus ATCC 4356, L. acidophilus NCFM/N2, L. fermentum NCDO1750, L. gasseri ATCC 33323, and L. johnsonii ATCC 33200 provided immunity to lactacin F. Furthermore, it was shown that lactacin F production by VPI 11088 could be used to select for L. fermentum NCDO1750 transformants containing the recombinant plasmid encoding LafI. The data demonstrate that lafI is functional in heterologous hosts, suggesting that it may be a suitable food-grade genetic marker for use in lactobacillus species.