Characterization of Lactobacillus curvatus G, a lactocin 705 and antilisteria-deficient mutant

Abstract

Lactobacillus curvatus CRL705 produces two bacteriocins, Lac705 and AL705, and harbors two plasmids, pRC18 and pRC12. The gene determinants of lactocin Lac705, genes #705/#705, imm705, abc705 and acc705, are encoded in pRC18, a plasmid of 18600 bp whose DNA sequence has been determined. In this study, the characterization of a Lac705- and AL705- deficient mutant, named strain G, is described. Strain G has been isolated after transforming L. curvatus CRL705 with plasmid pTV01::Tn917 and growing the transformants at high temperature (39C). Confirmation that strain G derived from CRL705 was determined by API50 analysis and by RAPD reactions, using primers RAPD1, RAPD2, REP1RI, REP2I and REPV. Agarose gel electrophoresis showed that plasmids pRC12 and pRC18 were absent in strain G. However, the presence pRC18 in strain G, and therefore of the lactocin Lac705 operon, was confirmed by PCR reactions using a set of three pRC18-specific primers (primers #1/#2, #3/#4 and #5/#6). The abundance and size of the amplified PCR products suggested that plasmid pRC18 is present in strain G in a low copy number and that the structural genes of lactocin Lac705 (705/705) are rearranged, which could explain the phenotype observed.