Characterization and exploitation of conjugation in Lactococcus lactis

Abstract

The sex factor in L. lactis MG1363 is a chromosomally-located element with an approximate 50kb size. The genetic study of this sex factor has previously been confined to the analysis of lactose plasmid cointegrates which often exhibit high transfer frequency and a constitutive cell aggregation phenotype. The cryptic nature of the sex factor limits its direct analysis. Accordingly, a tetracycline resistance gene (tet M) was integrated within the chromosomally-located sex factor and this allowed the demonstration of its independent capacity for high frequency transfer between a diverse group of lactococcal strains. Chromosomal genes were also shown to be transferred by the sex factor in a unidirectional polar manner reminiscent of an Escherichia coli Hfr strain. The sex factor was cloned from its chromosomal location and most of its sequence determined. Homology of proteins encoded by sex factor genes to known protein sequences revealed three of immediate interest. Tra D exhibits homology to the equivalent protein of the E. coli sex factor F as well as to the VirD4 protein of Agro-bacterium tumefaciens and it is likely to play a role in the transfer of DNA through the cell surface during conjugation. CluA is involved in the establishment of cell to cell contact and can induce a constitutive cell aggregation phenotype. A third protein exhibits strong homology to yeast mitochondrial intron maturases and the orf structure suggests that a type II intron is present in a sex factor gene. Sequence analysis also revealed the nature of sex factor integration and excision to be site-specific involving an identical 24 bp sequence on both the sex factor and the chromosome. The integration/ excision process resembled that of E. coli bacteriophage lambda.