INVIVO CLONING OF LAC-GENES IN STREPTOCOCCUS-LACTIS-ML3

The isolation and characterization of a S. lactis ML3 strain which possessed a recombinant lactose plasmid is described. The recombination events generating this plasmid occurred in vivo in a recombination-deficient strain and appeared to be mediated by transposition events. Restriction mapping revealed that the recombinant plasmid, pDA0307, contained a region of the lactose plasmid, pSK08, linked to another resident plasmid, pSK07. Copy number determinations indicated that the lac genes were present at .apprx. 20 copies/cell in pDA0307; the lac genes are normally present at .apprx. 10 copies/cell in pSK08. The strain containing pDA0307 displayed a 21-54% increase in the expression of the Lac enzyme phospho-.beta.-D-galactosidase. The strain containing pDA0307 grew and produced lactic acid in milk at rates identical to that of a strain containing pSK08. Thus, lac gene dosage of plasmid-linked lac genes was not limiting the rate at which these derivatives of S. lactis ML3 fermented milk.