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Cloning and Expression of theD-Galactosidase Gene from Streptococcus thermophilus in Escherichia colit

The P-D-galactosidase (,-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector. Chromosomal DNA from S. thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108. A j-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-o-D-galactoside. This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for ,8-gal activity. An extract from JM108(pRH116) contained a n-gal protein with the same electrophoretic mobility as the 3-gal from S. thermophilus 19258. Compared with the ,-gal from E. coli HB101, the S. thermophilus "-gal was of lower molecular weight. A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert. The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment. The ,-gal gene was further localized to a 3.85-kb region.