CLONING AND CHARACTERIZATION OF THE GALACTOKINASE GENE FROM STREPTOCOCCUS-THERMOPHILUS

The objective of this research was to clone and characterize the galac tokinase gene (galK) from Streptococcus thermophilus F410. Partially d igested genomic DNA was cloned into pBR322 and transformed into galK E scherichia coli, and a galactose-fermenting transformant was isolated. Restriction analysis revealed that the transformant resulted from a S au3A-HindIII 4.0-kb fragment. Galactokinase activity in the recombinan t was 10 times that of the parent: strain. Analysis of the DNA sequenc e showed the presence of a 1.3-kb open reading frame that had high hom ology with the galK gene from other organisms. A putative ribosome-bin ding site, start and stop codons, and -10 and -35 sequences were ident ified. The predicted protein had a molecular mass of 49 kDa, which cor responded to the estimated size of a band apparent by SDS-PAGE. Amino acid sequence homologies with other galactokinases ranged from 50 to 6 2% similarity. Northern blots were performed between the galK gene and mRNA from S. thermophilus. No hybridization signals were observed for cells grown in glucose, but cells grown in lactose or galactose gave moderate and strong signals. The results suggest that repression of th e galK gene by glucose may be responsible for the galactose-releasing phenotype in these strains.