Cloning and characterization of the galactokinase gene from Streptococcus thermophilus.
The objective of this research was to clone and characterize the galactokinase gene (galK) from Streptococcus thermophilus F410. Partially digested genomic DNA was cloned into pBR322 and transformed into galK Escherichia coli, and a galactose-fermenting transformant was isolated. Restriction analysis revealed that the transformant resulted from a Sau3A-HindIII 4.0-kb fragment. Galactokinase activity in the recombinant was 10 times that of the parent strain. Analysis of the DNA sequence showed the presence of a 1.3-kb open reading frame that had high homology with the galK gene from other organisms. A putative ribosome-binding site, start and stop codons, and -10 and -35 sequences were identified. The predicted protein had a molecular mass of 49 kDa, which corresponded to the estimated size of a band apparent by SDS-PAGE. Amino acid sequence homologies with other galactokinases ranged from 50 to 62% 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 the galK gene by glucose may be responsible for the galactose-releasing phenotype in these strains.
Mustapha
A
A
Department of Food Science and Technology, University of Nebraska-Lincoln 68583-0919, USA.
Hutkins
R W
RW
Zirnstein
G W
GW
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005686:Galactokinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013291:Streptococcus / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D005686:Galactokinase; D004262:DNA Restriction Enzymes