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Abstract
The physiology and genetics of carbohydrate metabolism in Streptococcus thermophilus have only recently been studied. S. thermophilus utilizes a lactose permease and the enzyme $\beta$-galactosidase to transport and hydrolyze lactose inside the cell. Additionally, most wild-type strains of this organism are also unable to ferment lactose completely and release the galactose portion back into the medium. Galactose-fermenting strains of S. thermophilus have been shown to metabolize galactose via the enzymes of the Leloir pathway, two of which (galactose permease and galactokinase) are rate-limiting. However, even these few Gal$\sp+$ strains release this sugar into the medium when grown on lactose.
It is economically and scientifically feasible to increase the metabolic diversity of this important thermophilic starter culture. The main intent of this work, therefore, was to achieve a greater understanding of galactose metabolism in S. thermophilus with regard to its basic physiological and genetic characteristics. The specific objectives of this work were to first isolate a strain of S. thermophilus which can ferment galactose without releasing it into the medium, and to isolate, identify, and purify the galactokinase gene (galK) from this organism. Next, the purified galK gene was cloned in Escherichia coli, and the gene was further analyzed by determining its size, restriction map, and nucleotide, as well as amino acid sequence. This galK gene was also compared with that of other organisms, and finally, flanking regions were analyzed in order to identify and locate any other adjacent genes.
The galK gene from S. thermophilus F410 was successfully cloned in E. coli in this study. The putative size of the gene was determined to be about 3.9 kb which translated to a protein monomer of about 49 kDa. The galactokinase from S. thermophilus F410 was found to exhibit significant homology to that of Lactobacillus helveticus, E. coli, Haemophilus influenzae, and Kluyveromyces lactis. Two additional genes, lacS and lacZ, have been proposed to be located downstream of this galK based on significant homology found between this region and the lacSZ genes and gene products from S. thermophilus A147.
