Lactose Transport System of Streptococcus thermophilus:a Hybrid Protein with Homology to the Melibiose Carrier and Enzyme III of Phosphoenolpyruvate-Dependent Phosphotransferase Systems

摘要

The gene responsible for the transport of lactose into Streptococcus thermophilus (lacS) was cloned in Escherichia coli as a 4.2-kilobase fragment from an EcoRI library of chromosomal DNA by using the vector pKK223-3. From deletion analysis, the gene for lactose transport mapped to two HindIII fragments with a total size of 2.8 kilobases. The gene was transcribed in E. coli from its own promoter. Functional expression of lactose transport activity was shown by assaying for the uptake and exchange of lactose both in intact cells and in membrane vesicles. The nucleotide sequence of lacS and 200 to 300 bases of 3' and 5' flanking regions were determined. The gene was 1,902 base pairs long, encoding a 69,454-dalton protein with an NH2-terminal hydrophobic region and a COOH-terminal hydrophilic region. The NH2-terminal end was homologous with the melibiose carrier of E. coli (23% similarity overall; 50% similarity for regions with at least 16 amino acids), whereas the COOH-terminal end showed 34 to 41% similarity with the enzyme III (domain) of three different phosphoenolpyruvate-dependent phosphotransferase systems. Among the conserved amino acids were two histidyl residues, of which one has been postulated to be phosphorylated by HPr. Since sugars are not phosphorylated during translocation by the lactose transport system, it is suggested that the enzyme III-like region serves a regulatory function in this protein. The lacS gene also appears similar to the partially sequenced lactose transport gene of Lactobacillus bulgaricus (lacL; 60% similarity). Furthermore, the 3' flanking sequence of the S. thermophilus lactose transport gene showed approximately 50% similarity with the N-terminal portion of the β-galactosidase gene of L. bulgaricus. In both organisms, the lactose transport gene and the β-galactosidase appear to be separated by a 3-base-pair intercistronic region.