Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12.
Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics*
D004262:DNA Restriction Enzymes; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D005696:Galactosidases; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C022361:6-phospho-beta-galactosidase