Cloning and expression of the phospho-beta-galactosidase genes on the lactose plasmid and the chromosome of Lactobacillus casei C257 in Escherichia coli.
A 4.7 kb BglII fragment of lactose plasmid pLY101 of Lactobacillus casei C257, which hybridized weakly to a fragment including a streptococcal phospho-beta-galactosidase (P-beta-gal) gene, was cloned into plasmid vectors of Escherichia coli. Transformant cells expressed P-beta-gal activity, confirming the presence of a P-beta-gal gene on pLY101. Deletion mapping showed that the structural gene of P-beta-gal was located on the 2.4 kb PvuI-EcoRV fragment. The direction of transcription was determined using an expression vector. L. casei MSK248 is a non-lactose-utilizing, pLY101-cured derivative of C257 and no P-beta-gal activity was detected. From MSK248, spontaneous lactose-utilizing revertants having P-beta-gal activity were isolated. Using the pLY101-derived P-beta-gal gene as a probe, a homologous sequence was detected in the chromosomal DNAs of MSK248 and MSK256, and cloned into plasmid vectors of E. coli. Both transformants carrying the homologous sequence from MSK248 and MSK256 expressed P-beta-gal activity. Restriction maps of the chromosomal P-beta-gal genes from MSK248 and MSK256 were identical with that derived from pLY101.
D002874:Chromosome Mapping; D003001:Cloning, Molecular; D015280:DNA Restriction-Modification Enzymes; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005696:Galactosidases / Q000235:genetics*; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics*; D007785:Lactose / Q000378:metabolism*; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics*
D015280:DNA Restriction-Modification Enzymes; D005696:Galactosidases; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C022361:6-phospho-beta-galactosidase; D007785:Lactose