Cloning, expression and location of the Streptococcus lactis gene for phospho-beta-D-galactosidase

Genes for lactose catabolism and proteinase production in Streptococcus lactis 712 are encoded by a 56.5 kb metabolic plasmid, pLP712. A lactose mini-plasmid of only 23.7 kb, pMG820, was constructed by introducing two deletions into pLP712, and was cloned as two segments of DNA into the Escherichia coli vector pAT153 using restriction endonuclease PstI. The lactose genetic region of pLP712, which has been defined by deletion and restriction mapping, was cut into two parts by this process. When the smaller 10.8 kb segment of pMG820 DNA was present, the key lactic streptococcal lactose splitting enzyme, phospho-beta-D-galactosidase, was expressed in E. coli. The gene for phospho-beta-D-galactosidase was more precisely located by introducing a series of deletions into cloned DNA by in vitro manipulations and then assaying for enzyme activity. The presence of this phospho-beta-D-galactosidase activity was correlated with the production of a 58 kDa 35S-labelled protein both by E. coli minicells and after coupled transcription and translation of cloned DNA. The product of a second gene, a 37 kDa protein ('protein X'), and a possible truncated phospho-beta-D-galactosidase protein of 16 kDa were also detected in minicells.