title: Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.
abstract: Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate.
authors.affiliation: Department of Biophysical Chemistry, Netherlands Institute for Dairy Research (NIZO), Ede.
authors.forename: G
authors.initials: G
authors.lastname: Simons
authors.forename: M
authors.initials: M
authors.lastname: Nijhuis
authors.forename: W M
authors.initials: WM
authors.lastname: de Vos
mesh_terms: D001692:Biological Transport; D002475:Cellobiose / Q000378:metabolism; D003429:Cross Reactions; D004790:Enzyme Induction; D005822:Genetic Vectors / Q000235:genetics; D006026:Glycoside Hydrolases; D007763:Lac Operon / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics*; D007785:Lactose / Q000378:metabolism; D016254:Mutagenesis, Insertional; D009598:Nitrophenylgalactosides / Q000378:metabolism; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D001616:beta-Galactosidase / Q000172:deficiency / Q000235:genetics* / Q000276:immunology
chemical_list: D002475:Cellobiose; D009598:Nitrophenylgalactosides; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C029022:phosphoenolpyruvate-lactose dependent phosphotransferase system; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C022361:6-phospho-beta-galactosidase; D007785:Lactose