Genetic localization and regulation of the maltose phosphorylase gene, malP, in Lactococcus lactis.
Maltose phosphorylase (MP) from Lactococcus lactis was purified and the corresponding gene was cloned and expressed in Escherichia coli. The isoelectric point of the pure enzyme was determined to be 7.0. According to zymogram analysis and SDS-PAGE, the native MP was shown to be a monomeric enzyme with a molecular mass of 75 kDa. A polyclonal antiserum was produced to assess the regulation of the gene encoding MP in LC: lactis. According to immunoblot analysis, synthesis of the enzyme was markedly repressed by both glucose and lactose in the growth medium. When the lactococci were cultivated in the presence of other sugars, including maltose, trehalose or galactose, there was a pronounced expression of the MP gene. In addition, when the cells were grown in media without any added sugar, there was also pronounced expression of the enzyme, according to immunoblot analysis and specific activity data. These results indicated that no particular sugar specifically induces the gene encoding MP. However, an effect of glucose on MP expression was demonstrated by performing fermentations in the presence of both maltose and glucose. When glucose was added to maltose-grown lactococci in the mid-exponential growth phase, both the specific activity and amount of MP per millilitre of cell extract decreased rapidly. The genetic locus for the MP gene was found to be in the vicinity of the region encoding a possible regulator belonging to the LacI-GalR family of transcriptional regulators. Furthermore, this genetic location was separated from the previously characterized maltose-inducible and glucose-repressible beta-phosphoglucomutase (beta-PGM) gene. The different genetic loci for the genes encoding MP and beta-PGM explains the different gene regulation behaviour.
Nilsson
Ulrika
U
Applied Microbiology, Centre for Chemistry and Chemical Engineering, Lund Institute of Technology, Lund University, PO Box 124, SE-221 00 Lund, Sweden1.
Rådström
Peter
P
Applied Microbiology, Centre for Chemistry and Chemical Engineering, Lund Institute of Technology, Lund University, PO Box 124, SE-221 00 Lund, Sweden1.
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D050260:Carbohydrate Metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D007106:Immune Sera; D015151:Immunoblotting; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D003470:Culture Media; D007106:Immune Sera; D005964:Glucosyltransferases; C021535:maltose phosphorylase