The gal genes for the Leloir pathway of Lactobacillus casei 64H

• Published in: Applied and environmental microbiology Vol. 64; no. 6; pp. 2013 - 2019
• Main Author: Bettenbrock, K. (Universitat Osnabruck, Osnabruck, Germany.)
• Format: Journal Article
• Language: English
• Published: Washington, DC American Society for Microbiology 01.06.1998
• Subjects: AMINO ACID SEQUENCES; ARN MENSAJERO; ARN MESSAGER; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Carbohydrate Epimerases - genetics; Carbohydrate Metabolism; CHEMICAL COMPOSITION; Cloning; Cloning, Molecular; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; Fermentation; Fundamental and applied biological sciences. Psychology; Galactokinase - genetics; Galactose - genetics; Galactose - metabolism; GENBANK/AF005933; GENE EXPRESSION; Genes; Genes, Bacterial; Genetic Complementation Test; Genetics; Genetics and Molecular Biology; LACTOBACILLUS CASEI; Lactobacillus casei - genetics; Lactobacillus casei - growth & development; Lactobacillus casei - metabolism; MESSENGER RNA; Microbiology; Mission oriented research; MOLECULAR SEQUENCE DATA; Mutation; NUCLEOTIDE SEQUENCE; Operon; Promoter Regions, Genetic; Repressor Proteins - genetics; SECUENCIA NUCLEOTIDICA; SEQUENCE NUCLEOTIDIQUE; UDPglucose 4-Epimerase - genetics; UTP-Hexose-1-Phosphate Uridylyltransferase - genetics; Lactobacillus casei; Nucleotide sequence; Operon; Gene organization; Metabolic pathway; Gene expression; Metabolism; Plasmid; Enzymatic activity; Bacteria; Lactobacillaceae; Molecular cloning; Galactose
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.64.6.2013-2019.1998

The gal genes from the chromosome of Lactobacillus casei 64H were cloned by complementation of the galK2 mutation of Escherichia coli HB101. The pUC19 derivative pKBL1 in one complementation-positive clone contained a 5.8-kb DNA HindIII fragment. Detailed studies with other E. coli K-12 strains indicated that plasmid pKBL1 contains the genes coding for a galactokinase (GalK), a galactose 1-phosphate-uridyltransferase (GalT), and a UDP-galactose 4-epimerase (GalE). In vitro assays demonstrated that the three enzymatic activities are expressed from pKBL1. Sequence analysis revealed that pKBL1 contained two additional genes, one coding for a repressor protein of the LacI-GalR-family and the other coding for an aldose 1-epimerase (mutarotase). The gene order of the L. casei gal operon is galKETRM. Because parts of the gene for the mutarotase as well as the promoter region upstream of galK were not cloned on pKBL1, the regions flanking the HindIII fragment of pKBL1 were amplified by inverse PCR. Northern blot analysis showed that the gal genes constitute an operon that is transcribed from two promoters. The galKp promoter is inducible by galactose in the medium, while galEp constitutes a semiconstitutive promoter located in galK