Cloning and Characterization of upp, a Gene Encoding Uracil Phosphoribosyltransferase from Lactococcus lactis

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    Abstract

    Uracil phosphoribosyltransferase catalyzes the key reaction in the salvage of uracil in many microorganisms. The gene encoding uracil phosphoribosyltransferase (upp) was cloned from Lactococcus lactis subsp. cremoris MG1363 by complementation of an Escherichia coli mutant. The gene was sequenced, and the putative amino add sequence was deduced. The promoter was mapped by both primer extension and analysis of beta-galactosidase expressed from strains carrying fusion between upp promoter fragments and the lacLM gene. The results showed that the upp gene was expressed from its own promoter. After in vitro construction of an internal deletion, a upp mutant was constructed by a double-crossover event. This implicated the utilization of a plasmid with a thermosensitive origin of replication and a new and easy way to screen for double crossover events in both gram-positive and gram-negative bacterial strains. The phenotype of the uracil phosphoribosyltransferase-deficient strain was established. Surprisingly, the upp strain is resistant only to very low concentrations of 5-fluorouracil. Secondary mutants in thymidine phosphorylase and thymidine kinase were isolated by selection for resistance to high concentrations of 5-fluorouracil.
    Original languageEnglish
    JournalJournal of Bacteriology
    Volume176
    Issue number21
    Pages (from-to)6457-6463
    Number of pages7
    ISSN0021-9193
    Publication statusPublished - 1994

    Keywords

    • Amino Acid Sequence
    • Base Sequence
    • Cloning, Molecular
    • Crossing Over, Genetic
    • Drug Resistance, Microbial
    • Fluorouracil
    • Gene Deletion
    • Gene Expression
    • Genes, Bacterial
    • Lactococcus lactis
    • Molecular Sequence Data
    • Pentosyltransferases
    • Promoter Regions, Genetic
    • Recombinant Fusion Proteins
    • Restriction Mapping
    • Sequence Analysis, DNA
    • Sequence Homology, Amino Acid
    • Uracil
    • 56HH86ZVCT Uracil
    • EC 2.4.2.- Pentosyltransferases
    • EC 2.4.2.9 uracil phosphoribosyltransferase
    • U3P01618RT Fluorouracil
    • CONCENTRATION DEPENDENT 5-FLUOROURACIL RESISTANCE
    • DOUBLE CROSSOVER SCREENING METHOD
    • PROMOTER MAPPING
    • THYMIDINE KINASE MUTANT
    • THYMIDINE PHOSPHORYLASE MUTANT
    • Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Gram-Positive Cocci [07700] gram-positive cocci Lactococcus lactis
    • 5-FLUOROURACIL 51-21-8
    • THYMIDINE KINASE 9002-06-6
    • THYMIDINE PHOSPHORYLASE 9030-23-3
    • URACIL PHOSPHORIBOSYLTRANSFERASE 9030-24-4
    • 10052, Biochemistry methods - Nucleic acids, purines and pyrimidines
    • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
    • 10064, Biochemistry studies - Proteins, peptides and amino acids
    • 10300, Replication, transcription, translation
    • 10506, Biophysics - Molecular properties and macromolecules
    • 10806, Enzymes - Chemical and physical
    • 13014, Metabolism - Nucleic acids, purines and pyrimidines
    • 31000, Physiology and biochemistry of bacteria
    • 31500, Genetics of bacteria and viruses
    • 32000, Microbiological apparatus, methods and media
    • Biochemistry and Molecular Biophysics
    • amino acid sequence
    • molecular sequence data
    • nucleotide sequence
    • X73329 EMBL
    • Enzymology
    • Genetics
    • Molecular Genetics
    • Physiology

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