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Published on: February 5, 2015

dfp Gene of Escherichia coli K-12, a locus affecting DNA synthesis, codes for a flavoprotein

E D Spitzer, B Weiss

    Journal of Bacteriology
    |December 1, 1985
    PubMed

    Insights

    The cloned dfp gene complements a DNA synthesis mutation in E. coli. The dfp gene product is a 45-kilodalton protein containing flavin mononucleotide.

    Area of Science:

    • Molecular Biology
    • Genetics
    • Microbiology

    Background:

    • A temperature-sensitive mutation, dfp-707, in Escherichia coli leads to a slow cessation of DNA synthesis at restrictive temperatures, while protein synthesis remains unaffected.
    • Previous studies indicated that this mutation did not present a specific defect in the initiation of DNA replication, and the shutdown of DNA synthesis was slower than typical initiation mutants like dnaA.

    Purpose of the Study:

    • To clone and characterize the dfp gene responsible for complementing the dfp-707 mutation.
    • To identify the protein product of the dfp gene and determine its properties.
    • To map the location and understand the transcriptional regulation of the dfp gene within the E. coli K-12 genome.

    Main Methods:

    • Complementation analysis using cloned dfp gene to rescue the temperature-sensitive dfp-707 mutation.
    • Construction and analysis of deletion and insertion mutants of dfp-containing plasmids to identify the gene and its product.
    • In vitro and in vivo experiments to assess DNA replication initiation and DNA synthesis.
    • Gene mapping on the E. coli K-12 linkage map and analysis of gene transcription.
    • Protein purification and characterization, including molecular weight determination and flavin mononucleotide (FMN) content analysis.

    Main Results:

    • The cloned dfp gene successfully complemented the dfp-707 mutation, confirming its role in DNA synthesis regulation.
    • The dfp gene was localized to the 81-minute region on the E. coli K-12 map, situated between the rpmB and dut genes, and transcribed clockwise, independently of dut.
    • Analysis of mutants revealed that the dfp gene encodes a 45-kilodalton polypeptide, and its expression correlated with complementation ability.
    • The purified dfp protein was found to contain one molecule of flavin mononucleotide (FMN) per polypeptide.

    Conclusions:

    • The dfp gene is essential for normal DNA synthesis in E. coli and encodes a 45 kDa FMN-binding protein.
    • The dfp gene's chromosomal location and independent transcription provide insights into its regulatory context within the E. coli genome.
    • Further research is warranted to elucidate the precise function of the DFP protein in DNA replication or related processes.

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