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Related Experiment Videos

An RNA polymerase mutation causing temperature-sensitive sporulation in Bacillus subtilis.

T J Leighton

    Proceedings of the National Academy of Sciences of the United States of America
    |April 1, 1973
    PubMed
    Summary

    A Bacillus subtilis mutant with temperature-sensitive sporulation was identified. This mutation, affecting RNA polymerase, blocks sporulation at stage III and disrupts RNA synthesis during specific growth phases.

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    Area of Science:

    • Microbiology
    • Molecular Biology
    • Bacterial Genetics

    Background:

    • Bacillus subtilis sporulation is a complex, multi-stage developmental process.
    • RNA polymerase plays a crucial role in gene regulation during bacterial development.
    • Understanding sporulation mutants aids in deciphering regulatory networks.

    Purpose of the Study:

    • To investigate the role of RNA polymerase in Bacillus subtilis sporulation.
    • To characterize a temperature-sensitive sporulation mutant.
    • To identify the specific stage of sporulation affected by the mutation.

    Main Methods:

    • Isolation and characterization of a single-site mutant in Bacillus subtilis.
    • Genetic analysis of temperature-sensitive sporulation.

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  • Assessment of RNA polymerase template specificity.
  • Enzyme activity assays (alkaline phosphatase, proteolysis).
  • Analysis of antibiotic production.
  • Pulse-chase RNA labeling and synthesis analysis.

    • Main Results:

    • A rifampin-resistant RNA polymerase mutation conferred temperature-sensitive sporulation.
    • The mutation affected sporulation during the middle third of the sequence, blocking at stage III.
    • Mutant cells showed normal RNA polymerase specificity, proteolysis, and antibiotic accumulation at non-permissive temperatures.
    • Alkaline phosphatase accumulation was abolished in mutant cells at non-permissive temperatures.
    • Pulse-labeled RNA synthesis was severely impaired during the post-exponential growth phase.

    Conclusions:

    • The identified mutation in RNA polymerase is critical for stage III of Bacillus subtilis sporulation.
    • Specific gene expression, including alkaline phosphatase, is dependent on functional RNA polymerase during sporulation.
    • Disruption of RNA synthesis is a key consequence of the mutation at non-permissive temperatures.