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Related Experiment Videos

Modulation of pPS10 host range by DnaA.

Beatriz Maestro1, Jesús M Sanz, Michel Faelen

  • 1Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.

Molecular Microbiology
|October 9, 2002
PubMed
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A triple mutation in the Escherichia coli dnaA gene enables the narrow-host-range plasmid pPS10 to replicate in E. coli. This discovery broadens plasmid host range and highlights DnaA

Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • The narrow-host-range plasmid pPS10, originating from Pseudomonas savastanoi, typically fails to replicate in Escherichia coli.
  • Plasmid replication and host range are often determined by interactions between plasmid-encoded proteins and host replication machinery.

Purpose of the Study:

  • To investigate the genetic basis for the inability of plasmid pPS10 to replicate in E. coli.
  • To identify specific mutations in the E. coli host that can enable pPS10 replication.
  • To elucidate the role of the DnaA protein in modulating plasmid establishment.

Main Methods:

  • Utilized a triple mutant of the E. coli dnaA gene (dnaA403) with specific amino acid substitutions (Q14amber, P297S, A412V).
  • Employed E. coli strains with amber suppressor mutations (supE, supF) to allow translation of the amber codon in dnaA403.

Related Experiment Videos

  • Conducted genetic analysis of DnaA protein variants to assess their impact on pPS10 establishment.

    • Main Results:

    • The dnaA403 triple mutant, particularly the P297S substitution, was found to be crucial for enabling pPS10 establishment in E. coli.
    • The P297S mutation exhibited toxicity, which was potentially mitigated by the other mutations in the dnaA403 background.
    • This study identified the first chromosomal mutant capable of broadening a plasmid's host range.

    Conclusions:

    • The DnaA protein plays a critical role in the establishment of the pPS10 replicon in E. coli.
    • Specific alterations in DnaA can overcome host range limitations for certain plasmids.
    • Interactions between RepA and DnaA are likely key modulators of pPS10 establishment in E. coli and potentially other bacterial species.