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Related Experiment Videos

Host-dependent requirement for specific DnaA boxes for plasmid RK2 replication.

K S Doran1, D R Helinski, I Konieczny

  • 1Department of Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634, USA.

Molecular Microbiology
|July 27, 1999
PubMed
Summary
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The broad-host-range plasmid RK2 origin of replication (oriV) shows varied DnaA box requirements across bacteria. Alternative initiation mechanisms may exist, particularly in Pseudomonas aeruginosa.

Area of Science:

  • Molecular Biology
  • Microbiology
  • Bacterial Genetics

Background:

  • The broad-host-range plasmid RK2 replication origin (oriV) contains DnaA boxes crucial for replication initiation.
  • DnaA protein binding to these boxes is essential for plasmid replication in many bacteria.

Purpose of the Study:

  • To investigate the differential requirement of DnaA boxes within oriV for replication in various Gram-negative bacteria.
  • To explore alternative mechanisms of oriV replication initiation.

Main Methods:

  • Transformation assays were used to analyze the function of mutated DnaA boxes in different bacterial species.
  • In vitro replication assays with E. coli extracts were performed to assess the impact of DnaA protein levels on origin activity.
  • Open complex formation and helicase loading were monitored under varying protein concentrations.

Related Experiment Videos

Main Results:

  • Mutational analysis revealed differential requirements for DnaA boxes 3 and 4 in E. coli, P. putida, A. vinelandii, and P. aeruginosa.
  • In vitro replication activity of oriV derivatives lacking DnaA boxes could be restored by increasing DnaA protein concentrations.
  • High levels of DnaA and TrfA proteins stimulated oriV initiation even without DnaA boxes, suggesting an alternative mechanism.

Conclusions:

  • The study highlights the variable importance of DnaA boxes for oriV replication across different bacterial hosts.
  • Evidence suggests an alternative replication initiation pathway for oriV, potentially utilized by P. aeruginosa and under high DnaA conditions.