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pTAR-encoded proteins in plasmid partitioning.
K Kalnin1, S Stegalkina, M Yarmolinsky
1Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892-4255, USA.
Journal of Bacteriology
|March 14, 2000
Summary
Bacterial plasmid partitioning relies on specific proteins binding to centromere-like sequences. Researchers identified essential partitioning genes (parA and parB) in Agrobacterium tumefaciens, revealing functional similarities with other bacterial systems.
Area of Science:
- Molecular Biology
- Genetics
- Microbiology
Background:
- Bacterial plasmids require partition cassettes for stable inheritance.
- These cassettes typically involve multiple proteins and cis-acting DNA sequences.
- The pTAR plasmid of Agrobacterium spp. was previously thought to have a unique, single-protein partition system.
Purpose of the Study:
- To re-evaluate the genetic organization of the pTAR plasmid partition region.
- To characterize the functions of the identified partition proteins (ParA and ParB).
- To investigate the functional similarities and differences of the pTAR partition system in a foreign host.
Main Methods:
- Gene resequencing to identify genes within the partition region.
- Protein purification and in vitro DNA-binding assays.
- ATPase activity assays for ParA.
- Functional analysis in the heterologous host Escherichia coli.
- Primer extension to identify transcription start sites.
Main Results:
- Resequencing revealed two genes, parB and orf-84, downstream of parA; only parB is essential for partitioning.
- Purified ParA showed weak ATPase activity, enhanced by non-specific DNA.
- ParB specifically bound to the parS region, which contains centromere and operator functions.
- ParB repressed the partition operon in both A. tumefaciens and E. coli.
- ParA augmented ParB-mediated repression in E. coli.
Conclusions:
- The pTAR partition system involves at least two proteins, ParA and ParB, binding to the parS site.
- ParB plays a key role in partitioning and operon repression.
- The pTAR partition system exhibits functional conservation with other bacterial plasmid partitioning mechanisms despite sequence and organizational differences.