Single-molecule analysis of proteinxDNA complexes formed during partition of newly replicated plasmid molecules in
Florencia Pratto1, Yuki Suzuki, Kunio Takeyasu
1Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, 28049 Madrid, Spain.
Abstract:
The Streptococcus pyogenes pSM19035 partition locus is ubiquitous among plasmids from vancomycin- or methicillin-resistant bacteria. An increasing understanding of this segregation system may highlight novel protein targets that could be blocked to curb bacterial proliferation. pSM19035 segregation depends on two homodimeric (delta(2) (ParA) and omega(2) (ParB)) proteins and six cis-acting centromeric noncurved parS sites. In the presence of ATPxMg(2+), delta(2) (delta x ATP x Mg(2+))(2) binds DNA in a sequence-independent manner. Protein omega(2) binds with high affinity and cooperatively to B-form parS DNA. Atomic force microscopy experiments indicate that about 10 omega(2) molecules bind parS, consisting of 10 contiguous iterons. Protein (delta x ATP x Mg(2+))(2), by interacting with the N terminus of omega(2) bound to parS, loses its association with DNA and relocalizes with omega(2).parS to form a ternary complex ((deltaxATPxMg(2+))(2) x omega(2) x parS) with the DNA remaining in straight B-form. Then, the interaction of two (delta x ATP x Mg(2+))(2).omega(2).parS complexes via delta(2) promotes pairing of a plasmid subfraction. (deltaD60A x ATP x Mg(2+))(2), which binds but does not hydrolyze ATP, leads to accumulation of pairing intermediates, suggesting that ATP hydrolysis induces plasmid separation. We propose that the molar omega(2):delta(2) ratio regulates the different stages of pSM19035 segregation, pairing, and delta(2) polymerization, before cell division.
Insights
The Streptococcus pyogenes pSM19035 plasmid segregation system uses ParA and ParB proteins to ensure accurate DNA partitioning. Understanding this process may reveal new targets to inhibit bacterial growth.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The Streptococcus pyogenes pSM19035 partition locus is found in plasmids of resistant bacteria.
- Understanding plasmid segregation is crucial for combating bacterial proliferation.
Purpose of the Study:
- To elucidate the molecular mechanism of pSM19035 plasmid segregation.
- To identify potential targets for inhibiting bacterial growth.
Main Methods:
- Atomic force microscopy to visualize protein-DNA interactions.
- Biochemical assays to study protein binding and activity.
- Mutational analysis of key proteins.
Main Results:
- ParA (delta(2)) and ParB (omega(2)) proteins, along with parS sites, mediate segregation.
- ParA binds DNA independently, while ParB binds parS sites with high affinity.
- ATP hydrolysis by ParA is essential for plasmid separation.
- The ratio of ParB to ParA regulates segregation stages.
Conclusions:
- The pSM19035 segregation system involves a complex interplay between ParA, ParB, and parS sites.
- ATP hydrolysis by ParA drives plasmid separation.
- The ParB:ParA ratio controls plasmid pairing and segregation dynamics.
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