Abstract
To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. We found that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmids segregating as a unit.
Highlights
In eubacteria and archaea, many biologically important processes are carried out by genes encoded on large, low-copy number plasmids
Segregation of R1 is driven by the par operon, which consists of a 150-bp centromeric sequence, a repressor protein (ParR) that binds to the centromeric sequence, and a divergent actin-like protein (ParM) that polymerizes into dynamically unstable filaments in the presence of ATP [5]
Binding of the ParR/parC complex to ParM filaments stabilizes them against catastrophic disassembly and promotes their elongation via insertional polymerization at the interface with the ParR/parC complex. When both ends of a ParM filament are bound to ParR/parC complexes, elongation of the filament pushes the attached plasmids in opposite directions
Summary
By forming a collar around the end of a filament, the ParR/parC complex would pose no barrier to elongation, and if the affinity of the complex for ParM depends on the nucleotide bound to the filament, hydrolysis of ATP within the filament could promote tracking of the complex with the elongating filament end. A collar encircling a ParM filament might prevent monomer dissociation, providing an attractive explanation for how the ParR/parC complex stabilizes filaments against catastrophic disassembly. This model appears to be at odds with in vivo studies of R1 spindles that suggest plasmids are segregated by spindles composed of multiple filaments [10]. We attached defined numbers of parC-containing DNA molecules to gold nanoparticles and counted the number of filaments associated with each particle. The high contrast of gold nanoparticles enabled us to identify the location of ParR/parC complexes relative to ParM filaments
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