Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

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.In eubacteria and archaea, many biologically important processes are carried out by genes encoded on large, low-copy number plasmids. These include genes conferring resistance to antibiotic and heavy metal toxicity, as well as genes involved in host cell invasion and pathogenicity. These large plasmids face a difficult challenge. To reduce the metabolic load they impose on the host cell, their copy numbers must be kept to a minimum (one to two per chromosome equivalent) (1). At such low-copy numbers, they can no longer rely on chance for their maintenance in the bacterial population. To be stably maintained, these low-copy number plasmids must, like chromosomes, be actively segregated to daughter cells before division. Two classes of plasmid segregation systems (Types I and II) have been known and studied for years (2), and recent work has uncovered several more (3, 4). Each system appears to be encoded on a single operon and to be composed of three pieces: 1) a centromeric DNA sequence, 2) a DNA-binding protein, and 3) a polymer-forming protein. The most well understood of these systems is the Type II segregation machinery encoded by the R1 drug-resistance plasmid. Segregation of R1 is driven by the par operon, which consists of a 150-bp centromeric sequence (parC), 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.High-resolution structures of components of the R1 spindle are available (6, 7), but provide little information about the ultrastructure of the R1 spindle itself. The atomic structure of ParM monomers reveals a basic similarity to convention...

The Structure and Function of Bacterial Actin Homologs

“…Also, why is it that ParM and actin filaments are structurally similar yet one polymerizes symmetrically at both ends while the other polymerizes asymmetrically? Along similar lines, EM studies both in vitro and in vivo suggest that both ends of the filament associate with the ParR/parC complex (Garner et al 2007;Salje et al 2009), and work by Choi and colleagues used very small gold beads to show that in a ParM spindle, a single copy of parC is bound to the end of a single ParM filament (Choi et al 2008). So how does the ParR/parC manage to interact with both ends of an asymmetric polymer?…”

The Bacterial Cytoskeleton

“…Plasmid-bound, fluorescently labeled ParM structures would sometimes get dimmer before completely depolymerizing, suggesting multiple filaments (28), a notion supported by ECT imaging (147). However, single, synthetic parC sequences coupled to gold nanoparticles and mixed with ParR label the ends of single ParM filaments in vitro (36,146). Similarly, purified ParM from plasmid R1 forms single filaments in vitro that are capped with circular ParR/parC structures (146).…”

Structural Polymorphism of the ParM Filament and Dynamic Instability

“…The stability operon par encodes three components sufficient for plasmid segregation: parR , and parM genes along with the cis -acting locus, parC (Gerdes et al, 1986). The current model for type II partitioning assumes that ParR binds cooperatively to several repeats of the parC locus while ParM bound to ParR pushes the two newly formed plasmids to the opposite poles of the bacterial cell via ATP-dependent polymerization (Moller-Jensen et al, 2003;Salje et al, 2008;Campbell et al, 2007;Choi et al, 2008). Sequence analysis of ParM (Jensen et al, 1997).…”

“…The filaments capped with the ParR/ParC complex are stable and can grow to be long in the cell (Campbell et al, 2007). The ParR/ParC complex may bind to the side of ParM and remain attached to the ATP cap of the growing filament, preventing the dissociation of the cap (Choi et al ., 2008). Alternatively, the ParR/ParC complex may form a protective cap on the end of the filament to maintain its integrity via a processive polymerization mechanism (Salje et al, 2008).…”