Single-molecule analysis of proteinxDNA complexes formed during partition of newly replicated plasmid molecules in Streptococcus pyogenes

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.

FIGURE 1.

Genome organization of plasmid pSM19035 and proposed structure of the ω₂·parS complex. A, pSM19035 duplicated sequences, which comprise ∼80% of the molecule, indicated by a thick line and unique nonrepeated sequences (NR1 and NR2) by a thin line. The replication origins (yellow boxes), direction of replication (denoted by arrows), the six resolution sites (orange boxes), and the parS sites (red boxes) are indicated. The outer thin arrows indicate the organization of the genes. The plasmid is divided in regions that direct (repS) and control (copS) replication (Rep region). Plasmid resolution (α, β, and γ genes) (SegA) and postsegregation growth inhibition (ϵ and ζ; brown arrows) and segregation (δ and ω; red arrows) (SegB) are indicated. The resistance to erythromycin (erm1 and erm2) and the set of poorly characterized genes in blue and purple arrows are also indicated. The upstream region of the promoters of the copS, δ, and ω genes constitute the six cis-acting centromere-like parS or parS′ sites. A parS site consists of a variable number of contiguous 7-bp heptad repeats (symbolized by ▶, 5′-WATCACW-3′, where W is A or T). The number of repeats and their relative orientations (direct ▶ or invert ◀ repeats) are enlarged. B, structural model of ω₂ bound to parS1 DNA. The overall structure of ω₂ forming a left-handed matrix around straight parS1 DNA based on the crystal structure determined for (ωΔ19)₂·▶ ▶ DNA and (ωΔ19)₂·▶ ◀ complexes (10, 17) is shown.

FIGURE 2.

AFM of the ω₂·parS DNA complexes. A, representative overview and selected images of single complexes on linear DNA. B, illustrated scheme of 70-bp parS DNA located 120 bp from one end of a 2.9-kb linear DNA molecule and ω₂-bound parS DNA. C, histogram of the protein binding position from the nearest DNA end. The mean distance is 31.2 nm. D, representative overview and selected images of single complexes on circular DNA.

FIGURE 3.

Volume of the ω₂·parS nucleoprotein complex. A, single ω₂·parS complex on linear DNA with the histograms showing the length of the nucleoprotein complex. The measured width of the DNA-bound protein presents a linear relationship with the measured width of DNA. B, single ω₂·parS complex on circular DNA with the histograms showing the length of the nucleoprotein complex.

FIGURE 4.

Structure of the parS·ω₂·(δ·ATP·Mg²⁺)₂ complex. A, number of plasmid molecules/complex observed when circular parS DNA is incubated with ω₂ and increasing concentrations of δ₂. Column 1 shows the number of monomeric DNA molecules (ternary complex); columns 2, 3, 4, and 5+ denote the percentage of complexes formed with 2, 3, 4 or more plasmid molecules (higher order complexes). B, summary of AFM images. Panel a, single pair mediated by a large nucleoprotein complex. Panel b, intermolecular pair showing two (panels c–e) or three or more (panel f) distinct nucleoprotein complexes interacting.

FIGURE 5.

Structure of the parS·ω₂·(δD60A·ATP·Mg²⁺)₂ complex. A, quantification of plasmid molecules/complex observed when circular parS DNA (25 nm) was incubated with 180 nm ω₂ and increasing concentrations of δ₂D60A (175 and 350 nm). The column indication is as in Fig. 4. B, two representative images showing two and three plasmids paired.