Molecular anatomy of the Streptococcus pyogenes pSM19035 partition and segrosome complexes

Abstract

Vancomycin or erythromycin resistance and the stability determinants, δω and ωεζ, of Enterococci and Streptococci plasmids are genetically linked. To unravel the mechanisms that promoted the stable persistence of resistance determinants, the early stages of Streptococcus pyogenes pSM19035 partitioning were biochemically dissected. First, the homodimeric centromere-binding protein, ω2, bound parS DNA to form a short-lived partition complex 1 (PC1). The interaction of PC1 with homodimeric δ [δ2 even in the apo form (Apo-δ2)], significantly stimulated the formation of a long-lived ω2·parS complex (PC2) without spreading into neighbouring DNA sequences. In the ATP·Mg2+ bound form, δ2 bound DNA, without sequence specificity, to form a transient dynamic complex (DC). Second, parS bound ω2 interacted with and promoted δ2 redistribution to co-localize with the PC2, leading to transient segrosome complex (SC, parS·ω2·δ2) formation. Third, δ2, in the SC, interacted with a second SC and promoted formation of a bridging complex (BC). Finally, increasing ω2 concentrations stimulated the ATPase activity of δ2 and the BC was disassembled. We propose that PC, DC, SC and BC formation were dynamic processes and that the molar ω2:δ2 ratio and parS DNA control their temporal and spatial assembly during partition of pSM19035 before cell division.

Cell-free study of F plasmid partition provides evidence for cargo transport by a diffusion-ratchet mechanism

“…The bridging activity of SopA and SopB studied here has features similar to the DNA-bridging complexes described for P1 and pSM19035 of Streptococcus pyogenes (14,24). For all systems, DNA bridging required both proteins and ATP, and bridge disassembly was coupled to ParB-stimulated ATP hydrolysis.…”

“…For P1, the complex was proposed to bridge plasmid and nucleoid and was referred to as the "nucleoid-adaptor complex" (NAC) (24). For pSM19035, the bridging was proposed to play a role in plasmid pairing as well as in forming an NAC-like complex (14). Likewise, the large plasmid clusters studied here likely are connected by the same protein-mediated contacts that tether plasmid to the DNA carpet.…”

“…4). A conserved basic patch of C-terminal residues has been implicated as the nsDNA-binding interface (13,14), and mutation of SopA at this interface damages the ATPdependent nsDNA-binding activity in vitro and plasmid stability in vivo (15).In vivo, nsDNA mainly takes the form of the nucleoid. Several fluorescent versions of plasmid and chromosomal ParAs display dynamic patterns on the nucleoid (reviewed in ref.…”

“…4). A conserved basic patch of C-terminal residues has been implicated as the nsDNA-binding interface (13,14), and mutation of SopA at this interface damages the ATPdependent nsDNA-binding activity in vitro and plasmid stability in vivo (15).…”

Functional Characterization of the Role of the Chromosome I Partitioning System in Genome Segregation in Deinococcus radiodurans

“…The light scattering approach was used previously to determine the dynamic variations in the sizes of macromolecular complexes. An increase in light scattering with time has been attributed to an increase in the sizes of ParA protein polymers (5), while a decrease in light scattering was suggested to be due to the disassembly of the ParA filament (44,48). Note that ParA, belonging to the type I category, e.g., Soj or pSM19035 delta, polymerizes in the presence of ATP and DNA; the interaction of this complex with Spo0J stimulates the intrinsic ATPase activity of ParA, leading to its depolymerization (40)(41)(42).…”