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.

Background: Small homodimeric ␦ 2 (ParA) and 2 (ParB) and parS mediate pSM19035 partitioning. Results: The ␦ 2 ATPase is a modular protein. Conclusion: ␦ 2 ⅐␦ 2 and ␦ 2 ⅐ 2 interacting domains are juxtaposed. Significance: ATP, nonspecific DNA, and 2 bound to parS induce multiple transitions on ␦ 2 .

In Firmicutes, small homodimeric ParA-like (δ2) and ParB-like (ω2) proteins, in concert with cis-acting plasmid-borne parS and the host chromosome, secure stable plasmid inheritance in a growing bacterial population. This study shows that (ω:YFP)2 binding to parS facilitates plasmid clustering in the cytosol. (δ:GFP)2 requires ATP binding but not hydrolysis to localize onto the cell’s nucleoid as a fluorescent cloud. The interaction of (δ:CFP)2 or δ2 bound to the nucleoid with (ω:YFP)2 foci facilitates plasmid capture, from a very broad distribution, towards the nucleoid and plasmid pairing. parS-bound ω2 promotes redistribution of (δ:GFP)2, leading to the dynamic release of (δ:GFP)2 from the nucleoid, in a process favored by ATP hydrolysis and protein-protein interaction. (δD60A:GFP)2, which binds but cannot hydrolyze ATP, also forms unstable complexes on the nucleoid. In the presence of ω2, (δD60A:GFP)2 accumulates foci or patched structures on the nucleoid. We propose that (δ:GFP)2 binding to different nucleoid regions and to ω2-parS might generate (δ:GFP)2 gradients that could direct plasmid movement. The iterative pairing and unpairing cycles may tether plasmids equidistantly on the nucleoid to ensure faithful plasmid segregation by a mechanism compatible with the diffusion-ratchet mechanism as proposed from in vitro reconstituted systems.

The Streptococcus pyogenes pSM19035 low-copy-number θ-replicating plasmid encodes five segregation (seg) loci that contribute to plasmid maintenance. These loci map outside of the minimal replicon. The segA locus comprises β2 recombinase and two six sites, and segC includes segA and also the γ topoisomerase and two ssiA sites. Recombinase β2 plays a role both in maximizing random segregation by resolving plasmid dimers (segA) and in catalyzing inversion between two inversely oriented six sites. segA, in concert with segC, facilitates replication fork pausing at ssiA sites and overcomes the accumulation of "toxic" replication intermediates. The segB1 locus encodes ω, ε, and ζ genes. The short-lived ε2 antitoxin and the long-lived ζ toxin form an inactive ζε2ζ complex. Free ζ toxin halts cell proliferation upon decay of the ε2 antitoxin and enhances survival. If ε2 expression is not recovered, by loss of the plasmid, the toxin raises lethality. The segB2 locus comprises δ and ω genes and six parS sites. Proteins δ2 and ω2, by forming complexes with parS and chromosomal DNA, pair the plasmid copies at the nucleoid, leading to the formation of a dynamic δ2 gradient that separates the plasmids to ensure roughly equal distribution to daughter cells at cell division. The segD locus, which comprises ω2 (or ω2 plus ω22) and parS sites, coordinates expression of genes that control copy number, better-than-random segregation, faithful partition, and antibiotic resistance. The interplay of the seg loci and with the rep locus facilitates almost absolute plasmid stability.

The ω gene is encoded in broad-host range and low-copy plasmids. It is genetically linked to antibiotic resistance genes of the major human pathogens of phylum Firmicutes. The homodimeric forms of ω (ω2) coordinate the plasmid copy number control, faithful partition (ω2 and δ2) and better-than-random segregation (ζϵ2ζ) systems. The promoter (P) of the ωϵζ operon (Pω) transiently interacts with ω2. Adding δ2 facilitates the formation of stable ω2·Pω complexes. Here we show that limiting ω2 interacts with the N-terminal domain of the β’ subunit of the Bacillus subtilis RNA polymerase (RNAP-σA) vegetative holoenzyme. In this way ω2 recruits RNAP-σA onto Pω DNA. Partial Pω occupancy by ω2 increases the rate at which RNAP-σA complex shifts from its closed (RPC) to open (RPO) form. This shift increases transcription activation. Adding δ2 further increases the rate of Pω transcription initiation, perhaps by stabilizing the ω2·Pω complex. In contrast, full operator occupancy by ω2 facilitates RPC formation, but it blocks RPO isomerization and represses Pω utilization. The stimulation and inhibition of RPO formation is the mechanism whereby ω2 mediates copy number fluctuation and stable plasmid segregation. By this mechanism, ω2 also indirectly influences the acquisition of antibiotic resistance genes.