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.