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.