Equipositioning of chromosomes in the polyploid archaeon Haloferax volcanii by HpaAB

Proper genome segregation is essential for cellular survival, yet this process is poorly understood in archaea. Archaea show considerable variability in genome ploidy and organization. Haloferax volcanii DS2 is a polyploid archaeon with a fragmented genome consisting of a main chromosome, three secondary chromosomes and a plasmid. Due to high ploidy, genomic segregation in H. volcanii is typically considered random. However, we identified four two-gene operons encoding ParA-like proteins and uncharacterized partners. Here we characterized HpaA and HpaB, encoded on the secondary chromosome pHV3. HpaA contains conserved ATPase walker motifs and is a ParA protein with a bacterial origin. Mutation of key residues in HpaA alters its localization, while HpaA is necessary for proper HpaB spatial distribution. HpaB forms cytosolic foci and functions as a centromere-binding protein, analogous to bacterial ParB. Deletion of the genes hpaA and hpaB results in severe growth defects when xylose is the sole carbon source, consistent with the location of xylose degradation genes on pHV3. It also reduces the pHV3 to main chromosome ratio. Single-particle tracking suggests that HpaB influences HpaA dynamics. Together, these findings demonstrate an equipositioning of pHV3, likely supporting maintenance in the different cell morphologies observed in H. volcanii.