Premrajka, K. | Search bioRxiv

bioRxiv2026

A conserved oscillatory system that positions the divisome in Archaea

Precise placement of the cell division machinery is essential for cell division in most organisms, yet the mechanisms responsible for this process vary substantially across the domains of life. In Eukaryotes, cell division is primarily driven by ESCRT-based systems, while many Bacteria rely on the Min system to ensure accurate positioning of the division septum. By contrast, the mechanisms by which Archaea spatially regulate divisome assembly remain largely unknown. Here, we identify a three-protein system, which we term divisome positioning proteins A, B and C (DipA, DipB and DipC) that is essential for correct divisome positioning in Haloferax volcanii. Deletion of any dip gene results in mislocalization of cell division proteins and the formation of abundant minicells, similar to Min defects in bacteria. Furthermore, we show that all three Dip proteins undergo pole-to-pole oscillation, and that DipB and DipC assemble into ring-like structures at midcell. Biochemical analyses demonstrate that DipA is a membrane binding GTPase whose association with the membrane is disrupted by DipB. Notably, Dip homologues are widely conserved across diverse archaeal lineages that employ FtsZ-based division, indicating that the Dip system is a broadly distributed key regulator of FtsZ-based cell division in Archaea. Despite its striking functional similarities to the bacterial Min system, the Dip system is entirely unrelated at the sequence level, representing a compelling example of convergent evolution of oscillatory spatial regulators in distinct domains of life.

microbiology↗

bioRxiv2025

Equipositioning of Chromosomes in the Polyploid Archaeon Haloferax volcanii by HpaAB

Proper genome segregation is essential for all life forms, but in archaea, this process is less characterized as compared to bacteria, or eukarya. Archaea show considerable variability in chromosome and plasmid copy number, and organization. Stable maintenance of chromosomes and low-copy plasmids in organisms requires an active segregation system. Haloferax volcanii DS2 is a polyploid archaeon that contains a fragmented genome composed of a main chromosome, three secondary chromosomes, pHV1, pHV3 and pHV4, as well as a plasmid pHV2. Due to the high copy number of all replicons, genomic segregation in H. volcanii is typically considered random. However, we identified four two-gene operons encoding a ParA-like homologue and an uncharacterized protein. Here we characterized one of these systems, termed here HpaA (ParA-homolog) and HpaB, that is encoded on the secondary chromosome pHV3. HpaA, showing conserved ATPase walker motifs, is a ParA protein with a bacterial origin acquired via horizontal gene transfer. Mutation of key residues in HpaA alters its localization, and HpaA is necessary for proper spatial distribution of HpaB. HpaB, an archaeal protein capable of forming foci in the cytosol, is shown to function as a centromere-binding protein, thereby acting as a functional ParB homologue. qPCR data confirm that HpaB is crucial for maintaining the plasmid-to-chromosome ratio, as deletion of hpaB reduces pHV3 copy number. Single particle tracking suggests that absence of HpaB leads to an altered population distribution of HpaA, based on protein dynamics. Deletion of the genes hpaA and hpaB results in severe growth defects when cells are grown on xylose as a sole carbon source, in line with the location of the genes for the xylose degradation pathway on chromosome pHV3. We propose that H. volcanii has an active chromosome segregation system, essential for the "two-dimensional" segregation and equipositioning of its replicons. This function is crucial for ensuring stable replicon maintenance across the distinct cell morphologies of H. volcanii, which transition between rod-shaped and disc-shaped forms at different stages of growth.

microbiology↗