In most bacteria, cell division depends on the tubulin homolog FtsZ and other proteins, such as SepF, that form a complex termed the divisome. Cell division also depends on FtsZ in many archaea, but other components of the divisome are unknown. Here, we demonstrate that a SepF homolog plays important roles in cell division in Haloferax volcanii, a halophilic archaeon that is known to have two FtsZ homologs with slightly different functions (FtsZ1 and FtsZ2). SepF co-localizes with both FtsZ1 and FtsZ2 at midcell. Attempts to generate a sepF deletion mutant were unsuccessful, suggesting an essential role. Indeed, SepF depletion leads to severe cell division defects and formation of large cells. Overexpression of FtsZ1-GFP or FtsZ2-GFP in SepF-depleted cells results in formation of filamentous cells with a high number of FtsZ1 rings, while the number of FtsZ2 rings is not affected. Pull-down assays support that SepF interacts with FtsZ2 but not with FtsZ1, although SepF appears delocalized in the absence of FtsZ1. Archaeal SepF homologs lack a glycine residue known to be important for polymerization and function in bacteria, and purified H. volcanii SepF forms dimers, suggesting that polymerization might not be important for the function of archaeal SepF.

Bacterial cell division has been studied for decades but reports on the different archaeal cell division systems are rare. In many archaea, cell division depends on the tubulin homolog FtsZ, but further components of the divisome in these archaea are unknown. The halophilic archaeon Haloferax volcanii encodes two FtsZ homologs with different functions in cell division and a putative SepF homolog. In bacteria, SepF is part of the divisome and is recruited early to the FtsZ ring, where it most likely stimulates FtsZ ring formation. H. volcanii SepF co-localized with FtsZ1 and FtsZ2 at midcell. Overexpression of SepF had no effect on cell morphology, but no sepF deletion mutants could be generated. SepF depletion led to a severe cell division defect, resulting in cells with a strongly increased size. Overexpression of FtsZ1- and FtsZ2-GFP in SepF-depleted cells resulted in filamentous cells with an increasing number of FtsZ1 rings depending on the cell length, whereas FtsZ2 rings were not increased. Pull-down assays with HA-tagged SepF identified an interaction with FtsZ2 but not with FtsZ1. Archaeal SepF homologs lack the conserved glycine residue important for polymerization in bacteria and the H. volcanii SepF was purified as a dimer, suggesting that in contrast to the bacterial SepF homologs, polymerization does not seem to be important for its function. A model is proposed where first the FtsZ1 ring is formed and where SepF recruits FtsZ2 to the FtsZ1 ring, resulting in the formation of the FtsZ2 ring. This study provides important novel insights into cell division in archaea and shows that SepF is an important part of the divisome in FtsZ containing archaea.