Multi-foci replication domains in Haloferax volcanii visualised by super-resolution microscopy

The archaeon Haloferax volcanii is a unique model organism to investigate replication dynamics and dissect the interplay between multiple origins, alternative replication pathways, and polyploidy. The combination of variable chromosome copy number and asynchronous replication across the cell population gives rise to pronounced phenotypic heterogeneity, further increasing system complexity. To investigate these mechanisms, we implemented (spt-)PALM and STORM microscopy to probe replication dynamics in different cell lines and growth conditions. We established the first STORM-based super-resolution imaging approach of a protein in Haloferax volcanii. Altogether, our results support a model in which each H. volcanii cell replicates a constant fraction of its chromosome copies, irrespective of total ploidy. Consequently, cells with a higher number of chromosomes exhibit proportionally more replication forks, resulting in pronounced ploidy heterogeneity across the population. Moreover, we show that replication foci are not randomly distributed but cluster into discrete Replication Domains containing multiple active sites. This organization persists across different growth conditions and is independent of canonical replication origins, as demonstrated in a strain lacking all four origins. Our findings suggest that replication coordination in H. volcanii is governed by spatial architecture rather than origin-dependent initiation, providing new insights into the evolutionary diversity of replication control mechanisms.