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Abstract

Bactofilins are rigid, non-polar bacterial cytoskeletal filaments that link cellular processes to specific curvatures of the cytoplasmic membrane. Although homologs of bactofilins have been identified in archaea and eukaryotes, functional studies have remained confined to bacterial systems. Here, we characterized representatives of two families of archaeal bactofilins from the pleomorphic archaeon Haloferax volcanii, halofilin A (HalA) and halofilin B (HalB). Unlike bacterial bactofilins, HalA polymerizes into polar filaments in vivo at positive membrane curvature, whereas HalB forms more static foci and accumulates in areas of local negative curvatures on the outer cell surface. Combining gene deletions, super-resolution, and single-cell microscopy showed that halofilins are critical in maintaining H. volcanii cell integrity during shape transition from disk (sessile) to rod (motile). Morphological defects in ΔhalA primarily affected rod-shaped cells from accumulating highly positive curvatures. Conversely, disk-shaped cells were exclusively affected by halB deletion, showing a decrease in positive and negative curvatures, resulting in flatter cells. Furthermore, while ΔhalA and ΔhalB cells displayed lower cell division placement precision, morphological defects arose predominantly during the disk-to-rod shape remodeling. We propose halofilins provide mechanical scaffolding, dynamically coupling the cytoplasmic membrane and the S-layer. We speculate that HalA filaments support rods under low S-layer lipidation (flexible, fast membrane diffusion). In contrast, HalB connects the S-layer to negative curvatures in disks under high lipidation levels (rigid, slow membrane diffusion).