Bacterial dynamin-like protein DynA mediates lipid and content mixing

Many bacterial species contain dynamin-like proteins (DLPs). However, so far the functional mechanisms of bacterial DLPs are poorly understood. DynA in Bacillus subtilis is a 2-headed DLP, mediating nucleotide-independent membrane tethering in vitro and contributing to the innate immunity of bacteria against membrane stress and phage infection. Here, we employed content mixing and lipid mixing assays in reconstituted systems to study if DynA induces membrane full fusion, characterize its subunits in membrane fusion, and further test the possibility that GTP hydrolysis of DynA may act on the fusion-through-hemifusion pathway. Our results based on fluorescence resonance energy transfer indicated that DynA could induce aqueous content mixing even in the absence of GTP. Moreover, DynA-induced membrane fusion in vitro is a thermo-promoted slow process, and it has phospholipid and membrane curvature preferences. The D1 part of DynA is crucial for membrane binding and fusion, whereas D2 subunit plays a role in facilitating membrane fusion. Surprisingly, digestion of DynA mediated an instant rise of content exchange, supporting the assumption that disassembly of DynA is a driving force for fusion-through-hemifusion. DynA is a rare example of a membrane fusion catalyst that lacks a transmembrane domain and hence sets this system apart from well-characterized fusion systems such as the soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes.