Role of FisB-Cardiolipin Interactions in Membrane Fission during Sporulation in Bacillus Subtilis

Abstract

Membrane fission is a fundamental process required for endocytosis, membrane trafficking, enveloped virus budding, phagocytosis, cell division and sporulation. Despite the diversity of fission reactions, there are only two fission machineries known in eukaryotes (dynamin and ESCRT-III), and none in bacteria. We describe FisB, a 254 amino acid protein which is conserved among spore-forming bacteria. FisB mediates membrane fission during sporulation in B. subtilis. Upon starvation, B. subtilis divides asymmetrically, producing a large mother cell and a small forespore. The mother cell then engulfs the forespore. Like in phagocytosis or endocytosis, engulfment ends with a fission event that releases the forespore into the mother cell cytoplasm. FisB possesses a short cytoplasmic N-terminus, one predicted transmembrane domain, and a large extracytoplasmic portion. In FisB knock-out cells, engulfment proceeds normally, but the fission step is severely impaired (Doan et al., Genes & Dev. 2013).The goal of our research is to understand how FisB mediates membrane fission. Therefore we investigate how FisB is able to form oligomers that translocate to the fission site and sever membranes. The extracytoplasmic domain (ECD) of FisB binds cardiolipin (CL) in a floatation assay and binding is highest at low salt and undetectable at 500 mM NaCl, suggesting electrostatic interactions between FisB and CL. FisB(173-220) was identified as the cardiolipin binding domain.FisB reconstituted into artificial liposomes efficiently mediates membrane mixing only in the presence of CL. Fluorescently tagged ECD binds to cardiolipin-containing giant unilammellar vesicles and induces aggregation, membrane deformation and collapse. CL is enriched at the poles of B. subtilis, presumably in microdomains that prefer negatively curved regions.We hypothesize that CL may regulate recruitment and oligomerization of FisB.