Synthetic motility and cell shape defects associated with deletions of flotillin/reggie paralogs in Bacillus subtilis and interplay of these proteins with NfeD proteins.

Flotillin/reggie proteins are membrane-associated proteins present in all kinds of cells and belong to the family of proteins carrying the SPFH (stomatin, prohibitin, flotillin, and HflK/HflC) domain. In addition to this domain of unknown function, flotillin proteins are characterized by the flotillin domain, which is rich in heptad repeats. Bacterial flotillin orthologs have recently been shown to be part of lipid rafts, like their eukaryotic counterparts, and to be involved in signaling events. Double deletions of floT and the gene encoding the second flotillin-like protein in Bacillus subtilis, floA, show strong synthetic defects in cell morphology, motility, and transformation efficiency. The lack of FloT resulted in a marked defect in motility. Using total internal reflection fluorescence (TIRF) microscopy, we show that both proteins localize in characteristic focal structures within the cell membrane, which move in a highly dynamic and random manner but localize independently of each other. Thus, flotillin paralogs act in a spatially distinct manner. Flotillin domains in both FloA and FloT are essential for focal assemblies and for the proper function of flotillins. Both flotillin genes are situated next to genes encoding NfeD proteins. FloT dramatically affects the localization of NfeD2: FloT apparently recruits NfeD2 into the focal assemblies, documenting a close interaction between flotillins and NfeDs in bacteria. In contrast, the localization of NfeD1b is not affected by FloA, FloT, or NfeD2. FloA does not show a spatial connection with the upstream-encoded NfeD1b (YqeZ). Our work establishes that bacterial flotillin-like proteins have overlapping functions in a variety of membrane-associated processes and that flotillin domain-mediated assembly and NfeD proteins play important roles in setting up the flotillin raft-like structures in vivo.