Assembly Order of Flagellar Rod Subunits in Bacillus subtilis

Bacterial flagella contain an axle-like rod that transits the cell envelope and connects the transmembrane basal body to the extracellular hook and filament. Although the rod is a crucial component of the flagellum, its structure and assembly are poorly understood. Previous reports defining the order of rod assembly in Gram-negative bacteria suggest that the rod requires five proteins to successfully assemble, but assembly intermediates have not been well characterized due to metastability and periplasmic proteolysis. Bacillus subtilis is a Gram-positive, genetically tractable model bacterium that synthesizes flagella and lacks a true periplasm. Here, we genetically, biochemically, and cytologically determine the assembly order of the flagellar rod proteins from cell proximal to distal as FliE, FlgB, FlgC, FlhO, and FlhP. We further show that, under conditions in which rod structure cannot be completed, assembly intermediates are both metastable and subject to proteolysis. Finally, we support previous results that FliE serves as both a structural assembly platform for the rod and as an enhancer of flagellar type III secretion. IMPORTANCE Bacteria rotate propeller-like flagella to find and colonize environmental niches. The flagellum is a complex machine, and the understanding of its structure is still incomplete. Here, we characterize and biochemically define the assembly order of the subunits that make up the axle-like rod. The rod is a critical structure for the assembly of subsequent components and is central to our understanding of how the flagellum is anchored but still free spinning within the context of the cell envelope.