Nascent flagellar basal bodies are immobilized by rod assembly in Bacillus subtilis

Flagella are complex, trans-envelope nanomachines that localize in species-specific patterns on the cell surface. Here, we study the localization dynamics of the earliest stage of basal body formation in Bacillus subtilis using a fluorescent fusion to the C-ring protein FliM. We find that B. subtilis basal bodies do not exhibit dynamic subunit exchange and are largely stationary at steady state, consistent with flagellar assembly through the peptidoglycan (PG). However, rare mobile basal bodies were observed, and the prevalence of mobile basal bodies is elevated both early in basal body assembly and when the rod is mutated. Thus, basal body mobility is a precursor to patterning, and we propose that rod polymerization probes the PG superstructure for pores of sufficient diameter to permit rod transit. Furthermore, mutation of the rod disrupts basal body patterning in a way that phenocopies mutation of the cytoplasmic flagellar patterning protein FlhF. We infer that rod synthesis and the cytoplasmic regulators coordinate flagellar assembly by interpreting a grid-like pattern of pores, pre-existent in the PG.

Bacteria insert flagella in a species-specific pattern on the cell body, but how patterns are achieved is poorly understood. In bacteria with a single polar flagellum, a marker protein localizes to the cell pole and nucleates the assembly of the flagellum at that site. Bacillus subtilis assembles ~25 basal bodies over the length of the cell in a grid-like pattern and lacks proteins required for their polar targeting. Here, we show that B. subtilis basal bodies are mobile soon after assembly and become immobilized when the flagellar rod transits the peptidoglycan (PG) wall. Moreover, defects in the flagellar rod lead to a more-random distribution of flagella and an increase in polar basal bodies. We conclude that the peritrichous patterning of flagella of B. subtilis is different from the polar patterning of other bacteria, and we infer that the B. subtilis rod probes the PG for holes that can accommodate the machine.