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

Flagella are complex, trans-envelope nanomachines that localize to species- specific cellular addresses. 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. Rare basal bodies were observed to be mobile however, and the frequency of basal body mobility 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 peptidoglycan superstructure for pores of sufficient diameter that permit rod completion. Furthermore, mutation of the rod also disrupts basal body patterning in a way that phenocopies mutation of the cytoplasmic flagellar patterning protein FlhF. We infer that conformational changes in the basal body exchange information between rod synthesis and the cytoplasmic patterning proteins to restrict assembly at certain pores established by a grid-like pattern pre-existent in the peptidoglycan itself.
Importance: 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 ∼15 flagella over the length of the cell body in a grid-like pattern and lacks all proteins associated with targeted assembly in polarly flagellated bacteria. Here we show that B. subtilis basal bodies are mobile soon after assembly and become immobilized when the flagellar rod transits the peptidoglycan wall. Moreover, defects in the flagellar rod lead to an asymmetric distribution of flagella with respect to the midcell. We conclude that the patterning of flagella is different in B. subtilis , and we infer that the B. subtilis rod probes the peptidoglycan for holes that can accommodate the machine.