Assembly dynamics and the roles of FliI ATPase of the bacterial flagellar export apparatus

Abstract

For construction of the bacterial flagellum, FliI ATPase forms the FliH2-FliI complex in the cytoplasm and localizes to the flagellar basal body (FBB) through the interaction of FliH with a C ring protein, FliN. FliI also assembles into a homo-hexamer to promote initial entry of export substrates into the export gate. The interaction of FliH with an export gate protein, FlhA, is required for stable anchoring of the FliI6 ring to the gate. Here we report the stoichiometry and assembly dynamics of FliI-YFP by fluorescence microscopy with single molecule precision. More than six FliI-YFP molecules were associated with the FBB through interactions of FliH with FliN and FlhA. Single FliI-YFP molecule exchanges between the FBB-localized and free-diffusing ones were observed several times per minute. Neither the number of FliI-YFP associated with the FBB nor FliI-YFP turnover rate were affected by catalytic mutations in FliI, indicating that ATP hydrolysis by FliI does not drive the assembly-disassembly cycle of FliI during flagellar assembly. We propose that the FliH2FliI complex and FliI6 ring function as a dynamic substrate carrier and a static substrate loader, respectively.

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK

“…Potential sources of error in step counting are misfolded eGFP, quenching, partial overlap of foci, malformed complexes, and the subjective nature of counting steps. Hence, a distribution of the number of steps counted is expected, and our data are consistent with prior studies in this respect (63)(64)(65)(66)(67). The highest number of steps we counted was 5.…”

“…To test this prediction in vivo, stepwise photobleaching was performed on SpoIVFB fused at its C terminus to eGFP and expressed from the native spoIVF promoter during sporulation. Photobleaching of a fluorescent protein can decrease its fluorescence intensity in a stepwise fashion over time, revealing the number of subunits in a multisubunit protein (63)(64)(65)(66)(67). We used TIRF microscopy to limit excitation to the evanescent field and only excite SpoIVFB-eGFP near the coverslip.…”

Insight into the flagella type III export revealed by the complex structure of the type III ATPase and its regulator

“…The FliI 6 ring has been identified to be located at the base of the flagellum by electron cryotomography (9). The FliI 6 ring associates with the basal body through the interactions of FliH with FlhA and FliN, a C-ring component of the basal body (10)(11)(12) (Fig. S1).…”

“…The chaperone-substrate complexes bind to the FliH 2 FliI complex through cooperative interactions among FliI, the chaperone, and the export substrate (23)(24)(25). FliI labeled with YFP shows rapid turnovers between the basal body and cytoplasmic pool in an ATP-independent manner (12), suggesting that the FliH 2 FliI complex acts as a dynamic carrier to deliver export substrates or the chaperone-substrate complexes to the docking platform made up of the C-terminal domain of FlhA.…”

“…The N-terminal region composed of residues 1-100 (FliH N ) is elongated, and the extreme N-terminal region is responsible for the interaction with FliN and FlhA to allow the FliI 6 ring to associate with the export gate (10)(11)(12)27). The middle region, residues 101-140, is essential for homodimer formation.…”