A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum

Abstract

Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP–FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.

In situ imaging of the bacterial flagellar motor disassembly and assembly processes

“…As the export of the rod proteins to the periplasm requires the T3SS export apparatus, this complex ought to be a disassembly product. In the ΔflgG strain, the inner‐membrane sub‐complex was observed in two examples (Fig Q and R) and no (sub‐)complexes related to the flagellar motor could be detected in cells lacking FlhA which is believed to be one of the first proteins to be assembled (Li & Sourjik, ; Fabiani et al , ; Fukumura et al , ).…”

“…This is likely because the process of flagellar assembly is so energetically demanding (Paradis et al , ). Our current understanding of flagellar assembly comes from studies of two enteric species of Gram‐negative bacteria, E. coli and Salmonella , which suggest an inside‐to‐outside sequential assembly process starting from the export apparatus, MS‐ring (FliF), followed by the C‐ring proteins, the rod, the P‐ and L‐rings, the hook, and finally the filament (Jones & Macnab, ; Kubori et al , ; Macnab, ; Li & Sourjik, ; Fabiani et al , ; Fukumura et al , ). The process is thought to be a cooperative one, whereby the addition of each new component stabilizes its antecedent (Li & Sourjik, ).…”

The structure of an injectisome export gate demonstrates conservation of architecture in the core export gate between flagellar and virulence type three secretion systems

“…Proteins associated with the cytoplasmic face of the basal body select proteins for export that are then transferred to a set of 5 membrane associated proteins located at the center of the inner-membrane ring. These components (FliP, FliQ, FliR, FlhB, FlhA in the flagellar system and SctR, SctS, SctT, SctU, SctV in 10 injectisomes) are collectively termed the export apparatus (EA) and are absolutely required for the translocation of substrates across the bacterial envelope (Wagner, Konigsmaier et al 2010, Fabiani, Renault et al 2017, Fukumura, Makino et al 2017.…”

A Polar Flagellar Transcriptional Program Mediated by Diverse Two-Component Signal Transduction Systems and Basal Flagellar Proteins Is Broadly Conserved in Polar Flagellates

“…Flagellar biogenesis begins by activating the transcription of genes encoding components essential for the initial steps in assembly, which include the flagellar type III secretion system (fT3SS), MS ring, and C ring rotor and switch proteins (14)(15)(16)(17)(18)(19)(20). MS and C ring formation around the fT3SS core completes biogenesis of a competent fT3SS for export and assembly of rod and hook components (21)(22)(23)(24)(25). Up to this point, the alternative factor 28 , which is required for transcription of flagellins and other motility genes, is inhibited by the antifactor FlgM (11,13,(26)(27)(28)(29).…”