Hook-basal-body assembly state dictates substrate specificity of the flagellar type-III secretion system.

The assembly of the bacterial flagellum is orchestrated by the secretion of distinct early and late secretion substrates via the flagellar-specific type-III secretion system (fT3SS). However, how the fT3SS is able to distinguish between the different (early and late) substrate classes during flagellar assembly remains poorly understood. In this study, we investigated the substrate selectivity and specificity of the fT3SS of Salmonella enterica at different assembly stages. For this, we developed an experimental setup that allowed us to synchronize hook-basal-body assembly and to monitor early and late substrate secretion of fT3SSs operating in either early or late secretion mode, respectively. Our results demonstrate that the fT3SS features a remarkable specificity for only the substrates required at the respective assembly stage. No crosstalk of substrates was observed for fT3SSs operating in the opposing secretion mode. We further found that a substantial fraction of fT3SS surprisingly remained in early secretion mode. Our results thus suggest that the secretion substrate specificity switch of the fT3SS is unidirectional and irreversible. The developed secretion substrate reporter system further provides a platform for future investigations of the underlying molecular mechanisms of the elusive substrate recognition of the T3SS.

Control of membrane barrier during bacterial type-III protein secretion.

Type-III secretion systems (T3SSs) of the bacterial flagellum and the evolutionarily related injectisome are capable of translocating proteins with a remarkable speed of several thousand amino acids per second. Here, we investigate how T3SSs are able to transport proteins at such a high rate while preventing the leakage of small molecules. Our mutational and evolutionary analyses demonstrate that an ensemble of conserved methionine residues at the cytoplasmic side of the T3SS channel create a deformable gasket (M-gasket) around fast-moving substrates undergoing export. The unique physicochemical features of the M-gasket are crucial to preserve the membrane barrier, to accommodate local conformational changes during active secretion, and to maintain stability of the secretion pore in cooperation with a plug domain (R-plug) and a network of salt-bridges. The conservation of the M-gasket, R-plug, and salt-bridge network suggests a universal mechanism by which the membrane integrity is maintained during high-speed protein translocation in all T3SSs.

Export Mechanisms and Energy Transduction in Type-III Secretion Machines.

The remarkably complex architecture and organization of bacterial nanomachines originally raised the enigma to how they are assembled in a coordinated manner. Over the years, the assembly processes of the flagellum and evolutionary-related injectisome complexes have been deciphered and were shown to rely on a conserved protein secretion machine: the type-III secretion system. In this book chapter, we demonstrate how individually evolved mechanisms cooperate in highly versatile and robust secretion machinery to export and assemble the building blocks of those nanomachines.

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

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.