Abstract
Type III Secretion Systems (T3SS) deliver subunits from the bacterial cytosol to nascent cell surface flagella. Early flagellar subunits that form the rod and hook substructures are unchaperoned and contain their own export signals. A gate recognition motif (GRM) docks them at the FlhBc component of the FlhAB-FliPQR export gate, but the gate must then be opened and subunits must be unfolded to pass through the flagellar channel. This induced us to seek further signals on the subunits. Here, we identify a second signal at the extreme N-terminus of flagellar rod and hook subunits and determine that key to the signal is its hydrophobicity. We show that the two export signal elements are recognised separately and sequentially, as the N-terminal signal is recognised by the flagellar export machinery only after subunits have docked at FlhBC via the GRM. The position of the N-terminal hydrophobic signal in the subunit sequence relative to the GRM appeared to be important, as a FlgD deletion variant (FlgDshort), in which the distance between the N-terminal signal and the GRM was shortened, 'stalled' at the export machinery and was not exported. The attenuation of motility caused by FlgDshort was suppressed by mutations that destabilised the closed conformation of the FlhAB-FliPQR export gate, suggesting that the hydrophobic N-terminal signal might trigger opening of the flagellar export gate.
Highlights
Type III Secretion Systems (T3SS) are multi-component molecular machines that deliver protein cargo from the bacterial cytosol either to their site of assembly in cell surface flagella or virulence factor injectisomes, or directly to their site of action in eukaryotic target cells or the extracellular environment [1-5]
FlhBN is connected via a linker (FlhBCN) to the cytoplasmic domain of FlhB (FlhBC), which is thought to sit between the FlhA N-terminal region (FlhAN) and FlhAC rings, where it functions as a docking site for early flagellar subunits [14, 22, 28]. 63 64 The ‘early’ flagellar subunits that assemble to form the rod and hook substructures are not chaperoned: instead, the signals for targeting and export are found within the early subunits themselves
We show that the extreme N-terminus of rod/hook subunits contains a hydrophobic export signal and investigate its functional relationship to the subunit gate recognition motif (GRM). 83 Results Identification of a hydrophobic export signal at the N-terminus of FlgD The N-terminal region of flagellar rod and hook subunits is required for their export [12, 14]
Summary
Type III Secretion Systems (T3SS) are multi-component molecular machines that deliver protein cargo from the bacterial cytosol either to their site of assembly in cell surface flagella or virulence factor injectisomes, or directly to their site of action in eukaryotic target cells or the extracellular environment [1-5]. During flagellum biogenesis, when the rod/hook structure reaches its mature length, the fT3SS switches export specificity from recognition of ‘early’ rod/hook subunits to ‘late’ subunits for filament assembly [9, 10]. This means that early and late flagellar subunits must be differentiated by the fT3SS machinery to ensure that they are exported at the correct stage of flagellum biogenesis. Structural studies suggest that FliPQR-FlhBN adopts an energetically favourable closed conformation, possibly to maintain the membrane permeability barrier [22, 25, 30, 31] This suggests that there must be a mechanism to trigger opening of the export gate when subunits dock at cytosolic face of the flagellar export machinery. We show that the extreme N-terminus of rod/hook subunits contains a hydrophobic export signal and investigate its functional relationship to the subunit gate recognition motif (GRM)
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