Chaperone mediated coupling of subunit availability to activation of proton motive force-driven flagellar Type III Secretion

Abstract Bacterial flagella are assembled from thousands of protein subunits that are unfolded and exported across the cell membrane by a specialized flagellar Type III Secretion System (fT3SS). Export of subunit cargo is fuelled by the proton motive force (pmf), facilitated by an export ATPase that is evolutionarily related to the F1 ATPase. The FliJ stalk component of the ATPase binds the flagellar export gate, enabling it to utilise the pmf electric potential (ΔΨ) to drive efficient export of subunit cargo. What is unclear is how FliJ activation of the export gate is regulated to prevent constitutive proton influx and wasteful dissipation of pmf when no subunit cargo is available. FliJ, in addition to activating the export gate, recruits unladen export chaperones and transfers them to cognate subunit cargo, creating a local cycle of chaperone-subunit binding and release at the fT3SS. To investigate whether chaperones might regulate FliJ activation of the export gate, we isolated chaperone variants that were defective for FliJ binding yet still able to bind cognate subunits and other export components. Disruption of the chaperone-FliJ interaction attenuated motility and cognate subunit export. In vitro and in vivo competitive binding assays showed that chaperones and the export gate component FlhA compete for binding FliJ. Our data indicate that unladen chaperones, which would be present in the cell when subunit levels are low, sequester FliJ to prevent activation of the export gate. This suggests a mechanism whereby chaperones couple availability of export cargo to utilization of pmf by the fT3SS. Significance Type III Secretion Systems (T3SS) are used by bacteria to build flagella, rotary nanomachines for cell motility, and related injectosomes, specialised molecular syringes that deliver bacterial effector proteins into host cells to subvert their function. T3SS activity uses two power sources – the high-energy ATP molecule and the proton motive force (pmf), chemical and electric potential stored in a proton gradient across the bacterial membrane. Here, we show that use of pmf by the flagellar T3SS is regulated by cytoplasmic chaperones that bind and deliver cargo proteins to the T3SS for export. We demonstrate that when cargo availability is low, the unladen chaperones bind the T3SS to reduce its activity, minimising energy use and preventing wasteful dissipation of the pmf.