The FlgN chaperone activates the Na+-driven engine of the Salmonella flagellar protein export apparatus

Abstract

The bacterial flagellar protein export machinery consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. The gate complex has two intrinsic and distinct H+-driven and Na+-driven engines to drive the export of flagellar structural proteins. Salmonella wild-type cells preferentially use the H+-driven engine under a variety of environmental conditions. To address how the Na+-driven engine is activated, we analyzed the fliJ(Δ13–24) fliH(Δ96–97) mutant and found that the interaction of the FlgN chaperone with FlhA activates the Na+-driven engine when the ATPase complex becomes non-functional. A similar activation can be observed with either of two single-residue substitutions in FlhA. Thus, it is likely that the FlgN-FlhA interaction generates a conformational change in FlhA that allows it to function as a Na+ channel. We propose that this type of activation would be useful for flagellar construction under conditions in which the proton motive force is severely restricted.

Membrane voltage-dependent activation mechanism of the bacterial flagellar protein export apparatus

“…We chose the hook-capping protein FlgD as a representative export substrate because the level of FlgD secretion by this mutant strain is even higher than the wild-type level ( 20 ). The transmembrane export gate complex of the ΔHI B * strain prefers Na + over H + as the coupling ion in an external pH range of 6.0–8.0 ( 17 , 18 ). Therefore, the ΔHI B * cells were grown in T-broth at external pH values of 7.0, 7.5, 8.0, or 8.5 in the absence of external Na + to examine the Δψ dependence of protein export.…”

“…A gain-of-function mutation, flhA(T490M) , located in FlhA C ( Fig. 4 A ), overcomes the FliJ defect to a considerable degree ( 18 , 26 ). Therefore, we investigated whether this gain-of-function mutation affects the Δψ dependence of flagellar protein export by the ΔHI B* mutant.…”

“…Flagellar protein export by the ΔHI B* mutant shows a clear dependence on the external Na + concentration over an external pH range of 6.0–8.0 ( 17 , 18 ). Consistently, the secretion level of FlgD increased considerably when 100 mM NaCl was present in the medium ( SI Appendix , Fig.…”

“…The flagellar protein export channel is intrinsically a dual-fuel engine that utilizes both H + and Na + as the coupling ion to drive flagellar protein export, and FlhA acts as a transmembrane ion channel to conduct both H + and Na + ( 17 , 18 ). In the wild-type protein export apparatus, neither the ΔpH nor ΔpNa component is essential; the Δψ component is sufficient for flagellar protein export ( 13 , 19 ).…”

“…S1 ) ( 16 ). When the cytoplasmic ATPase complex becomes nonfunctional, the FlgN chaperone activates the Na + -driven export engine of the export gate complex over a wide range of external pH, allowing the export gate complex to drive Na + -coupled protein export ( 17 , 18 ). The transmembrane domain of FlhA (FlhA TM ) acts as a transmembrane ion channel for the transit of both H + and Na + across the cytoplasmic membrane ( 17 ).…”