The Bacterial Flagellar Type III Export Gate Complex Is a Dual Fuel Engine That Can Use Both H+ and Na+ for Flagellar Protein Export

Abstract

The bacterial flagellar type III export apparatus utilizes ATP and proton motive force (PMF) to transport flagellar proteins to the distal end of the growing flagellar structure for self-assembly. The transmembrane export gate complex is a H+–protein antiporter, of which activity is greatly augmented by an associated cytoplasmic ATPase complex. Here, we report that the export gate complex can use sodium motive force (SMF) in addition to PMF across the cytoplasmic membrane to drive protein export. Protein export was considerably reduced in the absence of the ATPase complex and a pH gradient across the membrane, but Na+ increased it dramatically. Phenamil, a blocker of Na+ translocation, inhibited protein export. Overexpression of FlhA increased the intracellular Na+ concentration in the presence of 100 mM NaCl but not in its absence, suggesting that FlhA acts as a Na+ channel. In wild-type cells, however, neither Na+ nor phenamil affected protein export, indicating that the Na+ channel activity of FlhA is suppressed by the ATPase complex. We propose that the export gate by itself is a dual fuel engine that uses both PMF and SMF for protein export and that the ATPase complex switches this dual fuel engine into a PMF-driven export machinery to become much more robust against environmental changes in external pH and Na+ concentration.

High-Resolution pH Imaging of Living Bacterial Cells To Detect Local pH Differences

“…The transmembrane export gate conducts H + through the FlhA proton channel to drive flagellar protein export (14, 34). The FliM-FliN complex binds to FliG to form the C ring (35–37).…”

“…Interestingly, the export gate by itself utilizes Na + as the coupling ion in addition to H + when FliH and FliI are not functional. FlhA shows the H + and Na + channel activities, suggesting that FlhA may act as an energy transducer of the export gate, although its H + channel activity is quite low (14). …”

Structural stability of flagellin subunit affects the rate of flagellin export in the absence of FliS chaperone

“…This suggests that the export gate complex acts as a PMF-driven unfoldase to facilitate protein unfolding and translocation through the gate. A transmembrane export gate protein FlhA, which forms a nonameric ring structure in the export apparatus (Abrusci et al, 2013), not only provides binding-sites for FliH, FliI, FliJ and flagellar chaperone-substrate complexes (Minamino and Macnab, 2000;Minamino et al, 2003Minamino et al, , 2010Minamino et al, , 2012aMinamino et al, , 2016aKinoshita et al, 2013) but also acts as an energy transducer along with the cytoplasmic ATPase ring complex (Minamino et al, , 2016bIbuki et al, 2013). An N-terminal amino acid sequence of FliC functions as the export signal recognized by the flagellar type III export apparatus (V egh et al, 2006).…”

The role of intrinsically disordered C‐terminal region of FliK in substrate specificity switching of the bacterial flagellar type III export apparatus

“…The flagellar type III export apparatus is composed of a transmembrane export gate complex fueled by proton motive force across the cytoplasmic membrane and a cytoplasmic ATPase ring complex (Minamino and Namba, ; Paul et al ., ; Minamino et al ., ), and acts as a proton/protein antiporter to couple the proton influx through the gate with protein export (Minamino et al ., ; Morimoto et al ., ). The export gate complex is composed of five highly conserved transmembrane proteins, FlhA, FlhB, FliP, FliQ and FliR.…”