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

Date Published: March 4, 2016

Publisher: Public Library of Science

Author(s): Tohru Minamino, Yusuke V. Morimoto, Noritaka Hara, Phillip D. Aldridge, Keiichi Namba, Kelly T. Hughes.


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.

Partial Text

Many membrane-embedded biological nanomachines utilize proton motive force (PMF) across the membrane for their biological activities. In Escherichia coli and Salmonella enterica, PMF is utilized as the energy source for ATP synthesis, solute transport, nutrient uptake, protein transport, multidrug efflux pump and flagellar motility [1]. Alkaliphilic bacteria and hyperthermophilic bacteria utilize sodium motive force (SMF) instead of PMF [1]. The flagellar motor of E. coli and Salmonella uses H+ as the coupling ion to power flagellar motor rotation. In contrast, the flagellar motor of marine Vibrio and extremely alkalophilic Bacillus utilizes Na+ as the coupling ion instead of H+ [2]. It has been reported that some systems such as the melibiose permease of E. coli [3] and the flagellar motor of alkalophilic Bacillus clausii [4] can utilize both H+ and Na+ as their coupling ion. Interestingly, the flagellar motor of Bacillus alcalophilus Vedder 1934 can conduct K+ as well as Na+ [5]. Each biological system appears to have been optimized for the best use of specific ions according to the environmental conditions.

PMF is the primary driving force for the flagellar and non-flagellar type III export apparatus [10]. The flagellar export gate of S. enterica is intrinsically a H+–protein antiporter that requires both the Δψ and ΔpH components to couple the energy of proton influx with protein export in the absence of the ATPase complex [13]. The cytoplasmic ATPase complex switches the export gate into a highly efficient, Δψ-driven protein export apparatus, and an interaction between FliJ and FlhA is key in driving this switch [13]. In this study, we showed that, in addition to PMF, the export gate can use SMF to drive flagellar protein export over an external pH range of 6.0–8.0 in the absence of FliH, FliI and FliJ (Figs 1, 4 and 5). This suggests that without FliH, FliI and FliJ the export gate alone is a dual fuel export engine that can exploit both H+ and Na+ as the coupling ion (Fig 7). Interestingly, environmental changes significantly affected flagellar protein export by the ΔfliH-fliI flhB(P28T) but not that by wild-type cells (Figs 1 and 4). Therefore, we propose that the export apparatus is robust and has evolved to be able to maintain protein export activity against internal or external, genetic or environmental perturbations. To achieve this level of robustness the export gate has evolved to exploit both H+ and Na+ as the coupling ion rather than becoming an exclusive PMF or SMF dependent machine.




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