Sodium-driven energy conversion for flagellar rotation of the earliest divergent hyperthermophilic bacterium

Abstract

Aquifex aeolicus is a hyperthermophilic, hydrogen-oxidizing and carbon-fixing bacterium that can grow at temperatures up to 95 °C. A. aeolicus has an almost complete set of flagellar genes that are conserved in bacteria. Here we observed that A. aeolicus has polar flagellum and can swim with a speed of 90 μm s(-1) at 85 °C. We expressed the A. aeolicus mot genes (motA and motB), which encode the torque generating stator proteins of the flagellar motor, in a corresponding mot nonmotile mutant of Escherichia coli. Its motility was slightly recovered by expression of A. aeolicus MotA and chimeric MotB whose periplasmic region was replaced with that of E. coli. A point mutation in the A. aeolicus MotA cytoplasmic region remarkably enhanced the motility. Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+). As motor proteins from hyperthermophilic bacteria represent the earliest motor proteins in evolution, this study strongly suggests that ancient bacteria used Na(+) for energy coupling of the flagellar motor. The Na(+)-driven flagellar genes might have been laterally transferred from early-branched bacteria into late-branched bacteria and the interaction surfaces of the stator and rotor seem not to change in evolution.

Figure 1. Schematic cartoon of the flagellum and the stator proteins.

(A) The flagellum is a large complex composed of many proteins and consists of a filament, a hook and a basal body. A. aeolicus has most genes for flagellar component except for FliM. (B) The stator is composed of two membrane proteins, MotA (blue) and MotB (red). MotA is a four TM protein and MotB is single TM protein. The spontaneous mutated Alanine residue is indicated as ‘A’ in the black circle. Black arrowhead, border of chimeric MotB: OM, outer membrane; PG, peptidoglycan layer; IM, inner membrane.

Figure 2. Transmission electron micrograph of the cells of A. aeolicus.

(A) Image of the whole cell and its flagellum. (B) and (C) Partial enlargements of the flagellated cell pole. Cells were negatively stained with 1% uranyl acetate.

Figure 3. Swimming speed of A. aeolicus in solution.

(A) Photograph of the variable-temperature chamber used in this study. (B) Histogram of swimming speed of the cells at 85 °C, the optimal growth temperature. (C) Thermo-dependent swimming speed of the A. aeolicus cells. The cells were grown at 85 °C and observed at each temperature.

Figure 4. Motility assay of E. coli cells producing MotA and MotB of A. aeolicus in soft-agar plates.

(A) Schematics of primary structures of MotA, MotB of E. coli and A. aeolicus and chimera MotB. We switched the sequence at the middle of the plug region for chimera MotB. (B) and (C) Motilities of E. coli cells in soft-agar plate. MotA and MotB were expressed from two compatible plasmids which were constructed from pBAD24 and pSBETa, respectively, in a E. coli ΔmotAB strain. Plates were incubated at 30 °C for the indicated number of hours. ^Ec, protein of E. coli; ^Aa, protein of A. aeolicus; ^AE, chimera fused proteins of A. aeolicus and E. coli.

Figure 5. Na⁺ dependent motor function of E. coli cells producing MotA and chimera MotB proteins.

Rotation speeds of E. coli (ΔmotAB) cells producing chimeric stators with the MotA-A225D mutation (MotA^Aa(A225D)/MotB₁^AE for (A), and MotA^Aa(A225D)/MotB₂^AE for (B)) or the native E. coli stator (C) were measured in various Na⁺ concentrations as noted.

Figure 6. Schematic showing the acquisition and derivation of stator genes through bacterial evolution.

First, the ancestral bacteria acquired the Na⁺-driven stator, and earliest branched bacteria have the Na⁺-driven stator. The stator then was converted into the H⁺-driven type through the middle period of bacterial evolution. The Na⁺-driven stator of the late-branched bacteria seems to be provided from early-branched bacteria (Aquificae or Thermotogae) by lateral gene transfer.