Hybrid motor with H(+)- and Na(+)-driven components can rotate Vibrio polar flagella by using sodium ions

Abstract

The bacterial flagellar motor is a molecular machine that converts ion flux across the membrane into flagellar rotation. The coupling ion is either a proton or a sodium ion. The polar flagellar motor of the marine bacterium Vibrio alginolyticus is driven by sodium ions, and the four protein components, PomA, PomB, MotX, and MotY, are essential for motor function. Among them, PomA and PomB are similar to MotA and MotB of the proton-driven motors, respectively. PomA shows greatest similarity to MotA of the photosynthetic bacterium Rhodobacter sphaeroides. MotA is composed of 253 amino acids, the same length as PomA, and 40% of its residues are identical to those of PomA. R. sphaeroides MotB has high similarity only to the transmembrane region of PomB. To examine whether the R. sphaeroides motor genes can function in place of the pomA and pomB genes of V. alginolyticus, we constructed plasmids including both motA and motB or motA alone and transformed them into missense and null pomA-paralyzed mutants of V. alginolyticus. The transformants from both strains showed restored motility, although the swimming speeds were low. On the other hand, pomB mutants were not restored to motility by any plasmid containing motA and/or motB. Next, we tested which ions (proton or sodium) coupled to the hybrid motor function. The motor did not work in sodium-free buffer and was inhibited by phenamil and amiloride, sodium motor-specific inhibitors, but not by a protonophore. Thus, we conclude that the proton motor component, MotA, of R. sphaeroides can generate torque by coupling with the sodium ion flux in place of PomA of V. alginolyticus.

FIG. 1

(a) Amino acid alignments of V. alginolyticus PomA and R. sphaeroides MotA. White letters in black boxes, diamond, and star show identical residues, the mutation site of the PomA mutant VIO586 (G154R), and the threonine residue that is highly conserved among all species of MotA, respectively. Dotted bars indicate putative transmembrane regions. (b) Restriction map of plasmids. White boxes and the direction of the arrowheads indicate the vector part of pSU41 and the direction of translation from the lac promoter, respectively. Insertional fragments are indicated by solid lines. The bold lines in the inserted fragments indicate the region of the native promoter. The open arrows show the coding regions of PomA, PomB, MotA, and MotB. Abbreviations: B, BamHI; H, HindIII; E, EcoRI; Sp, SphI; X, XhoI; Sm, SmaI; Sl, SalI.

FIG. 2

Swarming abilities of transformants. Fresh colonies were inoculated in 0.3% agar VPG plates containing 100 μg of kanamycin per ml and incubated at 30°C for 5 h. (a) Motility of the pomA mutant (VIO586) and the pomB mutant (NMB161) as the host strains were transformed with plasmids pYA301, pYA603, pYA701, pSU41, and pYA603. The introduced plasmids and the host strains are noted on the left and right, respectively. (b) Effect of the T186C mutation of R. sphaeroides MotA. Two pomA mutants, VIO586 and NMB190, were transformed with plasmids pYA701 and pYA701-T186C.

FIG. 3

Detection of the R. sphaeroides MotA protein by the antipeptide antibody. NMB190 cells harboring each plasmid (lanes 1 and 5, pSU41; lanes 2 and 6, pYA301; lanes 3 and 7, pYA701; lane 4, pYA701-T186I) were cultured until the mid-log phase, harvested, and suspended in distilled-deionized water. Immunoblottings were done with anti-RsMotA antibody (a) and anti-PomA antibody (b).

FIG. 4

Swimming speed of NMB190 cells harboring pYA301 or pYA701. (a) The cells suspended in Tris motility buffer were diluted 100-fold into buffer containing various concentration of sodium ions, and the swimming speed was measured. (a′) Enlargement of the low-Na⁺-concentration part of panel a. (b and c) The swimming speed was measured at various concentrations of the specific inhibitor of the sodium motor, phenamil (b) or amiloride (c). The sodium concentration was 50 mM in all measurements.