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

Takekawa N, Nishiyama M, Kaneseki T, Kanai T, Atomi H, Kojima S, Homma M - Sci Rep (2015)

Bottom Line: 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.Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+).

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f4: 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.

Mentions: A. aeolicus harbors one motA and two motB genes that constitute an operon (motAAa/motB1Aa/motB2Aa)7, whereas other bacteria commonly have one set (one motA/motB gene) or two sets (two motA/motB genes) of stator genes. The MotA protein of A. aeolicus (MotAAa) shares 30.5% sequence similarity with MotA of E. coli (MotAEc). MotB1Aa and MotB2Aa share 35.6% and 36.3% sequence similarity with MotBEc, respectively. Conserved charged residues mainly important for stator-rotor interaction, R90 and E98 in MotAEc, are conserved as R88 and E96 in MotAAa. The secondary important charged residue, E150 in MotAEc, is not conserved in MotAAa (Fig. S2A). When the sequences of the two MotB proteins of A. aeolicus are compared, the TM and N-terminal regions are very similar (sequence identity is 97%) except for one residue (Gly20 in MotB1Aa is Ser20 in MotB2Aa), whereas the C-terminal periplasmic region is vastly different (sequence identity is 23.0%) (Fig. S2B). When the predicted secondary structures of MotBEc, MotB1Aa and MotB2Aa are compared, the flexible linker sequences between the plug region and the OmpA-like domain, which works as a PG-binding domain, are very different (Fig. 4A), and their linker lengths are 82, 70 and 56, respectively.


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

Takekawa N, Nishiyama M, Kaneseki T, Kanai T, Atomi H, Kojima S, Homma M - Sci Rep (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525482&req=5

f4: 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.
Mentions: A. aeolicus harbors one motA and two motB genes that constitute an operon (motAAa/motB1Aa/motB2Aa)7, whereas other bacteria commonly have one set (one motA/motB gene) or two sets (two motA/motB genes) of stator genes. The MotA protein of A. aeolicus (MotAAa) shares 30.5% sequence similarity with MotA of E. coli (MotAEc). MotB1Aa and MotB2Aa share 35.6% and 36.3% sequence similarity with MotBEc, respectively. Conserved charged residues mainly important for stator-rotor interaction, R90 and E98 in MotAEc, are conserved as R88 and E96 in MotAAa. The secondary important charged residue, E150 in MotAEc, is not conserved in MotAAa (Fig. S2A). When the sequences of the two MotB proteins of A. aeolicus are compared, the TM and N-terminal regions are very similar (sequence identity is 97%) except for one residue (Gly20 in MotB1Aa is Ser20 in MotB2Aa), whereas the C-terminal periplasmic region is vastly different (sequence identity is 23.0%) (Fig. S2B). When the predicted secondary structures of MotBEc, MotB1Aa and MotB2Aa are compared, the flexible linker sequences between the plug region and the OmpA-like domain, which works as a PG-binding domain, are very different (Fig. 4A), and their linker lengths are 82, 70 and 56, respectively.

Bottom Line: 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.Using this system in E. coli, we demonstrate that the A. aeolicus motor is driven by Na(+).

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

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.

No MeSH data available.


Related in: MedlinePlus