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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

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 (MotAAa(A225D)/MotB1AE for (A), and MotAAa(A225D)/MotB2AE for (B)) or the native E. coli stator (C) were measured in various Na+ concentrations as noted.
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f5: 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 (MotAAa(A225D)/MotB1AE for (A), and MotAAa(A225D)/MotB2AE for (B)) or the native E. coli stator (C) were measured in various Na+ concentrations as noted.

Mentions: To investigate the function of the chimeric stators for the rotation of a single motor, we performed the tethered cell assay. We attached a flagellar filament to a cover glass and the fractions and speeds of rotation of the E. coli cells were measured (Movie S4). Regarding the MotAAa-A225D mutation, the E. coli cells expressing chimeric MotAAa/MotBAE increased the fractions of rotation but not the speed (Fig. S4A and B). The rotation speeds of E. coli cells expressing chimeric stators decreased with lower concentrations of NaCl in the buffer and the rotations completely stopped in the absence of NaCl (Fig. 5A,B). This indicates that the stator of A. aeolicus is a Na+-driven stator. The Michaelis constants (Km) for Na+ are 12 ± 2 mM or 13 ± 4 mM in the E. coli cells expressing chimeric MotAAa/B1AE or MotAAa/B2AE, respectively. Those values are a little higher than the previously reported Km of other Na+-driven flagellar motor (ca. 2.2 mM)32. In contrast, the rotation speed of E. coli cells expressing native MotAEc/MotBEc was not affected by the NaCl concentration (Fig. 5C).


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)

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 (MotAAa(A225D)/MotB1AE for (A), and MotAAa(A225D)/MotB2AE for (B)) or the native E. coli stator (C) were measured in various Na+ concentrations as noted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: 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 (MotAAa(A225D)/MotB1AE for (A), and MotAAa(A225D)/MotB2AE for (B)) or the native E. coli stator (C) were measured in various Na+ concentrations as noted.
Mentions: To investigate the function of the chimeric stators for the rotation of a single motor, we performed the tethered cell assay. We attached a flagellar filament to a cover glass and the fractions and speeds of rotation of the E. coli cells were measured (Movie S4). Regarding the MotAAa-A225D mutation, the E. coli cells expressing chimeric MotAAa/MotBAE increased the fractions of rotation but not the speed (Fig. S4A and B). The rotation speeds of E. coli cells expressing chimeric stators decreased with lower concentrations of NaCl in the buffer and the rotations completely stopped in the absence of NaCl (Fig. 5A,B). This indicates that the stator of A. aeolicus is a Na+-driven stator. The Michaelis constants (Km) for Na+ are 12 ± 2 mM or 13 ± 4 mM in the E. coli cells expressing chimeric MotAAa/B1AE or MotAAa/B2AE, respectively. Those values are a little higher than the previously reported Km of other Na+-driven flagellar motor (ca. 2.2 mM)32. In contrast, the rotation speed of E. coli cells expressing native MotAEc/MotBEc was not affected by the NaCl concentration (Fig. 5C).

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