Charged residues in the cytoplasmic loop of MotA are required for stator assembly into the bacterial flagellar motor

Authors

  • Yusuke V. Morimoto,

    1. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Shuichi Nakamura,

    1. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
    Search for more papers by this author
    • These authors contributed equally to this work.

  • Nobunori Kami-ike,

    1. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
    Search for more papers by this author
  • Keiichi Namba,

    Corresponding author
    1. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
    Search for more papers by this author
  • Tohru Minamino

    Corresponding author
    1. Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
    2. PRESTO, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
    Search for more papers by this author

E-mail tohru@fbs.osaka-u.ac.jp; Tel. (+81) 6 6879 4625; Fax (+81) 6 6879 4652;

E-mail keiichi@fbs.osaka-u.ac.jp; Tel. (+81) 6 6879 4625; Fax (+81) 6 6879 4652.

Summary

MotA and MotB form a transmembrane proton channel that acts as the stator of the bacterial flagellar motor to couple proton flow with torque generation. The C-terminal periplasmic domain of MotB plays a role in anchoring the stators to the motor. However, it remains unclear where their initial binding sites are. Here, we constructed Salmonella strains expressing GFP-MotB and MotA-mCherry and investigated their subcellular localization by fluorescence microscopy. Neither the D33N and D33A mutations in MotB, which abolish the proton flow, nor depletion of proton motive force affected the assembly of GFP-MotB into the motor, indicating that the proton translocation activity is not required for stator assembly. Overexpression of MotA markedly inhibited wild-type motility, and it was due to the reduction in the number of functional stators. Consistently, MotA-mCherry was observed to colocalize with GFP-FliG even in the absence of MotB. These results suggest that MotA alone can be installed into the motor. The R90E and E98K mutations in the cytoplasmic loop of MotA (MotAC), which has been shown to abolish the interaction with FliG, significantly affected stator assembly, suggesting that the electrostatic interaction of MotAC with FliG is required for the efficient assembly of the stators around the rotor.

Ancillary