Disulphide cross-linking between the stator and the bearing components in the bacterial flagellar motor.
The flagellar motor is composed of the stator and the rotor, and the interaction between the stator and the rotor at the cytoplasmic region is believed to produce mechanical force for the rotation of flagella. The periplasmic region of the stator has been proposed to play an important role in assembly around and incorporation into the motor. In this study, we provide evidence suggesting that the periplasmic region of the stator component MotB interacts with the P-ring component FlgI, which functions as a bearing for the rotor along with the L-ring protein FlgH, from a site-directed disulphide cross-linking approach. First, we prepared four FlgI and three MotB cysteine-substituted mutant proteins and co-expressed them in various combinations in Escherichia coli. We detected cross-linked combinations of FlgI G11C and MotB S248C when treated with the oxidant Cu-phenanthroline or bismaleimide cross-linkers. Furthermore, we performed Cys-scanning mutagenesis around these two residues and found additional combinations of cross-linked residues. Treatment with a protonophore CCCP significantly reduced the cross-linking efficiency between FlgI and MotB in flagellated cells, but not in non-flagellated cells. These results suggest a direct contact between MotB and FlgI upon assembly of the stator into a motor.
Hizukuri
Yohei
Y
Division of Biological Science, Graduate School of Science, Nagoya University, Furo-Cho, Chikusa-Ku, Nagoya 464-8602, Japan.
Kojima
Seiji
S
Homma
Michio
M
D001426:Bacterial Proteins / Q000378:metabolism; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005407:Flagella / Q000737:chemistry*; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins; D011485:Protein Binding
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004220:Disulfides; D029968:Escherichia coli Proteins; D020409:Molecular Motor Proteins; C064779:MotB protein, Bacteria; D050505:Mutant Proteins; C074899:flgI protein, Bacteria