Disulfide 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 disulfide 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 R248C 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.