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Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body

By Seiji Kojima, Hiroki Kajino, Keiichi Hirano, Yuna Inoue, Hiroyuki Terashima, Michio Homma

Posted 05 Jan 2021
bioRxiv DOI: 10.1101/2021.01.04.425360

The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues at the N-terminus ({Delta}N30 and {Delta}N50), and 83 ({Delta}C83) or 110 residues ({Delta}C110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing {Delta}N30FliF or {Delta}N50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without an additional factor or FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for {Delta}C83FliF as with wild-type FliF, although further deletion of the second transmembrane segment ({Delta}C110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly. ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

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