Kojima Seiji, Kajino Hiroki, Hirano Keiichi, Inoue Yuna, Terashima Hiroyuki, Homma Michio
Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.
J Bacteriol. 2021 May 1;203(9). doi: 10.1128/JB.00009-21. Epub 2021 Feb 22.
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 species. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues from the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF 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 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 ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) 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.The 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.
MS环是鞭毛基体的一部分，由34个FliF亚基组成，FliF由一个大的周质区域和两个跨膜段组成，这两个跨膜段分别连接到面向细胞质的N端和C端区域。一种细胞质蛋白FlhF，它决定基体的位置和数量，在某些物种中支持膜中MS环的形成。在本研究中，我们构建了FliF缺失突变体，这些突变体在N端缺失30或50个残基（ΔN30和ΔN50），在C端缺失83个（ΔC83）或110个残基（ΔC110）。N端缺失的突变体具有功能并赋予细胞运动性，而C端缺失的突变体无功能。在[具体物种]中表达这些突变体以确定是否仍能组装MS环。当将ΔN30FliF或ΔN50FliF与FlhF共表达时，在MS环组分中观察到的MS环比野生型FliF表达时少，这表明N端与FlhF相互作用。没有FlhF时，MS环的形成可能效率低下。C端细胞质区域的缺失不影响FliF形成MS环的能力，因为与野生型FliF相比，ΔC83FliF观察到的MS环数量相似，尽管进一步缺失第二个跨膜段（ΔC110FliF）会消除MS环的形成。这些结果表明FliF的末端区域具有不同的作用；N端区域用于高效的MS环形成，C端区域用于MS环功能。第二个跨膜段对于MS环组装是不可或缺的。细菌鞭毛是一种参与细胞运动的超分子结构。在鞭毛基部，一个在细胞质膜中开始构建MS环的旋转马达由34个跨膜蛋白（FliF）组成。在这里，我们研究了作为MS环的FliF的N端和C端区域的作用。出乎意料的是，FliF的细胞质区域对于MS环的形成不是不可或缺的，但N端似乎通过招募对鞭毛形成至关重要的FlhF来协助环的形成。C端对于马达的形成或功能是必不可少的。
