Oligomerization of the FliF Domains Suggests a Coordinated Assembly of the Bacterial Flagellum MS Ring.

Giuseppina Mariano,Soi Bui,Weilong Zhao,Raquel Faba-Rodriguez,Julien R C Bergeron,Svetomir B Tzokov,James Ross

doi:10.3389/fmicb.2021.781960

Giuseppina Mariano, Soi Bui + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2021.781960

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Abstract

The bacterial flagellum is a complex, self-assembling macromolecular machine that powers bacterial motility. It plays diverse roles in bacterial virulence, including aiding in colonization and dissemination during infection. The flagellum consists of a filamentous structure protruding from the cell, and of the basal body, a large assembly that spans the cell envelope. The basal body is comprised of over 20 different proteins forming several concentric ring structures, termed the M- S- L- P- and C-rings, respectively. In particular, the MS rings are formed by a single protein FliF, which consists of two trans-membrane helices anchoring it to the inner membrane and surrounding a large periplasmic domain. Assembly of the MS ring, through oligomerization of FliF, is one of the first steps of basal body assembly. Previous computational analysis had shown that the periplasmic region of FliF consists of three structurally similar domains, termed Ring-Building Motif (RBM)1, RBM2, and RBM3. The structure of the MS-ring has been reported recently, and unexpectedly shown that these three domains adopt different symmetries, with RBM3 having a 34-mer stoichiometry, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring. This observation raises some important question on the assembly of the MS ring, and the formation of this symmetry mismatch within a single protein. In this study, we analyze the oligomerization of the individual RBM domains in isolation, in the Salmonella enterica serovar Typhimurium FliF ortholog. We demonstrate that the periplasmic domain of FliF assembles into the MS ring, in the absence of the trans-membrane helices. We also report that the RBM2 and RBM3 domains oligomerize into ring structures, but not RBM1. Intriguingly, we observe that a construct encompassing RBM1 and RBM2 is monomeric, suggesting that RBM1 interacts with RBM2, and inhibits its oligomerization. However, this inhibition is lifted by the addition of RBM3. Collectively, this data suggest a mechanism for the controlled assembly of the MS ring.

Highlights

The flagellum is a complex macromolecular motor, whose role is to allow swimming motility, through the rotation of a long filament at the bacterium cell surface
We demonstrate that the RBM2 and RBM3 domains oligomerize in isolation, and form ring-like structures, with symmetry corresponding to that of these domains within the basal body
Given that RBM1-RBM2 (FliF50–229) was shown to be strictly monomeric, while RBM1-RBM2-RBM3 (FliF50–438) assembled into the MS ring (Figure 2; Table 1), we further investigated whether addition to RBM3 (FliF231–438) would prompt RBM1RBM2 (FliF50–229) to oligomerize

Summary

Introduction

The flagellum is a complex macromolecular motor, whose role is to allow swimming motility, through the rotation of a long filament at the bacterium cell surface. In-between the two transmembrane helices, FliF possesses a large periplasmic region consisting of three globular domains termed Ring-Building Domains (RBM1, RBM2, and RBM3, respectively; Figure 1A; Bergeron, 2016) Those RBMs possess a common fold (Spreter et al, 2009), and show structural homology with components of the Type III Secretion System (T3SS) injectisome, and in particular RBM1 and RBM2 have sequence similarity with the T3SS protein SctJ (Yip et al, 2005; Bergeron et al, 2015; Bergeron, 2016). RBM3 shows homology to the SpoIIIAG protein (Bergeron, 2016; Zeytuni et al, 2017), a macromolecular complex involved in spore formation

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