Insights into the evolution of bacterial flagellar motors from high-throughput in situ electron cryotomography and subtomogram averaging.

Florian M Rossmann,Morgan Beeby

doi:10.1107/s2059798318007945

Florian M Rossmann, Morgan Beeby

Open Access

https://doi.org/10.1107/s2059798318007945

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Abstract

In situ structural information on molecular machines can be invaluable in understanding their assembly, mechanism and evolution. Here, the use of electron cryotomography (ECT) to obtain significant insights into how an archetypal molecular machine, the bacterial flagellar motor, functions and how it has evolved is described. Over the last decade, studies using a high-throughput, medium-resolution ECT approach combined with genetics, phylogenetic reconstruction and phenotypic analysis have revealed surprising structural diversity in flagellar motors. Variations in the size and the number of torque-generating proteins in the motor visualized for the first time using ECT has shown that these variations have enabled bacteria to adapt their swimming torque to the environment. Much of the structural diversity can be explained in terms of scaffold structures that facilitate the incorporation of additional motor proteins, and more recent studies have begun to infer evolutionary pathways to higher torque-producing motors. This review seeks to highlight how the emerging power of ECT has enabled the inference of ancestral states from various bacterial species towards understanding how, and `why', flagellar motors have evolved from an ancestral motor to a diversity of variants with adapted or modified functions.

Highlights

Understanding how molecular machines evolve is important for reasons ranging from antibiotic design to synthetic biology
The flagellar motor powers the rotation of bacterial flagella, which are helical proteinaceous filaments extending from the bacterial cell body that act as propellers for bacterial propulsion through liquid medium or swarming across surfaces (Jarrell & McBride, 2008)
We start with an overview of electron cryotomography (ECT) and subtomogram averaging (STA), outline how sample preparation, data collection and data analysis have been optimized for the problem, and describe how ECT has contributed to the understanding of flagellar motor diversity and evolution

Summary

Introduction

Understanding how molecular machines evolve is important for reasons ranging from antibiotic design to synthetic biology. Electron cryomicroscopy (cryo-EM) has become the technique of choice to gain structural insights into such large macromolecular complexes. While single-particle analysis cryo-EM (SPA) involves the purification of protein complexes for imaging (Bai et al, 2015; Passmore & Russo, 2016), a related technique called electron cryotomography (ECT) together with subtomogram averaging (STA) can be used to obtain three-dimensional structures of molecular machines in situ without requiring a large number of particles (Ferreira et al, 2018; Oikonomou & Jensen, 2017; Briggs, 2013). We review how ECT has contributed to our understanding of bacterial flagellar evolution by describing ECT, how the ECT workflow has been optimized to image these relatively low-abundance particles, and the resulting insights into motor diversity and evolution. We start with an overview of ECT and STA, outline how sample preparation, data collection and data analysis have been optimized for the problem, and describe how ECT has contributed to the understanding of flagellar motor diversity and evolution

Electron cryotomography and subtomogram averaging

Sample selection and preparation

Optimization of data acquisition and processing

Reconstruction

A central assay: deletion mutants to understand motor architecture

Structural diversity provides a clear selective benefit: higher torque

Future prospects