Abstract
Bacterial flagellar motility is controlled by the binding of CheY proteins to the cytoplasmic switch complex of the flagellar motor, resulting in changes in swimming speed or direction. Despite its importance for motor function, structural information about the interaction between effector proteins and the motor are scarce. To address this gap in knowledge, we used electron cryotomography and subtomogram averaging to visualize such interactions inside Caulobacter crescentus cells. In C. crescentus, several CheY homologs regulate motor function for different aspects of the bacterial lifestyle. We used subtomogram averaging to image binding of the CheY family protein CleD to the cytoplasmic Cring switch complex, the control center of the flagellar motor. This unambiguously confirmed the orientation of the motor switch protein FliM and the binding of a member of the CheY protein family to the outside rim of the C ring. We also uncovered previously unknown structural elaborations of the alphaproteobacterial flagellar motor, including two novel periplasmic ring structures, and the stator ring harboring eleven stator units, adding to our growing catalog of bacterial flagellar diversity.
Highlights
Bacterial flagella are propulsive helical filaments rotated by intricate nanomachines for motility, which quickly respond to environmental conditions by binding various effector proteins
We overexpressed one CheY-like protein, CleD, in the CheY-gutted strain and imaged the putative binding location of this protein in detail. This improves our understanding of the mechanism of action of these c-di-GMP-binding proteins, and we present the first structure of a CheY-homolog bound to the flagellar motor at nanometer resolution
We aimed to image the structure of the C. crescentus cytoplasmic ring (C ring) when a CheY-like effector protein is bound
Summary
Bacterial flagella are propulsive helical filaments rotated by intricate nanomachines for motility, which quickly respond to environmental conditions by binding various effector proteins. The observation that this strain showed lower motility on semisolid agar plates (Figure S4a) and that CleD-GFP localized to the pole opposite the stalk (Figure S4b) confirmed that this CleD variant is able to interfer with flagellar motility at high c-di-GMP concentrations (Nesper et al, 2017) This overexpression strain ensured maximal occupation of CleD binding sites, increasing the chances to successfully detect additional densities derived from bound effector proteins. A difference map of radially averaged CleD-GFP and empty vector structures in isosurface representation with a defined threshold of 2.7 σ helps to visualize the location of the CleD binding interface (Figure 4a) Together, this data strongly supports a model in which CleD binds to the outside of the C ring likely via the N-terminal CheY-binding motif of FliM
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