The rapid evolution of flagellar ion-selectivity in experimental populations of<i>E. coli</i>

Ridone, Pietro; Sowa, Yoshiyuki; Baker, Matthew A. B.

doi:10.1101/2021.01.26.427765

Cited by 4 publications

(7 citation statements)

References 93 publications

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“…Different triple mutations of residues at the periplasmic side of MotB TM switched the dual-functional stator unit into either a proton-driven or sodium-driven one, indicating that ion specificity is located along the channel at the region preceding the critically conserved aspartate [65]. Introduction of sodium-driven PomAB into E. coli through CRISPR/ Cas-mediated genome engineering showed that, when sodium is lacking, PomAB can spontaneously mutate, which allows it to use the proton gradient as driving force [66]. Thus, additional studies are needed to fully understand the molecular basis underlying the ion selectivity mechanism of the stator unit.…”

Section: Open Accessmentioning

confidence: 99%

Structural basis of torque generation in the bi-directional bacterial flagellar motor

Santiveri

Wadhwa

et al. 2022

Trends in Biochemical Sciences

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The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA 5 B 2 ) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryoelectron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor. The bacterial flagellum and its rotary motorMany bacteria, including Escherichia coli, Salmonella, and Bacillus spp., use flagella (see Glossary) to move through liquid environments and across surfaces. The flagellum is a supramolecular nanomachine that protrudes from the cell envelope and measures~5-20 μm in length. It is able to rotate in both clockwise (CW) and counterclockwise (CCW) directions to propel the bacterial cell body in different living environments [1,2]. Rotational switching between these two modes is regulated by chemotactic signaling, which is a rapid process that responds to environmental stimuli and biases movement of the cell toward attractants and away from repellents. Flagella-mediated chemotaxis further enables pathogenic bacteria to move toward cells to establish in vivo niches. [3,4]. Thus, flagella have fundamental roles in bacterial locomotion and virulence [5].The flagellum comprises more than 25 kinds of building blocks, which assemble in a highly ordered manner. The flagellar structure can be divided into three morphologically distinguishable parts: a cell envelope-spanning motor (basal body), a universal joint (hook), and a long, thin helical filament [6,7] (Figure 1). Among them, the most intricate part is the basal body, containing the components responsible for assembly of the flagellum [the flagellar-specific type-III secretion system (T3SS) [8]], torque generation (the stator units [9]), and rotational switching (binding of the response regulator CheY-P to the cytoplasmic C-ring [10,11]). Cryo-ET studies of the motor from different bacterial species show the variation of its structure, while the core components are conserved [7,12,13]. For example, in the Gram-negative bacteria Salmonella and E. coli, the flagellar motor contains four ring-like structures based on their distributions relative to the cell surface layers [lipopolysaccharide (L-)ring, peptidoglycan (P-)ring, inner membrane/ supramembrane (MS-)ring, and cytoplasmic (C-)ring] surrounding a central rigid rod [14][15][16][17]. Additional ring-like structures, H-and T-rings, located in the periplasmic space, have also been observed in Vibrio spp [18]. It i...

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Section: Open Accessmentioning

confidence: 99%

Structural basis of torque generation in the bi-directional bacterial flagellar motor

Santiveri

Wadhwa

et al. 2022

Trends in Biochemical Sciences

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“…We evaluated the ionic power source (H + or Na + ) of our functional MotA-ASRs in low Na + condition (~1 mM) using a minimal swim plate without tryptone or yeast extract and where NaCl had been substituted with KCl (Islam et al, 2020; Ridone et al, 2021). Here, we checked whether the tested strains could generate swim rings or not in the low sodium environment.…”

Section: Resultsmentioning

confidence: 99%

“…The copyright holder for this this version posted October 18, 2022. ; https://doi.org/10.1101/2022.10.17.512626 doi: bioRxiv preprint (Ridone et al, 2022) may mean that there may be many combinations of A and B subunits which are able to power rotation and many combinations which cannot. It is therefore suitable to resurrect MotBs that co-existed with MotAs.…”

Section: Discussionmentioning

confidence: 99%

“…This indicates that there is specificity between MotA and MotB proteins. The promiscuity of stators (Ito and Takahashi, 2017) and the modular nature of the stator complex (Ridone et al, 2022) may mean that there may be many combinations of A and B subunits which are able to power rotation and many combinations which cannot. It is therefore suitable to resurrect MotBs that co-existed with MotAs.…”

Section: Discussionmentioning

confidence: 99%

“…We used a minimal swim plate in low Na + condition (~1 mM) without tryptone or yeast extract and where NaCl had been substituted with KCl (Islam et al, 2020; Ridone et al, 2021), to determine which one between sodium (Na + ) and proton (H + ) was the power source of our MotA-ASRs. The minimal swim plates were inoculated with a fresh single colony of MotA-ASR transformed Δ motA E. coli with a sterile toothpick and incubated at 30°C for 16h.…”

Section: Methodsmentioning

confidence: 99%

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Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility

Islam

Ridone

Lin

et al. 2022

Preprint

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The bacterial flagellar motor (BFM) is a rotary nanomachine powered by the translocation of ions across the inner membrane through the stator complex. The stator complex consists of two membrane proteins: MotA and MotB (in H+ powered motors), or PomA and PomB (in Na+ powered motors). In this study we used ancestral sequence reconstruction (ASR) to probe which residues of MotA correlate with function and may have been conserved to preserve motor function. We reconstructed ten ancestral sequences of MotA and found four of them were functional in combination with contemporary E. coli MotB and in combination with our previously published functional ancestral MotBs. Sequence comparison between wild-type (WT) E. coli MotA and MotA-ASRs revealed 30 critical residues across multiple domains of MotA that were conserved among all functional stators. These conserved residues included pore-facing, cytoplasm-facing and MotA-MotA interfacing sites. Overall, this work demonstrates the role of ASR in assessing conserved variable residues in a subunit of a molecular complex.

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Mechanisms of ion selectivity and rotor coupling in the bacterial flagellar sodium-driven stator unit

Santiveri

Eguiara

et al. 2022

Preprint

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Bacteria swim using a flagellar motor that is powered by stator units. These stator units are energized by an ionic gradient across the membrane, typically proton or sodium. The presumed monodirectional rotation of the stator units allows the bidirectional rotation of the flagellar motor. However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the motor remain largely unclear. Here we have determined by cryo-electron microscopy the structure of the Na+-driven type stator unit PomAB from the gram-negative bacterium Vibrio alginolyticus in both lipidic and detergent environments, at a resolution up to 2.5 Angstrom. The structure is in a plugged, auto-inhibited state consisting of five PomA subunits surrounding two PomB subunits. The electrostatic potential map uncovers sodium ion binding sites within the transmembrane domain, which together with functional experiments and explicit solvent molecular dynamics simulations, suggest a mechanism for ion translocation and selectivity. Resolved conformational isomers of bulky hydrophobic residues from PomA, in the vicinity of key determinant residues for sodium ion coupling of PomB, prime PomA for clockwise rotation. The rotation is tightly blocked by the trans-mode organization of the PomB plug motifs. The structure also reveals a conformationally dynamic helical motif at the C-terminus of PomA, which we propose regulates the distance between PomA subunit cytoplasmic domains and is involved in stator unit-rotor interaction, concomitant stator unit activation, and torque transmission. Together, our studies provide mechanistic insight for understanding flagellar stator unit ion selectivity and incorporation of the stator units into the motor.

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The rapid evolution of flagellar ion-selectivity in experimental populations ofE. coli

Cited by 4 publications

References 93 publications

Structural basis of torque generation in the bi-directional bacterial flagellar motor

Structural basis of torque generation in the bi-directional bacterial flagellar motor

Ancestral reconstruction of the MotA stator subunit reveals that conserved residues far from the pore are required to drive flagellar motility

Mechanisms of ion selectivity and rotor coupling in the bacterial flagellar sodium-driven stator unit

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