Measurements of the Rotation of the Flagellar Motor by Bead Assay

Kasai, Taishi; Sowa, Yoshiyuki

doi:10.1007/978-1-4939-6927-2_14

Cited by 4 publications

(4 citation statements)

References 28 publications

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“…Most importantly, it is also has the largest surface area in motion, which potentially increases its susceptibility to surface based affects such as increased stickiness. To examine the mechanism of inhibition, and to rule out potentially confounding cell-surface effects that may have influenced earlier experiments using tethered cells, we used a 1 µm polystyrene bead assay (44) to examine the effects of BB2-50F and HM2-16F on rotation. We observed that phenamil in some instances slowed, but did not stop, rotation, whereas BB2-50F and HM2-16F stopped rotation and fixed the location of the bead (Fig.…”

Section: Discussionmentioning

confidence: 99%

Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types

et al. 2021

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The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H + ) and sodium ions (Na + ). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we characterised two possible motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. We used three approaches: effect on rotation of tethered cells, effect on free swimming bacteria and effect on rotation of marker beads. We showed that both HM2-16F and BB2-50F stopped rotation of tethered cells driven by Na + motors comparable to phenamil at matching concentrations, and could also stop rotation of tethered cells driven by H + motors. Bead measurements in presence and absence of stators confirmed that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F stopped swimming in both Na + and H + stator types, and in pathogenic and non-pathogenic strains. Importance: Here we characterised two novel amiloride derivatives in the search for antimicrobial compounds that target bacterial motility. Our two compounds were shown to inhibit flagellar motility at 10 μM across multiple strains, from non-pathogenic E. coli with flagellar rotation driven by proton or chimeric sodium-powered stators, to proton-powered pathogenic E. coli (EHEC/UPEC) and lastly in sodium-powered Vibrio alginolyticus . Broad anti-motility compounds such as these are important tools in our efforts control virulence of pathogens in health and agricultural settings.

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Section: Discussionmentioning

confidence: 99%

Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types

et al. 2021

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“…The tethered cell assay is an excellent approach for measuring variations in the response of individual cells to treatment and for initial compound screening, however it does not provide positional information for the rotor or assess performance at low load (41). To examine the mechanism of inhibition, and to rule out potentially confounding cell-surface effects that may have influenced earlier experiments using tethered cells, we used a 1 µm polystyrene bead assay (42) to examine the effects of BB2-50F and HM2-16F on rotation. We observed that phenamil in some instances slowed, but did not stop, rotation, whereas BB2-50F and HM2-16F periodically stopped rotation (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Most importantly, it is also has the largest surface area in motion, which potentially increases its susceptibility to surface based affects such as increased stickiness. To examine the mechanism of inhibition, and to rule out potentially confounding cell-surface effects that may have influenced earlier experiments using tethered cells, we used a 1 μm polystyrene bead assay (44) to examine the effects of BB2-50F and HM2-16F on rotation. We observed that phenamil in some instances slowed, but did not stop, rotation, whereas BB2-50F and HM2-16F stopped rotation and fixed the location of the bead (Fig.…”

Section: Discussionmentioning

confidence: 99%

Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

Islam

Bae

Ishida

et al. 2021

Preprint

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The bacterial flagellar motor (BFM) is a protein complex that confers motility to cells and contributes to survival and virulence. The BFM consists of stators that are ion-selective membrane protein complexes and a rotor that directly connects to a large filament, acting as a propeller. The stator complexes couple ion transit across the membrane to torque that drives rotation of the motor. The most common ion gradients that drive BFM rotation are protons (H+) and sodium ions (Na+). The sodium-powered stators, like those in the PomAPomB stator complex of Vibrio spp, can be inhibited by sodium channel inhibitors, in particular, by phenamil, a potent and widely used inhibitor. However, relatively few new sodium-motility inhibitors have been described since the discovery of phenamil. In this study, we discovered two motility inhibitors HM2-16F and BB2-50F from a small library of previously reported amiloride derivatives. Using a tethered cell assay, we showed that both HM2-16F and BB2-50F had inhibition comparable to that of phenamils on Na+ driven motors at matching concentrations, with an additional ability to inhibit rotation in H+ driven motors. The two compounds did not exhibit adverse effects on bacterial growth at the motility-inhibiting concentration of 10 uM, however toxicity was seen for BB2-50F at 100 uM. We performed higher resolution measurements to examine rotation inhibition at moderate (1 um polystyrene bead) and low loads (60 nm gold bead) and in both the presence and absence of stators. These measurements suggested that the compounds did not inhibit rotation via direct association with the stator, in contrast to the established mode of action of phenamil. Overall, HM2-16F and BB2-50F showed reversible inhibition of motility across a range of loads, in both Na+ and H+ stator types, and in pathogenic and non-pathogenic strains.

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“…Cells were incubated in a sample chamber for 15 min to attach on the coverslips via their sticky filament. After flushing the unbound cells with the same buffer, spinning cells, which attach on the coverslips via single filaments, were captured at 60 fps by CMOS camera for 10 s [24]. To observe the effect of the electrochemical potential of Mg 2+ on the motor speed, the buffer in a sample chamber was exchanged by running the buffer containing appropriate concentrations of EDTA, MgCl 2 , or CCCP (carbonyl cyanide m-chlorophenyl hydrazone).…”

Section: Rotation Measurement By Tethered Cell Assaysmentioning

confidence: 99%

Coupling Ion Specificity of the Flagellar Stator Proteins MotA1/MotB1 of Paenibacillus sp. TCA20

et al. 2020

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The bacterial flagellar motor is a reversible rotary molecular nanomachine, which couples ion flux across the cytoplasmic membrane to torque generation. It comprises a rotor and multiple stator complexes, and each stator complex functions as an ion channel and determines the ion specificity of the motor. Although coupling ions for the motor rotation were presumed to be only monovalent cations, such as H+ and Na+, the stator complex MotA1/MotB1 of Paenibacillus sp. TCA20 (MotA1TCA/MotB1TCA) was reported to use divalent cations as coupling ions, such as Ca2+ and Mg2+. In this study, we initially aimed to measure the motor torque generated by MotA1TCA/MotB1TCA under the control of divalent cation motive force; however, we identified that the coupling ion of MotA1TCAMotB1TCA is very likely to be a monovalent ion. We engineered a series of functional chimeric stator proteins between MotB1TCA and Escherichia coli MotB. E. coli ΔmotAB cells expressing MotA1TCA and the chimeric MotB presented significant motility in the absence of divalent cations. Moreover, we confirmed that MotA1TCA/MotB1TCA in Bacillus subtilis ΔmotABΔmotPS cells generates torque without divalent cations. Based on two independent experimental results, we conclude that the MotA1TCA/MotB1TCA complex directly converts the energy released from monovalent cation flux to motor rotation.

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Measurements of the Rotation of the Flagellar Motor by Bead Assay

Cited by 4 publications

References 28 publications

Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types

Novel Amiloride Derivatives That Inhibit Bacterial Motility across Multiple Strains and Stator Types

Novel amiloride derivatives that inhibit bacterial motility across multiple strains and stator types

Coupling Ion Specificity of the Flagellar Stator Proteins MotA1/MotB1 of Paenibacillus sp. TCA20

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