The bacterial flagellar motor powers the rotation that propels the swimming bacteria. Rotational torque is generated by harnessing the flow of ions through ion channels known as stators which couple the energy from the ion gradient across the inner membrane to rotation of the rotor. Here, we used error-prone PCR to introduce single point mutations into the sodiumpowered Vibrio alginolyticus /Escherichia coli chimeric stator PotB and selected for motors that exhibited motility in the presence of the sodiumchannel inhibitor phenamil. We found single mutations that enable motility under phenamil occurred at two sites: (i) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus and (ii) near the peptidoglycan binding region that corresponds to the C-terminal region of the MotB stator from E. coli. Single cell rotation assays confirmed that individual flagellar motors could rotate in up to 100 µM phenamil. Using phylogenetic logistic regression, we found correlation between natural residue variation and ion source at positions corresponding to PotB F22Y, but not at other sites. Our results demonstrate that it is not only the pore region of the stator that moderates motility in the presence of ion-channel blockers.
Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants that rescue function. The bacterial flagellar motor (BFM) is an excellent candidate—an ancient and highly conserved molecular complex for bacterial propulsion toward favorable environments. Motor rotation is often powered by H + or Na + ion transit through the torque-generating stator subunit of the motor complex, and ion selectivity has adapted over evolutionary time scales. Here, we used CRISPR engineering to replace the native Escherichia coli H + -powered stator with Na + -powered stator genes and report the spontaneous reversion of our edit in a low-sodium environment. We followed the evolution of the stators during their reversion to H + -powered motility and used both whole-genome and RNA sequencing to identify genes involved in the cell’s adaptation. Our transplant of an unfit protein and the cells’ rapid response to this edit demonstrate the adaptability of the stator subunit and highlight the hierarchical modularity of the flagellar motor.
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.
Most motile bacteria are propelled by rigid, helical, flagellar filaments and display distinct swimming patterns to explore their favorable environments. Escherichia coli cells have a reversible rotary motor at the base of each filament. They exhibit a run-tumble swimming pattern, driven by switching of the rotational direction, which causes polymorphic flagellar transformation. Here we report a novel swimming mode in E. coli ATCC10798, which is one of the original K-12 clones. High-speed tracking of single ATCC10798 cells showed forward and backward swimming with an average turning angle of 150°. The flagellar helicity remained right-handed with a 1.3 μm pitch and 0.14 μm helix radius, which is consistent with the feature of a curly type, regardless of motor switching; the flagella of ATCC10798 did not show polymorphic transformation. The torque and rotational switching of the motor was almost identical to the E. coli W3110 strain, which is a derivative of K-12 and a wild-type for chemotaxis. The single point mutation of N87K in FliC, one of the filament subunits, is critical to the change in flagellar morphology and swimming pattern, and lack of flagellar polymorphism. E. coli cells expressing FliC(N87K) sensed ascending a chemotactic gradient in liquid but did not spread on a semi-solid surface. Based on these results, we concluded that a flagellar polymorphism is essential for spreading in structured environments.
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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