Biology subjects

Sowa, Y.

Publications and source records attributed to Sowa, Y..

9 recordsLinked to original sources

bioRxiv2026

A Specific Ancestral Sequence Reconstruction of Flagellin Can Drive Motility in Salmonella enterica but not Escherichia coli

The bacterial flagellar filament, built from thousands of flagellin subunits, is essential for flagellar motility. Here, we aimed to use ancestral sequence reconstruction (ASR) to generate conservative, ancestral flagellin sequences that surround the divergency point between Escherichia and Salmonella species and tested their ability to restore motility in flagellin deleted Escherichia coli and Salmonella Typhimurium. Only one (N131) out of three candidates restored motility in S. Typhimurium but not E. coli, despite high structural similarity to native FliC/FljB. S. Typhimurium expressing N131 showed reduced motility relative to wild-type Salmonella FliC, alongside increased tumbling frequency. Serial passage across swim plates produced a derivative, N131.10, containing a single flagellin mutation (M144I), with a larger swim ring diameter but also increased tumbling frequency. Comparison of structural interaction networks among the residues of S. Typhimurium LT2 FliC with those of other filament proteins showed that non-motile candidates (N132 and N134) carried substitutions that disrupted key core-domain atomic interactions. These results demonstrate that flagellar filament assembly tolerates only limited sequence variation and that this capacity for tolerance differs between E. coli and S. Typhimurium despite their close phylogenetic relationship.

microbiology↗

bioRxiv2026

Body-wrapping anterior flagella drive ultrafast swimming in bacterial zoospores

Most bacteria swim at [~]10 body lengths per second (bl s-1), yet some microorganisms move far faster, and the physical design principles enabling such extreme motility remain poorly understood. Here we uncover an ultrafast swimming strategy in Actinoplanes missouriensis zoospores, which reach up to 560 m s-1 ([~]500 bl s-1), the fastest relative swimmer reported for any microorganism. High-speed imaging shows that propulsion is driven by an anterior, body-wrapping bundle of short flagella that rotate synchronously as a right-handed helix at [~]150 Hz. This architecture generates high thrust without extreme motor speeds and contrasts with the canonical rear-bundle paradigm of bacterial swimming. Microfluidic assays further demonstrate that this propulsion mode enhances dispersal across the flow interface, providing a mechanism for rapid colonization during the transient motile phase of the life cycle. Furthermore, chemotaxis-dependent reorientation is observed, suggesting that zoospore swimming can be directionally regulated. These results identify a new locomotion principle in which supramolecular organization of multiple flagella, rather than motor speed alone, sets the upper limits of bacterial swimming and offers inspiration for the design of high-performance microswimmers.

microbiology↗

bioRxiv2024

Rescue of bacterial motility using two and three-species FliC chimeras

The bacterial flagellar filament acts as a propeller to drive most bacterial swimming. The filament is made of flagellin, known as FliC in Escherichia coli. FliC consists of four domains, the highly conserved core D0 and D1 domains and the hypervariable outer D2 and D3 domains. The size and structure of the outer domains varies, being completely absent in some bacterial species. Here, we sought to identify outer domains from various species that are compatible with the ability of E. coli K-12 FliC to form filaments capable of supporting motility. We calculated a phylogeny of 210 representative flagellin amino acid sequences and generated a series of FliC variants, including outer-domain-deleted forms and eleven chimeric FliC mutants using domains from E. coli K-12, Salmonella Typhimurium, Pseudomonas aeruginosa, Collimonas fungivorans, Helicobacter mustelae and Mesorhizobium sp. ORS3359 in various combinations. Notably, two of the chimeric fliC mutants rescued motility in a fliC-disrupted E. coli K-12 strain, both of which contained the S. Typhimurium D2 domain. Overall, we demonstrate that, while most FliC chimeras did not support motility, interchangeability of the outer domains can produce filaments that provide motility, providing insights to guide the design of synthetic flagellins. ImportanceFlagellin is a key protein forming the filament of the bacterial flagellar motor which powers most bacterial swimming. Flagellin can have hypervariable domains which can alter motility in different environments and provide immune evasion. Here we engineered two flagellin chimeras that could drive motility. This indicates that the flagellin outer domains can be exchanged, to some degree, allowing us to refine rational design approaches for engineering of bacterial swimming. Our work shows the challenges to overcome when combining flagellins from different species and provides evidence that domain switched flagellins can form filaments.

microbiology↗

bioRxiv2021

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

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. ImportanceHere 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 M 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.

microbiology↗

bioRxiv2021

Spontaneous adaptation of ion selectivity in a bacterial flagellar motor

Determining which cellular processes facilitate adaptation requires a tractable experimental model where an environmental cue can generate variants which rescue function. The Bacterial Flagellar Motor (BFM) is an excellent candidate - an ancient and highly conserved molecular complex for propulsion which navigates bacteria towards favourable environments. In most species, rotation is powered by H+ or Na+ ion transit through the torque-generating stator subunit of the motor complex. The ion that drives the rotor has changed over evolutionary timescales but the molecular basis of this selectivity remains unknown. Here we used CRISPR engineering to replace the native Escherichia coli H+-powered stator with Na+-powered stator genes and report the rapid and 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 whole genome and transcriptome sequencing to identify both flagellar- and non-flagellar-associated genes involved in the cells adaptation. Our transplant of an unfit protein and the cells rapid response to this edit demonstrates the adaptability of the stator subunit and highlights the hierarchical modularity of the flagellar motor.

microbiology↗

bioRxiv2020

Injectable prevascularized mature adipose tissues (iPAT) to achieve long-term survival in soft tissues regeneration

Soft tissue regeneration remains a challenge in reconstructive surgery. Current autologous fat implantations lead to high fat absorption ratios, while artificial implants can be associated with lymphoma occurrence. To overcome these limitations, our aim was to reproduce adipose tissue vasculature structure before implantation. Here, we developed injectable prevascularized adipose tissues (iPAT), using physiological collagen microfibers (CMF) mixed with human mature adipocytes, adipose-derived stem cells (ADSC) and human umbilical vein endothelial cells (HUVEC). Following murine subcutaneous implantation, higher cell survival (84{+/-}6% viability) and volume maintenance were shown after 3 months for the iPAT (up to twice heavier than the non-prevascularized balls). This higher survival can be explained by the greater amount of blood vessels (up to 1.6 folds increase), with balanced host anastomosis (51{+/-}1% of human/mouse lumens), also involving infiltration by the lymphatic and neural vasculature networks. These iPAT tissues allowed non-invasive soft tissue reconstruction for long-term outcomes, and the ability to cryopreserve them with maintained viability and functionality also enables a later reinjection usually required before reaching the final patient desired graft volume.

bioengineering↗

bioRxiv2020

Distinct chemotactic behavior in the original Escherichia coli K-12 depending on forward-and-backward swimming, not on run-tumble movements

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 rotatory 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{degrees}. The flagellar helicity remained right-handed with a 1.3 m pitch and 0.14 m helix radius, which is assumed to be 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 form rings on a semi-solid surface. Based on these findings, we propose a flagellar polymorphism-dependent migration mechanism in structured environments.

microbiology↗

bioRxiv2019

High pressure inhibits signaling protein binding to the flagellar motor and bacterial chemotaxis through enhanced hydration

In the chemotaxis of Escherichia coli, the cells behavioral switch involves binding of the phosphorylated form of the chemotaxis signaling protein CheY (CheYp) to the flagellar motor protein FliM, which induces the motor to rotate clockwise; otherwise, the motor rotates counterclockwise. To investigate high-pressure effects on CheYp-FliM binding at atomic resolution, we conduct molecular dynamics simulations of monomeric CheYp, the N-terminal fragment of the FliM (FliMN) that binds to CheYp, and the complex that forms between those proteins at pressures ranging from 0.1 to 100 MPa. The results show that the active form of monomeric CheYp is maintained even at 100 MPa but high pressure increases the water density in the first hydration shell and can cause conformational change of the C-terminal helix. The dissociation process of the complex is investigated by parallel cascade selection molecular dynamics (PaCS-MD), revealing that high pressure considerably induces water penetration into the complex interface. Pressure dependence of standard binding free energy calculated by the Markov state model indicates that the increase of pressure from 0.1 to 100 MPa weakens the binding by [~] 10 kcal/mol. Using high-pressure microscopy, we observed that high hydrostatic pressure reversibly fixes the motor rotation in the counter-clockwise orientation, which supports the notion that high pressure inhibits the binding of CheYp to FliM. We conclude that high pressure induces water penetration into the complex interface, which interferes with CheYp-FliM binding and prevents motor reversal.

biophysics↗

bioRxiv2018

Mutations close to the peptidoglycan-binding region of the stators of the bacterial flagellar motor influence phenamil resistance

The bacterial flagellar motor (BFM) is a molecular complex which powers the rotation of the filament that propels 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 sodium-powered Vibrio alginolyticus/Eschrichia Coli chimeric stator PotB. We then selected for motors that exhibited resistance to the sodium-channel inhibitor phenamil. We found that single mutations that inferred resistance to phenamil occurred at two sites: 1) the transmembrane domain of PotB, corresponding to the TM region of the PomB stator from V. alginolyticus, and 2) near the peptidoglycan (PG) binding region that corresponds to the C-terminal region of the MotB stator from E. coli. We corroborated our swim plate observations with single cell rotation assays to confirm that individual cells could drive rotation of flagellar motors in the presence of up to 100 M phenamil. Our results demonstrate that it is not only the pore region of the stator that moderates the effect of motility in the presence of ion-channel blockers. We hypothesise that mutations in the PG region can allow motors to function in the presence of phenamil by allowing multiple semi-functioning stators to persist on the motor and drive flagellar rotation.

microbiology↗