Biology subjects

Baker, M. A.

Publications and source records attributed to Baker, M. A..

At least 19 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

Evolutionary Coupling of Flagellar Motility and Type VI Secretion Systems Across Bacteria

Bacteria use both motility and antagonism to compete in spatially structured environments, but whether these traits evolve together across broad bacterial diversity remains unclear. We developed a spatial kin-competition model predicting that motility should couple robustly with contact-dependent weapons by increasing encounter rates with competitors, whereas coupling with diffusible weapons should be weaker and context-dependent. To test these predictions, we performed a comprehensive analysis of 11,365 bacterial genomes across the Tree of Life (ToL). By utilising large-scale homology-based searches, we annotated flagellar, T6SS, and bacteriocin components and then applied phylogenetic comparative models to examine evolutionary associations. T6SS presence was strongly associated with flagellar motility: T6SS-positive lineages were predominantly flagellated, and BayesTraits supported a dependent model of FliC and T6SS evolution. In contrast, bacteriocins showed no detectable evolutionary coupling with flagellar motility. Transition-rate analyses further indicated that T6SS gain was strongly biased towards motile lineages, even though T6SS loss was common overall. These results support an asymmetric macroevolutionary relationship between bacterial motility and antagonism, in which flagellar motility is robustly coupled to contact-dependent competition but not to diffusible antagonistic systems.

evolutionary biology↗

bioRxiv2025

Phage-encoded sRNA counteracts xenogenic silencing in pathogenic E. coli

Horizontal gene transfer introduces foreign DNA that can disrupt cellular processes and is therefore subject to xenogenic silencing by nucleoid-associated proteins such as H-NS and Hha. In Enterohaemorrhagic Escherichia coli (EHEC), prophages make up a large fraction of the accessory genome and encode many virulence factors, yet to be expressed they must overcome this silencing. We identify a prophage-encoded small RNA (sRNA), HnrS, that functions as an anti-silencing factor by targeting the H-NS paralogue Hha. HnrS is a short (66-nt) sRNA present in multiple copies (up to nine) in EHEC and Enteropathogenic Escherichia coli (EPEC) genomes and is enriched in E. coli strains that carry the locus of enterocyte effacement (LEE). We show that HnrS directly base-pairs with the ribosome-binding site of the hha mRNA, repressing its translation and thereby reducing Hha-enhanced H-NS silencing. This counter-silencing de-represses the LEE type III secretion system and concomitantly represses motility. Transcriptomic profiling further revealed that HnrS indirectly activates genes involved in nitrate/nitrite respiration and nitric oxide resistance, metabolic pathways that contribute to survival in the inflamed gastrointestinal tract. Deletion of hnrS reduced expression of nitrate reductase genes and impaired actin pedestal formation on host epithelial cells. Our results indicate that prophage-encoded, multicopy hnrS provides a counter-silencing mechanism that reduces Hha-H-NS repression at specific virulence loci. This likely enables expression of horizontally acquired genes without broadly disrupting the core H-NS regulon. HnrS illustrates how mobile genetic elements deploy sRNAs to counteract xenogenic silencing and promote virulence gene expression, enhancing colonisation of the host. Importance statementHorizontally acquired genes are often silenced to protect bacterial genomes, but this defence also limits the expression of new traits. We identify a prophage-encoded small RNA, HnrS, that counteracts this restriction by repressing the xenogenic silencer Hha, lifting Hha-H-NS-mediated repression of virulence and metabolic genes. HnrS activates the locus of enterocyte effacement, nitrate/nitrite respiration, and nitric oxide resistance--pathways that help E. coli survive and colonise the inflamed gut. Our findings reveal an RNA-based counter-silencing mechanism encoded by mobile genetic elements, showing how phages can reprogram bacterial regulatory networks to promote adaptation and pathogenicity.

microbiology↗

bioRxiv2025

A compact vector for scarless gene editing in E. coli

Here we present pCASKD, a single-plasmid system for scarless chromosomal editing in Escherichia coli. Our plasmid pCASKD integrates CRISPR-Cas9-mediated counterselection, Lambda-Red recombineering, and temperature-sensitive plasmid curing into a 12 Kb vector to enable kilobase-scale insertions and deletions using a linear dsDNA donor and homologous recombination. Using the flagellar stator motAB locus as a model, we demonstrate that pCASKD enables efficient knock-in and knock-out edits with lower donor DNA input and reduced false positives compared with its parent, multi-plasmid system, No-SCAR. Using a single plasmid reduces transformation steps, accelerates screening, and increases the frequency of correctly edited clones. The protocol can be completed in five days, with potential for further optimization, offering a compact and efficient alternative for microbial genome engineering.

microbiology↗

bioRxiv2025

The effect of PEGylation on surface tethering of liposomes via DNA nanotechnology.

Polyethylene glycol (PEG) is widely used in liposome formulation due to its blocking properties and ability to prolong circulation in vivo, to create biomimetic liposomes and drug delivery devices. Similarly, membrane-embedded DNA nanotechnology is increasingly used to modulate cellular behaviour and communication. However, there is a gap in knowledge in how PEG-lipid formulations can be optimised for both liposome properties and control of selective DNA hybridisation. To address this, we systematically investigated the effect of liposome PEG content on DNA mediated tethering of liposomes to glass surfaces. We formulated liposomes of two different lipid compositions (DOPE/DOPC or DPhPC), with varying amounts of PEGylated lipid (0-50%). We measured the effect of increased PEG content on liposome size and polydispersity through dynamic light scattering (DLS). Small amounts of PEG (0-20%) introduced repulsive forces that reduced size, while large amounts of PEG (30-50%) increased polydispersity. PEG-liposomes were then decorated with cholesterol-DNA strands and labelled with either intercalating lipid dyes or fluorescently labelled lipids. Binding to surfaces via complementary DNA strands was quantified using total internal reflection fluorescence (TIRF) microscopy. We found that PEGylation of DNA-liposomes could either block or enhance surface binding, depending on the amount of PEG. DNA-liposomes with reduced surface binding included DPhPC/DiD with 10% or 20% PEG-lipid. In contrast, DNA-liposome surface binding increased for DOPE/DOPC/DiD with increasing PEG%. This study highlights that while PEG can act to stabilise liposome formulations, its ability to block specific DNA binding interactions on membranes is variable and dependent on membrane composition. Table of contents figure O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/663613v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@183b215org.highwire.dtl.DTLVardef@1208fd6org.highwire.dtl.DTLVardef@c343ccorg.highwire.dtl.DTLVardef@1982f80_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Acute testicular hyperthermia leads to a rapid loss of global piRNA levels and a consequent increase in transcript abundance, including LINE1 activity within heat-sensitive male germ cells.

ObjectiveTo understand the immediate impact that testicular heat stress has on isolated populations of precursor male germ cells including Spermatocytes and Spermatids. DesignMice were given testicular heat stress and pre-cursor male germ cells were immediately isolated. RNA sequencing was performed and validated using qPCR. SubjectsThis work was carried out in adult male CD1 mice. ResultsUsing next-generation RNA sequencing 134 differentially expressed transcripts were found to be differentially expressed upon exposure to testicular hyperthermia, 93% of which were upregulated. In addition, testicular hyperthermia induced 395 differential splicing events and altered the usage of 61 polyadenylation sites. To explain these observations and understand why transcript abundance appears to favour upregulation following testicular hyperthermia, we assessed global piRNA levels and found an overall, rapid reduction. Concomitantly, we observed an increase in transposable element RNA (LINE1) and protein (ORF1p) abundance. Furthermore, increased LINE1 expression appeared to be correlated with DNA damage in the male germline. At 24 hours post heat stress, piRNA levels recovered close to control levels, coincident with a significant reduction in LINE1 transcript expression within spermatocytes. ConclusionTesticular hyperthermia needs to be considered in the context of all reproductive outcomes. Affected spermatozoa are likely to be genetically compromised, leading to adverse outcomes such as infertility or loss of embryo following fertilization.

molecular biology↗

bioRxiv2025

The architecture, assembly, and evolution of a complex flagellar motor

Bacterial flagella drive motility in many species, likely including the last bacterial common ancestor 1,2. Knowledge of flagellar assembly and function has mainly come from studies of Escherichia coli and Salmonella enterica, which have simple flagellar motors 3-7. However, most flagellated bacteria possess complex motors with unique, species-specific adaptations whose mechanisms and evolution remain largely unexplored 8-10. Here, we deploy a multidisciplinary approach to build a near-complete model of the flagellar motor in Campylobacter jejuni, revealing its remarkable complexity in architecture and composition. We identify an E-ring around the MS-ring, a periplasmic cage with two distinctive conformations, and an intricate interaction network between the E-ring and cage. These scaffolds play critical roles in stabilizing and regulating 17 torque-generating stator complexes for optimal motility. In-depth evolutionary analyses uncover the ancient origin and prevalence of the E-ring in flagellated species of the domain Bacteria as well as a unique exaptation of type IV pili components PilMNOPQF in the ancestral motor of the phylum Campylobacterota. Collectively, our studies reveal novel mechanisms of assembly and function in complex flagellar motors and shed light on the evolution of flagella and modern bacterial species.

microbiology↗

bioRxiv2024

Easy come, easier go: mapping the loss of flagellar motility across the tree of life

Bacterial swimming is mostly powered by the bacterial flagellar motor and the number of proteins involved in the flagellar motor can vary. Quantifying the proteins present in flagellar motors from a range of species delivers insight into how motility has changed throughout history and provides a platform for estimating from its genome whether a species is likely to be motile. We conducted sequence and structural homology searches for 54 flagellar pathway proteins across 11,365 bacterial genomes and developed a classifier with up to 95% accuracy that could predict whether a strain was motile or not. We then mapped the evolution of flagellar motility across the GTDB bacterial tree of life. We confirmed that the last common bacterial ancestor had flagellar motility and that the rate of loss of this motility was four-fold higher than the rate of gain. We showed that the presence of filament protein homologues was highly phylogenetically correlated with motility and that all species classified as motile contained at least one filament homologue. We calculated the rate of gain and loss for each flagellar protein and that the filament protein FliC was highly correlated with motility across the tree of life. We then measured the correlation of each flagellar motor protein with FliC and showed that the filament, rotor, and rod and hook proteins were all highly correlated with FliC, and thus with motility. We calculated the differential rates of gain and loss for each flagellar protein and quantified which genomes encoded for partial sets of flagellar proteins, indicating potential pathways by which motility could be lost. Overall, we show that filament, rod and hook and rotor proteins are conserved when flagellar motility is preserved and that the presence or absence of a FliC homologue is a good, simple predictor of whether or not a species has flagellar motility.

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↗

bioRxiv2024

Insertion of fluorescent proteins near the plug domain of MotB generates functional stator complex.

Many bacteria swim by the rotation of the bacterial flagellar motor (BFM). The BFM is powered by proton translocation across the inner membrane through the hetero-heptameric MotA5MotB2 protein complex. Two periplasmic domains of MotB are critical in activating BFM rotation: (1) the peptidoglycan binding (PGB) domain that anchors MotB in the peptidoglycan layer and (2) the plug domain that modulates the proton flow. Existing cytoplasmic fluorescent probes have been shown to negatively affect motor rotation and switching. Here we inserted a fluorescent probe in the periplasm near the plug of MotB in an attempt to circumvent issues with cytoplasmic probes and for possible use in observing the mechanism of plug-based regulation of proton flow. We inserted green fluorescent protein (GFP) and iLOV, a fluorescent version of the light-oxygen-voltage (LOV) domain, in four periplasmic locations in MotB. Insertions near the plug retained motility but showed limited fluorescence for both fluorophores. Additional short, flexible glycine-serine (GS) linkers improved motility but did not improve brightness. Further optimization is necessary to improve the fluorescence of these periplasmic probes.

microbiology↗

bioRxiv2024

Molecular and structural innovations of the stator motor complex at the dawn of flagellar motility

The rotation of the bacterial flagellum is powered by the MotAB stator complex, which converts ion flux into torque. The origin and evolution of this remarkable complex is understudied. Here, we perform the first phylogenetic and structural characterisation and classification of MotAB and nonflagellar relatives. Using 193 genomes sampled across 27 bacterial phyla, we estimated phylogenies and ancestral sequences, and generated AlphaFold predictions for all extant and reconstructed proteins. We then mapped them onto the phylogeny to determine patterns of diversity and distribution of structural innovations. We identify two discrete groups: the Flagellar Ion Transporters (FIT) and the Generic Ion Transporters (GIT). The FIT proteins are structurally conserved and have a square fold domain and a torque-generating interface (TGI). FIT proteins are divided into two clades, termed TGI4 and TGI5, referring to whether there have 4 or 5 short helices in the TGI. TGI5 motors are predominantly found in Proteobacteria and include the well-studied E. coli K12 system, while TGI4 motors are found in diverse phyla and include the Na+-powered polar motors of Vibrio (PomAB). The GIT proteins, on the other hand, are structurally diverse and lack these attributes. The interaction between the A and B subunits is conserved across the FIT and GIT proteins. The two subunits are jointly necessary for function, with the genes typically adjacent within an operon. Motility assays in E. coli show that the structural elements unique to FIT play an important role in flagellar motility. Our results indicate that the stator motor complex has a single origin and shares unique motility-related structural traits. Significance StatementFlagellar motility is a key feature in bacterial pathogenicity and survival. It allows bacteria to propel themselves and direct movement according to environmental conditions. We investigated the molecular and structural diversity of the stator motor proteins that provide the ion motive force to power flagellar rotation. This study integrates phylogenetics, 3D protein structure modeling, motility assays and ancestral state reconstruction (ASR) to provide insights into the structural mechanisms that first powered the flagellar motor. We provide the first phylogenetic and structural characterisation and classification of MotAB and relatives.

evolutionary biology↗

bioRxiv2024

Comparison of the CDC2-like kinase family across eukaryotes highlights the functional conservation of these unique biological thermometers

The family of CDC2-like kinases (CLKs) play a crucial role in regulating alternative splicing (AS), a process fundamental to eukaryotic gene expression and adaptation. Of particular interest, these enzymes exhibit unique responsiveness to minor temperature shifts, enabling them to modulate AS accordingly. Dysregulated CLK expression is linked to a wide variety of human diseases, establishing them as promising therapeutic targets. Despite the importance of CLKs, limited research has explored the genetic and functional diversification of this gene family. This report investigates the evolutionary origins, diversification, and functional implications of CLKs across major eukaryotic lineages through phylogenetic and structural comparisons. Our data demonstrate these kinases are prevalent throughout eukaryotes, with the original gene (which shares orthology to human CLK2), dating back to the Last Eukaryotic Common Ancestor. We identified three key duplication events in vertebrates, highlighting how this gene family has expanded and diversified in complex metazoans. Despite two instances of CLK paralog loss in vertebrate lineages, CLKs remain prevalent throughout metazoans, suggesting they are essential for complex eukaryotic life. Structural comparisons across diverse eukaryotes demonstrate kinase domain conservation, which is in line with their maintained function in AS regulation. While their N-terminal regions vary significantly in amino acid sequence, the function of this domain to regulate phosphorylation of AS factors is conserved, albeit in a species-specific manner. CLKs exhibit unique thermo-sensitive properties across diverse species, challenging conventional enzymatic behaviour. This temperature regulation, mediated by their kinase activation segment, is characterised by increased activity at lower physiological temperatures. The conservation of this structure, and a thermo-sensitive amino acid motif within it, suggests this was an ancient adaptation for responding to environmental cues. Species-specific temperature profiles highlight the adaptive evolution of CLKs, enabling organisms to thrive in diverse environmental conditions including extreme temperatures. Our analysis expands the understanding of CLK biology across diverse eukaryotes and connects insights from model organisms to human biology.

evolutionary biology↗

bioRxiv2024

CetZ1-dependent assembly and positioning of the motility machinery in haloarchaea

The archaeal tubulin-like cytoskeletal protein CetZ1 is required for rod-cell morphogenesis during the development of motility in Haloferax volcanii. This is expected to improve swimming speed and directionality. Here, we found that deletion of cetZ1 or expression of a GTPase-defective mutant caused a substantial defect in the assembly of the motility machinery, including the archaellum base marker protein ArlD1, the chemotaxis sensory array adapter CheW1, and signal transducer CheY. Furthermore, overexpression of cetZ1 reduced the assembly and polar placement of the motility machinery without detectably affecting the rod shape of motile cells. In contrast, deletion of the conserved paralog cetZ2 caused no defects in swimming or rod shape, although expression of the cetZ2 GTPase-defective mutant reduced motility whereas cetZ2 overexpression caused mild hyper-motility; these effects were dependent on the presence of cetZ1. A functional CetZ1-mTq2 fusion strongly localized at the poles of mature motile cells, where it partially co-localized with the motility machinery markers. These results suggest that CetZ1 has another role in the organisation or structure of the cell poles that promotes the assembly of the motility machinery. The multiple roles and locations of CetZ1 during motile cell development are reminiscent of the multiple functions of eukaryotic cytoskeletal proteins.

microbiology↗

bioRxiv2024

Allosteric adaptation in the stator complex rescues bacterial motility in Exb/Mot chimeras.

Powered by ion transport across the cell membrane, conserved ion-powered rotary motors (IRMs) drive bacterial motility by generating torque on the rotor of the bacterial flagellar motor. Homologous heteroheptameric IRMs have been structurally characterized in ion channels such as Tol/Ton/Exb/Gld, and most recently in phage defense systems such as Zor. Functional stator complexes synthesized from chimeras of PomB/MotB (PotB) have been used to study flagellar rotation at low ion-motive force achieved via reduced external sodium concentration. The function of such chimeras is highly sensitive to the location of the fusion site, and these hybrid proteins have thus far been arbitrarily designed. To date, no chimeras have been constructed using interchange of components from Tol/Ton/Exb/Gld and other ion powered motors with more distant homology. Here we synthesised chimeras of MotAB, PomAPotB and ExbBD to assess their capacity for cross-compatibility. We generated motile strains powered by stator complexes with B-subunit chimeras. This motility was further optimised by directed evolution. Whole genome sequencing of these strains revealed that motility-enhancing residue changes occurred in the A-subunit and at the peptidoglycan binding domain of the B-unit, which could improve motility. Overall, our work highlights the complexity of stator architecture and identifies the challenges associated with rational design of chimeric IRMs. ImportanceIon-powered rotary motors (IRMs) underpin the rotation of one of natures oldest wheels, the flagellar motor. Recent structures have shown that this complex drives ion conduction and even phage defence and thus appears to be a fundamental molecular module with diverse biological utility where electrical energy can be coupled to rotational force to execute work. Here, we attempted to rationally design chimeric IRMs to explore the cross-compatibility of these ancient motors. We succeeded in making one working chimera of a flagellar motor and a non-flagellar transport system protein. This had only a short hybrid stretch in the ion-conducting channel, and function was subsequently significantly improved through additional substitutions at sites distant from this hybrid pore region. Our goal was to test the cross-compatibility of these homologous systems and highlight challenges arising when engineering new rotary motors from this fundamental nanomotor.

microbiology↗

bioRxiv2024

DIB-BOT: An open-source hardware approach for high throughput droplet interface bilayer deposition

Droplet interface bilayers (DIBs) provide a controlled lipid environment for the single-molecule investigation of a range of biologically relevant membrane-bound processes and have garnered attention for their potential applications in bottom-up artificial cells, biosensing, and biophysics. However, the fabrication of DIBs is currently hindered by time-consuming processes and specialized equipment. These fabrication limitations prevent the scale-up of DIB assays, making it difficult to generate the large data sets required to achieve statistically significant conclusions in single-molecule biological assays where heterogeneous behaviour is often observed. This research describes an open-source solution, dubbed "DIB-BOT," constructed by coupling a nanoinjector with an entry-level 3D printer. We present DIB-BOT as a platform to achieve rapid, reproducible, and reliable fabrication of large numbers of DIBs, addressing the limitations of manual methods. Leveraging commercially available off-the-shelf components, DIB-BOT exhibits high spatial reproducibility, minimal user input, and the ability to scale experiments rapidly. Here we demonstrate the utility of the system by integrating pairwise droplet assembly with a fluorescence plate-reader to execute a biologically relevant assay. When compared with manual DIB fabrication, the DIB-BOT had a tenfold reduction in droplet volume error, a threefold reduction in positional error, and 100% droplet yield. Overall, this method has potential to reduce entry barriers to the use of DIB methods, broadening the applications of DIB research, and generating higher quality data sets.

synthetic biology↗

bioRxiv2023

Tuning the stator subunit of the flagellar motor with coiled-coil engineering.

Many bacteria swim driven by an extracellular filament rotated by the bacterial flagellar motor. This motor is powered by the stator complex, MotA5MotB2, a heterodimeric complex which forms an ion channel which couples energy from the ion motive force to torque generation. Recent structural work revealed that stator complex consists of a ring of five MotA subunits which rotate around a central dimer of MotB subunits. Transmembrane (TM) domains TM3 and TM4 from MotA combine with the single TM domain from MotB to form two separate ion channels within this complex. Much is known about the ion binding site and ion specificity; however, to date, no modelling has been undertaken to explore the MotB-MotB dimer stability and the role of MotB conformational dynamics during rotation. Here, we modelled the central MotB dimer using coiled-coil engineering and modelling principles and calculated free energies to identify stable states in the operating cycle of the stator. We found 3 stable coiled-coil states with dimer interface angles of 28{degrees}, 56{degrees} and 64{degrees}. We tested the effect of strategic mutagenesis on the comparative energy of the states and correlated motility with a specific hierarchy of stability between the three states. In general, our results indicate agreement with existing models describing a 36{degrees} rotation step of the MotA pentameric ring during the power stroke and provide an energetic basis for the coordinated rotation of the central MotB dimer based on coiled-coil modelling.

microbiology↗

bioRxiv2023

Microbial stir bars: light activated rotation of tethered bacterial cells to enhance mixing in stagnant fluids.

Microfluidics devices are gaining significant interest in biomedical applications. However, in a micron-scale device, reaction speed is often limited by the slow rate of diffusion of the reagents. Several active and passive micro-mixers have been fabricated to enhance mixing in microfluidic devices. Here, we demonstrate external control of mixing by rotating a rodshaped bacterial cell. This rotation is driven by ion transit across the bacterial flagellar stator complex. We first measured the flow fields generated by rotating a single bacterial cell rotationally locked to rotate either clockwise (CW) or counterclockwise (CCW). Micro-Particle Image Velocimetry (PIV) and Particle Tracking Velocimetry results showed that a bacterial cell of ~ 2.75 m long, rotating at 5.75 {+/-} 0.39 Hz in a counterclockwise direction could generate distinct micro-vortices with circular flow fields with a mean velocity of 4.72 {+/-} 1.67 m/s and maximum velocity of 7.90 m/s in aqueous solution. We verified our experimental data with a numerical simulation at matched flow conditions which revealed vortices of similar dimensions and speed. We observed that the flow-field diminished with increasing z-height above the plane of the rotating cell. Lastly, we showed we could activate and tune rotational mixing remotely using strains engineered with Proteorhodopsin (PR), where rotation could be activated by controlled external illumination using green laser light (561 nm).

microbiology↗

bioRxiv2022

Establishment and characterisation of oviductal organoids from farm and companion animals

Organoid technology has provided us with a unique opportunity to study early human development and decipher various steps involved in the pathogenesis of human diseases. The technology is already used in clinics to improve human patient outcomes. However, limited knowledge of the methodologies required to establish organoid culture systems in domestic animals has slowed the advancement and application of organoid technology in veterinary medicine. Here, we have developed a platform to grow organoids from animal tissue samples and characterized oviductal organoids from five domestic animal species. Organoids were grown progressively from single cells derived from the enzymatic digestion of freshly collected equine, bovine, feline, canine, and porcine oviducts. The addition of WNT, TGFB, BMP, Rock, and Notch signalling pathway activators or inhibitors in the culture medium suggested remarkable conservation of the molecular signals involved in oviductal epithelial development and differentiation across species. The gross morphology of organoids from all the domestic species was initially similar. However, some differences in size, complexity, and growth rate were observed and described. Well-defined and synchronised motile ciliated cells were observed in differentiated organoids in mature populations. Histopathologically, oviductal organoids mimicked their respective native tissue. In summary, we have developed a detailed cross-species comparison of oviductal organoid models, which will be valuable for advancing assisted reproductive technologies and fertility studies in these animal species in the future. Summary sentenceOrganoids can be derived from the oviductal epithelium of cow, cat, dog, horse, and pig to advance assisted reproductive technologies in animals.

developmental biology↗