Biology subjects

Thomas, G. H.

Publications and source records attributed to Thomas, G. H..

At least 19 recordsLinked to original sources

bioRxiv2026

The YjfJ protein is the effector of a second PspA/VIPP1 system that is widespread in the Gammaproteobacteria

Maintaining the integrity of the cytoplasmic membrane is essential for bacterial cell survival. Unsurprisingly, prokaryotes have evolved conserved mechanisms by which they sense, maintain, and repair membrane integrity in response to stress. The phage shock protein (Psp) is one such system, understood primarily through work undertaken in the Gammaproteobacterial species Escherichia coli and Yersinia enterocolitica. The system utilises an effector, PspA, alongside accessory proteins that sense stress and recruit PspA to damaged membranes. Homologues of PspA - part of the PspA/VIPP1 family - are found in a variety of bacterial phyla. Although most work in the Gammaproteobacteria has concerned the canonical Psp response, there have been descriptions of non-canonical PspA/VIPP1-containing systems that warrant further genomic analysis. By analysing the PspA/VIPP1 content of a diverse selection of Gammaproteobacterial genomes, we reveal the existence of two phylogenetically distinct subtypes of PspA/VIPP1 proteins, PspA- and YjfJ-like, which are found at similar frequencies. The YjfJ-like proteins are not only a novel subtype of PspA/VIPP1, having their own unique set of accessory proteins, but in some species, such as the ESKAPE pathogen Pseudomonas aeruginosa, comprise the only PspA/VIPP1-containing system. Our study positions the YjfJ-like containing systems on equal footing with the PspA system in terms of their conservation and distribution in the Gammaproteobacteria. This suggests that studies into the biological functions of YjfJ-like proteins would be well-justified from their unique presence in some important human pathogens.

microbiology↗

bioRxiv2026

The avian colourscape is disproportionately threatened by species extinctions

The impact of projected extinctions on global animal colour diversity remains unknown. Combining citizen science with self-supervised deep learning, we built novel representations of bird plumage colour patterning based on >125,000 museum specimen images covering 9,143 species. We demonstrate that losing currently threatened bird species will drive a disproportionate reduction in avian plumage diversity, with the most severe losses occurring in tropical and subtropical regions. Furthermore, while humans generally find non-typical plumage phenotypes more aesthetically attractive, threatened species are unexpectedly deemed less visually appealing despite their comparatively unusual plumages. Overall, our results highlight severe, imminent threats to the existing avian colourscape and raise critical questions about the future of animal colour diversity in a changing world.

ecology↗

bioRxiv2026

Disparity analyses are robust to ancestral state estimation uncertainty

Morphological trait datasets and phylogenies are routinely paired to investigate macroevolutionary patterns during disparity analyses. However, incomplete fossil sampling can distort disparity estimates, obscuring true evolutionary signals. Ancestral state estimation can be used for both continuous and discrete traits to extend these analyses beyond incomplete fossil data, such as investigations into disparity through time. However, when ancestral state estimation occurs in the disparity pipeline, and the inevitable uncertainty in these estimates, complicate their integration. Determining the most robust workflow for integrating ancestral state estimation in disparity analyses remains a critical methodological challenge. Using simulations to attain a ground-truth disparity value, we evaluated different approaches to performing ancestral state estimation and incorporating uncertainty across varying continuous and discrete trait models, fossil sampling densities and disparity metrics. Ancestral state estimation generally improved recovery of true disparity relative to tip-only analyses, though the optimal approach depended on the interaction between trait model and fossil sampling density. For continuous traits, probabilistic approaches were most accurate, but were sensitive to model misspecification under low fossil sampling density. For discrete traits, pre-ordination methods were most reliable and probabilistic approaches outperformed point estimates under low sampling, while point estimates became increasingly accurate as sampling density increased. Fossil sampling density was a stronger predictor of disparity accuracy than estimation method choice, underscoring that methodologies are only as powerful as the data provided. Our findings offer a practical decision framework for selecting the most appropriate workflow given the sampling density and trait characteristics of a dataset.

evolutionary biology↗

bioRxiv2025

Itaconate utilisation by the human pathogen Pseudomonas aeruginosa requires uptake via the IctPQM TRAP transporter

Pseudomonas aeruginosa PA01 is one of the major causes of disease persistence and mortality in patients with lung pathologies, relying on various host metabolites as carbon and energy sources for growth. The ict-ich-ccl operon (pa0878, pa0882, pa0883) in PAO1 is required for growth on the host molecule itaconate, a C5-dicarboxylate. However, it is not known how itaconate is taken up into P. aeruginosa. Here we demonstrate that a genetically linked tripartite ATP-independent periplasmic (TRAP) transporter (pa0884-pa0886), which is homologous to the known C4-dicarboxylate binding TRAP system, is essential for growth on itaconate, but not for the closely related C4-dicarboxylate succinate. Using tryptophan fluorescence spectroscopy we demonstrate that the substrate binding protein, IctP (PA0884), binds itaconate, but still retains higher affinity for the related C4-dicarboxylates. The structures of IctP bound to itaconate (1.80 [A]) and succinate (1.75 [A]), revealed an enclosed ligand binding pocket with ion pairing interactions with the ligand carboxylates. The C2 methylene group that is the distinguishing feature of itaconate compared to succinate is accommodated by a unique change in the IctP binding site from a Leu to Val, which distinguishes it from closely related C4-dicarboxylate binding SBPs. Together these data suggest that this transporter, which we name IctPQM, has duplicated from a canonical C4-dicarboxylate transporter and its evolution towards itaconate specificity enables this pathogen to now access a key metabolite for persistence in the host.

microbiology↗

bioRxiv2025

High-resolution Staphylococcus profiling reveals intra-species diversity in a single skin niche

The skin microbiome is dominated by a few key genera, among which Staphylococcus is one of the most well-characterised. Recent studies have examined the roles of various Staphylococcus species such as S. epidermidis and S. hominis within broader skin microbial communities. However, these investigations often rely on isolates from multiple individuals and hence, limiting their ability to capture intra-community interactions. In this study, we focused on the axillary microbiome of a single healthy individual to characterise the genetic and functional diversity of resident Staphylococcus isolates. Using a low-cost, high-throughput DNA extraction and long-read whole genome sequencing pipeline, we generated complete genomes for 93 isolates spanning seven genetically distinct lineages across three major skin species. These comprised of one dominant with three additional lineages of S. epidermidis, two of S. hominis and one of S. capitis. Functional and metabolic analyses revealed species- and strain-specific features, suggesting potential metabolic cross-feeding and specialisation within this community, including within strains of S. epidermidis. These findings highlight the metabolic complexity and potential interdependence of staphylococci inhabiting a single skin site and the need for strain-level resolution of the community. The strains form part of the York Skin Microbiome (YSM) collection, a growing open biobank of genetically diverse skin isolates from matched individuals.

microbiology↗

bioRxiv2024

Distinct roles for the Staphylococcus aureus POT transporter DtpT in di/tripeptide uptake and glutathione utilisation inside human macrophages

Peptides available in biological niches inhabited by the human pathogen Staphylococcus aureus serve as a rich source of amino acids required for growth and successful host colonisation. Uptake of peptides by S. aureus involves at least two transport systems: the di/tripeptide permease DtpT and the oligopeptide ABC transporter Opp3. Here we study the individual and combined functions of DtpT and Opp3 in enabling utilisation of diverse di-/tripeptides via a high-throughput phenotypic screen. We reveal that DtpT is the primary route of uptake for dipeptides, and although many peptides can be utilised via either of the two transport systems, we demonstrate a clear preference for Asp/Glu-containing peptides among DtpT substrates. To better understand the substrate preferences of DtpT, the protein was purified and reconstituted into proteoliposomes. Active transport of diverse di- and tripeptides was demonstrated, supporting the conclusions of the phenotypic screen. During this in vitro analysis, we discovered that DtpT could transport the biologically prevalent tripeptide glutathione (GSH). Bacterial growth assays demonstrate that dtpT is essential for GSH utilisation in the absence of the known glutathione transporter, Gis, identifying DtpT as the second GSH uptake system of S. aureus. We demonstrate that GSH transport is required by S. aureus for complete fitness during in vitro macrophage infection experiments. Finally, based on analysis of the DtpT structure and identification of key residues needed for GSH binding and transport, we suggest that GSH transport may be conserved in the DtpT orthologue of Listeria monocytogenes. Together, these data reveal important new functions for DtpT in the utilisation of diverse peptides and point toward a novel role for DtpT (and, potentially, other bacterial POT proteins) in glutathione acquisition during intracellular infection. Author summaryThe environments where bacterial pathogens thrive are often rich in proteins and their degradation products, including oligopeptides, which can be taken up by the bacterium and used as nutrients. Understanding how this occurs could help us find ways to tackle the growth of these pathogens during infection. Here we examine how the major human pathogen Staphylococcus aureus takes up oligopeptides. We demonstrate that a membrane transporter protein, DtpT, is the major route of dipeptide uptake and reveal over 100 new di-/tripeptide targets for this protein. Our findings highlight a defined role for DtpT in the accumulation of Aspartate- and Glutamate-containing peptides, which may serve as relevant nitrogen sources during infection. We also provide the first evidence that DtpT transports the prevalent human metabolite reduced glutathione, and demonstrate that DtpT functions alongside the previously identified Gis glutathione transport system to support intracellular survival of this pathogen inside macrophages. Overall, our findings provide a clear example of how substrate selectivity allows DtpT to fulfil specific biological roles in S. aureus, and this functional specialisation may be a common feature of homologous peptide transporters in other bacterial pathogens and across the Tree of Life.

microbiology↗

bioRxiv2024

Revealing the diversity of commensal corynebacteria from a single human skin site

Our understanding of the skin microbiome has dramatically improved since the pioneering studies and the improvements in sequencing technologies. Species of the genus Corynebacterium are known to form a major part of the human skin microbiome but most detailed studies have focussed on other similarly prevalent genera like Staphylococcus and Cutibacteria. Prior to this study, there were few complete genomes for skin commensals of the genus Corynebacterium, with only 9 complete genomes available for the most commonly identified species C. tuberculostearicum. In this study we explored the genus Corynebacterium from a single body site by swabbing the axilla/underarm of 4 individuals and using a selective media to enrich for corynebacteria. We then generated whole genome sequencing data of these corynebacteria enriched isolates using long-read sequencing and subsequent bioinformatics analysis to reveal an unparalleled diversity of this genus from a single skin site. Through this approach, we obtained the closed genomes of 215 isolates, 154 derived from a single individual. With this genetic information, we were able to identify 7 different species including species previously not associated to the skin and two novel species provisionally named C. axilliensis and C. jamesii. We used pangenome analysis on 30 genetically distinct isolates spanning the 7 species to identify putative metabolic differences, antimicrobial resistance profiles, novel biosynthetic gene clusters (BGCs), prophages and phage defence systems. Our culture-based Nanopore sequencing approach has dramatically improved our overall knowledge of skin corynebacteria, and uniquely here also providing in-depth analysis from a single skin site, revealing a multitude of differences between the isolates. Here we not only improved our knowledge of axillary corynebacteria but also greatly expanded the publicly available number of cutaneous corynebacterial genomes complementing recent studies seeking to understand the diversity of skin corynebacteria.

microbiology↗

bioRxiv2024

Structural unification of diverse membrane-boundacyltransferases reveals a conserved fold that defines the Transmembrane Acyl Transferase (TmAT) superfamily

The movement of acyl groups across biological membranes is essential for many cellular processes. One major family of proteins catalysing this reaction are the acyl transferase family 3 (AT3) proteins, which form a pore to allow acyl-CoA to penetrate the membrane for transfer onto an extracytosolic acceptor molecule. Recent structures of the sequence-unrelated human heparan--glucosaminide N-acetyltransferase (HGSNAT) support a similar transmembrane acyl-group transfer mechanism. Here we demonstrate that both protein families contain a conserved 10-transmembrane helical fold with high structural and detectable sequence conservation around the acyl-CoA pore, supporting the previously proposed Transmembrane Acyl Transferase (TmAT) protein superfamily. In addition, we identify TmAT proteins, including the human Golgi sialate-O-acetyltransferase (CASD1), the human/fungal PIG-W/GWT1 enzymes and the bacterial vancomycin resistance protein VanTG, where the TmAT domains function has been largely unrecognised. We conclude that the TmAT fold represents an ancient architecture for transmembrane acyl-group transfer with important roles in the dynamic modification of glycans in diverse processes across the three domains of life.

biochemistry↗

bioRxiv2024

Structure, substrate selectivity determinants and membrane interactions of a Glutamate-specific TAXI TRAP binding protein rom Vibrio cholerae.

Tripartite ATP independent periplasmic (TRAP) transporters are widespread in prokaryotes and are responsible for the transport of a variety of different ligands, primarily organic acids. TRAP transporters are secondary active transporters that employ a substrate binding protein to bind and present the substrate to membrane embedded translocation component. TRAP transporters can be divided into two subclasses; DctP-type and TAXI type, which share the same overall architecture and requirement of the SBP for transport, but their SBPs share no similarity. The DctP-type transporters are very well studied and have been shown to transport a range of compounds including dicarboxylates, keto acids, sugar acids. However, the TAXI type transporters are relatively poorly understood, with the range of transportable compounds still to be discovered and selectivity requirements for binding unknown. To address these shortfalls in our understanding, we have structurally and biochemically characterized VC0430 from Vibrio cholerae revealing it to be a monomeric high affinity glutamate binding protein. VC0430 is stereoselective, binding the L-isomer preferentially, and can also bind L-glutamine and L-pyroglutamate, but with low affinity relative to L-glutamate. Structural characterization of ligand bound VC0430 reveals details of the binding site and biophysical characterization of binding site mutant reveal the substrate binding determinants, which differ substantially from the DctP-type TRAPs. Finally, we have analysed in silico the interaction between VC0430 and its cognate membrane component revealing an architecture hitherto unseen. To our knowledge, this is the first transporter in V. cholerae to be identified as specific to glutamate, which plays a key role in osmoadaptation of V. cholerae, making this transporter a potential therapeutic target.

biochemistry↗

bioRxiv2024

Characterisation of anhydro-sialic acid transporters from mucosa-associated bacteria

Sialic acid (Sia) transporters are critical to the capacity of host-associated bacteria to utilise sialic acid for growth and/or cell-surface modification. While N-acetyl-neuraminic acid (Neu5Ac)-specific transporters have been studied extensively, little is known on transporters dedicated to anhydro-sialic acid forms such as 2,7-anhydro-Neu5Ac (2,7-AN) or 2,3-dehydro-2-deoxy-Neu5Ac (Neu5Ac2en). Here, we used a Sia-transport-null strain of Escherichia coli to investigate the function of members of anhydro-Sia transporter families previously identified by computational studies. First, we showed that the transporter NanG, from the Glycoside-Pentoside-Hexuronide:cation symporter family, is a specific 2,7-AN transporter, and identified by mutagenesis a crucial functional residue within the putative substrate-binding site. We then demonstrated that NanX transporters, of the Major Facilitator Superfamily, also only transport 2,7-AN and not Neu5Ac2en nor Neu5Ac. Finally, we provided evidence that SiaX transporters, of the Sodium-Solute Symporter superfamily, are promiscuous Neu5Ac/Neu5Ac2en transporters able to acquire either substrate equally well. The characterisation of anhydro-Sia transporters expands our current understanding of prokaryotic Sia metabolism within host-associated microbial communities.

microbiology↗

bioRxiv2023

The mechanistic basis of evolutionary transitions between grey, slate, and blue colour in Tanagers (Thraupidae)

Both pigmentary and structural colours share many common elements of their feather anatomy, i.e. keratin, air and melanin packed in the melanosomes, despite utilizing different mechanisms of the colour production. This means that evolutionary transitions between pigmentary and structural colours can be achieved through a simple adjustment of these elements. Recently, an evolutionary hypothesis for the transition between pigmentary grey, through slate and finally to structural blue colour has been proposed and confirmed in the clade Tanagers on a macroevolutionary level. Here, we investigate mechanistic basis of this evolutionary pathway. By using SAXS (small-angle X-ray scattering) we have quantified important elements of spongy layer in medullary cells that is crucial for colour production by coherent scattering of light wavelengths. We have quantified five elements of the spongy layer: nanostructure complexity, average hard block thickness, average soft block thickness, filling fraction and Io value. We report that across different categories of feather colour, i.e. blue, slate and grey, nanostructure complexity, filling fraction and Io value explained variation in the chromatic component of the colour (between the three colour categories). Chromatic variation within the colour category was explained by filling fraction in the case of slate colour and by nanostructure complexity and average hard block thickness in the case of blue colour. We propose that variation in different elements or combination of elements of the spongy nanostructure has been utilised in feather colour evolution, both within and between colour categories, to overcome developmental constraints imposed by self-assembly processes.

evolutionary biology↗

bioRxiv2023

Evolutionary dynamics of pigmentary grey and non- iridescent structural blue colouration in Tanagers (family: Thraupidae)

3.1Birds are one of the most colourful animal groups in the world and there are multiple ways by which they achieve this feature. Mechanisms of colour production range from pigmentary (pigment deposition) to structural (nanostructural arrangements), or the combination of both. Despite the huge breadth of colour gamut, the basic components of feathers are shared across all of them (keratin, air and presence of pigments in accordance with the colour produced). It has been shown that in some instances, colour evolution between pigmentary and structural colours can proceed by rearrangement of the nano-structural elements of feathers. Here, we investigated evolutionary transitions between pigmentary grey and non-iridescent structural blue. We focus on the Thraupidae (tanagers and allies) that display a variety of blues and greys including a potential transition state that we refer to as slate. We used digitally calibrated images of birds to quantify colour and determine the distinctiveness of slate colour in colourspace. Following, we identify the most likely pathway for the evolution of the colour blue: from grey via slate colour. Our research reveals a new pathway in the evolution of blue colour.

evolutionary biology↗

bioRxiv2023

Interrogation of RNA-protein interaction dynamics in bacterial growth

Characterising RNA-protein interaction dynamics is fundamental to understand how bacteria respond to their environment. In this study, we have analysed the dynamics of 91% of the Escherichia coli expressed proteome and the RNA-interaction properties of 271 RNA-binding proteins (RBPs) at different growth phases. We find that 68% of RBPs differentially bind RNA across growth phases and characterise 17 previously unannotated proteins as bacterial RBPs including YfiF, a ncRNA-binding protein. While these new RBPs are mostly present in Proteobacteria, two of them have human orthologs in the form of mitochondrial proteins associated with rare metabolic disorders. Moreover, we reveal novel RBP functions for proteins such as the chaperone HtpG, a new stationary phase tRNA-binding protein. Altogether, we provide the first dynamic RBPome of a bacterium, showcasing how this approach can reveal the function of uncharacterised proteins, and identify critical RNA-protein interactions for cell growth which could inform new antimicrobial therapies.

microbiology↗

bioRxiv2023

Conformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP

The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, was locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, was locked in either its predicted inward- or outward-facing states. We characterized the disulfide-locked variants and used single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa.

biochemistry↗

bioRxiv2022

The phase separated CO2-fixing pyrenoid proteome determined by TurboID

Phase separation underpins many biologically important processes such as RNA metabolism, signaling and CO2 fixation. However, determining the composition of a phase separated organelle is often challenging due to their sensitivity to environmental conditions which limits the application of traditional proteomics techniques like organellar purification or affinity purification mass spectrometry to understand their composition. In Chlamydomonas reinhardtii, Rubisco is condensed into a crucial phase separated organelle called the pyrenoid that improves photosynthetic performance by supplying Rubisco with elevated concentrations of CO2. Here, we developed a TurboID based proximity labeling technique in Chlamydomonas chloroplasts, where proximal proteins are labeled by biotin radicals generated from the TurboID-tagged protein. Through the expression of two core pyrenoid components fused with the TurboID tag, we have generated a high confidence pyrenoid proxiome that contains the majority of known pyrenoid proteins plus a number of novel pyrenoid candidates. Fluorescence protein tagging of 8 previously uncharacterized TurboID-identified proteins showed 7 were localized to a range of sub-pyrenoid regions. The resulting proxiome also suggests new secondary functions for the pyrenoid in RNA-associated processes and redox sensitive iron-sulfur cluster metabolism. This developed pipeline opens the possibility of investigating a broad range of biological processes in Chlamydomonas especially at a temporally resolved sub-organellar resolution.

plant biology↗

bioRxiv2022

Overconfidence undermines global wildlife abundance trends

In the face of rapid global change and an uncertain fate for biodiversity, it is vital to quantify trends in wild populations. These trends are typically estimated from abundance time series for suites of species across large geographic and temporal scales. Such data implicitly contain phylogenetic, spatial, and temporal structure which, if not properly accounted for, may obscure the true magnitude and direction of biodiversity change. Here, using a novel statistical framework to simultaneously account for all three of these structures, we show that the majority of current abundance trends estimates among 10 high-profile datasets, representing millions of abundance observations, are likely unreliable or incorrect. Our new approach suggests that previous models are too simplistic, incorrectly estimating global abundance trends and often dramatically underestimating uncertainty, an aspect that is critical when translating global assessments into policy outcomes. Further, our approach also results in substantial improvements in abundance forecasting accuracy. Whilst our results do not improve the outlook for biodiversity, our framework does allow us to make more robust estimates of global wildlife abundance trends, which is critical for developing policy to protect our biosphere.

ecology↗

bioRxiv2022

Innovation and elaboration on the avian tree of life

1Widely documented, megaevolutionary jumps in phenotypic diversity continue to perplex researchers because it remains unclear whether these dramatic changes can emerge from microevolutionary processes. Here we tackle this question using new approaches for modeling multivariate traits to evaluate the magnitude and distribution of elaboration and innovation in the evolution of bird beaks. We find that elaboration, evolution along the major axis of phenotypic change, is common at both macro- and megaevo-lutionary scales whereas innovation, evolution away from the major axis of phenotypic change, is more prominent at megaevolutionary scales. Indeed, the major axis of phenotypic change among species beak shapes at megaevolutionary scales is an emergent property of innovation across clades. Our analyses suggest that the reorientation of phenotypes via innovation is a ubiquitous route for divergence that can arise through gradual change alone, opening up new avenues for evolution to explore.

evolutionary biology↗

bioRxiv2022

A novel fold for acyltransferase-3 (AT3) proteins provides a framework for transmembrane acyl-group transfer

Acylation of diverse carbohydrates occurs across all domains of life and can be catalysed by proteins with a membrane bound acyltransferase-3 (AT3) domain (PF01757). In bacteria, these proteins are essential in processes including symbiosis, resistance to viruses and antimicrobials, and biosynthesis of antibiotics, yet their structure and mechanism is largely unknown. In this study, evolutionary co-variance analysis was used to build a computational model of the structure of a bacterial O-antigen modifying acetyltransferase, OafB. The resulting structure exhibited a novel fold for the AT3 domain, which molecular dynamics simulations demonstrated is stable in the membrane. The AT3 domain contains 10 transmembrane helices arranged to form a large cytoplasmic cavity lined by residues known to be essential for function. Further molecular dynamics simulations support a model where the acyl-coA donor spans the membrane through accessing a pore created by movement of an important loop capping the inner cavity, enabling OafB to present the acetyl group close to the likely catalytic resides on the extracytoplasmic surface. Limited but important interactions with the fused SGNH domain in OafB are identified and modelling suggests this domain is mobile and can both accept acyl-groups from the AT3 and then reach beyond the membrane to reach acceptor substrates. Together this new general model of AT3 function provides a framework for the development of inhibitors that could abrogate critical functions of bacterial pathogens.

biochemistry↗