Biology subjects

Mulligan, C.

Publications and source records attributed to Mulligan, C..

10 recordsLinked to original sources

bioRxiv2026

Effects of Reduced Watering Regimes on Hops Growth and Rhizospheric Microbial Communities

Marked by shifts in historic climatic patterns, the effects of climate change have begun to disrupt biological life worldwide. In agricultural systems, the abiotic effects of climate change such as increased temperatures and reduced rainfall extend beyond crop yield and can be felt at the ecological level through microbial community shifts associated at the interface of the root/soil matrix, known as the rhizosphere. Shifts in rhizospheric community composition have been known to greatly impact crop health, making common garden studies examining the totality of climate related impacts on crops of the utmost importance. Using a combination of both phenotypic and metabarcoding data collected from the rhizosphere, we present a multi-month drought tolerance experiment using the cash crop Humulus lupulus (hops) and five of its cultivars; Cascade, Centennial, Chinook, Comet and CTZ. Statistical analyses of phenotypic data revealed that hops displayed a differential impact on reduced watering strategies while rhizospheric data revealed more consistent patterns of association between microbes and cultivar type. We hope results from our study can be used to further expand our knowledge on the impact of future climate change and necessary reduced watering strategies on agricultural systems, providing insight into potential mechanisms of response for drought tolerance in crop systems.

plant biology↗

bioRxiv2026

Structural modelling and functional analysis support lipid binding by the dimeric E. coli DedA protein YqjA

Maintenance of membrane homeostasis is essential for bacterial viability, yet the molecular functions of many membrane proteins involved in this process remain poorly understood. The widely distributed DedA superfamily of integral membrane proteins has been implicated in membrane homeostasis, with deletion of DedA genes resulting in sensitivities to temperature, pH and a range of antimicrobial compounds. Several bacterial DedA proteins have been linked to lipid transport, providing a potential connection between function and phenotype. However, a lack of direct functional and structural data means that the precise role of DedA proteins in bacterial membrane homeostasis remains unclear. Here, using analytical ultracentrifugation (AUC), we show that the Escherichia coli DedA protein YqjA exists predominantly as a dimer and, by combining structural modelling with site-specific cysteine crosslinking, we have identified the most likely dimer interface. Modelling of dimeric YqjA in the presence of lipids predicts an interfacial lipid-binding site located in close proximity to several conserved, functionally important residues. Consistent with lipid binding, addition of lipid substantially increased the thermal stability of YqjA. Furthermore, mutation of residues associated with the predicted binding site impaired YqjA function. Together, these findings provide new structural and functional insights into the DedA family and support a role for lipid binding in the activity of YqjA.

biochemistry↗

bioRxiv2026

COPAL: An ensemble of protein-ligand co-folding models enriches preferred ligand predictions for the LuxR-family of quorum sensing receptors

Acyl-homoserine lactone (AHL) quorum sensing enables many species of proteobacteria to coordinate collective behaviors. In such systems, a synthase produces an AHL signal, which is sensed by a LuxR-family receptor. Despite extensive genomic annotation of LuxR homologs, preferred AHLs for most receptors remain unknown, limiting functional understanding of quorum sensing across diverse bacteria. Here, we present the COPAL (combining ordered predictions of audited ligands) pipeline, which integrates multiple protein-ligand co-folding models to identify preferred AHLs for a specific LuxR. Benchmarking on a leakage-controlled subset of 96 experimentally characterized LuxR-AHL pairs shows that COPAL places the preferred AHL within the top-6 candidates (out of 58) for 68% of receptors, outperforming every individual co-folding model. Further, inter-model agreement correlates with ranking accuracy, offering an indication of confidence. We show that COPAL resolves the specificity shift induced by three-point mutations in LasR and correctly nominates C8-HSL as the preferred ligand for the previously uncharacterized Mesorhizobium sp. NJ3 receptor, which we verified experimentally. Finally, we release the Ranked AHL-LuxR Prediction Hub (RALPH), comprising precomputed rankings for about 10,000 unique LuxR homologs. More broadly, COPAL shows that unweighted rank aggregation of complementary co-folding models offers a general strategy for predicting receptor-ligand specificity in data-scarce biological systems.

biochemistry↗

bioRxiv2026

Monitoring seagrass meadow health using coastal bird fecal fatty acid biomarkers

Lipids have the potential to trace trophic interactions through an ecosystem, but their use has been largely limited to predator/prey-scale studies. Carnivore fecal lipids offer an unexplored way to strategically monitor the health and spatial distribution of ecosystems that are difficult to access. This hypothesis is explored here in seagrass meadows, which are a globally significant long-term carbon storage reservoir. This study tested the hypothesis that lipids can be used to passively monitor seagrass meadow health by tracing fatty acids transferred up the food chain and onto nearby beaches via coastal bird stool. By comparing the lipids produced by seagrasses and those made by diverse microbial eukaryotes, such as diatoms and dinoflagellates, our analysis could distinguish between healthy and collapsed meadows in a pilot dataset. A metabolite that is produced by the seagrass wasting disease pathogen was also recovered in our data, providing a potential diagnostic for seagrass health. The advantages to this non-invasive monitoring approach using lipids are that beaches are far more accessible than seagrass meadows, and birds spatially sample a larger area than researchers could do efficiently. Although this pilot study was limited in samples and field sites, our results provide compelling reasons to pursue carnivorous fecal dietary lipids as a source of ecosystem data.

ecology↗

bioRxiv2024

A new class of binding protein-dependent solute transporter exemplified by the TAXI-GltS system from Bordetella pertussis

Tripartite ATP-dependent periplasmic (TRAP) transporters are widespread in prokaryotes, but absent in eukaryotes, and facilitate the uptake of a wide variety of substrates. TRAP transporters are composed of a substrate binding protein (SBP) and two unequally sized membrane components that exist as either separate proteins or are fused into a single polypeptide. Almost all TRAP SBPs exist as monomeric globular proteins that bind substrate and present it to the membrane component. Here, we describe the discovery and characterisation of a novel TRAP SBP from the TAXI subfamily with a previously unidentified architecture. BP0403 from the human pathogen Bordetella pertussis is a predicted lipoprotein composed of 3 distinct domains; an /{beta} globular domain with a unique fold, a long helical domain and a C-terminal TAXI SBP domain. Expression and purification of the full-length protein reveals that it forms a stable dimer. Structural modelling of the dimer interface and molecular weight analysis using size exclusion chromatography of the individual domains reveals that the interdomain helical region is solely responsible for dimerization. Differential scanning fluorimetry (DSF) and intrinsic tyrosine fluorescence reveal that BP0403 binds L-glutamate with nanomolar affinity. Unexpectedly, analysis of the genome context of BP0403 reveals the complete absence of characteristic genes for TRAP membrane components but co-localisation and translational coupling with gltS, encoding a Na+/glutamate symporter. In other bacteria, we identified fused BP0403-GltS homologues, strongly suggesting that this constitutes a completely novel SBP-dependent secondary active transporter. Structural comparisons suggest GltS operates by an elevator-type mechanism, like TRAP transporters; the association of an SBP with this class of secondary transporter is an emerging theme.

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↗

bioRxiv2022

Elevator mechanism dynamics in a sodium-coupled dicarboxylate transporter

VcINDY, the sodium-dependent dicarboxylate transporter from Vibrio cholerae, is responsible for C4- and C5-carboxylate uptake into cells. The molecular mechanism of how VcINDY physically moves substrates across the membrane, and does so in an energetically efficient manner, is unclear. Here, we use single-molecule fluorescence resonance energy transfer experiments to directly observe the individual mechanistic steps that VcINDY takes to translocate substrates across a lipid bilayer, and then test key predictions of transport cycle mechanistic models. Our data provide the first direct evidence that VcINDY undergoes stochastic, elevator-type conformational motions that enable substrate translocation. Kinetic analysis suggests that the two protomers of the VcINDY homodimer undergo those motions in a non-cooperative manner, and thus catalyze two independent transport reactions. The relative substrate independence of those motions supports the notion that the VcINDY transport cycle maintains strict co-substrate coupling using a cooperative binding mechanism. Finally, thermodynamic modeling provides insight into how such a cooperative binding mechanism provides a generalized approach to optimizing transport for many secondary active transporters. Significance StatementTransporter proteins use energy to move molecular materials into and out of cells. To be efficient, the transporter motions responsible for moving the molecules must be tightly choreographed to avoid wasting energy without transporting anything. By measuring the motions and kinetics of a prototypical transporter (VcINDY) at the single-molecule level, this study finds the first evidence that transporters like VcINDY achieve efficient transport by coordinating constantly dynamic, "elevator-type" motions while sitting in the cellular membrane. The efficiency of these surprisingly dynamic transporters is then revealed by thermodynamic modeling, which explains the molecular basis behind how highly cooperative, substrate binding reactions may have evolved as the optimal strategy for maximizing transporter efficiency.

biophysics↗

bioRxiv2022

Structural basis of ion - substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY

The Na+-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na+ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen induced-fit mechanism. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HPinb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDYs induced-fit mechanism is a result of the sodium-dependent formation of the substrate binding site.

biophysics↗

bioRxiv2021

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance.

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake, and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we have performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli. By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we have mapped the topology of the protein. Using these data, we have experimentally validated a structural model of YqjA generated using evolutionary co-variance, which consists of an -helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggests that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

biochemistry↗

bioRxiv2020

Substrate-dependent accessibility changes in the Na+-dependent C4-dicarboxylate transporter, VcINDY, suggest differential substrate effects in a multistep mechanism.

Members of the divalent anion sodium symporter (DASS) family (SLC13 in humans) play critical roles in metabolic homeostasis, influencing many processes including fatty acid synthesis, insulin resistance, adiposity, and lifespan determination. DASS transporters catalyse the Na+-driven concentrative uptake of Krebs cycle intermediates and sulfate into cells; disrupting their function can protect against age-related metabolic diseases and can extend lifespan. An inward-facing crystal structure and an outward-facing model of a bacterial DASS family member, VcINDY from Vibrio cholerae, predict an elevator-like transport mechanism involving a large rigid body movement of the substrate binding site. How substrate binding influences the conformational state of VcINDY is currently unknown. Here, we probe the interaction between substrate binding and VcINDY conformation using a site-specific alkylation strategy to probe the solvent accessibility of several broadly distributed positions in VcINDY in the presence and absence of substrates (Na+ and succinate). Our findings reveal that accessibility to all positions tested can be modulated by the presence of substrates, with the majority becoming less accessible in the presence of Na+. Mapping these solvent accessibility changes onto the known structures of VcINDY suggests that Na+ binding drives the transporter into an as-yet-unidentified intermediate state. We also observe substantial, separable effects of Na+ and succinate binding at several amino acid positions suggesting distinct effects of the two substrates. Furthermore, analysis of a solely succinate-sensitive residue indicates that VcINDY binds its substrate with a low affinity and proceeds via an ordered process in which one or more Na+ ions must bind prior to succinate. These findings provide insight into the mechanism of VcINDY, which is currently the only structural-characterised representative of the entire DASS family.

biochemistry↗