Biology subjects

Scott, F. J.

Publications and source records attributed to Scott, F. J..

5 recordsLinked to original sources

bioRxiv2026

Precursor-Dependent Routing of Aromatic Amino Acids Determines Lignin Structure in Grasses by Sensitivity-Enhanced Solid-State NMR

Lignin biosynthesis in grasses exhibits unique metabolic flexibility, yet the precursor-specific routing of carbon into lignin polymers remains poorly resolved in planta. Here, we combine 13C-isotope labeling with solid-state NMR under sensitivity-enhancement by dynamic nuclear polarization (DNP), to directly track phenylalanine- and tyrosine-derived carbon incorporation into the lignin polymer in Brachypodium distachyon. Precursor-specific 13C labeling reveals that phenylalanine is the dominant contributor to canonical guaiacyl and syringyl lignins, whereas tyrosine preferentially enriches hydroxyphenyl lignin and hydroxycinnamates, including ferulates characteristic of grass cell walls. Two-dimensional 13C-13C correlation NMR resolves distinct lignin moieties arising from each precursor. Disruption of p-coumarate 3-hydroxylase (C3H) selectively impairs phenylalanine-derived lignification, while tyrosine-derived lignin remains comparatively unchanged, maintaining polymer assembly through alternative metabolic routes. These findings show precursor-dependent control of lignin composition and reveal tyrosine-mediated lignification as a compensatory pathway in grasses. This work also establishes precursor-resolved solid-state NMR and DNP as a powerful framework for dissecting lignin biosynthesis and metabolic plasticity in plant cell walls. SIGNIFICANCE STATEMENTLignin is a complex plant polymer that strengthens cell walls but also limits the efficiency of biomass processing for agriculture and bioenergy. Grasses possess a unique lignin biosynthetic flexibility that is not well understood. By combining stable isotope labeling with solid-state NMR spectroscopy, we directly traced how the aromatic amino acids, phenylalanine and tyrosine, contribute differently to lignin formation in intact grass cell walls. We show that phenylalanine primarily builds conventional lignin structures, whereas tyrosine supplies alternative phenolic components and maintains lignin synthesis even when a key biosynthetic enzyme is disrupted. This metabolic flexibility helps explain the unique structural aspects of grass cell walls and identifies precursor-level control as a promising strategy for engineering lignin composition to improve biomass utilization.

plant biology↗

bioRxiv2025

Nutrient-limited conditions reveal the activity of a minor groove binder, MGB-BP-3, against Escherichia coli

BackgroundAntimicrobial resistance demands new antibiotics, but conventional screening in nutrient-rich media often fails to predict in vivo efficacy, especially against Gram-negative bacteria. Minor groove binders (MGB), such as MGB-BP-3, are a promising new antibacterial with potent activity against Gram-positive bacteria but thought to have limited potency against Gram-negative bacteria. ObjectivesThis study evaluated the antibacterial activity of MGB-BP-3 under nutrient-limited, physiologically relevant conditions to better mimic in vivo environments. MethodsMinimum inhibitory concentrations (MICs) were determined against a panel of ESKAPEE pathogens in both rich (Cation Adjusted Mueller-Hinton Broth, CA-MHB) and nutrient-limited media (Roswell Park Memorial Institute, RPMI-1640 + 10% Luria-Bertani, LB). Synergy with efflux pump inhibitors and membrane permeabilizers was evaluated via checkerboard assays. Permeability was assessed using a fluorescent DNA-binding cell permeant (Hoechst 33342) accumulation assay. MICs were determined in the presence of different concentrations of ions, using both wild-type and efflux-compromised strains. In vivo efficacy was assessed using the Galleria mellonella infection model. ResultsThe activity of MGB-BP-3 against Gram-negative bacteria was improved by shifting the medium from rich to nutrient-limited conditions. Specifically, it showed potent activity against E. coli in nutrient-limited media (MIC 3.1 {micro}M) but was inactive in rich media (MIC 700 {micro}M). Synergy with efflux inhibitors occurred mainly in rich media, indicating barriers to intracellular accumulation were not present in nutrient-limited conditions. Increased permeability correlated with enhanced susceptibility and efflux-deficient strains were more susceptible, confirming the role of efflux pumps in resistance. MGB-BP-3 provided dose-dependent protection in G. mellonella larvae infected with E. coli or S. aureus. For example, at concentrations of 12.5 {micro}M and 100 {micro}M, MGB-BP-3 was able to protect the larvae by more than 70% over 5 days of treatment, respectively. ConclusionsNutrient-limited media better reveal MGB-BP-3s activity against Gram-negative bacteria and align with in vivo efficacy, highlighting the need for host-mimicking conditions in antibiotic screening.

microbiology↗

bioRxiv2025

Elucidation of Radical Degradation in Native Biofilms by EPR Sheds Light on Bacterial Resistance and Efficient DNP Solid-state NMR

Bacterial biofilms exhibit enhanced antimicrobial resistance, yet the mechanisms determining molecular transport and reactivity within these complex communities remain poorly understood. Here, we combine electron paramagnetic resonance (EPR), solid-state NMR (ssNMR), and dynamic nuclear polarization (DNP) ssNMR to determine how native Pseudomonas fluorescens Pf0-1 colony biofilms regulate nitroxide radicals. EPR measurements reveal that radical reduction depends on biofilm morphology, hydration, and composition of extracellular matrix (ECM). Wild-type biofilms exhibit slower nitroxide reduction than planktonic cells, while isolated ECM and dehydrated biofilms show no radical reduction, indicating that bacterial cells primarily drive radical reduction. Complementary ssNMR measurements identify polysaccharides and lipids within the ECM as primary interaction sites for nitroxide radicals. DNP-enhanced ssNMR further reveals compositional differences across biofilm morphologies, with increasingly polysaccharide-rich ECM environments correlating with slower reduction kinetics. These findings support a mechanism in which the ECM acts as a diffusion barrier and selective interaction region for nitroxide radicals to regulate cellular penetration. This work showcases an integrated magnetic resonance approach that provides molecular insight into how biofilm structure determines the fate of toxic redox-active small molecules. We also set the stage for high-sensitivity measurements of structure-function relationships in these medically relevant assemblies.

biophysics↗

bioRxiv2024

Unveiling Cell Wall Structure and Echinocandin Response in Candida auris and Candida albicans via Solid-State NMR

Invasive candidiasis affects 1.6 million people annually, implicating high mortality and morbidity in immunocompromised and hospitalized patients. Echinocandins, inhibitors of {beta}-1,3-glucan synthesis, are used as a first-line treatment; however, their efficacy is increasingly compromised by resistance and tolerance. To understand how echinocandins remodel Candida cell wall structures, thereby reducing drug effectiveness, this study compares the effects of echinocandin exposure on the cell walls of the prevalent pathogen Candida albicans and the recently emerged multidrug-resistant superbug Candida auris. High-resolution solid-state NMR analysis revealed a conserved cell wall structure in both species, with a rigid inner layer composed of closely associated chitin microfibrils and {beta}-1,3-glucans, supported by a flexible network of {beta}-1,6-glucans and additional {beta}-1,3-glucans. Despite the presence of N-mannan fibrils in the outer layer, mannan components are mobile and rely on -1,2-linked mannoside sidechains to maintain contact with chitin and {beta}-1,3-glucans. Caspofungin treatment rigidifies certain mannan sidechains and {beta}-1, 6-glucans to reinforce the cell wall in response to the depletion of most {beta}-1,3-glucans. While caspofungin treatment reduced water permeability in both species, only C. albicans responded by inducing cell wall thickening and changes in chitin and {beta}-1,3-glucan dynamics. Furthermore, the deletion of KRE6 genes encoding {beta}-1,6-glucan synthase reduced the echinocandin susceptibility of C. auris, and the impaired {beta}-1,6-glucan biosynthesis were offset by compensatory upregulation of this wall component due to caspofungin treatment. The profound alterations induced by caspofungin in Candida cell wall architecture suggest that cell wall structural contribute substantially to drug resistance and tolerance.

microbiology↗

bioRxiv2024

Comparative Analysis of Polysaccharide and Cell Wall Structure in Aspergillus nidulans and Aspergillus fumigatus by Solid-State NMR

Invasive aspergillosis poses a significant threat to immunocompromised patients, leading to high mortality rates associated with these infections. Targeting the biosynthesis of cell wall carbohydrates is a promising strategy for antifungal drug development and will be advanced by a molecular-level understanding of the native structures of polysaccharides within their cellular context. Solid-state NMR spectroscopy has recently provided detailed insights into the cell wall organization of Aspergillus fumigatus, but genetic and biochemical evidence highlights species-specific differences among Aspergillus species. In this study, we employed a combination of 13C, 15N, and 1H-detection solid-state NMR, supplemented by Dynamic Nuclear Polarization (DNP), to compare the structural organization of cell wall polymers and their assembly in the cell walls of A. fumigatus and A. nidulans, both of which are key model organisms and human pathogens. The two species exhibited a similar rigid core architecture, consisting of chitin, -glucan, and {beta}-glucan, which contributed to comparable cell wall properties, including polymer dynamics, water retention, and supramolecular organization. However, differences were observed in the chitin, galactosaminogalactan, protein, and lipid content, as well as in the dynamics of galactomannan and the structure of the glucan matrix.

biophysics↗