Sustainable biofuels can be produced using agricultural feedstocks rich in lignocellulose, such as corn stalks and barley straw—low-cost, renewable feedstocks that are typically discarded. However, lignocellulose is both physically and chemically challenging to degrade. Anaerocellum bescii, a thermophilic bacterium that thrives at ~75°C, is a promising candidate for biorefining processes due to its powerful arsenal of carbohydrate-active enzymes (CAZymes), which can efficiently break down lignocellulose and convert it to ethanol and other biofuels. However, efforts to metabolically engineer A. bescii are hindered by a limited understanding of its ATP-binding cassette (ABC) sugar transport systems, which play a vital role in importing extracellular oligosaccharides into the cell. The Xylan Degradation Locus (XDL) and Conserved Xylan Utilization Locus (CXUL) are involved in xylose utilization and together encode three putative ABC sugar transporters. The XDL includes AxoFGE (Athe_0174–0176) and XloEFG (Athe_0179–0181), with Athe_0174 and Athe_0181 as the associated substrate binding proteins, while the CXUL gene cluster xynUVW (Athe_0614–0616) encodes Athe_0614. By heterologously expressing and purifying these proteins, followed by ligand screening using differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC), we identified their hemicellulosic sugar preferences and roles in transport. Additionally, ROSIE docking simulations, contact residue mapping, and sequence alignments with homologous proteins revealed conserved amino acids involved in xylo-oligosaccharide recognition. This work advances our understanding of how A. bescii utilizes diverse hemicellulosic substrates, accelerating its development as a platform for sustainable bioproduction of biofuels and other valuable chemicals.

Plant root exudates shape the structure and function of the rhizosphere microbiome by providing chemical cues and substrates that influence microbial survival, colonization, and interaction with the host. These exudates can select for beneficial microbes that support plant growth or suppress immunity, making them critical in mediating plant-microbe interactions. This study focuses on two Arabidopsis thaliana (Arabidopsis) associated, immunity-suppressive bacterial strains, Dyella japonica MF79 (MF79) and Brevundimonas sp. MF374 (MF374), which use distinct genetic mechanisms to suppress root immune responses. To evaluate how the root exudate composition influences bacterial fitness, transposon mutant libraries of MF374 and MF79 were grown in exudates derived from Arabidopsis Col-0, fls2 mutant, and cyp79b2/b3 mutant lines. Genome-wide fitness profiling using random barcode transposon-site sequencing (RB-TnSeq) and Random Forest classification revealed minimal fitness changes in response to flg22 treatment, but strong genotype-specific fitness differences driven by root exudate composition. In MF374, candidate genes involved in osmotic regulation, cell wall synthesis, oxidative stress response, and translation were identified as major contributors to fitness in root exudate environments. Targeted gene deletions confirmed reduced fitness in immune-active or metabolite-rich exudates; however, root colonization assays demonstrated that these fitness effects did not always translate to differences in colonization capacity. This framework enabled the identification and functional assessment of bacterial genes important for exudate-mediated fitness and adaptation. The findings offer insight into how variation in root exudate composition influences the assembly, persistence, and adaptation of immunity-suppressive bacteria within the root microbiome and have broad implications for understanding plant health and developing strategies for microbiome-based agricultural applications.

Agricultural systems around the world are using increasingly large amounts of nitrogen, phosphate, and potassium synthetic fertilizers to support modern-day demands. Unfortunately, these fertilizers contribute to greenhouse gas emissions and wash off into natural waterways, posing environmental harm. Climate change further exacerbates the stresses on agricultural systems, leaving them susceptible to crop disease and reduced productivity. A consequence of excessive synthetic fertilizer use and climate change stress is plant nutrient imbalance. Nutrients are essential for a variety of necessary functions; without them, the plant will face stunted growth, low yield production, and other negative side effects. While the Earth’s soil is abundant in nutrients, most of them are bound in soil minerals. Geochemical release processes like mineral weathering are very slow, but minerals can also be processed for plant uptake via microbial mineralization mechanisms. Rhizospheric microbes play a crucial role in making nutrients available for plant uptake. Nitrogen-fixing, phosphate-solubilizing, and potassium-solubilizing bacteria are essential in making these macronutrients bioavailable to support plant growth. This project sought to uncover the potassium-solubilizing bacteria (KSB) within the Arabidopsis and Brachypodium root-microbiome and elucidate the key genes and mechanisms that these KSB employ to make nutrients available for plant uptake. Furthermore, using transposon mutagenesis, this project constructed a mutant library of a select strain, Rhizobacterium sp. PA090, to further investigate the KSB genome. This work will be used to inform further research and guide the development of biofertilizers as a sustainable alternative to the environmentally harmful chemical fertilizers.

Global agricultural production must increase by up to 70% by 2050 to meet the demands of a growing population (1). Sustainable agriculture, harnessing the power of naturally occurring plant-associated microbes, offers a promising way to improve agricultural productivity. Optimizing the performance of beneficial microbes will require a deeper understanding of how these bacteria colonize roots and interact with the plant immune system. Traditional tools for microbial tracking such as 16S rRNA sequencing, fluorescence markers, or antibiotic resistance are often limited by resolution, bias, and poor compatibility with soil environments (17-19). This study applies Wild-Type Isogenic Standardized Hybrid (WISH) tagging, a novel microbial barcoding technique developed by Daniel et al. (2024), to track and quantify root colonization dynamics with (20). In this study, WISH tags were inserted into the following bacterial strains: Pseudomonas simiae WCS417, Dyella japonica MF79 wildtype, and its mutant, a knockout of the immunosuppressive subtilase A (IssA) gene. Sequencing of WISH-tagged populations revealed a competitive advantage of the ΔIssA mutant over the wild-type, indicating that immune suppression traits may incur fitness trade-offs. Overall, this work demonstrates the power of WISH-tagging for high-resolution analysis of plant–microbe interactions and underscores its potential as a tool for future microbiome engineering and sustainable agricultural innovation.