Biology subjects

Geddes, B. A.

Publications and source records attributed to Geddes, B. A..

5 recordsLinked to original sources

bioRxiv2025

Host-Specific Fluorescence Dynamics in Legume-Rhizobia Symbiosis During Nodulation

The legume-rhizobia symbiosis is a cornerstone of sustainable agriculture due to its ability to facilitate biological nitrogen fixation. Still, real-time visualization and quantification of this interaction remain technically challenging, especially across different host backgrounds. In this study, we systematically evaluate the efficacy of the nitrogenase system nifH promoter (PnifH) in driving expression of distinct fluorescent reporters; superfolder yellow fluorescent protein (sfYFP), superfolder cyan fluorescent protein (sfCFP), and various red fluorescent proteins (RFPs) within root nodules of determinate (Lotus japonicus-Mesorhizobium japonicum) and indeterminate (Pisum sativum-Rhizobium leguminosarum) systems. We show that PnifH-driven sfYFP and sfCFP yield strong, uniform, and reproducible fluorescence in nodules of both systems, facilitating reliable quantification of nodulation traits and strain occupancy. In contrast, RFPs including monomeric (mScarlet-I, mRFP1, mARs1) and multimeric (AzamiRed1.0) variants exhibited weak or inconsistent signals in pea. Notably, fluorescent labeling did not impair rhizobial competitiveness for root nodule occupancy, and PnifH-driven sfYFP and sfCFP reporters enabled robust multiplexed imaging in single-root and split-root assays. In the lotus, mScarlet-I worked robustly and facilitated a tripartite strain labeling system. Complementing our molecular toolkit, we established a deep learning-based analytical pipeline for high-throughput, automated quantification of nodulation traits, validated against standard ImageJ analysis. Altogether, our results identify PnifH-driven sfYFP and sfCFP as robust, broadly applicable reporters for legume-rhizobia symbiosis studies, while highlighting the need for optimized red fluorophores in some contexts. The integration of validated promoter-reporter constructs with state-of-the-art computational approaches provides a scalable framework for dissecting the spatial and competitive dynamics of plant-microbe mutualisms. IMPORTANCEThe legume-rhizobia symbiosis is central to sustainable agriculture through its capacity for biological nitrogen fixation, yet tools for real-time, quantitative visualization of this interaction remain limited. Here, we demonstrate that the nifH promoter (PnifH) effectively drives expression of superfolder yellow (sfYFP) and cyan (sfCFP) fluorescent proteins in both determinate (Lotus japonicus-Mesorhizobium japonicum) and indeterminate (Pisum sativum-Rhizobium leguminosarum) nodules. These reporters enable robust, reproducible fluorescence without impairing rhizobial competitiveness, supporting multiplexed imaging and quantitative nodulation analyses. By contrast, red fluorescent proteins exhibited host-dependent variability, underscoring the need for improved red fluorophores. Integration of validated promoter-reporter constructs with a deep learning-based image analysis pipeline establishes a scalable framework for high-throughput assessment of nodule occupancy and symbiotic dynamics. This work provides a practical molecular and computational toolkit for dissecting plant-microbe mutualisms across diverse host systems.

bioengineering↗

bioRxiv2025

Culturomics from field-grown crop plants using dilution-to-extinction, two-step library preparation and amplicon sequencing

Culturomics approaches have advanced microbial research by enabling the high-throughput isolation and characterization of a broader range of bacterial taxa, including some previously considered unculturable. Here, we present the testing and optimization of a protocol for isolating and identifying hundreds of cultivable microbes from field-grown plants. This protocol was tested and optimized using the root microbiomes of field-grown corn and pea plants under varying environmental conditions in North Dakota, USA. By employing dilution-to-extinction culturing and a two-step barcoding PCR strategy targeting the V4 region of the 16S rRNA gene, we identified over 200 unique bacterial isolates. The optimized bioinformatic pipeline, built around the DADA2 package, ensured accurate amplicon sequence variant (ASV) detection and taxonomy assignment. The resulting bacterial isolates span diverse phylogenetic groups, including plant-associated taxa known for promoting plant growth and mitigating stress. Our findings highlight the value of culturomics in generating microbial collections for synthetic community design and advancing plant-microbe interaction research. The protocols scalability, cost-effectiveness, and robust performance demonstrate its potential for widespread application in agricultural microbiome studies. Impact statementHigh-throughput isolation and characterization of cultivable microbes from plant microbiomes is crucial for advancing microbiome research. However, efficiently recovering a diverse range of bacterial taxa remains a challenge due to high costs and labor-intensive protocols. Our optimized culturomics protocol integrates dilution-to-extinction culturing with a two-step barcoding PCR strategy, enhancing recovery rates and reducing costs while maintaining high accuracy. By employing next-generation sequencing (NGS) and a streamlined bioinformatic pipeline built around the robust DADA2 workflow for amplicon sequencing, the method enables the scalable recovery of hundreds of unique bacterial isolates. This approach makes significant advancements over traditional culturing methods and other high-throughput cultivation protocols, providing an efficient, cost-effective platform for generating microbial collections essential for synthetic communities, comparative genomics, and agricultural microbiome applications. Data summaryAll protocols, sequence data, and analysis codes have been made publicly available in open-access repositories. The authors confirm all supporting data, code, and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2025

Adaptation of Plasmid-ID Technology for Evaluation of N2-Fixing Effectiveness and Competitiveness for Root Nodulation in the Sinorhizobium-Medicago System

Maximizing the nitrogen fixation occurring in rhizobia-legume associations represents an opportunity to sustainably reduce nitrogen fertilizer inputs in agriculture. High-throughput measurement of symbiotic traits has the potential to accelerate the identification of elite rhizobium/legume associations and enable novel research approaches. Plasmid-ID technology, recently deployed in Rhizobium leguminosarum, facilitates the concurrent assessment of rhizobium nitrogen-fixing effectiveness and competitiveness for root nodulation. This study adapts Plasmid-ID technology to function in Sinorhizobium species that are central models for studying rhizobium-legume associations and form economically important symbioses with alfalfa. New Sino-Plasmid-IDs were developed and tested for stability and their ability to measure competitiveness for root nodulation and nitrogen-fixing effectiveness. Rhizobial competitiveness is measured by identifying strain-specific nucleotide barcodes using Next-Generation Sequencing while effectiveness is measured by GFP fluorescence driven by the synthetic nifH promoter. Sino-Plasmid-IDs allow researchers to efficiently study competitiveness and effectiveness in a multitude of Sinorhizobium strains simultaneously.

microbiology↗

bioRxiv2024

Improved Efficiency of Two-Step Amplicon PCR Using an Acoustic Liquid Handler

2.The improvement in next-generation sequencing technologies has reduced the costs of sequencing significantly. However, library preparation costs for amplicon sequencing have remained largely unchanged - which is ultimately the cost-limiting step in processing large numbers of microbiome samples. Acoustic liquid handlers can transfer volumes as low as 2.5 nL and have been used to miniaturize several different molecular and cellular assays, including single-step polymerase chain reaction (PCR) amplicon library preparations. However, there are no current methods available for a two-step library preparation process using an acoustic liquid handler. In this study, we tested the efficiency of an acoustic liquid handler to automate the PCRs and library quantification while also incorporating automated library bead cleanup. We compared the material usage and costs for library preparation and sequencing results of this automated method to the standard, manual method. The automated protocol was able to reduce both PCR reaction volumes five-fold and increased efficiency for library preparation by [~]32% without affecting bacterial community compositions. The associated increase in efficiency of our automated method will allow for greater throughput in sequencing hundreds of microbiome samples without affecting the quality of those sequences. 3. Impact statementLibrary preparation for amplicon sequencing in microbiome studies remains a significant cost constraint, even while sequencing costs have decreased in the last few decades. Acoustic liquid handling robots have been utilized to miniaturize several different assays, including a one-step PCR library preparation process, to reduce costs. However, there are currently no methods available for utilizing acoustic liquid handlers for two-step PCR library preparation. Here, we show that the incorporation of an acoustic liquid handler and automated purification instruments into a two-step library preparation protocol led to a reduction in costs and time to prepare libraries. This method is comparable to the standard manual method and will lead to significant cost savings for the preparation of hundreds of samples required for microbiome studies. 4. Data summaryAll protocols, sequence data, and analysis codes have been made publicly available in open-access repositories. The authors confirm all supporting data, code, and protocols have been provided within the article or through supplementary data files. Sequencing data was deposited in the NCBI SRA under the BioProject PRJNA1190462. R scripts used for data analysis are available at https://github.com/NDSU-Geddes-Lab/brb-libprep doi: 10.5281/zenodo.14194699. Protocols for automated library preparation have been published on protocols.io and are available at dx.doi.org/10.17504/protocols.io.e6nvwb5r2vmk/v2 and dx.doi.org/10.17504/protocols.io.e6nvwb5w2vmk/v2.

microbiology↗

bioRxiv2024

BEVA2.0: Modular Assembly of Golden Gate-Compatible Vectors with Expanded Utility for Genetic Engineering

This expansion for the modular vector assembly platform BEVA (Bacterial Expression Vector Archive) introduces 11 new BEVA parts including two new cloning site variants, two new antibiotic resistance modules, three new origins of replication, and four new accessary modules. As a result, the modular system is now doubled in size and expanded in its capacity to produce diverse replicating plasmids. Furthermore, it is now amenable to genetic engineering methods involving genome-manipulation of target strains through deletions or integrations. In addition to introducing the new modules, we provide several BEVA-derived Golden Gate cloning plasmids that are used to validate parts and that may be useful for genetic engineering of proteobacteria and other bacteria. We also introduce new parts to allow compatibility with the CIDAR MoClo parts libraries.

synthetic biology↗