Nitrogen fixing soil bacteria can provide plants with reduced nitrogen, an essential nutrient, and offer an ecological alternative to synthetic fertilizers, reducing leaching, denitrification, and volatilization. Our group has previously characterized two plant associated N-fixing bacteria, Kosakonia sacchari and Klebsiella variicola, and edited key gene targets to enhance nitrogen fixation and excretion, even in the presence of excess nitrogen. Greenhouse and field studies have demonstrated that these remodeled strains enhance plant growth, nitrogen uptake, and yields compared to untreated crops.
To improve on this initial success and identify better microbes that deliver higher levels of fixed nitrogen at the root tips, it is critical to identify the molecular drivers of the microbial establishment and activity on the plant roots. However, the complex nature of the soil matrix complicates the downstream analyses of root-microbe interactions using analytical and biological tools. The synthetic ecosystems developed by EMSL provide a powerful platform for characterizing the mechanical and molecular interactions of these nitrogen fixing microbes with plant roots and plant exudates. Computational simulations on the “rhizosphere on a chip”, which enables growing small plants on transparent soil, demonstrated that the physical soil structure leads to exudate “hotspots” that occur at relevant timescales to microbial processes. We hypothesize that these metabolite rich hotspots will show increased microbial colonization and nitrogenase activity, which will lead to higher nitrogen content in the surrounding root tissue. To see if there is an overlap between exudate hotspots and microbial colonization and activity, we propose inoculating seeds with fluorescently tagged K. sacchari and K. variicola strains and imaging them using fluorescent microscope and MALDI-MS. We hypothesize that microbes engineered to enhance nitrogen fixation may have a colonization defect due to the increased metabolic burden of constitutively expressing pathway. We will test this hypothesis by measuring total microbial fluorescence on, near, and within plant roots from the wildtype strain compared to an enhanced nitrogen fixer. We additionally hypothesize that engineered microbes will provide more N to the plant compared to wildtype or nifH knock-out strains, and that quantity of N is proportional to the colonization density. Confocal Raman spectroscopy will be employed to test this hypothesis by monitoring plant nitrogen status to determine if the root regions colonized with nitrogen fixing bacteria show higher nitrogen content. To further characterize and differentiate microbial molecular responses from those of plants, we will encapsulate the microbes in EMSL’s synthetic soil and run multi-omics analysis of microbes in the presence and absence of a growing root. Transcriptomic and metabolomic analyses on the encapsulated microbes will uncover the microbial pathways activated during root colonization, while the reciprocal analyses on the root samples will be used to identify plant biomarkers and metabolites that are indicative of microbial presence.
In sum, this work will help characterize microbial and plant molecular pathways that facilitate a mutualistic relationship where microbes thrive on root exudates and deliver fixed nitrogen to the plant roots. Elucidating these mechanisms will enable us isolate and engineer better bacterial inoculants that will improve soil-plant nutrient management.