(728d) Engineering a Purple Non-Sulfur Bacterium to Expand Symbiotic Nitrogen Fixation

The production of nearly 50% of the world’s food relies on ammonia fertilizer. While this source of bioavailable nitrogen has led to a marked increase in the number of people that can be fed, the impact on the environment is striking. Ammonia production requires more energy and releases more carbon dioxide than any other industrial reaction, and up to 90% of the product is used to manufacture fertilizers. Furthermore, soil microbes quickly convert ammonia to nitrate, which is easily washed into our water supply. Elevated levels of nitrates can be toxic to humans and animals. Sustainable innovations are required to maintain nutritional security while reducing the environmental damage. When nitrogen-stressed, legumes form an endosymbiotic relationship with bacteria that can fix the critical nutrient. Unraveling the mechanisms responsible for this symbiosis could lead to the expansion of the relationship to cereal crops and a decrease in agriculture’s reliance on nitrogen fertilizers. Using the more basic Nod factor-independent relationship between the legume Aeschynomene evenia (A. evenia) and the photosynthetic bacterium Bradyrhizobium sp. ORS278 as a template, the long-term goal is to engineer the closely-related, non-endosymbiotic Rhodopseudomonas palustris CGA009 (R. palustris) to nodulate the legume while resolving the prerequisites for triggering the symbiotic signal transduction pathway employed by both rhizobacteria and arbuscular mycorrhizal fungi.

Determining R. palustris’ response to A. evenia’s Nodule-specific Cysteine-Rich (NCR) peptides is the first step towards this goal and the focus of this work. NCR peptides are expressed in the nodules of some legumes, such as A. evenia, and cause their symbiotic partner to differentiate into nitrogen-producing bacteroids. It has been shown that the NCR peptides from some legumes induce signs of differentiation in their symbiotic partner in vitro, but kill other bacteria. Since R. palustris harbors the proteins that have been shown to be essential for the Bradyrhizobium sp. ORS278’s differentiation in A. evenia, the hypothesis is that R. palustris and Bradyrhizobium sp. ORS278 will respond similarly to the in vitro treatment with the legume’s NCR peptides. Changes in both bacteria’s ploidy, morphology, membrane permeability, and gene expression, all signs of differentiation, will be presented, in addition to work towards consistently creating knockout strains in the polyploid R. palustris using CRISPR.