(489a) Creating Stable Mutants in the Plant Growth-Promoting Polyploid Rhodopseudomonas Palustris CGA009

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.

The nitrogen-fixing, plant-growth promoting Rhodopseudomonas palustris CGA009 is closely related to multiple endosymbiotic bacteria but has not been found within the cell walls of any plant. The long-term goal is to engineer this bacterium to nodulate the legume Aeschynomene evenia while resolving the prerequisites for triggering the symbiotic signal transduction pathway employed by both rhizobacteria and arbuscular mycorrhizal fungi. A. evenia is nodulated by Bradyrhizobium sp. ORS278, R. palustris’ closest photosynthetic neighbor, through an elementary Nod factor-independent process. The novel strategy of adding and removing the few genes that separate R. palustris from Bradyrhizobium sp. ORS278 could help reveal the requirements of symbiosis. Sucrose counterselection has been used to successfully alter R. palustris’ chromosome; although, tens of colonies must be screened to find a single stable mutant. The CRISPR gene editing system utilizes a less demanding workflow with the promise of higher efficiency. Yet, R. palustris' intrinsic resistance to antibiotics, affinity for homologous recombination, and previously unreported multiple chromosomes introduce challenges to engineering this bacterium by any method. The results from multiple strategies for editing R. palustris’ chromosomes with the CRISPR system will be presented. The lessons learned from creating stable mutants of this metabolically versatile soil bacterium could also be applied to other recalcitrant yet potentially powerful non-model microorganisms.