Artificial selection of Stutzerimonas stutzeri MBI-RS3 towards enhanced nitrogen fixation in presence of ammonia and oxygen

Abstract

Food security is a primary concern for every region of the world. Today, the agricultural sector relies heavily on artificial fertilizers to maintain crop production and yield. This has an unsustainable dependency that has exceeded the planetary boundary for nitrogen fertilizer usage for decades. Biologically, nitrogen nutrition can be provided by nitrogen fixation carried out by bacteria in the rhizosphere of crops. Nitrogen fixation is done by an enzyme called nitrogenase. This enzyme converts biologically inaccessible N2 from the atmosphere and into NH4, which is then readily available in the soil. Nitrogenase expression and activity are heavily downregulated by oxygen and biologically available nitrogen, making it challenging for bacteria to balance respiration, proliferation, and nitrogen fixation when subject to levels of oxygen and nitrogen. Bacteria capable of nitrogen fixation can be endosymbiotic or free-living. Endosymbiotic nitrogen-fixing bacteria interact specifically with legume plants by triggering nodulation. Free-living nitrogen-fixing bacteria can colonize legume and non-legume rhizospheres, opening possible environments where free-living nitrogen-fixing bacteria can survive and fix nitrogen. Free-living nitrogen-fixing bacteria are exposed to a wide range of oxygen and nitrogen concentrations, forcing them to adapt more extensively than endosymbiotic nitrogen-fixing bacteria. The genetic modification of free-living nitrogen-fixing bacteria can give an advantage in the race against artificial fertilizers, contributing to a more sustainable and resilient agriculture. Stutzerimonas stutzeri MBI-RS3 is a free-living bacterium that is capable of fixing nitrogen. This thesis focuses on exposing strain MBI-RS3 to UV energy, targeting a 99% kill rate and generating random mutations in the 1% survivors. A plasmid construct carrying a fusion of nif promoter and gus was developed. The UV-irradiated culture was recovered, transformed with a reporter gene plasmid pFT1NP and screened for blue colonies. Forty-three isolates were recovered and cryopreserved. Six candidates (FT11, FT16, FT22, FT34, FT38, FT40) were characterized by β-Glucuronidase assay, identifying mutant FT11 as the sample with the strongest nif gene expression. MBI-RS3 wildtype and mutants were inoculated in tomatoes, soybeans, and canola to assess their plant growth-promoting activity in different conditions. This showed a stronger performance of the mutant groups (FT11, FT16, FT22, FT38) compared to the wild-type in tomatoes, with higher shoot dry weight production. Mutant FT11 and FT22 showed some potential to make up for the decrease in nitrogen fertilizer use while maintaining yield. Mutants FT16 and FT 40 showed more pod and shoot dry weight in soybeans than wildtype in the presence of Bradyrhizobium sp. The MBI-RS3 wild-type was the best performer in terms of dry weight production of canola pods and shoots. These results demonstrate that not only MBI-RS3 wild-type but also some mutants exhibit plant growth-promoting activity, with a particular focus on nitrogen nutrition. Furthermore, in the tomato and soybean rhizosphere, the mutants induce higher yields and biomass production compared to the wild-type. All MBI-RS3 6 mutants and wild type were assessed by acetylene reduction assay to confirm nitrogenase activity, but only the MBI-RS3 wildtype strain showed ethylene production.