Creating a Genetic Toolbox for the Carbon-Fixing, Nitrogen-Fixing and Dehalogenating Bacterium <i>Xanthobacter autotrophicus</i>

<i>Xanthobacter autotrophicus</i> is a metabolically flexible microorganism with two key features: (1) The organism has adapted to grow on a wide variety of carbon sources including CO₂, methanol, formate, propylene, haloalkanes and haloacids; and (2) <i>X. autotrophicus</i> was the first chemoautotroph identified that could also simultaneously fix N₂, meaning the organism can utilize CO₂, N₂, and H₂ for growth. This metabolic flexibility has enabled use of <i>X. autotrophicus</i> for gas fixation, the creation of fertilizers and foods from gases, and the dehalogenation of environmental contaminants. Despite the wide variety of applications that have already been demonstrated for this organism, there are few genetic tools available to explore and exploit its metabolism. Here, we report a genetic toolbox for use in <i>X. autotrophicus</i>. We first identified suitable origins of replication and quantified their copy number, and identified antibiotic resistance cassettes that could be used as selectable markers. We then tested several constitutive and inducible promoters and terminators and quantified their promoter strengths and termination efficiencies. Finally, we demonstrated that gene expression tools remain effective under both autotrophic and dehalogenative metabolic conditions to show that these tools can be used in the environments that make <i>X. autotrophicus</i> unique. Our extensive characterization of these tools in <i>X. autotrophicus</i> will enable genetic and metabolic engineering to optimize production of fertilizers and foods from gases, and enable bioremediation of halogenated environmental contaminants.