Site-directed mutagenesis to deactivate two nitrogenase isozymes of Kosakonia radicincitans DSM16656T

Abstract

Biological nitrogen fixation (BNF) is considered one of the key plant-growth-promoting (PGP) factors for diazotrophic organisms. Whether the iron and iron–molybdenum nitrogenases of Kosakonia radicincitans contribute to its PGP effect is yet to be proven. Hence, for the first time, we conducted site-directed mutagenesis in K. radicincitans to knock out anfH and (or) nifH as a mean to deactivate BNF in this strain. We used 15N2-labeled air to trace BNF activities in ΔanfH, ΔnifH, and ΔanfHΔnifH mutants. Assessing bacterial growth, nitrogen content, and 15N incorporation revealed that BNF is impaired in K. radicincitans DSM16656T ΔnifH and ΔanfHΔnifH. However, we detected no significant contribution of the Fe nitrogenase to biological dinitrogen assimilation under our pure bacterial culture experimental conditions. Such nondiazotrophic K. radicincitans DSM16656T mutants represent excellent tools for investigating nitrogen nutrition in K. radicincitans-inoculated plants.