Generation of Tn5 insertion mutations in nitrogen-fixing bacterium Kosakonia radicincitans GXGL-4A associated with maize

Abstract:

[Background] The long-term excessive use of chemical nitrogen fertilizers in agriculture has already caused ecological destruction of farmland. The direct consequences are soil compaction, secondary salinization and heavy metal pollution. Moreover, a large amount of nitrogen leaching loss of field could lead to eutrophication and groundwater pollution. The nitrate content of agricultural products may exceed standard, and eventually human health would be threatened due to the food chain. Thus, developing the biological nitrogen-fixing (NF) bacterial agents to reduce the use of chemical nitrogen fertilizer is of great significance for environmental protection and sustainable agriculture. [Objective] This study aims at creating abundant mutants of an associative NF bacterial strain, Kosakonia radicincitans GXGL-4A, isolated from maize root by Tn5 insertional mutagenesis and the results will benefit for further exploration of nitrogen fixation mechanism, nitrogen metabolism and related regulatory network, and lay a solid foundation for the application of GXGL-4A in agricultural production. [Methods] The nitrite reductase gene nirBD was cloned from K. radicincitans GXGL-4A by PCR. Meanwhile, a Tn5 transposon plasmid pMOD-egfp-tet was constructed and then transferred into the wild type GXGL-4A bacterial cells by electroporation to generate Tn5 insertional mutants. [Results] Large quantities of mutants were obtained, and four mutants with significant decrease in nitrite reductase activities comparing with the wild type strain GXGL-4A were identified. Subsequently, DNA sequence flanking integration site in the genome of mutant M36 were also isolated. [Conclusion] It is feasible and applicable for Tn5 transposon mutagenesis in the associative NF K. radicincitans strain GXGL-4A, and an effective and stable insertional mutagenesis system of this microorganism has been successfully established in this study.