Siderophore Synthesis Ability of the Nitrogen-Fixing Bacterium (NFB) GXGL-4A is Regulated at the Transcriptional Level by a Transcriptional Factor (trX) and an Aminomethyltransferase-Encoding Gene (amt).

Kosakonia radicincitans GXGL-4A, a gram-negative nitrogen-fixing (NF) bacterial strain is coated with a thick capsulatus on the surface of cell wall, which becomes a physical barrier for exogenous DNA to enter the cell, so the operation of genetic transformation is difficult. In this study, an optimized Tn5 transposon mutagenesis system was established by using a high osmotic HO-1 medium combined with the electroporation transformation. Eventually, a mutant library containing a total of 1633 Tn5 insertional mutants were established. Of these mutants, the mutants M81 and M107 were found to have an enhanced capability to synthesize siderophore through the CAS agar plate assay and the spectrophotometric determination. The bacterial cells of two mutants were applied in cucumber growth-promoting experiment. Cucumber seedlings treated with M81 and M107 cells had a significant increase in biomass including seedling height, seedling fresh weight, root fresh weight, and root length. The whole genome sequencing of the mutants M81 and M107 showed that the integration sites of Tn5 transposon element were located in MmyB-like helix-turn-helix transcription regulator (locus tag: A3780_19720, trX) and aminomethyltransferase-encoding genes (locus tag: A3780_01680, amt) in the genome of GXGL-4A, respectively. The ability of siderophore synthesis of the target mutants was improved by Tn5 insertion mutagenesis, and the mutants obtained showed a good plant growth-promoting effect when applied to the cucumber seedlings. The results suggest that the identified functional genes regulates the biosynthesis of siderophore in azotobacter GXGL-4A, and the specific mechanism needs to be further investigated.