Nitrogen fixation and transcriptome of a new diazotrophic Geomonas from paddy soils

ABSTRACTNitrogen gas (N2) fixation driven by diazotrophs is a crucial process for supplying nitrogen to paddy soil ecosystems. The genus Geomonas has been considered to be an important potential diazotroph in paddy soils, but direct experimental evidence of the nitrogen-fixing ability of Geomonas in pure culture is still lacking. Hence, we aimed to demonstrate this nitrogen-fixing capability and shed light on how this process was regulated in response to ammonium (NH4+) in Geomonas. In this study, we determined that a key nitrogenase gene (nifH) was present in 50 isolates from paddy soils. Members of Geomonas contained the minimum nitrogen fixation gene cluster (nifBHDKEN) based on genomic analysis, implying Geomonas species had the potential to fix nitrogen. Acetylene reduction assay (ARA), 15N2 isotope labeling, and total nitrogen accumulation assays validated that Geomonas was, indeed, able to fix nitrogen in pure culture. Under nitrogen-fixing conditions, the cell morphology of Geomonas changed from short rod-shaped (with NH4+) to long rod-shaped and flagella became longer and thicker. The expression of genes correlated to nitrogen fixation in the Geomonas transcriptome was quantified in response to NH4+. Expression of genes associated with nitrogenase, flavin-based electron bifurcation complexes (such as the FixAB system), NH4+ uptake, and transformation (e.g., glutamine and glutamate synthetases) were significantly upregulated under nitrogen-fixing conditions, suggesting these mechanisms might be involved in N2 fixation in Geomonas. These results were verified by RT-qPCR. Taken together, our results demonstrate that Geomonas species possess the ability to fix N2 and expand our understanding on the ecological significance and potential applications of Geomonas in paddy soil ecosystems.IMPORTANCEThe ability of Geomonas species to fix nitrogen gas (N2) is an important metabolic feature for its application as a plant growth-promoting rhizobacterium. This research is of great importance as it provides the first comprehensive direct experimental evidence of nitrogen fixation by the genus Geomonas in pure culture. We isolated a number of Geomonas strains from paddy soils and determined that nifH was present in these strains. This study demonstrated that these Geomonas species harbored genes encoding nitrogenase, as do Geobacter and Anaeromyxobacter in the same class of Deltaproteobacteria. We demonstrated N2-dependent growth of Geomonas and determined regulation of gene expression associated with nitrogen fixation. The research establishes and advances our understanding of nitrogen fixation in Geomonas.