Diazotrophic <i>Anaeromyxobacter</i> Isolates from Soils

Biological nitrogen fixation is an essential reaction in a major pathway for supplying nitrogen to terrestrial environments. Previous culture-independent analyses based on soil DNA/RNA/protein sequencing could globally detect the nitrogenase genes/proteins of <i>Anaeromyxobacter</i> (in the class <i>Deltaproteobacteria</i>), commonly distributed in soil environments and predominant in paddy soils; this suggests the importance of <i>Anaeromyxobacter</i> in nitrogen fixation in soil environments. However, direct experimental evidence is lacking; there has been no research on the genetic background and ability of <i>Anaeromyxobacter</i> to fix nitrogen. Therefore, we verified the diazotrophy of <i>Anaeromyxobacter</i> based on both genomic and culture-dependent analyses using <i>Anaeromyxobacter</i> sp. strains PSR-1 and Red267 isolated from soils. Based on the comparison of <i>nif</i> gene clusters, strains PSR-1 and Red267 as well as strains Fw109-5, K, and diazotrophic <i>Geobacter</i> and <i>Pelobacter</i> in the class <i>Deltaproteobacteria</i> contain the minimum set of genes for nitrogenase (<i>nifBHDKEN</i>). These results imply that <i>Anaeromyxobacter</i> species have the ability to fix nitrogen. In fact, <i>Anaeromyxobacter</i> PSR-1 and Red267 exhibited N₂-dependent growth and acetylene reduction activity (ARA) <i>in vitro</i> Transcriptional activity of the <i>nif</i> gene was also detected when both strains were cultured with N₂ gas as a sole nitrogen source, indicating that <i>Anaeromyxobacter</i> can fix and assimilate N₂ gas by nitrogenase. In addition, PSR-1- or Red267-inoculated soil showed ARA activity and the growth of the inoculated strains on the basis of RNA-based analysis, demonstrating that <i>Anaeromyxobacter</i> can fix nitrogen in the paddy soil environment. Our study provides novel insights into the pivotal environmental function, i.e., nitrogen fixation, of <i>Anaeromyxobacter</i>, which is a common soil bacterium.<b>IMPORTANCE</b><i>Anaeromyxobacter</i> is globally distributed in soil environments, especially predominant in paddy soils. Current studies based on environmental DNA/RNA analyses frequently detect gene fragments encoding nitrogenase of <i>Anaeromyxobacter</i> from various soil environments. Although the importance of <i>Anaeromyxobacter</i> as a diazotroph in nature has been suggested by culture-independent studies, there has been no solid evidence and validation from genomic and culture-based analyses that <i>Anaeromyxobacter</i> fixes nitrogen. This study demonstrates that <i>Anaeromyxobacter</i> harboring nitrogenase genes exhibits diazotrophic ability; moreover, N₂-dependent growth was demonstrated <i>in vitro</i> and in the soil environment. Our findings indicate that nitrogen fixation is important for <i>Anaeromyxobacter</i> to survive under nitrogen-deficient environments and provide a novel insight into the environmental function of <i>Anaeromyxobacter</i>, which is a common bacterium in soils.