Accurate prediction of nitrogen fixation in cyanobacteria reveals the dynamic evolution driving high retention rate with mosaic distribution

Abstract Cyanobacteria are the only prokaryotic group capable of performing oxygenic photosynthesis and nitrogen fixation (catalyzed by extremely oxygen-sensitive nitrogenase) simultaneously. A systematic understanding of the phylogenetic distribution of nitrogen-fixing ability remains incomplete due to difficulties in accurately identifying genes essential for nitrogen fixation from genomic data, caused by occasional confusion with nitrogenase-like enzymes and fragmentation of nif genes in heterocystous species. In this study, we comprehensively evaluated the distribution of the minimal nitrogen fixation gene set ( nifHDKENB ) across 586 major cyanobacterial strains. Overall, 48% of the strains were predicted to be nitrogen-fixing with a mosaic distribution in non-heterocystous strains. Using the results as reference data, we evaluated nitrogen fixation across a broader range of cyanobacterial lineages, including uncultured strains and metagenomes. We predicted that approximately 20% of 2,718 strains are nitrogen fixers, revealing a high nif retention rate of nif genes in the Cyanobacteria comparing with other bacterial phyla. While most cyanobacteria carry Group I nif genes, we identified widely distributed lineages with Group II nif genes, some of which are associated with distinct CnfR proteins. We discuss the dynamic evolutionary history of nif genes in cyanobacteria.