Methanosarcina acetivorans simultaneously produces molybdenum, vanadium, and iron-only nitrogenases in response to fixed nitrogen and molybdenum depletion

All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N2) to ammonia. Some diazotrophs also contain alternative nitrogenases that lack Mo: vanadium (V) and iron-only (Fe) nitrogenases. Among diazotrophs, the regulation and usage of the alternative nitrogenases in methanogens is largely unknown. Methanosarcina acetivorans contains nif, vnf, and anf gene clusters encoding putative Mo-, V-, and Fe-nitrogenases, respectively. This study investigated the effect of fixed nitrogen and Mo/V availability on nitrogenase expression and growth by M. acetivorans. The availability of Mo and V did not affect growth of M. acetivorans with fixed nitrogen but significantly affected growth with N2. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N2 in medium containing Mo. Depletion of Mo (Fe-only condition) resulted in a significant decrease in growth rate and cell yield. The addition of V to Mo-depleted medium stimulated diazotrophic growth but was still less than growth in Mo-replete medium. qPCR analysis revealed transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo. However, vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being the key factor. Immunoblot analysis revealed Mo-nitrogenase is produced when fixed nitrogen is depleted regardless of Mo availability, while V- and Fe-nitrogenases are produced only in the absence of fixed nitrogen and Mo. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and that all three nitrogenases can be simultaneously produced. IMPORTANCEMethanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. As such, methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, even simultaneously. Surprisingly, Mo-nitrogenase was produced in cells grown in the absence of Mo, indicating components of Mo-nitrogenase regulate or are needed to produce V- and Fe-nitrogenases in methanogens. The results provide a foundation to understanding the assembly, regulation, and activity of the alternative nitrogenases in methanogens.