Abstract
The nitrogenase enzyme, which catalyzes the reduction of N2 gas to NH4+, occurs as three separate isozyme that use Mo, Fe-only, or V. The majority of global nitrogen fixation is attributed to the more efficient ‘canonical’ Mo-nitrogenase, whereas Fe-only and V-(‘alternative’) nitrogenases are often considered ‘backup’ enzymes, used when Mo is limiting. Yet, the environmental distribution and diversity of alternative nitrogenases remains largely unknown. We searched for alternative nitrogenase genes in sequenced genomes and used PacBio sequencing to explore the diversity of canonical (nifD) and alternative (anfD and vnfD) nitrogenase amplicons in two coastal environments: the Florida Everglades and Sippewissett Marsh (MA). Genome-based searches identified an additional 25 species and 10 genera not previously known to encode alternative nitrogenases. Alternative nitrogenase amplicons were found in both Sippewissett Marsh and the Florida Everglades and their activity was further confirmed using newly developed isotopic techniques. Conserved amino acid sequences corresponding to cofactor ligands were also analyzed in anfD and vnfD amplicons, offering insight into environmental variants of these motifs. This study increases the number of available anfD and vnfD sequences ∼20-fold and allows for the first comparisons of environmental Mo-, Fe-only, and V-nitrogenase diversity. Our results suggest that alternative nitrogenases are maintained across a range of organisms and environments and that they can make important contributions to nitrogenase diversity and nitrogen fixation.
Highlights
Nitrogen fixation is a biogeochemically important process that shapes the fertility of marine and terrestrial environments
Using a newly developed technique, the Isotopic Acetylene Reduction Assay (ISARA), which distinguishes between canonical and alternative N2fixation by measuring 13C isotopes in the acetylene reduction assay, we recently reported the activity of alternative nitrogenases in Sippewissett Marsh (Zhang et al, 2016)
In order to understand the taxonomic distribution of alternative nitrogenases, we searched for NifHDKENB, as well as AnfHDKG and VnfHDKG protein sequences in publically available genomes (GenBank, June 2015)
Summary
Nitrogen fixation is a biogeochemically important process that shapes the fertility of marine and terrestrial environments. Biological nitrogen fixation is catalyzed by the nitrogenase enzyme, which can occur as three different isozymes, that use either Mo, Fe-only, or V at the active site (Bishop et al, 1986; Robson et al, 1986). While all known diazotrophs encode a canonical Mo-nitrogenase, some encode an additional Fe-only nitrogenase, V-nitrogenase, or both. Due to their lower efficiency (Eady and Robson, 1984) compared with canonical nitrogenases, these ‘alternative’ nitrogenases are typically viewed as ‘backup’ enzymes, used only when Mo is not available. The exact conditions under which these enzymes are used in the environment remain unknown
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