Distribution of nitrogen fixation and nitrogenase-like sequences amongst microbial genomes

Abstract

Background: The metabolic capacity for nitrogen fixation is known to be present in several prokaryotic species scattered across taxonomic groups. Experimental detection of nitrogen fixation in microbes requires species-specific conditions, making it difficult to obtain a comprehensive census of this trait. The recent and rapid increase in the availability of microbial genome sequences affords novel opportunities to re-examine the occurrence and distribution of nitrogen fixation genes. The current practice for computational prediction of nitrogen fixation is to use the presence of the nifH and/or nifD genes.

Results: Based on a careful comparison of the repertoire of nitrogen fixation genes in known diazotroph species we propose a new criterion for computational prediction of nitrogen fixation: the presence of a minimum set of six genes coding for structural and biosynthetic components, namely NifHDK and NifENB. Using this criterion, we conducted a comprehensive search in fully sequenced genomes and identified 149 diazotrophic species, including 82 known diazotrophs and 67 species not known to fix nitrogen. The taxonomic distribution of nitrogen fixation in Archaea was limited to the Euryarchaeota phylum; within the Bacteria domain we predict that nitrogen fixation occurs in 13 different phyla. Of these, seven phyla had not hitherto been known to contain species capable of nitrogen fixation. Our analyses also identified protein sequences that are similar to nitrogenase in organisms that do not meet the minimum-gene-set criteria. The existence of nitrogenase-like proteins lacking conserved co-factor ligands in both diazotrophs and non-diazotrophs suggests their potential for performing other, as yet unidentified, metabolic functions.

Conclusions: Our predictions expand the known phylogenetic diversity of nitrogen fixation, and suggest that this trait may be much more common in nature than it is currently thought. The diverse phylogenetic distribution of nitrogenase-like proteins indicates potential new roles for anciently duplicated and divergent members of this group of enzymes.

Figure 1

Genes involved in nitrogen fixation. Top- A. vinelandii nif gene regions. Gray-shaded trapezoids are essential genes in Mo-dependent nitrogen fixation that were used as queries for the in silico identification of nitrogen fixing species described in this study. Bottom –The proposed minimum set of genes required for nitrogen fixation. All species with sequenced genomes that are known diazotrophs and all the species proposed to be diazotrophs based on genetic content contain the minimum gene set.

Figure 2

Taxonomic diversity of nitrogen fixing species. Species with fully sequenced genomes (999 Bacteria and 93 Archaea genomes) were analyzed for the minimum set of nitrogen fixation ortholog genes. Taxonomic distribution of diazotrophic species based on experimental evidence (gray bars) and in silico prediction of nitrogen fixation (black bars) is displayed by phylum. The ratio of the number of proposed species versus the number of total distinct species with sequenced genomes within each phylum is indicated.

Figure 3

Alignment of residues flanking conserved FeMoco ligands in NifD/VnfD/AnfD proteins. Alignment of residues flanking the conserved co-factor ligands, Cys275 and His442, in the alpha subunit of Mo-dependent and alternative nitrogenases. (The sequence numbering refers to A. vinelandii NifD, Avin_01390.) Protein groups labeled A and B correspond to subfamilies 2 and 1 respectively, previously identified by Kechris et al. [23]. Group C represent the additional sub family described in the text. Group V corresponds to AnfD and VnfD sequences.

Figure 4

Maximum-likelihood phylogenetic tree of conventional nitrogenases and nitrogenase-like sequences. The tree is represented by a core set of 73 sequences, selected from a larger tree of 472 sequences. Shimodaira-Hasegawa local support values were >0.6 except for those nodes marked with a red star. The clade coloring reflects sequences that are co-located in genomes and likely to correspond to the alpha and beta subunits of nitrogenase, with the exception of those shown in light gray, which are single subunit enzymes (NflD). Dark blue clades are conventional nitrogenases, labeled as NifD/E and NifK/N respectively. Clades colored in light-green are NifD/E and NifK/N-like sequences in which the FeMoco ligand Cys 275 in the alpha component, is either present (dark green nodes) or absent (yellow nodes). In all other cases known FeMoco ligands are absent. The number of conserved Cys residues in each subunit that correspond to P cluster ligands in conventional nitrogenases are indicated for each clade.