Diazotroph Genomes and Their Seasonal Dynamics in a Stratified Humic Bog Lake

Abstract

Aquatic N-fixation is generally associated with the growth and mass development of Cyanobacteria in nitrogen-deprived photic zones. However, sequenced genomes and environmental surveys suggest active aquatic N-fixation also by many non-cyanobacterial groups. Here, we revealed the seasonal variation and genomic diversity of potential N-fixers in a humic bog lake using metagenomic data and nif gene clusters analysis. Groups with diazotrophic operons were functionally divergent and included Cholorobi, Geobacter, Desulfobacterales, Methylococcales, and Acidobacteria. In addition to nifH (a gene that encodes the dinitrogenase reductase component of the molybdenum nitrogenase), we also identified sequences corresponding to vanadium and iron-only nitrogenase genes. Within the Chlorobi population, the nitrogenase (nifH) cluster was included in a well-structured retrotransposon. Furthermore, the presence of light-harvesting photosynthesis genes implies that anoxygenic photosynthesis may fuel nitrogen fixation under the prevailing low-irradiance conditions. The presence of rnf genes (related to the expression of H⁺/Na⁺-translocating ferredoxin: NAD+ oxidoreductase) in Methylococcales and Desulfobacterales suggests that other energy-generating processes may drive the costly N-fixation in the absence of photosynthesis. The highly reducing environment of the anoxic bottom layer of Trout Bog Lake may thus also provide a suitable niche for active N-fixers and primary producers. While future studies on the activity of these potential N-fixers are needed to clarify their role in freshwater nitrogen cycling, the metagenomic data presented here enabled an initial characterization of previously overlooked diazotrophs in freshwater biomes.

Keywords: N-fixation; diazotrophs; hypolimnion; lake; nifH gene.

FIGURE 1

Comparison between nitrogenase gene abundances calculated from epilimnion and hypolimnion and environmental data during 2008 sampling campaign. (A) Nitrogenase gene percentages calculated using IMG annotations (bar plot: in red epilimnion and blue hypolimnion). The approximate depth and date when the DNA samples were collected are depicted with triangles (red = epilimnion and blue = hypolimnion). Dissolved oxygen (mg/L) is showed as a continuous heat map. (B) The relationship between TN:TP ratio (measured in part per billion) and the abundance of nitrogenase genes calculated from epilimnion and hypolimnion using IMG annotations. The polynomial function of non-linear regression is shown in red for hypolimnion and in black for epilimnion.

FIGURE 2

Relative contributions of different taxonomic groups to the potential diazotrophic community in Trout Bog Lake. The nitrogenase phylotype abundances were calculated using nifH reads identified from IMG annotations of assembled sequences and genomes using DNA data collected from hypolimnion.

FIGURE 3

Alignment of nitrogenase gene clusters of two MAGs corresponding to potential N-fixers classified as Methylococcales (I) Composite genome 3552 [scaffolds: (A) TH3552DRAFT TBhypo metabat 3552 10001230.12, (B) TH3552DRAFT TBhypo metabat 3552 10006655.34] and Composite genome 2062v2 [scaffolds: (A) TH02062DRAFT TH02062 TBL comb47 HYPODRAFT 10005551.84, (B) TH02062DRAFT TH02062 TBL comb47 HYPODRAFT 10000828.15]. (A) The nifHDK gene cluster and (B) accessory N-fixation genes. Coding region sequences (CDS) are colored according to GenBank and KO annotation, with orange representing unknown or unclassified genes. Details of the general functions attributed to the CDS are listed in the color legend.

FIGURE 4

Alignment of nitrogenase gene clusters of five MAGs corresponding to potential N-fixers classified as Deltaproteobacteria (three scaffolds) and Betaproteobacteria (two scaffolds). Deltaproteobacteria: (I) Composite genome 4645 (scaffold: TH4645DRAFT TBhypo metabat 4645 10001890.329), (II) Composite genome 2922v2 (scaffold: TH02922DRAFT TH02922 TBL comb47 HYPODRAFT 10000417.24), (III) Composite genome 433 (scaffold: TH433DRAFT TBhypo metabat 433 10016042.145). Betaproteobacteria: (I) Composite genome 2160 (scaffold: TH2160DRAFT TBhypo metabat 2160 10003883.171), (II) Composite genome 2159 (scaffold: TH2159DRAFT TBhypo metabat 2159 10000110.67). Coding region sequences (CDS) are colored according to GenBank and KO annotation, with orange representing genes unknown or unclassified. Details of the general functions attributed to the CDS are listed in the color legend.

FIGURE 5

Alignment of nitrogenase gene clusters of four MAGs corresponding to potential N-fixers classified as Chlorobi (I) Composite genome 111 (scaffold: TH111DRAFT TBhypo metabat 111 10000140.1), (II) Composite genome 211 (scaffold: TE211DRAFT TBepi metabat 211 1000175.54), (III) Composite genome 2493 (scaffold: TE2493DRAFT TBepi metabat 2493 1001483.68), (IV) Composite genome 3520v2 (scaffold: TH03520DRAFT TH03520 TBL comb47 HYPODRAFT 10004746.48). In the upper panel (Chlorobi I and II) is represented the nif gene clusters and in the lower panel (Chlorobi III and IV) the anf gene clusters. Coding region sequences (CDS) are colored according to GenBank and KO annotation, with orange representing genes unknown or unclassified. Details of the general functions attributed to the CDS are listed in the color legend.