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.
Highlights
Recent studies in deep marine waters have revealed active N-fixation and a wide diversity of non-cyanobacterial N-fixers (Farnelid et al, 2013; Bombar et al, 2016; Gradoville et al, 2017)
The prevalence of nif genes seemed to be coupled to photosynthetic bacteria, since nifH phylotype associated with Chlorobi were abundant in many samples (Figure 2)
Strategies for carbon fixation varied from WL pathway in Deltaproteobacteria to reductive tricarboxylic acid (rTCA) and reductive pentose phosphate cycle in Acidobacteria, while carbon fixation genes linked to photosynthesis were detected in Chlorobi
Summary
Recent studies in deep marine waters have revealed active N-fixation and a wide diversity of non-cyanobacterial N-fixers (Farnelid et al, 2013; Bombar et al, 2016; Gradoville et al, 2017). The metabolic process by which atmospheric dinitrogen gas (N2) is transformed to biologically more reactive forms is known as diazotrophy (Howarth et al, 1988). Very little is known about non-cyanobacterial diazotrophs in freshwater systems, while at the same time, many of these ecosystems feature steep redox gradients and anoxic zones that might provide unique habitats for previously unrecognized N-fixers. Select Archaea and Bacteria have the ability to transform and assimilate N2 gas, and the key enzyme to mediate this process is the nitrogenase (Canfield et al, 2010). Three nitrogenases co-exist in nature: molybdenumiron (Mo-nitrogenase), vanadium-iron, and iron-only (Fe-only nitrogenase). The corresponding gene clusters involved are nifHDK (molybdenum-iron), vnfHGDK (vanadium-iron), and anfHGDK (Fe-only). Vanadiumiron nitrogenase is present in some microorganisms and may be expressed when Mo is scarce. The other alternative nitrogenase, Fe-only, has dual enzymatic functions and may simultaneously reduce N2 and CO2 to CH4 and H2 in a single enzymatic step (Zheng et al, 2018)
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