Table_1_Diazotroph Genomes and Their Seasonal Dynamics in a Stratified Humic Bog Lake.DOCX

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.

水生固氮作用通常与氮限制透光带中的蓝细菌（Cyanobacteria）生长及大规模增殖相关。然而，已测序基因组与环境调查结果显示，诸多非蓝细菌类群也能介导活跃的水生固氮作用。本研究借助宏基因组数据与固氮基因簇（nif gene cluster）分析，揭示了腐殖质沼泽湖中潜在固氮微生物的季节动态与基因组多样性。携带固氮操纵子的类群功能分化显著，涵盖绿菌门（Chlorobi）、地杆菌属（Geobacter）、脱硫杆菌目（Desulfobacterales）、甲基球菌目（Methylococcales）以及酸杆菌门（Acidobacteria）。除编码钼固氮酶二氮酶还原酶亚基的nifH基因外，本研究还鉴定出对应钒固氮酶与铁铁固氮酶的基因序列。在绿菌门类群中，固氮酶（nifH）基因簇整合于结构完整的反转录转座子（retrotransposon）内部。此外，光捕获光合基因的存在表明，在低辐照主导的环境中，不产氧光合作用可为固氮过程提供能量支持。甲基球菌目与脱硫杆菌目中存在rnf基因（该基因编码氢/钠转运型铁氧还蛋白-NAD+氧化还原酶），这表明在缺乏光合作用的情况下，其他能量生成过程可驱动高能耗的固氮作用。特劳特沼泽湖（Trout Bog Lake）缺氧底层的强还原环境，可为活跃固氮微生物与初级生产者提供适宜的生态位。尽管未来需针对这些潜在固氮微生物的活性开展研究，以明确其在淡水氮循环中的作用，但本研究提供的宏基因组数据已首次对淡水生物群落中此前被忽视的固氮微生物进行了初步表征。