Department of Civil and Environmental Engineering, University of Wisconsin-Madison, Madison, WI, USA.
Department of Microbiology, Institute for Water and Wetland Research, Radboud University, Nijmegen, the Netherlands.
ISME J. 2021 Mar;15(3):673-687. doi: 10.1038/s41396-020-00805-w. Epub 2020 Oct 20.
Anaerobic ammonium-oxidizing (anammox) bacteria mediate a key step in the biogeochemical nitrogen cycle and have been applied worldwide for the energy-efficient removal of nitrogen from wastewater. However, outside their core energy metabolism, little is known about the metabolic networks driving anammox bacterial anabolism and use of different carbon and energy substrates beyond genome-based predictions. Here, we experimentally resolved the central carbon metabolism of the anammox bacterium Candidatus 'Kuenenia stuttgartiensis' using time-series C and H isotope tracing, metabolomics, and isotopically nonstationary metabolic flux analysis. Our findings confirm predicted metabolic pathways used for CO fixation, central metabolism, and amino acid biosynthesis in K. stuttgartiensis, and reveal several instances where genomic predictions are not supported by in vivo metabolic fluxes. This includes the use of the oxidative branch of an incomplete tricarboxylic acid cycle for alpha-ketoglutarate biosynthesis, despite the genome not having an annotated citrate synthase. We also demonstrate that K. stuttgartiensis is able to directly assimilate extracellular formate via the Wood-Ljungdahl pathway instead of oxidizing it completely to CO followed by reassimilation. In contrast, our data suggest that K. stuttgartiensis is not capable of using acetate as a carbon or energy source in situ and that acetate oxidation occurred via the metabolic activity of a low-abundance microorganism in the bioreactor's side population. Together, these findings provide a foundation for understanding the carbon metabolism of anammox bacteria at a systems-level and will inform future studies aimed at elucidating factors governing their function and niche differentiation in natural and engineered ecosystems.
厌氧氨氧化（anammox）细菌介导了生物地球化学氮循环中的一个关键步骤，并且已经在全球范围内被用于从废水中高效去除氮。然而，除了核心能量代谢之外，对于驱动厌氧氨氧化细菌合成代谢的代谢网络以及除了基于基因组预测之外利用不同碳和能源底物的情况，人们知之甚少。在这里，我们使用时间序列 C 和 H 同位素示踪、代谢组学和同位素非稳态代谢通量分析，实验解析了厌氧氨氧化细菌 Candidatus 'Kuenenia stuttgartiensis' 的中心碳代谢。我们的研究结果证实了 K. stuttgartiensis 中用于 CO 固定、中心代谢和氨基酸生物合成的预测代谢途径，并揭示了一些基因组预测与体内代谢通量不相符的情况。这包括使用不完全三羧酸循环的氧化分支来合成α-酮戊二酸，尽管基因组中没有注释的柠檬酸合酶。我们还证明，K. stuttgartiensis 能够通过 Wood-Ljungdahl 途径直接同化细胞外的甲酸盐，而不是将其完全氧化为 CO 后再进行再同化。相比之下，我们的数据表明，K. stuttgartiensis 不能在原位将乙酸作为碳源或能源利用，并且乙酸氧化是通过生物反应器侧流群中低丰度微生物的代谢活性发生的。总之，这些发现为在系统水平上理解厌氧氨氧化细菌的碳代谢提供了基础，并将为未来阐明其在自然和工程生态系统中的功能和生态位分化的因素的研究提供信息。
