McRose Darcy L, Zhang Xinning, Kraepiel Anne M L, Morel François M M
Department of Geosciences, Princeton University, Princeton NJ, USA.
Department of Chemistry, Princeton University, Princeton NJ, USA.
Front Microbiol. 2017 Feb 28;8:267. doi: 10.3389/fmicb.2017.00267. eCollection 2017.
The nitrogenase enzyme, which catalyzes the reduction of N gas to NH, occurs as three separate isozyme that use Mo, Fe-only, or V. The majority of global nitrogen fixation is attributed to the more efficient 'canonical' Mo-nitrogenase, whereas Fe-only and V-('alternative') nitrogenases are often considered 'backup' enzymes, used when Mo is limiting. Yet, the environmental distribution and diversity of alternative nitrogenases remains largely unknown. We searched for alternative nitrogenase genes in sequenced genomes and used PacBio sequencing to explore the diversity of canonical () and alternative ( and ) nitrogenase amplicons in two coastal environments: the Florida Everglades and Sippewissett Marsh (MA). Genome-based searches identified an additional 25 species and 10 genera not previously known to encode alternative nitrogenases. Alternative nitrogenase amplicons were found in both Sippewissett Marsh and the Florida Everglades and their activity was further confirmed using newly developed isotopic techniques. Conserved amino acid sequences corresponding to cofactor ligands were also analyzed in and amplicons, offering insight into environmental variants of these motifs. This study increases the number of available and sequences ∼20-fold and allows for the first comparisons of environmental Mo-, Fe-only, and V-nitrogenase diversity. Our results suggest that alternative nitrogenases are maintained across a range of organisms and environments and that they can make important contributions to nitrogenase diversity and nitrogen fixation.
固氮酶可催化将氮气还原为氨，它以三种不同的同功酶形式存在，分别使用钼、仅含铁或钒。全球大部分的固氮作用归因于效率更高的“典型”钼固氮酶，而仅含铁和含钒（“替代”）固氮酶通常被视为“备用”酶，在钼含量有限时发挥作用。然而，替代固氮酶的环境分布和多样性在很大程度上仍不为人知。我们在已测序的基因组中搜索替代固氮酶基因，并使用PacBio测序技术来探究两种沿海环境——佛罗里达大沼泽地和锡普维西特沼泽（马萨诸塞州）中典型（钼固氮酶）和替代（仅含铁和含钒固氮酶）固氮酶扩增子的多样性。基于基因组的搜索鉴定出另外25个物种和10个属，这些物种和属之前并不知晓可编码替代固氮酶。在锡普维西特沼泽和佛罗里达大沼泽地均发现了替代固氮酶扩增子，并使用新开发的同位素技术进一步证实了它们的活性。还对仅含铁和含钒固氮酶扩增子中与辅因子配体相对应的保守氨基酸序列进行了分析，从而深入了解这些基序的环境变体。这项研究使可用的仅含铁和含钒固氮酶序列数量增加了约20倍，并首次实现了对环境中钼、仅含铁和含钒固氮酶多样性的比较。我们的结果表明，替代固氮酶在一系列生物体和环境中均得以保留，并且它们可为固氮酶多样性和固氮作用做出重要贡献。
