Expression of V-nitrogenase and Fe-nitrogenase in Methanosarcina acetivorans is controlled by molybdenum, fixed nitrogen, and the expression of Mo-nitrogenase
Expression of V-nitrogenase and Fe-nitrogenase in Methanosarcina acetivorans is controlled by molybdenum, fixed nitrogen, and the expression of Mo-nitrogenase
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DOI：
10.1128/aem.01033-23
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发表时间：
2023-09-28
影响因子：
4.4
通讯作者：
Lessner, Daniel J.
中科院分区：
文献类型：
--
作者：
Chanderban, Melissa;Hill, Christopher A.;Lessner, Daniel J.
所有固氮细菌和古细菌（固氮菌）都使用钼 (Mo) 固氮酶将二氮 (N-2) 还原为氨，有些还含有缺乏 Mo 的钒 (V) 和纯铁 (Fe) 固氮酶。在固氮菌中，甲烷菌中替代 V-固氮酶和 Fe-固氮酶的调节和使用 很大程度上不为人所知。乙酰甲烷八叠球菌含有 nif、vnf 和 anf 基因簇，分别编码推定的 Mo-固氮酶、V-固氮酶和 Fe-固氮酶。本研究调查了 M. acetivorans 对固定氮、Mo/V 可用性以及 nif、vnf 和/或 anf 基因簇的 CRISPRi 抑制的固氮酶表达和生长。 Mo 和 V 的可用性显着影响 N-2 中的 M. acetivorans 生长，但 NH4Cl 中则没有。 M. acetivorans 在含有 Mo 的培养基中使用 N-2 生长期间，表现出最快的生长速率和最高的细胞产量，而在缺乏 Mo 和 V 的培养基中生长最慢。 qPCR 分析显示，nif 操纵子的转录仅受固定氮和 Mo 消耗的中度影响，而当固定氮和 Mo 消耗时，vnf 和 anf 转录显着增加，其中 Mo 的去除是关键。免疫印迹分析显示，无论 Mo 的可用性如何，当固定氮耗尽时，都会检测到 Mo 固氮酶，而 V 固氮酶和 Fe 固氮酶仅在固定氮和 Mo 不存在的情况下才能检测到。CRISPRi 抑制研究表明，V 固氮酶和/或 Fe 固氮酶是不依赖 Mo 的固氮营养所必需的，并且出乎意料的是，Mo 固氮酶的表达是 也需要。这些结果表明，M. acetivorans 中替代固氮酶的产生受到严格控制，并依赖于 Mo 固氮酶的表达。 重要性 产甲烷菌和密切相关的甲烷氧化菌是唯一已知或预测具有固氮酶的古细菌。产甲烷菌在全球生物氮和碳循环中发挥着关键作用。此外，产甲烷菌是一种古老的微生物谱系，固氮酶可能起源于产甲烷菌。了解固氮酶在产甲烷菌中的用途和特性可以为固氮的演变提供新的见解，并有助于开发基于固氮酶的生物技术。这项研究提供了第一个证据，证明产甲烷菌可以产生所有三种形式的固氮酶，包括同时产生。结果揭示了产甲烷菌中钼固氮酶的成分可以调节或产生 V 固氮酶和铁固氮酶，这是在细菌中未发现的结果。总的来说，这项研究为了解产甲烷菌中替代固氮酶的组装、调节和活性奠定了基础。
All nitrogen-fixing bacteria and archaea (diazotrophs) use molybdenum (Mo) nitrogenase to reduce dinitrogen (N-2) to ammonia, with some also containing vanadium (V) and iron-only (Fe) nitrogenases that lack Mo. Among diazotrophs, the regulation and usage of the alternative V-nitrogenase and Fe-nitrogenase in methanogens are largely unknown. Methanosarcina acetivorans contains nif, vnf, and anf gene clusters encoding putative Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase, respectively. This study investigated nitrogenase expression and growth by M. acetivorans in response to fixed nitrogen, Mo/V availability, and CRISPRi repression of the nif, vnf, and/or anf gene clusters. The availability of Mo and V significantly affected growth of M. acetivorans with N-2 but not with NH4Cl. M. acetivorans exhibited the fastest growth rate and highest cell yield during growth with N-2 in medium containing Mo, and the slowest growth in medium lacking Mo and V. qPCR analysis revealed the transcription of the nif operon is only moderately affected by depletion of fixed nitrogen and Mo, whereas vnf and anf transcription increased significantly when fixed nitrogen and Mo were depleted, with removal of Mo being key. Immunoblot analysis revealed Mo-nitrogenase is detected when fixed nitrogen is depleted regardless of Mo availability, while V-nitrogenase and Fe-nitrogenase are detected only in the absence of fixed nitrogen and Mo. CRISPRi repression studies revealed that V-nitrogenase and/or Fe-nitrogenase are required for Mo-independent diazotrophy, and unexpectedly that the expression of Mo-nitrogenase is also required. These results reveal that alternative nitrogenase production in M. acetivorans is tightly controlled and dependent on Mo-nitrogenase expression.IMPORTANCE Methanogens and closely related methanotrophs are the only archaea known or predicted to possess nitrogenase. Methanogens play critical roles in both the global biological nitrogen and carbon cycles. Moreover, methanogens are an ancient microbial lineage and nitrogenase likely originated in methanogens. An understanding of the usage and properties of nitrogenases in methanogens can provide new insight into the evolution of nitrogen fixation and aid in the development nitrogenase-based biotechnology. This study provides the first evidence that a methanogen can produce all three forms of nitrogenases, including simultaneously. The results reveal components of Mo-nitrogenase regulate or are needed to produce V-nitrogenase and Fe-nitrogenase in methanogens, a result not seen in bacteria. Overall, this study provides a foundation to understand the assembly, regulation, and activity of the alternative nitrogenases in methanogens.