Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans.
Control of nitrogen fixation and ammonia excretion in Azorhizobium caulinodans.
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DOI：
10.1371/journal.pgen.1010276
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发表时间：
2022-06
期刊：
影响因子：
4.5
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中科院分区：
文献类型：
--
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由于固氮 (N) 的能量需求昂贵，固氮细菌进化出了复杂的调控网络，仅在缺氮条件下才允许催化剂固氮酶的表达，而相同的条件会刺激谷氨酰胺合成酶 (GS) 对高亲和力氨 (NH3) 同化的上调，从而防止植物过量释放过量的 NH3。固氮细菌可以通过干扰 GS 进行改造以分泌 NH3，但是需要进行控制以最大程度地减少生长损失并防止无意中向非目标植物提供 NH3。在这里，我们在我们的模型谷物共生体 Azorhizobium caulinodans AcLP（ORS571 的衍生物）中测试了两种控制 GS 调节和 NH3 排泄的策略。我们首先尝试回顾之前的工作，其中 PII 同源物 glnB 和 glnK 的突变刺激 GS 关闭，但发现其中一个基因对于生长至关重要。其次，我们在 AcLP 的 ΔglnE 突变体中表达单向腺苷酸转移酶（uAT），该突变体允许强烈的 GS 关闭和 N2 固定产生的 NH3 的排泄，并完全减轻对固氮酶表达的负反馈调节。我们将 uAT 等位基因置于 NifA 依赖性启动子 PnifH 的控制下，允许 GS 关闭和 NH3 排泄，特别是在微氧条件下，这与启动 N2 固定的提示相同，然后删除 nifA 并将根氮平 nifAL94Q/D95Q-rpoN 控制质粒转移到该菌株中，从而允许 N2 的耦合根氮平依赖性激活 固定和 NH3 排泄。这种高度复杂的多层控制电路使我们离“合成共生体”的发展又近了一步，其中N2固定和NH3排泄可以在定殖于转基因根氮平生产谷物的固氮细菌中被特异性激活，从而将固定氮输送到作物，同时防止与非目标植物相互作用。给谷类作物接种可将大气中的氮 (N2) 转化为氨 (NH3) 的缔合固氮细菌，可用于可持续地改善作物的氮输送。然而，由于固定 N2 需要昂贵的能量，细菌限制了 NH3 的过量生产并释放到植物中。可以对固氮菌进行改造，以产生和释放过量的 NH3，但是需要进行遗传控制，以最大限度地减少生长损失并防止无意中向非目标杂草物种提供 NH3。在这里，我们设计了 N2 固定和 NH3 释放的耦合控制，以响应体外补充的信号分子根佐平。该控制电路代表了“合成共生”未来发展的原型，其中细菌的 N2 固定和 NH3 排泄可以在生产转基因根氮平的谷物在田间定植后被特异性激活，从而最大限度地减少细菌的能量需求并防止向非目标植物提供 NH3。
Due to the costly energy demands of nitrogen (N) fixation, diazotrophic bacteria have evolved complex regulatory networks that permit expression of the catalyst nitrogenase only under conditions of N starvation, whereas the same condition stimulates upregulation of high-affinity ammonia (NH3) assimilation by glutamine synthetase (GS), preventing excess release of excess NH3 for plants. Diazotrophic bacteria can be engineered to excrete NH3 by interference with GS, however control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target plants. Here, we tested two strategies to control GS regulation and NH3 excretion in our model cereal symbiont Azorhizobium caulinodans AcLP, a derivative of ORS571. We first attempted to recapitulate previous work where mutation of both PII homologues glnB and glnK stimulated GS shutdown but found that one of these genes was essential for growth. Secondly, we expressed unidirectional adenylyl transferases (uATs) in a ΔglnE mutant of AcLP which permitted strong GS shutdown and excretion of NH3 derived from N2 fixation and completely alleviated negative feedback regulation on nitrogenase expression. We placed a uAT allele under control of the NifA-dependent promoter PnifH, permitting GS shutdown and NH3 excretion specifically under microaerobic conditions, the same cue that initiates N2 fixation, then deleted nifA and transferred a rhizopine nifAL94Q/D95Q-rpoN controller plasmid into this strain, permitting coupled rhizopine-dependent activation of N2 fixation and NH3 excretion. This highly sophisticated and multi-layered control circuitry brings us a step closer to the development of a "synthetic symbioses” where N2 fixation and NH3 excretion could be specifically activated in diazotrophic bacteria colonising transgenic rhizopine producing cereals, targeting delivery of fixed N to the crop while preventing interaction with non-target plants. Inoculation of cereal crops with associative diazotrophic bacteria that convert atmospheric nitrogen (N2) into ammonia (NH3) could be used to sustainably improve delivery of nitrogen to crops. However, due to the costly energy demands of N2 fixation, bacteria restrict excess production of NH3 and release to the plants. Diazotrophs can be engineered for excess NH3 production and release, however genetic control is required to minimise growth penalties and prevent unintended provision of NH3 to non-target weed species. Here, we engineer coupled control of N2 fixation and NH3 release in response to the signalling molecule rhizopine supplemented in vitro. This control circuitry represents a prototype for the future development of a “synthetic symbiosis” where bacterial N2 fixation and NH3 excretion could be specifically activated following colonisation of transgenic rhizopine producing cereals in the field, minimising bacterial energy requirements and preventing provision of NH3 to non-target plants.
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