Heterotrophic nitrifiers are able to oxidize and remove ammonia from nitrogen-rich wastewaters but the genetic elements of heterotrophic ammonia oxidation are poorly understood. Here, we isolated and identified a novel heterotrophic nitrifier, Alcaligenes ammonioxydans sp. nov. strain HO-1, oxidizing ammonia to hydroxylamine and ending in the production of N₂ gas. Genome analysis revealed that strain HO-1 encoded a complete denitrification pathway but lacks any genes coding for homologous to known ammonia monooxygenases or hydroxylamine oxidoreductases. Our results demonstrated strain HO-1 denitrified nitrite (not nitrate) to N₂ and N₂O at anaerobic and aerobic conditions respectively. Further experiments demonstrated that inhibition of aerobic denitrification did not stop ammonia oxidation and N₂ production. A gene cluster (dnfT1RT2ABCD) was cloned from strain HO-1 and enabled E. coli accumulated hydroxylamine. Sub-cloning showed that genetic cluster dnfAB or dnfABC already enabled E. coli cells to produce hydroxylamine and further to ¹⁵N₂ from (¹⁵NH₄)₂SO₄. Transcriptome analysis revealed these three genes dnfA, dnfB and dnfC were significantly upregulated in response to ammonia stimulation. Taken together, we concluded that strain HO-1 has a novel dnf genetic cluster for ammonia oxidation and this dnf genetic cluster encoded a previously unknown pathway of direct ammonia oxidation (Dirammox) to N₂.

中文翻译：

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新型产碱菌 ammonioxydans sp。十一月 来自废水处理污泥的氨氧化成 N2，途径是以前未知的

异养硝化菌能够从富氮废水中氧化和去除氨，但对异养氨氧化的遗传元素知之甚少。在这里，我们分离并鉴定了一种新型异养硝化菌Alcaligenes ammonioxydans sp。十一月 菌株HO-1，将氨氧化成羟胺并最终产生N ₂气体。基因组分析显示，菌株 HO-1 编码了一条完整的反硝化途径，但缺乏任何编码与已知氨单加氧酶或羟胺氧化还原酶同源的基​​因。我们的结果表明菌株 HO-1 将亚硝酸盐（不是硝酸盐）反硝化为 N ₂和 N ₂O 分别在厌氧和好氧条件下。进一步的实验表明，抑制好氧反硝化并不能阻止氨氧化和 N ₂的产生。从菌株 HO-1中克隆了一个基因簇 ( dnfT1RT2ABCD ) 并启用了E. 大肠杆菌积累羟胺。亚克隆显示基因簇dnfAB或dnfABC已经启用了E。大肠杆菌细胞产生羟胺并进一步由( ¹⁵ NH ₄ ) ₂ SO _4产生¹⁵ N ₂。转录组分析揭示了这三个基因dnfA、dnfB和dnfC在氨刺激下显着上调。总之，我们得出结论，菌株 HO-1 具有用于氨氧化的新型 dnf 遗传簇，并且该dnf遗传簇编码了以前未知的直接氨氧化( Dirammox ) 到 N ₂的途径。