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Strains and plasmids	Description	Resource
Wild-type (WT)	Cereibacter azotoformans ATCC 17025	Lab preservation
ΔntrY	ntrY markerless deletion mutant	Lab preservation
WT/op	WT harboring pBBR1MCS-2	Lab preservation
ΔntrY/ntrY	ΔntrY harboring pBBR1MCS-2::ntrY	Lab preservation
pBBR1MCS-2	Ori pBBR1, lacZa, Km^r, used for gene overexpression	Lab preservation

Strains and plasmids	Description	Resource
Wild-type (WT)	Cereibacter azotoformans ATCC 17025	Lab preservation
ΔntrY	ntrY markerless deletion mutant	Lab preservation
WT/op	WT harboring pBBR1MCS-2	Lab preservation
ΔntrY/ntrY	ΔntrY harboring pBBR1MCS-2::ntrY	Lab preservation
pBBR1MCS-2	Ori pBBR1, lacZa, Km^r, used for gene overexpression	Lab preservation

Primer names	Primer sequences (5′→3′)
16S-F	AGAGTTTGATCCTGGCTCAG
16S-R	TACGGCTACCTTGTTACGACTT
ntrY-F	CTTTGACGATGTGACCGATCT
ntrY-R	GTCAGCGGGTTCTTGATCTC
ntrX-F	GCTCGAGAAGGTCACGAAAT
ntrX-R	CCCGAGTTGGAATGGATGAA

Primer names	Primer sequences (5′→3′)
16S-F	AGAGTTTGATCCTGGCTCAG
16S-R	TACGGCTACCTTGTTACGACTT
ntrY-F	CTTTGACGATGTGACCGATCT
ntrY-R	GTCAGCGGGTTCTTGATCTC
ntrX-F	GCTCGAGAAGGTCACGAAAT
ntrX-R	CCCGAGTTGGAATGGATGAA

Gene name	Login ID	Function
nirK	Rsph17025_1595	Nitrite reductase mediates the key denitrification reaction by catalyzing the reduction of NO₂^- to NO
glnA	Rsph17025_1344	L-glutamine synthetase: involved in ammonia assimilation, and helps maintain the overall carbon and nitrogen metabolic balance during the DPA process
norB/C	Rsph17025_0970	Nitric oxide reductase subunit C: catalyzes NO reduction and maintains the operation of denitrification electron transport chain
phaC	Rsph17025_0254	PHAs synthase: synthesizes PHAs and supplies electrons (NADH) and energy reserves for nitrogen and phosphorus removal
sdhA	Rsph17025_0088	Succinate dehydrogenase subunit A: participates in TCA cycle and provides crucial reducing power for nitrogen and phosphorus removal
glgB	Rsph17025_1783	1,4-α-glucan branching enzyme: participates in glycogen synthesis
nuoD	Rsph17025_2004	NADH-quinone oxidoreductase subunit D: involves in electron transport chain and pumps protons, acting as a core hub driving ATP synthesis
gltB, gltD	Rsph17025_2781, Rsph17025_2778	Glutamate synthase II: Cooperates with glnA to mediate efficient ammonia assimilation pathway
nifD, nifK1, nifH	Rsph17025_1247, Rsph17025_1248, Rsph17025_1246	Participates in the assembly of nitrogenase complex

Gene name	Login ID	Function
nirK	Rsph17025_1595	Nitrite reductase mediates the key denitrification reaction by catalyzing the reduction of NO₂^- to NO
glnA	Rsph17025_1344	L-glutamine synthetase: involved in ammonia assimilation, and helps maintain the overall carbon and nitrogen metabolic balance during the DPA process
norB/C	Rsph17025_0970	Nitric oxide reductase subunit C: catalyzes NO reduction and maintains the operation of denitrification electron transport chain
phaC	Rsph17025_0254	PHAs synthase: synthesizes PHAs and supplies electrons (NADH) and energy reserves for nitrogen and phosphorus removal
sdhA	Rsph17025_0088	Succinate dehydrogenase subunit A: participates in TCA cycle and provides crucial reducing power for nitrogen and phosphorus removal
glgB	Rsph17025_1783	1,4-α-glucan branching enzyme: participates in glycogen synthesis
nuoD	Rsph17025_2004	NADH-quinone oxidoreductase subunit D: involves in electron transport chain and pumps protons, acting as a core hub driving ATP synthesis
gltB, gltD	Rsph17025_2781, Rsph17025_2778	Glutamate synthase II: Cooperates with glnA to mediate efficient ammonia assimilation pathway
nifD, nifK1, nifH	Rsph17025_1247, Rsph17025_1248, Rsph17025_1246	Participates in the assembly of nitrogenase complex

一种具有同步反硝化聚磷能力的不产氧光合细菌固氮蜡色杆菌及其分子调控机制解析

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王世龙 , 刘书良 , 周波 , 邢宪春 , 亓正良 ^* , 刘新利

微生物学报 | 研究报告 2026,66(10): 5093-5106

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微生物学报 |研究报告 2026 , 66 (10) : 5093 -5106

一种具有同步反硝化聚磷能力的不产氧光合细菌固氮蜡色杆菌及其分子调控机制解析

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王世龙, 刘书良, 周波, 邢宪春, 亓正良^*, 刘新利

作者信息

齐鲁工业大学(山东省科学院) 山东省生物传感与智能代谢调控重点实验室，山东济南

通讯作者:

亓正良

作者简介:

作者贡献声明

王世龙：研究构思和设计、实验操作、论文撰写与修改；刘书良：数据收集；周波：数据分析；邢宪春：资料收集；亓正良：研究设计、研究指导、论文修改；刘新利：项目管理。

An anoxygenic phototrophic bacterium Cereibacter azotoformans with simultaneous denitrifying and polyphosphate-accumulating capabilities and its molecular regulation mechanism

Shilong WANG, Shuliang LIU, Bo ZHOU, Xianchun XING, Zhengliang QI^*, Xinli LIU

Affiliations

Shandong Provincial Key Laboratory of Biosensing and Microbial Intelligent Metabolic Regulation, Qilu University of Technology (Shandong Academy of Sciences), Jinan, Shandong, China

Corresponding Author:

^*E-mail: qizhengliang@qlu.edu.cn

出版时间: 2026-10-04 doi: 10.13343/j.cnki.wsxb.20260132

文章导航

摘要

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固氮蜡色杆菌(Cereibacter azotoformans)具备高效的氮、磷去除能力，且对水生动物有益生功能，被广泛应用于水产养殖水质调节与渔业病害预防。目前，该菌是否具备同步反硝化聚磷(denitrifying polyphosphate-accumulating, DPA)功能尚属未知。 【目的】 明确固氮蜡色杆菌的DPA表型，并解析其分子调控机制。 【方法】 以固氮蜡色杆菌ATCC 17025菌株为研究对象，基于DPA产生条件对其代谢特点进行表征，明确是否具有DPA表型；通过基因敲除、DNA亲和纯化测序(DNA affinity purification sequencing, DAP-seq)及生理生化分析等手段系统研究双组分系统NtrX/Y在ATCC 17025菌株DPA代谢中的调控作用。 【结果】 固氮蜡色杆菌ATCC 17025表现出典型DPA表型：厌氧阶段释放磷酸盐，缺氧阶段过量吸收磷酸盐并与反硝化协同，实验结束时硝酸盐降解率达到95%，磷酸盐去除率则超过90%。敲除ntrY导致该菌丧失DPA表型，特别是环境中的磷酸盐浓度不仅未下降反而持续上升，且胞内聚羟基脂肪酸酯(polyhydroxyalkanoates, PHAs)的合成峰值较野生型(0.11 mg/mL)显著降低。DAP-seq分析表明，NtrX可直接调控该菌反硝化关键基因(nirK、norB/C)和聚羟基脂肪酸酯合成基因phaC，从而协同调节其反硝化与聚磷代谢。 【结论】 本研究证实不产氧光合细菌固氮蜡色杆菌具有优异的DPA能力，并揭示了NtrX/Y双组分系统在该过程中的重要调控作用机制，有望为同步脱氮除磷微生物资源开发提供新的理论依据。

关键词

固氮蜡色杆菌 / 反硝化聚磷 / 双组分系统 / 分子调控机制

Abstract

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Cereibacter azotoformans has been widely applied in the water quality regulation and disease prevention of aquaculture owing to its efficient nitrogen and phosphorus removal capacity as well as its probiotic effects on aquatic animals. However, whether this bacterium possesses both capabilities of denitrification and polyphosphate accumulation (DPA) remains unclear. [Objective] To identify the DPA phenotype of C. azotoformans and to elucidate its underlying molecular regulation mechanism. [Methods] Using the C. azotoformans strain ATCC 17025 as a model, we first examined its metabolic characteristics under DPA-inducing conditions to determine the presence of a DPA phenotype. Subsequently, gene deletion, DNA affinity purification sequencing (DAP-seq), and physiological and biochemical analyses were employed to systematically investigate the regulatory role of the NtrX/Y two-component system in DPA of the model strain. [Results] The C. azotoformans strain exhibited a typical DPA phenotype: releasing phosphate during the anaerobic stage and excessively absorbing phosphate in synergy with denitrification during the anoxic stage. At the end of the experiment, the nitrate degradation rate reached 95%, and the phosphate removal rate exceeded 90%. The deletion of ntrY resulted in the loss of the DPA phenotype. Particularly, the environmental phosphate concentration continuously increased rather than decreased, and the peak synthesis of intracellular polyhydroxyalkanoates (PHAs) was significantly impaired compared with that of the wild type (0.11 mg/mL). DAP-seq analysis revealed that NtrX directly targeted key denitrification genes (nirK and norB/C) as well as the PHAs synthesis gene phaC, thereby coordinately regulating denitrification and polyphosphate metabolism. [Conclusion] This study demonstrates that the anoxygenic phototrophic bacterium C. azotoformans possesses excellent DPA capabilities, and it reveals a central regulatory role of the NtrX/Y two-component system in this process. These findings offer a new theoretical basis for the development of microbial resources for simultaneous nitrogen and phosphorus removal.

Key words

Cereibacter azotoformans / denitrification and polyphosphate accumulation / two-component system / molecular regulation mechanism

引用本文

王世龙, 刘书良, 周波, 邢宪春, 亓正良, 刘新利. 一种具有同步反硝化聚磷能力的不产氧光合细菌固氮蜡色杆菌及其分子调控机制解析. 微生物学报, 2026 , 66 (10) : 5093 -5106 . DOI: 10.13343/j.cnki.wsxb.20260132

Shilong WANG, Shuliang LIU, Bo ZHOU, Xianchun XING, Zhengliang QI, Xinli LIU. An anoxygenic phototrophic bacterium Cereibacter azotoformans with simultaneous denitrifying and polyphosphate-accumulating capabilities and its molecular regulation mechanism[J]. Acta Microbiologica Sinica, 2026 , 66 (10) : 5093 -5106 . DOI: 10.13343/j.cnki.wsxb.20260132

正文

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随着全球陆基集约化水产养殖的快速发展，水产养殖尾水已逐渐成为一种棘手的新型污染源^[1]。此类微污染水体总体污染负荷较低，主要污染物为氮、磷及有机物；其污染物组成相对稳定，但具有废水产生量大、排放时段集中的特点。研究表明，生物处理技术尤其是以微生物为核心的处理工艺非常适用于此类污水的治理^[2]。水产养殖污水处理的核心目标在于降低氮、磷污染负荷。传统治理手段受相关微生物代谢特性的限制，污水中的反硝化与除磷过程通常被设计为相互独立的处理单元^[3]。反硝化聚磷微生物(denitrifying polyphosphate-accumulating organisms, DPAOs)的发现，使污水处理过程中反硝化与除磷的同步实现成为可能^[4]。将DPAOs应用于污水处理具有显著优势：其可实现“一碳两用”，从而大幅降低外加有机碳源的需求^[5]；同时，该过程在厌氧-缺氧条件下运行，无需额外供氧，可有效降低运行成本^[6]。因此，基于DPAOs的废水同步脱氮除磷技术有望成为一种新型、高效且经济的水产养殖污水治理策略。

目前，DPAOs主要分离自假单胞菌属(Pseudomonas)、索氏菌属(Thauera)和嗜水气单胞菌属(Aeromonas)^[7]等，而在不产氧光合细菌(anoxygenic phototrophic bacteria, APB)中尚未有DPAOs的报道。固氮蜡色杆菌(Cereibacter azotoformans)属于紫色非硫细菌，根据KEGG数据库对固氮蜡色杆菌ATCC 17025全基因组序列的注释，其基因组内具有完整且成簇排布的不产氧光合作用完整基因簇，包括编码光反应中心的puf和puh操纵子、负责捕光天线合成的puc操纵子以及参与细菌叶绿素合成的bch基因簇，证实固氮蜡色杆菌为不产氧光合细菌(APB)^[8-9]。因此，本研究旨在探究固氮蜡色杆菌是否具有同步反硝化聚磷(denitrifying polyphosphate-accumulating, DPA)能力。本研究的开展有望进一步拓展DPAOs的微生物资源库，从而为水产养殖污水的生物处理提供了一种新的潜在功能菌。

双组分系统(two-component system, TCS)广泛存在于细菌中，是许多细菌感知环境变化并启动相关代谢反应(如反硝化作用)的关键调控元件^[10]。典型的TCS由成对的组氨酸激酶(histidine kinase, HK)和响应调节蛋白(response regulator, RR)构成。HK的活性及其催化方向决定RR的磷酸化水平，进而调控靶基因转录强度^[11]。NtrX/Y双组分系统最早在茎瘤固氮根瘤菌(Azorhizobium caulinodans)中被鉴定，随后发现其在多种细菌中广泛分布并具有多样化的生理功能^[12]。例如，在草螺菌(Herbaspirillum seropedicae)中，NtrX/Y系统参与硝酸盐还原代谢调控^[12]；在荚膜红细菌(Rhodobacter capsulatus)中，该系统不仅调控氮代谢，还控制光合生长所必需的光反应中心的合成^[13]。本课题组前期研究已证实NtrX/Y系统可调控固氮蜡色杆菌的反硝化过程，但该双组分系统能否介导其DPA功能仍有待阐明。

本研究主要目的是阐明NtrX/Y双组分系统调控固氮蜡色杆菌ATCC 17025菌株DPA代谢的分子机制。首先，验证固氮蜡色杆菌是否具有DPA表型，其次明确NtrX/Y系统对固氮蜡色杆菌DPA能力是否具有调控作用；在此基础上，采用遗传操作技术、生理生化分析以及DNA亲和纯化测序(DNA affinity purification sequencing, DAP-seq)等技术解析其调控机制。本研究有望为蜡色杆菌属APB菌株在水产养殖尾水处理中实现氮磷同步去除的应用奠定理论基础。

1 材料与方法

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1.1 菌株、培养基及培养条件

固氮蜡色杆菌ATCC 17025菌株及其相应突变株接种于NH1培养基中，于30 ℃、200 r/min条件下培养12 h作为种子液^[14]。本研究所使用的菌株及质粒信息见表1。种子制备方法如下：从甘油保藏管中挑取一环固氮蜡色杆菌ATCC 17025菌苔，在NH1固体培养基上划线分离纯种，平板倒置后置于30 ℃恒温培养箱中培养，待形成红色单菌落(2-4 d)。随后挑取单个菌落接种于NH1液体培养基中，于30 ℃、200 r/min条件下振荡培养，直至菌液OD₆₀₀>1.0 (通常为1-2 d)，作为种子菌液。接种前，将种子菌液于6 000×g离心10 min，弃上清后用质量分数0.85%的无菌生理盐水重悬。在固氮蜡色杆菌突变菌培养中需要加入卡那霉素，其终浓度为25 μg/mL。

1.2 固氮蜡色杆菌在合成污水中的反硝化聚磷代谢分析

采用两阶段(厌氧/缺氧)培养策略研究固氮蜡色杆菌ATCC 17025的DPA代谢，实验装置如图1A所示。厌氧阶段：用N₂充分置换反应器中的溶解氧(dissolved oxygen, DO)，随后紧固夹具以建立厌氧条件，此时培养基中溶解氧(用电极测定)<0.2 mg/L。缺氧阶段：松开夹具以允许空气进入，通过开闭排气管调节培养液中溶解氧维持在0.2-0.7 mg/L。

采用人工合成污水培养基开展上述研究^[15]。其中，无水乙酸钠、NH₄Cl/KNO₃和K₂HPO₄分别模拟污水中的碳、氮和磷污染物。ΔntrY突变株在谷氨酰胺为唯一氮源的条件下生长良好，因此培养ΔntrY时将NH₄Cl替换为谷氨酰胺(ntrY缺失使细胞无法利用胞外NH₄Cl，否则将完全抑制菌体生长)。

厌氧阶段操作：取体积分数10%种子菌接种于800 mL人工合成污水培养基中(反应器工作体积1 000 mL)，30 ℃培养48 h。缺氧阶段操作：向培养液中加入KNO₃，使其终浓度达到30 mg/L，继续30 ℃培养60 h。培养过程中，每12 h取样10 mL，8 000 r/min离心5 min，上清液可立即分析或-20 ℃保存备用。同时，整个实验维持12 h光照/12 h黑暗循环，光照由全光谱LED灯提供，光照强度控制在5 000-6 000 lx。

1.3 生物信息学分析

从KEGG数据库(https://www.genome.jp/kegg/)中获取固氮蜡色杆菌ATCC 17025的NtrB、NtrC、NtrY和NtrX氨基酸序列。利用SMART保守结构域数据库(https://smart.embl.de/)对上述蛋白的主要结构域及螺旋-转角-螺旋(helix-turn-helix, HTH)基序进行鉴定。采用Predict Protein数据库(https://predictprotein.org/)预测NtrY的跨膜结构，同时利用SWISS-MODEL数据库(https://swissmodel.expasy.org/interactive)对蛋白质三级结构进行预测。NtrX的磷酸化位点及NtrY的组氨酸残基通过Pfam软件(InterPro)进行分析鉴定。

1.4 RNA提取及实时荧光定量逆转录PCR (RT-qPCR)

在缺氧阶段，于选定时间点收集ATCC 17025菌体，常温6 000 r/min离心10 min去除上清后，于-80 ℃保存备用。采用TRIzol法提取菌体总RNA，并使用First cDNA Synthesis Kit (北京鼎国昌盛生物技术有限公司)反转录合成cDNA。荧光定量逆转录PCR (reverse transcription quantitative PCR, RT-qPCR)反应体系：cDNA模板1.0 μL，2×SuperReal PreMix Plus 5.0 μL，正、反向引物(10 μmol/L)各0.4 μL，ddH₂O补充体系至10.0 μL。混合均匀后使用实时荧光定量PCR仪(ThermoFisher Scientific公司)进行检测。扩增条件：95 ℃ 15 min；95 ℃ 10 s，60 ℃ 30 s，共40个循环。以16S rRNA基因为内参基因，对不同样品间的基因表达水平进行标准化处理，具体引物序列见表2。通过2^-ΔΔ^C^t方法对数据进行相对定量分析。

1.5 DNA亲和纯化测序

DNA亲和纯化测序(DNA affinity purification sequencing, DAP-seq)是一种用于研究转录因子与DNA相互作用的高效技术。与染色质免疫共沉淀测序(chromatin immunoprecipitation sequencing, ChIP-seq)相比，该方法无需制备转录因子特异性抗体^[16]。DAP-seq的基本流程如下：首先，提取固氮蜡色杆菌ATCC 17025的基因组DNA，并构建NtrX表达载体；其次，对部分基因组DNA进行片段化处理，通过琼脂糖凝胶电泳筛选100-500 bp片段，构建亲和测序文库；将纯化的NtrX蛋白与DNA文库在体外孵育，利用磁珠特异性富集与NtrX蛋白结合的靶DNA片段；最后，将洗脱回收的DNA片段(DAP-seq文库)进行高通量测序。下机数据比对至ATCC 17025参考基因组后，采用MACS2软件进行全基因组结合峰(peak calling)识别，并利用MEME Suite工具包进行核心结合基序(motif)的预测分析^[17]。

1.6 细胞内多聚磷酸盐及多聚羟基脂肪酸酯测定

分别在实验过程的48、72、96 h取野生型(WT)和ΔntrY菌株的菌悬液各1.5 mL。样品于4 ℃、10 000 r/min离心15 min，弃去上清。将菌体沉淀置于1.0 mL 20 mmol/L HEPES缓冲液(pH 7.0)中充分涡旋重悬后，再次于4 ℃、10 000 r/min离心15 min^[18]，弃去上清。随后将菌体沉淀重悬于1.0 mL HEPES缓冲液中。取0.3 mL菌悬液加入1.5 mL离心管中，加入0.1 mL 100 μmol/L DAPI溶液^[19]，充分涡旋混匀后，于室温避光条件下孵育10 min；随后于4 ℃、10 000 r/min离心3 min，弃去上清^[20]。菌体沉淀用HEPES缓冲液洗涤1次，并重悬于1.0 mL HEPES缓冲液中。取10 μL重悬液滴加于载玻片上，加入香柏油后，在荧光显微镜下使用100×油镜物镜和10×目镜进行观察。以488 nm为激发波长，在500-560 nm波段采集绿色荧光信号，用于表征DAPI-多聚磷酸盐复合物的荧光强度^[21]。

尼罗红可快速穿透悬浮细胞，实现细胞内多聚羟基脂肪酸酯(polyhydroxyalkanoates, PHAs)的快速检测，其染色后细胞的荧光强度与PHAs含量呈正相关关系^[22]。以二甲基亚砜为溶剂配制尼罗红储备液，终浓度为80 μg/mL，用于菌体染色。在0、24、48、72、96 h取适量菌液，12 000 r/min离心5 min收集菌体沉淀，并通过涡旋振荡以纯水重悬，将所有时间点细胞悬液调整为OD₆₀₀=0.8。随后向1.0 mL菌悬液中加入50 μL尼罗红染色液，室温孵育30 min以完成染色；染色结束后，于12 000 r/min离心5 min，弃去上清。菌体沉淀以纯水重悬并洗涤1次，最终重悬于1.0 mL纯水中。荧光测定时，取200 μL重悬后的菌液加入96孔板中，每个样品设置不少于2个重复孔。采用荧光酶标仪进行检测，激发波长为535 nm，发射波长为595 nm。通过与PHAs标准曲线对比计算样品中的PHAs含量^[23]。

1.7 生物量、脱氮除磷理化指标测定及数据分析

细胞密度采用UV2300Ⅱ分光光度计(Techcomp公司)在600 nm波长下测定(OD₆₀₀)。细胞干重(dry cell weight, DCW)与吸光度的关系为：1 OD₆₀₀对应0.40 g DCW/L。硝酸盐氮(NO₃^--N)采用紫外分光光度法测定(分别在220 nm和275 nm处测量吸光度)；亚硝酸盐氮(NO₂^--N)采用N-(1-萘基)-乙二胺分光光度法测定。上述指标检测的具体操作步骤参照文献[15]。磷酸盐(PO₄^3--P)采用铵-钼酸盐比色法测定。检测前，所有样品均以10 000 r/min离心10 min。所有数据分析均使用SAS Ver 8.01软件(SAS公司)，结果以均值±标准差形式表示。

2 结果与讨论

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2.1 固氮蜡色杆菌具备典型的同步反硝化聚磷代谢表型

如图1B所示，实验过程中硝酸盐(NO₃^--N)、亚硝酸盐(NO₂^--N)和磷酸盐(PO₄^3--P)的浓度变化符合典型的DPA代谢特征。具体来说，厌氧阶段固氮蜡色杆菌释放出一定量的磷酸盐，导致环境中PO₄^3--P浓度升高；随后，在缺氧阶段呈现PO₄^3--P快速下降的现象，这是多聚磷酸盐(poly-P)过量积累所致，最终PO₄^3--P去除率超过90%。缺氧阶段开始时加入硝酸盐，其间NO₃^--N浓度迅速下降，到实验结束(96 h)时降至<1.0 mg/L。同时，实验过程中NO₂^--N浓度先短暂升高随后下降，最终降至<0.2 mg/L，硝酸盐去除率达到95%。这种变化趋势在污水反硝化处理中较为常见，通常是由于处理初期细菌反硝化酶合成能力的不平衡所致。此外，通过荧光显微镜观察到细菌细胞内DAPI-poly-P的荧光强烈积累(图1C)，证实了缺氧阶段细胞内储存poly-P。

DPAOs驱动的氮、磷同步去除在水产养殖污水处理中的应用前景广阔^[24]。现有研究表明，大多数DPAOs属于假单胞菌属(Pseudomonas)、索氏菌属(Thauera)和嗜水气单胞菌属(Aeromonas)^[7]，但来源于这3个属中的部分DPAOs菌株可能对水产养殖物种构成潜在的致病风险。本研究证实，固氮蜡色杆菌具有典型DPA特征。同时，考虑到蜡色杆菌属APB在水产养殖中具有多种有益功能，可维持水质并抑制病害。因此，固氮蜡色杆菌ATCC 17025作为推进水产养殖尾水处理中DPA处理策略的候选菌株，展现出极高的应用潜力与安全性。

2.2 NtrY突变抑制固氮蜡色杆菌反硝化聚磷代谢

细菌通过多种代谢调控机制适应环境变化。其中，双组分系统(TCS)是细菌快速响应外界刺激的重要机制。研究表明，双组分系统NtrX/Y可以调控多种细菌的反硝化过程。例如，已证实NtrX/Y系统在固氮蜡色杆菌ATCC 17025中正向调控反硝化代谢^[15]。鉴于在DPAOs中反硝化和聚磷代谢密切协同，本研究提出了一个重要问题：NtrX/Y系统是否也调控固氮蜡色杆菌的这种同步代谢？

为解答这一问题，本研究对野生型WT和ntrY基因敲除株(ΔntrY)进行了DPA能力测试。本研究监测了硝酸盐(NO₃^--N)、亚硝酸盐(NO₂^--N)和磷酸盐(PO₄^3--P)的浓度变化(图2)。结果显示ΔntrY与WT之间存在显著差异。关于PO₄^3--P代谢：ATCC 17025 WT菌株在缺氧阶段显示出快速吸收磷酸盐的表型，但对于接种了ΔntrY菌株的实验组，在缺氧阶段PO₄^3--P浓度并未下降，反而持续上升(图2A)，表明敲除ntrY后细菌poly-P合成功能显著受损。关于NO₃^--N还原：ΔntrY突变株的硝酸盐还原能力显著受到抑制(图2B)。关于NO₂^--N积累：ΔntrY菌株在缺氧阶段的NO₂^--N积累明显高于野生型菌株(图2C)，这一现象与之前的研究结果一致^[15]。此外，ΔntrY在0 h时仅显示微弱荧光，且随着培养时间的延长始终未出现绿色荧光，进一步证实其无法积累poly-P。接下来，本研究使用导入空质粒的突变菌株(WT/op)作为对照，测试ntrY基因回补株(ΔntrY/ntrY)是否恢复DPA能力。结果表明，回补株成功恢复了DPA表型(图2D、2E)，进一步证实了ntrY基因的缺失显著损害了固氮蜡色杆菌的聚磷能力。同时，该结果揭示NtrX/Y系统通过直接或间接的方式正向调控该代谢过程。上述研究表明，NtrX/Y双组分系统在固氮蜡色杆菌的DPA代谢中发挥关键的调控作用。

2.3 固氮蜡色杆菌反硝化聚磷代谢的调控机制

2.3.1 NtrX/Y蛋白结构解析

在固氮蜡色杆菌ATCC 17025基因组中，编码NtrX/Y蛋白的基因位于另一双组分系统NtrB/C的下游(图3)。其中，响应调节蛋白NtrX属于Fis家族，是一种σ⁵⁴依赖型转录因子。其结构域组成包括N端的受体结构域(REC，通过磷酸化调节蛋白活性)以及核心AAA⁺ ATPase结构域(在核苷酸结合/水解、寡聚化以及与σ⁵⁴的直接相互作用中发挥关键作用)^[25]。REC结构域的磷酸化状态受到严格调控，是控制AAA⁺结构域寡聚化的关键开关^[26]。磷酸化引发蛋白构象发生改变，从而促进寡聚体组装或解除REC与AAA⁺结构域之间的自抑制相互作用，从而形成转录激活所必需的功能性六聚体^[27]。

NtrY作为NtrX/Y系统的组氨酸激酶，含有组氨酸激酶二聚体/磷酸受体结构域(HisKA)、类组氨酸激酶ATP酶结构域(HATPase_c)以及PAS信号感知结构域。HisKA与HATPase_c结构域协同介导NtrY在保守组氨酸残基上的自磷酸化及随后向NtrX的磷酸基团转移。HAMP结构域通过响应PAS域传递的信号，在调控NtrY同源二聚体构象与活性中发挥关键作用。值得注意的是，与其相类似的激酶NtrB不同，NtrY具有跨膜区和HAMP信号转导结构域。在α-变形菌中，NtrB/C系统主要调控氮代谢，通过转录调控靶基因实现对有限氮源(如硝酸盐)的利用^[28]。然而，先前研究发现，固氮蜡色杆菌中的NtrB/C系统对反硝化起负调控作用，而NtrX/Y系统则发挥激活作用，这一调控模式在草螺菌等细菌中也有体现^[12]。更为重要的是，结构分析揭示了NtrY与NtrB之间的一个关键差异：NtrY为膜结合型传感激酶，具有跨膜结构域和HAMP结构域，而NtrB缺乏上述结构。该结构特征使NtrY能够感知胞外或周质信号，而胞质型的NtrB则不具备此功能。

综合功能差异(NtrX/Y系统与NtrB/C系统在反硝化调控中发挥相反作用)与结构特征(NtrY为膜整合结合型传感激酶)等分析结果，可以推断出固氮蜡色杆菌在进化过程中更倾向于以NtrX/Y系统而非NtrB/C系统作为调控反硝化代谢的信号传导通路。

2.3.2 ntrX/Y 基因的转录动态变化

为明确固氮蜡色杆菌ATCC 17025在DPA过程中ntrX和ntrY的转录动态变化，采用RT-qPCR方法进行分析。在48-72 h期间，每隔6 h采集3个生物学重复样品(图4)。结果显示，在48、54、60 h时ntrY/X的转录水平持续上调，表明NtrX/Y双组分系统在该耦合代谢过程中处于活跃状态。

值得注意的是，在66 h出现了明显的转录转折点：ntrX/Y的表达水平急剧下降。与此同时，NO₂^--N浓度出现峰值(图1)。NO₂^--N浓度升高可能抑制细菌的反硝化活性，从而导致NtrX/Y表达下调。此外，ntrX/Y的表达水平在72 h时再次回升。考虑到NtrX/Y系统作为一种全局转录调控因子，推测固氮蜡色杆菌可能通过反馈调控机制重新激活ntrX/Y的表达，以满足新的代谢需求或适应资源变化，从而维持自身的生存适应性。

2.3.3 细胞内多聚羟基脂肪酸酯含量变化

多聚羟基脂肪酸酯(polyhydroxyalkanoates, PHAs)是DPAOs中驱动DPA代谢的重要能量与还原力储备物质^[29]。在厌氧阶段，细胞内poly-P水解释放PO₄^3--P同时生成ATP；与此同时，乙酰辅酶A (acetyl-CoA)作为前体合成并储存PHAs。随后在缺氧阶段，PHAs发生分解再生成乙酰辅酶A，产生ATP和NADH，从而为聚磷代谢与反硝化的耦合过程提供关键的电子和能量^[30]。

与上述代谢模型一致，固氮蜡色杆菌在厌氧条件下表现出显著的PHAs合成能力(图5)。结果显示，在48 h时细胞内PHAs含量达到峰值(0.11 mg/mL)，该时间点对应厌氧阶段磷酸盐释放的后期。进入缺氧阶段并发生聚磷积累后，细胞内PHA含量迅速下降，符合DPAOs的经典代谢动态特征。值得注意的是，ΔntrY菌株的PHA生物合成能力显著受损，进一步表明NtrX/Y双组分系统在PHA代谢调控中发挥关键作用。

2.3.4 NtrX靶基因的全基因组规模鉴定

本研究采用DAP-seq技术对固氮蜡色杆菌全基因组范围内的NtrX结合靶位点进行了鉴定[相关数据已上传ScienceDB数据库(www.scidb.cn)，CSTR编号为31253.11.sciencedb.j00231.00072)]。2次技术重复中峰型分布高度一致，表明实验样品具有良好的重复性和数据可靠性。利用depTools2软件分析发现，NtrX的结合峰主要聚集于靶基因编码区起始位点附近，其中46.30%的结合位点位于转录起始位点(transcription start site, TSS)上游2 000 bp以内，证实NtrX作为转录因子主要通过结合启动子区域发挥调控作用。MEME-chip分析进一步揭示49.10%的结合峰序列中存在一个保守序列基序(YGTGGCVNWNBYGCM，命名为MEME-1)，该序列被确定为NtrX的核心结合位点。对目标基因进行GO富集分析发现，其主要参与DNA结合、整合及转录调控等功能，反映了NtrX/Y系统的调控特征。KEGG富集分析结果显示，排名前20的通路主要集中于氮代谢相关通路，包括酪氨酸代谢，表明NtrX/Y系统可能通过调控氮代谢及氧化磷酸化相关基因来介导固氮蜡色杆菌的DPA代谢。

截至目前，细菌DPA代谢的转录调控机制仍不清楚。本研究将NtrX/Y双组分系统与固氮蜡色杆菌的DPA代谢过程直接联系起来。表3总结了部分鉴定的NtrX靶基因，其中包括反硝化关键基因nirK (亚硝酸盐还原酶)和norB/C (一氧化氮还原酶)，与先前研究结果一致^[15]。此外，电泳迁移率实验(electrophoretic mobility shift assay, EMSA)进一步验证了DAP-seq的结果(图6A)。

值得注意的是，DAP-seq结果显示，聚磷代谢的核心基因(如ppk和ppx)并未出现在NtrX的直接靶基因列表中，提示NtrX并不直接作用于聚磷途径。然而，NtrX却直接作用于PHAs合酶基因(phaC)以及NADH-醌氧化还原酶亚基D基因(nuoD)。在经典的DPA模型中，PHAs是反硝化和生成ATP的重要内碳源与能量前体；而nuoD编码的复合物I直接参与电子传递链并建立跨膜质子梯度，是驱动ATP合成的关键枢纽。基于上述靶基因网络，结合WT在缺氧阶段积累高达0.11 mg/mL的PHAs且将硝酸盐降至<1 mg/L，而ΔntrY突变体反硝化与PHA合成受阻且完全丧失聚磷能力的宏观表型，推测NtrX蛋白可能通过直接激活反硝化与能量代谢途径(phaC、nuoD)，以电子传递和ATP供应为桥梁，间接驱动聚磷代谢的完成。

综上所述，NtrX/Y系统通过直接调控反硝化代谢和PHAs合成，从而协同调节整个DPA过程(图6B)。与前人报道的经典DPAOs相比，固氮蜡色杆菌在代谢调控网络上展现出了鲜明的特性。传统理论认为DPAOs (如假单胞菌属、候选累积杆菌等)的聚磷代谢通常是“以磷为驱动”的，其核心调控信号主要来源于环境磷浓度感受器(如经典的PhoB/PhoR双组分系统)，通过直接结合聚磷核心基因启动相关代谢^[31-32]。然而，本研究证实固氮蜡色杆菌ATCC 17025展现出一种“以氮驱动”的全局调控策略：其高度依赖感应环境氮素信号的NtrX/Y系统，以反硝化模块作为关键中枢枢纽，连接PHAs与聚磷代谢途径。这种跨网络的调控模式，不仅在转录层面上丰富了微生物同步反硝化聚磷代谢的分子机制理论框架，也表明了固氮蜡色杆菌在应对复杂水环境时的代谢灵活性。

3 结论

收起

本研究以一种具有DPA能力的不产氧光合细菌固氮蜡色杆菌为研究对象，系统研究了其DPA代谢特征及分子调控机制。结果表明，固氮蜡色杆菌表现出典型的DPA表型，能够在厌氧/缺氧交替条件下实现氮、磷的协同去除。通过构建ntrY缺失突变株并开展对比分析发现，双组分调控系统NtrX/Y在固氮蜡色杆菌DPA代谢中发挥重要的调控作用。ntrY缺失显著削弱了菌株的反硝化能力和聚磷能力，并导致胞内PHAs合成受阻，表明NtrX/Y系统是连接反硝化、PHAs代谢与聚磷过程的重要调控纽带。DAP-seq结果进一步揭示，NtrX可直接结合并调控多种反硝化及能量代谢相关基因(如nirK、norB/C、phaC等)，从转录水平上协调反硝化与PHAs合成过程，而聚磷代谢可能通过能量代谢间接受到调控。

综上所述，本研究从分子水平阐明了NtrX/Y系统在固氮蜡色杆菌DPA代谢中的重要调控作用，加深了对DPA代谢调控网络的认识，为DPA功能菌的筛选、调控及其在水产养殖污水氮磷协同去除中的应用提供了重要理论基础。

基金

收起

山东省自然科学基金(ZR2024MC069)
国家自然科学基金(31800108)
济南市市校融合发展战略工程项目(JNSX2025030)

参考文献

收起

文献

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参考文献引证文献

排序方式：

[1]

Wang

, Xu

, Chen

, Li

, Zhang

. Microbial nitrogen removal in synthetic aquaculture wastewater by fixed-bed baffled reactors packed with different biofilm carrier materials[J]. Bioresource Technology, 2021, 331: 125045.

[2]

Tom

, Jayakumar

, Biju

, Somarajan

, Ibrahim

. Aquaculture wastewater treatment technologies and their sustainability: a review[J]. Energy Nexus, 2021, 4: 100022.

[3]

Delgadillo-Mirquez

, Lopes

, Taidi

, Pareau

. Nitrogen and phosphate removal from wastewater with a mixed microalgae and bacteria culture[J]. Biotechnology Reports, 2016, 11: 18-26.

[4]

, Yang

, Zhong

, Pang

, Zhang

, Xia

, Yang

, Xie

, Liu

, Ren

, Ding

. Simultaneous nitrification, denitrification and phosphorus removal: what have we done so far and how do we need to do in the future[J]. Science of the Total Environment, 2023, 856: 158977.

[5]

Dai

, Sun

, Wan

, Abbasi

, Guo

, Geng

, Wang

, Chen

. Simultaneous denitrification and phosphorus removal: a review on the functional strains and activated sludge processes[J]. Science of the Total Environment, 2022, 835: 155409.

[6]

Mishra

, Singh

, Cheng

, Hussain

, Ormeci

. Nitrogen removal from wastewater: a comprehensive review of biological nitrogen removal processes, critical operation parameters and bioreactor design[J]. Journal of Environmental Chemical Engineering, 2022, 10(3): 107387.

[7]

Singleton

, Petriglieri

, Wasmund

, Nierychlo

, Kondrotaite

, Petersen

, Peces

, Dueholm

, Wagner

, Nielsen

. The novel genus, ‘Candidatus Phosphoribacter’, previously identified as Tetrasphaera, is the dominant polyphosphate accumulating lineage in EBPR wastewater treatment plants worldwide[J]. The ISME Journal, 2022, 16(6): 1605-1616.

[8]

Hiraishi

, Muramatsu

, Ueda

. Molecular genetic analyses of Rhodobacter azotoformans sp. nov. and related species of phototrophic bacteria[J]. Systematic and Applied Microbiology, 1996, 19(2): 168-177.

[9]

Suresh

, Lodha

, Indu

, Sasikala

, Ramana

. Corrigendum: taxogenomics resolves conflict in the genus Rhodobacter: a two and half decades pending thought to reclassify the genus Rhodobacter [J]. Frontiers in Microbiology, 2020, 11: 1111.

[10]

Lazar

, Tabor

. Bacterial two-component systems as sensors for synthetic biology applications[J]. Current Opinion in Systems Biology, 2021, 28: 100398.

[11]

Cruz-Bautista

, Ruíz-Villafán

, Romero-Rodríguez

, Rodríguez-Sanoja

, Sánchez

. Trends in the two-component system’s role in the synthesis of antibiotics by Streptomyces [J]. Applied Microbiology and Biotechnology, 2023, 107(15): 4727-4743.

[12]

Bonato

, Alves

, Osaki

, Rigo

, Pedrosa

, Souza

, Zhang

, Schumacher

, Buck

, Wassem

, Chubatsu

. The NtrY-NtrX two-component system is involved in controlling nitrate assimilation in Herbaspirillum seropedicae strain SmR1[J]. The FEBS Journal, 2016, 283(21): 3919-3930.

[13]

Xing

, Zheng

, An

, Huang

, Yang

, Zeng

, Li

, Ouenzar

, Yu

, Luo

. Transcription regulation of cell cycle regulatory genes mediated by NtrX to affect Sinorhizobium meliloti cell division[J]. Genes, 2022, 13(6): 1066.

[14]

Yang

, Li

, Sun

, Li

, Qi

, Liu

. Improving CoQ10 productivity by strengthening glucose transmembrane of Rhodobacter sphaeroides [J]. Microbial Cell Factories, 2021, 20: 207.

[15]

, Gao

, Li

, Zhang

, Dong

, Liu

. A novel strategy to improving Rhodobacter azotoformans denitrification efficiency: insight into the role of a two-component system NtrX/Y in denitrification regulation[J]. Bioresource Technology, 2023, 368: 128349.

[16]

Zhang

, Wang

, Cao

, Jiao

, Ku

, Dou

, Liu

, Fu

, Xie

, Zhu

, Chong

, Wei

. Molecular mechanism analysis of ZmRL6 positively regulating drought stress tolerance in maize[J]. Stress Biology, 2023, 3: 47.

[17]

Bartlett

, O’Malley

, Huang

, Galli

, Nery

, Gallavotti

, Ecker

. Mapping genome-wide transcription-factor binding sites using DAP-seq[J]. Nature Protocols, 2017, 12(8): 1659-1672.

[18]

, Han

, Wang

, Wan

. A fluorometric method for direct detection of inorganic polyphosphate in enterohemorrhagic Escherichia coli O157:H7[J]. Journal of Southern Medical University, 2019, 39(3): 344-350.

[19]

Allan

, Miller

. Influence of S-adenosylmethionine on DAPI-induced fluorescence of polyphosphate in the yeast vacuole[J]. Canadian Journal of Microbiology, 1980, 26(8): 912-920.

[20]

Gomes

, Ramos

, Wendt

, Girard-Dias

, de Souza

, Machado

, Miranda

. New insights into the in situ microscopic visualization and quantification of inorganic polyphosphate stores by 4′,6-diamidino-2-phenylindole (DAPI)-staining[J]. European Journal of Histochemistry, 2013, 57(4): 34.

[21]

Omelon

, Georgiou

, Habraken

. A cautionary (spectral) tail: red-shifted fluorescence by DAPI-DAPI interactions[J]. Biochemical Society Transactions, 2016, 44(1): 46-49.

[22]

Spiekermann

, Rehm

BHA

, Kalscheuer

, Baumeister

, Steinbüchel

. A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds[J]. Archives of Microbiology, 1999, 171(2): 73-80.

[23]

Zhao

, Luo

, Huang

, Chen

, Xiong

, Li

, Chen

. Determining intracellular lipid content of different oleaginous yeasts by one simple and accurate Nile Red fluorescent method[J]. Preparative Biochemistry and Biotechnology, 2019, 49(6): 597-605.

[24]

Patel

, Nerurkar

. Thauera sp. for efficient nitrate removal in continuous denitrifying moving bed biofilm reactor[J]. Bioprocess and Biosystems Engineering, 2024, 47(3): 429-442.

[25]

Puchades

, Sandate

, Lander

. The molecular principles governing the activity and functional diversity of AAA⁺ proteins[J]. Nature Reviews Molecular Cell Biology, 2020, 21(1): 43-58.

[26]

Bhaskara

, Wong

, Verslues

. The flip side of phospho-signalling: regulation of protein dephosphorylation and the protein phosphatase 2Cs[J]. Plant, Cell & Environment, 2019, 42(10): 2913-2930.

[27]

Bush

, Dixon

. The role of bacterial enhancer binding proteins as specialized activators of σ⁵⁴-dependent transcription[J]. Microbiology and Molecular Biology Reviews, 2012, 76(3): 497-529.

[28]

Yang

, Li

, Yan

, Ke

, Han

, Wu

, Lv

, Shao

, Jiang

, Lin

, Zhang

, Zhan

. Master regulator NtrC controls the utilization of alternative nitrogen sources in Pseudomonas stutzeri A1501[J]. World Journal of Microbiology and Biotechnology, 2021, 37(10): 177.

[29]

郑春霞, 王侧容, 张漫漫, 吴启凤, 陈梦苹, 丁晨雨, 何腾霞. 反硝化聚磷菌及其脱氮除磷机理研究进展[J]. 生物工程学报, 2023, 39(3): 1009-1025.

Zheng

, Wang

, Zhang

, Wu

, Chen

, Ding

, He

. Denitrifying phosphate accumulating organisms and its mechanism of nitrogen and phosphorus removal[J]. Chinese Journal of Biotechnology, 2023, 39(3): 1009-1025 (in Chinese).

[30]

Jing

, Liu

, Zhang

, Yin

. Phosphorus release and uptake behavior and microbial metabolic activity in reversed A2O process: importance of influent VFAs/COD and anaerobic duration[J]. Biochemical Engineering Journal, 2023, 200: 109098.

[31]

Mukherjee

, Chowdhury

, Begam

, Ganguli

, Basak

, Chaudhuri

, Ray

. Effect of varying nitrate concentrations on denitrifying phosphorus uptake by DPAOs with a molecular insight into pho regulon gene expression[J]. Frontiers in Microbiology, 2019, 10: 2586.

[32]

Kondrotaite

, Petersen

, Singleton

, Peces

, Petriglieri

, Jensen

TBN

, Sereika

, Daugberg

AOH

, Wagner

, Dueholm

MKD

, Nielsen

. Ecophysiology and niche differentiation of three genera of polyphosphate-accumulating bacteria in a full-scale wastewater treatment plant[J]. mSystems, 2025, 10(9): e00322-25.

2026年第66卷第10期

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doi: 10.13343/j.cnki.wsxb.20260132

接收时间：2026-02-12
首发时间：2026-10-13
出版时间：2026-10-04

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收稿日期：2026-02-12
录用日期：2026-04-29

基金

Natural Science Foundation of Shandong Province(ZR2024MC069)

山东省自然科学基金(ZR2024MC069)

National Natural Science Foundation of China(31800108)

国家自然科学基金(31800108)

Jinan City-University Integration Development Strategic Project(JNSX2025030)

济南市市校融合发展战略工程项目(JNSX2025030)

作者信息

齐鲁工业大学(山东省科学院) 山东省生物传感与智能代谢调控重点实验室，山东济南

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Strains and plasmids	Description	Resource
Wild-type (WT)	Cereibacter azotoformans ATCC 17025	Lab preservation
ΔntrY	ntrY markerless deletion mutant	Lab preservation
WT/op	WT harboring pBBR1MCS-2	Lab preservation
ΔntrY/ntrY	ΔntrY harboring pBBR1MCS-2::ntrY	Lab preservation
pBBR1MCS-2	Ori pBBR1, lacZa, Km^r, used for gene overexpression	Lab preservation

Strains and plasmids

Description

Resource

Wild-type (WT)

Cereibacter azotoformans ATCC 17025

Lab preservation

ΔntrY

ntrY markerless deletion mutant

Lab preservation

WT/op

WT harboring pBBR1MCS-2

Lab preservation

ΔntrY/ntrY

ΔntrY harboring pBBR1MCS-2::ntrY

Lab preservation

pBBR1MCS-2

Ori pBBR1, lacZa, Km^r, used for gene overexpression

Lab preservation

Primer names	Primer sequences (5′→3′)
16S-F	AGAGTTTGATCCTGGCTCAG
16S-R	TACGGCTACCTTGTTACGACTT
ntrY-F	CTTTGACGATGTGACCGATCT
ntrY-R	GTCAGCGGGTTCTTGATCTC
ntrX-F	GCTCGAGAAGGTCACGAAAT
ntrX-R	CCCGAGTTGGAATGGATGAA

Primer names

Primer sequences (5′→3′)

16S-F

AGAGTTTGATCCTGGCTCAG

16S-R

TACGGCTACCTTGTTACGACTT

ntrY-F

CTTTGACGATGTGACCGATCT

ntrY-R

GTCAGCGGGTTCTTGATCTC

ntrX-F

GCTCGAGAAGGTCACGAAAT

ntrX-R

CCCGAGTTGGAATGGATGAA

Gene name	Login ID	Function
nirK	Rsph17025_1595	Nitrite reductase mediates the key denitrification reaction by catalyzing the reduction of NO₂^- to NO
glnA	Rsph17025_1344	L-glutamine synthetase: involved in ammonia assimilation, and helps maintain the overall carbon and nitrogen metabolic balance during the DPA process
norB/C	Rsph17025_0970	Nitric oxide reductase subunit C: catalyzes NO reduction and maintains the operation of denitrification electron transport chain
phaC	Rsph17025_0254	PHAs synthase: synthesizes PHAs and supplies electrons (NADH) and energy reserves for nitrogen and phosphorus removal
sdhA	Rsph17025_0088	Succinate dehydrogenase subunit A: participates in TCA cycle and provides crucial reducing power for nitrogen and phosphorus removal
glgB	Rsph17025_1783	1,4-α-glucan branching enzyme: participates in glycogen synthesis
nuoD	Rsph17025_2004	NADH-quinone oxidoreductase subunit D: involves in electron transport chain and pumps protons, acting as a core hub driving ATP synthesis
gltB, gltD	Rsph17025_2781, Rsph17025_2778	Glutamate synthase II: Cooperates with glnA to mediate efficient ammonia assimilation pathway
nifD, nifK1, nifH	Rsph17025_1247, Rsph17025_1248, Rsph17025_1246	Participates in the assembly of nitrogenase complex

Gene name

Function

nirK

Rsph17025_1595

Nitrite reductase mediates the key denitrification reaction by catalyzing the reduction of NO₂^- to NO

glnA

Rsph17025_1344

L-glutamine synthetase: involved in ammonia assimilation, and helps maintain the overall carbon and nitrogen metabolic balance during the DPA process

norB/C

Rsph17025_0970

Nitric oxide reductase subunit C: catalyzes NO reduction and maintains the operation of denitrification electron transport chain

phaC

Rsph17025_0254

PHAs synthase: synthesizes PHAs and supplies electrons (NADH) and energy reserves for nitrogen and phosphorus removal

sdhA

Rsph17025_0088

Succinate dehydrogenase subunit A: participates in TCA cycle and provides crucial reducing power for nitrogen and phosphorus removal

glgB

Rsph17025_1783

1,4-α-glucan branching enzyme: participates in glycogen synthesis

nuoD

Rsph17025_2004

NADH-quinone oxidoreductase subunit D: involves in electron transport chain and pumps protons, acting as a core hub driving ATP synthesis

gltB, gltD

Rsph17025_2781, Rsph17025_2778

Glutamate synthase II: Cooperates with glnA to mediate efficient ammonia assimilation pathway

nifD, nifK1, nifH

Rsph17025_1247, Rsph17025_1248, Rsph17025_1246

Participates in the assembly of nitrogenase complex