Development of a xylose-inducible promoter and riboswitch combination system for manipulating gene expression in Fusobacterium nucleatum.
Development of a xylose-inducible promoter and riboswitch combination system for manipulating gene expression in Fusobacterium nucleatum.
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开发用于操纵具核梭杆菌基因表达的木糖诱导型启动子和核糖开关组合系统。
DOI：
10.1128/aem.00667-23
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发表时间：
2023
影响因子：
4.4
通讯作者：
Wu,Chenggang
中科院分区：
文献类型：
--
作者：
GC,Bibek;Zhou,Peng;Naha,Arindam;Gu,Jianhua;Wu,Chenggang
诱导型基因表达系统是研究细菌基因功能的重要手段，但大多数都存在渗漏现象。在这项研究中，我们通过整合木糖诱导的表达系统和茶碱响应的核糖开关，设计了一个无泄漏的杂交系统，用于精确控制inFusobacterium nucleatum的基因表达。这种创新的方法能够在转录和翻译起始水平上同时控制靶基因的表达。使用荧光素酶和吲哚产生酶色氨酸酶（TnaA）作为报告者，我们证明了杂交系统在缺乏诱导剂的情况下几乎没有可观察到的信号。我们利用该系统在抗FtsX突变株中表达FtsX， FtsX是一种与梭菌细胞分裂相关的蛋白，揭示了FtsX产生的剂量依赖性。没有诱导剂，细胞形成长丝，而通过增加诱导剂浓度增加FtsX水平导致细胞长度逐渐减少，直到恢复正常形态。至关重要的是，该系统促进了必需基因的研究，确定了信号肽sleepbgene对f至关重要。nucleatum。LepB的重要性源于耗竭，影响外膜生物发生和细胞分裂。这种新型杂交系统具有推进核仁必需基因和精确基因调控研究的潜力。重要意义核杆菌是一种在人类口腔中普遍存在的厌氧细菌，与牙周炎密切相关，并可在口腔以外的区域定植，如胎盘和胃肠道，导致不良妊娠结局并促进结直肠癌的生长。GivenF。鉴于Nucleatum的临床意义，研究人员正在开发有针对性的治疗方法来抑制其生长或专门根除这种细菌。对细菌生存、生长和繁殖至关重要的基本基因是有希望的药物靶点。研究这些基因需要一个无泄漏的诱导基因表达系统，使条件基因敲除和阐明这些必要基因的重要性成为可能。我们的研究首先通过产生条件基因突变nf . nucleatum确定了lepbas的必要基因。木糖诱导系统与核糖体开关的结合促进了核核体必需基因的分析，为针对该细菌的各种临床应用的潜在药物开发铺平了道路。
Inducible gene expression systems are important for studying bacterial gene function, yet most exhibit leakage. In this study, we engineered a leakage-free hybrid system for precise gene expression controls inFusobacterium nucleatumby integrating the xylose-inducible expression system with the theophylline-responsive riboswitch. This innovative method enables concurrent control of target gene expression at both transcription and translation initiation levels. Using luciferase and the indole-producing enzyme tryptophanase (TnaA) as reporters, we demonstrated that the hybrid system displays virtually no observable signal in the absence of inducers. We employed this system to express FtsX, a protein related to fusobacterial cytokinesis, in anftsXmutant strain, unveiling a dose-dependent manner in FtsX production. Without inducers, cells form long filaments, while increasing FtsX levels by increasing inducer concentrations led to a gradual reduction in cell length until normal morphology was restored. Crucially, this system facilitated essential gene investigation, identifying the signal peptidaselepBgene as vital forF. nucleatum. LepB’s essentiality stems from depletion, affecting outer membrane biogenesis and cell division. This novel hybrid system holds the potential for advancing research on essential genes and accurate gene regulation inF. nucleatum.IMPORTANCEFusobacterium nucleatum, an anaerobic bacterium prevalent in the human oral cavity, is strongly linked to periodontitis and can colonize areas beyond the oral cavity, such as the placenta and gastrointestinal tract, causing adverse pregnancy outcomes and promoting colorectal cancer growth. GivenF. nucleatum’s clinical significance, research is underway to develop targeted therapies to inhibit its growth or eradicate the bacterium specifically. Essential genes, crucial for bacterial survival, growth, and reproduction, are promising drug targets. A leak-free-inducible gene expression system is needed for studying these genes, enabling conditional gene knockouts and elucidating the importance of those essential genes. Our study identifiedlepBas the essential gene by first generating a conditional gene mutation inF. nucleatum. Combining a xylose-inducible system with a riboswitch facilitated the analysis of essential genes inF. nucleatum, paving the way for potential drug development targeting this bacterium for various clinical applications.
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影响因子：
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DOI：
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发表时间：
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影响因子：
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作者：
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