Genetic Manipulation of Wild Human Gut Bacteroides
Genetic Manipulation of Wild Human Gut Bacteroides
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DOI：
10.1128/jb.00544-19
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发表时间：
2020-02-01
影响因子：
3.2
通讯作者：
Goodman, Andrew L.
中科院分区：
文献类型：
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作者：
Bencivenga-Barry, Natasha A.;Lim, Bentley;Goodman, Andrew L.
拟杆菌属是人类肠道微生物组中最突出的属之一，对该细菌群的研究提供了对肠道微生物生态学和发病机制的见解。在这份报告中，我们介绍了一种阴性选择系统，用于在野生拟杆菌属物种中进行快速有效的等位基因交换，该系统不需要对遗传背景或营养成分确定的培养基进行任何改变。在这种方法中，通常通过VI型分泌递送的双重抗菌效应物在严格调节的脱水四环素（aTC）诱导型启动子的控制下靶向细菌周质。aTC的引入选择产生所需遗传修饰的重组事件，并且双效应子设计允许跨可能对一种反选择效应子具有免疫力的菌株的广泛适用性。我们证明了这种方法在21个代表不同物种的人类肠道拟杆菌分离株中的实用性，包括直接从人类供体中分离的菌株。我们使用这个系统来建立，抗菌肽耐药性的普通拟杆菌是由一个基因的产品，不包括在基因组中以前的人类肠道微生物的基因组成员的易处理的重要性人类肠道拟杆菌物种表现出菌株水平的差异，在他们的生理，生态和对人类健康和疾病的影响。然而，用于遗传操作的现有方法通常需要为每种感兴趣的微生物或确定成分的培养基制剂构建遗传修饰的亲本菌株。在这份报告中，我们介绍了一个强大的和有效的战略，有针对性的遗传操作的不同野生型拟杆菌物种从人类肠道。该系统能够对现有模式生物以外的人类和动物微生物组成员进行遗传研究。
Bacteroides is one of the most prominent genera in the human gut microbiome, and study of this bacterial group provides insights into gut microbial ecology and pathogenesis. In this report, we introduce a negative selection system for rapid and efficient allelic exchange in wild Bacteroides species that does not require any alterations to the genetic background or a nutritionally defined culture medium. In this approach, dual antibacterial effectors normally delivered via type VI secretion are targeted to the bacterial periplasm under the control of tightly regulated anhydrotetracycline (aTC)-inducible promoters. Introduction of aTC selects for recombination events producing the desired genetic modification, and the dual effector design allows for broad applicability across strains that may have immunity to one counterselection effector. We demonstrate the utility of this approach across 21 human gut Bacteroides isolates representing diverse species, including strains isolated directly from human donors. We use this system to establish that antimicrobial peptide resistance in Bacteroides vulgatus is determined by the product of a gene that is not included in the genomes of previously genetically tractable members of the human gut microbiome.IMPORTANCE Human gut Bacteroides species exhibit strain-level differences in their physiology, ecology, and impact on human health and disease. However, existing approaches for genetic manipulation generally require construction of genetically modified parental strains for each microbe of interest or defined medium formulations. In this report, we introduce a robust and efficient strategy for targeted genetic manipulation of diverse wild-type Bacteroides species from the human gut. This system enables genetic investigation of members of human and animal microbiomes beyond existing model organisms.