Abstract：
Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiont Snodgrassella alvi. We are also able to engineer the genomes of multiple strains of S. alvi and another species, Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont, Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout in S. alvi using this approach is recA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods.
OBJECTIVE: Honey bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.
OBJECTIVE: Honey bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health.
摘要：
对许多宿主-微生物系统中相互作用的机械理解，包括蜜蜂微生物组，受限于缺乏易于使用的基因组工程方法。为此,我们展示了一种一步法的基因组工程方法，用于在蜜蜂肠道细菌共生体的染色体中进行基因缺失和插入。包含抗生素抗性盒的线性或非复制质粒DNA的电穿孔可靠地导致染色体整合，该抗生素抗性盒的两侧与共生体基因组具有同源性。这种轻量级方法不需要表达任何外源重组机制。使用现代DNA合成和组装方法可以容易地产生使该方法有效所需的具有长同源区的高浓度大DNA。我们用这种方法敲除基因，包括参与生物膜形成的基因，并将荧光蛋白基因插入到β蛋白细菌蜂肠共生体Snodgrassellaalvi的染色体中。我们还能够设计S.alvi和另一个物种的多个菌株的基因组，Snodgrassellacommunis,在大黄蜂肠道微生物组中发现。最后,我们用同样的方法来设计另一只蜜蜂共生体的染色体，巴尔通菌,是一种变形杆菌.不出所料,使用这种方法在Alvi中的基因敲除是recA依赖性的，这表明这个简单的程序可以应用于其他缺乏方便的基因组工程方法的微生物。
目的：蜜蜂是生态和经济上重要的作物传粉者，具有影响其健康的细菌肠道共生体。用于研究或改善蜜蜂健康的基于微生物组的策略已经利用野生型或质粒工程化细菌。我们证明了一个简单的，单步法可用于在多个蜂肠细菌的染色体中插入盒和替换基因。此方法可用于研究蜜蜂肠道群落中宿主-微生物相互作用的机制，并稳定地改造有利于传粉者健康的共生体。
目的：蜜蜂是生态和经济上重要的作物传粉者，具有影响其健康的细菌肠道共生体。用于研究或改善蜜蜂健康的基于微生物组的策略已经利用野生型或质粒工程化细菌。我们证明了一个简单的，单步法可用于在多个蜂肠细菌的染色体中插入盒和替换基因。此方法可用于研究蜜蜂肠道群落中宿主-微生物相互作用的机制，并稳定地改造有利于传粉者健康的共生体。
