Conjugation and transposon mutagenesis of Xenorhabdus griffiniae HGB2511, the bacterial symbiont of the nematode Steinernema hermaphroditum (India).
Conjugation and transposon mutagenesis of Xenorhabdus griffiniae HGB2511, the bacterial symbiont of the nematode Steinernema hermaphroditum (India).
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DOI：
10.17912/micropub.biology.000772
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发表时间：
2023
影响因子：
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通讯作者：
Heppert, Jennifer K
中科院分区：
文献类型：
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作者：
Alani, Omar S;Cao, Mengyi;Goodrich-Blair, Heidi;Heppert, Jennifer K
共生是两个有机体之间的有益互动，是地球上所有生命的普遍特征，包括动物和细菌之间的联系。然而，动物和细菌之间形成不同伙伴关系的具体分子和细胞机制仍在探索中。昆虫病原线虫在昆虫宿主之间传播细菌，共同杀死昆虫，细菌消耗昆虫，并作为线虫的食物来源。这些线虫，包括斯氏线虫属的线虫，是研究共生分子机制的有效实验室模型，因为它们与杆菌形成自然伙伴关系，并且它们直接饲养。斯氏线虫雌雄同体线虫及其共生菌是研究共生的遗传模式对。我们在这个项目中的目标是开始识别可能对与线虫宿主共生相互作用重要的细菌基因。为此，我们修改和优化了一种传递和插入LacZ-启动子-探针转座子的协议，用于互花链霉菌共生菌X.Griffiniae HGB2511(曹等人，2022)。我们评估了我们获得外接子、代谢营养缺陷型突变体和活性启动子-LacZ融合的频率。我们的数据表明，TN 10转座子的插入是相对随机的，因为4.7%的突变体表现出营养缺陷型。在47%的菌株中，启动子与转座子编码的LacZ发生融合，导致β-半乳糖苷酶活性的表达。据我们所知，这是第一个针对该细菌物种产生的突变方案，并将促进对格里菲亚氏菌共生和其他感兴趣的表型进行大规模筛选。
Symbiosis, the beneficial interactions between two organisms, is a ubiquitous feature of all life on Earth, including associations between animals and bacteria. However, the specific molecular and cellular mechanisms which underlie the diverse partnerships formed between animals and bacteria are still being explored. Entomopathogenic nematodes transport bacteria between insect hosts, together they kill the insect, and the bacteria consume the insect and serve as food source for the nematodes. These nematodes, including those in the Steinernema genus, are effective laboratory models for studying the molecular mechanisms of symbiosis because of the natural partnership they form with Xenorhabdus bacteria and their straightforward husbandry. Steinernema hermaphroditum nematodes and their Xenorhabdus griffiniae symbiotic bacteria are being developed as a genetic model pair for studying symbiosis. Our goal in this project was to begin to identify bacterial genes that may be important for symbiotic interactions with the nematode host. Towards this end, we adapted and optimized a protocol for delivery and insertion of a lacZ- promoter-probe transposon for use in the S. hermaphroditum symbiont, X. griffiniae HGB2511 (Cao et al., 2022). We assessed the frequencies at which we obtained exconjugants, metabolic auxotrophic mutants, and active promoter- lacZ fusions. Our data indicate that the Tn 10 transposon inserted relatively randomly based on the finding that 4.7% of the mutants exhibited an auxotrophic phenotype. Promoter-fusions with the transposon-encoded lacZ , which resulted in expression of β-galactosidase activity, occurred in 47% of the strains. To our knowledge, this is the first mutagenesis protocol generated for this bacterial species, and will facilitate the implementation of large scale screens for symbiosis and other phenotypes of interest in X. griffiniae .