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首页> 外文期刊>Applied Microbiology >Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense
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Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense

机译：基于Cre-lox的方法在gspirphiswaldense的基因组磁小体岛内产生大量缺失

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Magnetosome biomineralization and magnetotaxis in magnetotactic bacteria are controlled by numerous, mostly unknown gene functions that are predominantly encoded by several operons located within the genomic magnetosome island (MAI). Genetic analysis of magnetotactic bacteria has remained difficult and requires the development of novel tools. We established a Cre- lox -based deletion method which allows the excision of large genomic fragments in Magnetospirillum gryphiswaldense . Two conjugative suicide plasmids harboring lox sites that flanked the target region were subsequently inserted into the chromosome by homologous recombination, requiring only one single-crossover event, respectively, and resulting in a double cointegrate. Excision of the targeted chromosomal segment that included the inserted plasmids and their resistance markers was induced by trans expression of Cre recombinase, which leaves behind a scar of only a single loxP site. The Cre helper plasmid was then cured from the deletant strain by relief of antibiotic selection. We have used this method for the deletion of 16.3-kb, 61-kb, and 67.3-kb fragments from the genomic MAI, either in a single round or in subsequent rounds of deletion, covering a region of approximately 87 kb that comprises the mamAB , mms6 , and mamGFDC operons. As expected, all mutants were Mag~(?) and some were Mot~(?); otherwise, they showed normal growth patterns, which indicates that the deleted region is not essential for viability in the laboratory. The method will facilitate future functional analysis of magnetosome genes and also can be utilized for large-scale genome engineering in magnetotactic bacteria.
机译：趋磁细菌中的磁小体生物矿化和趋磁性受众多（主要是未知基因）基因功能的控制，这些功能主要由位于基因组磁小体岛（MAI）内的几个操纵子编码。趋磁细菌的遗传分析仍然很困难，需要开发新的工具。我们建立了基于Crelox的删除方法，该方法可以切除Gspirphiswaldense的Magspirospirillum gryphiswaldense中的大基因组片段。随后通过同源重组将两个携带有位于靶区域侧翼的lox位点的自杀质粒插入到染色体中，分别仅需要一个单交换事件，并导致双共整合。 Cre重组酶的反式表达诱导了包括插入质粒及其抗性标记物在内的靶向染色体片段的切除，而Cre重组酶的反式表达仅留下了单个loxP位点的疤痕。然后通过减轻抗生素选择，从缺失菌株中治愈Cre辅助质粒。我们已经使用这种方法在单轮或后续轮次删除中从基因组MAI中删除了16.3kb，61kb和67.3kb片段，覆盖了包含mamAB的约87 kb区域，mms6和mamGFDC操纵子。不出所料，所有的突变体都是Mag〜（？），有些是Mot〜（？）。否则，它们显示正常的生长模式，这表明缺失的区域对于实验室的生存力不是必需的。该方法将促进磁小体基因的未来功能分析，也可用于趋磁细菌的大规模基因组工程。

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