Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense
Cre-lox-Based Method for Generation of Large Deletions within the Genomic Magnetosome Island of Magnetospirillum gryphiswaldense
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基于 Cre-lox 的 Magnetospirillum gryphiswaldense 基因组磁小体岛内大缺失生成方法
DOI：
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发表时间：
2010
影响因子：
4.4
通讯作者：
D. Schüler
中科院分区：
文献类型：
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作者：
S. Ullrich;D. Schüler
摘要趋磁细菌的磁小体生物矿化和趋磁性是由许多未知的基因功能控制的，这些基因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。我们建立了一个基于Cre-lox的删除方法，该方法可以切除大的基因组片段在磁藻gryphiswaldense。随后通过同源重组将两个接合性自杀质粒插入染色体中，所述接合性自杀质粒携带位于靶区域侧翼的lox位点，所述接合性自杀质粒分别仅需要一个单交换事件，并且导致双共整合。通过Cre重组酶的反式表达诱导包括插入的质粒及其抗性标记的靶向染色体区段的切除，其留下仅单个loxP位点的瘢痕。然后通过消除抗生素选择从缺失菌株中消除Cre辅助质粒。我们已经使用该方法从基因组MAI中缺失16.3-kb、61-kb和67.3-kb片段，在单轮或后续缺失轮中，覆盖包含mamAB、mms 6和mamGFDC操纵子的约87 kb的区域。正如预期的那样，所有突变体都是Mag-，有些是Mot-;否则，它们表现出正常的生长模式，这表明缺失的区域对于实验室中的生存能力不是必需的。该方法将有助于磁小体基因的功能分析，也可用于趋磁细菌的大规模基因组工程。
ABSTRACT 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.
影响因子：
2.7
作者：
Michael E. Kovach;R. Phillips;P. Elzer;R. Roop;K. Peterson
通讯作者：
Michael E. Kovach;R. Phillips;P. Elzer;R. Roop;K. Peterson
影响因子：
2.7
作者：
Marx, CJ;Lidstrom, ME
通讯作者：
Lidstrom, ME