Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling.
Bacterial genome reduction using the progressive clustering of deletions via yeast sexual cycling.
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DOI：
10.1101/gr.182477.114
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发表时间：
2015-03
期刊：
影响因子：
7
通讯作者：
Venter JC
中科院分区：
文献类型：
--
作者：
Suzuki Y;Assad-Garcia N;Kostylev M;Noskov VN;Wise KS;Karas BJ;Stam J;Montague MG;Hanly TJ;Enriquez NJ;Ramon A;Goldgof GM;Richter RA;Vashee S;Chuang RY;Winzeler EA;Hutchison CA 3rd;Gibson DG;Smith HO;Glass JI;Venter JC
具有简单基因组的遗传上易处理的生物体的可用性对于基本生物过程的快速、系统级理解至关重要。在自由生活的细胞生物体中，支原体细菌具有最小的已知基因组，是实现这一目的的理想模型，但这些细胞的自然版本具有基因组复杂性仍然太大，无法提供基本生命形式的全面视图。在这里，我们描述了一种有效的方法，用于减少这些生物体的基因组，通过使用转座子诱变和逐步聚类删除的基因组片段之间的减数分裂重组的细菌基因组中窝藏在酵母中识别个别可删除的区域。支原体基因组经过这一过程并移植到受体细胞中产生了两种支原体菌株。第一个同时缺少基因组的8个单一可删除区域，代表总共91个基因和原始基因组的10%。第二个菌株缺少八个区域中的七个，代表84个基因。生长测定数据显示，在测试条件下，91个基因之间不存在遗传相互作用。尽管预测的糖代谢和蛋白质组的缺失的影响，生长速率不受基因缺失的七缺失菌株。这些结果支持了使用单基因破坏数据来设计和构建缺乏多个基因的可行基因组的可行性，为基因组最小化铺平了道路。这种渐进聚类方法有望有效地重组酵母中克隆的任何巨型DNA分子，促进微生物中设计基因组的构建以及高等真核生物基因工程的基因组片段。
The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes.
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影响因子：
56.9
作者：
Hillenmeyer, Maureen E.;Fung, Eula;Giaever, Guri
通讯作者：
Giaever, Guri
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Gibson, Daniel G.;Young, Lei;Smith, Hamilton O.
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Karas, Bogumil J.;Tagwerker, Christian;Smith, Hamilton O.
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作者：
Allam AB;Brown MB;Reyes L
通讯作者：
Reyes L
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14.9
作者：
Benders GA;Noskov VN;Denisova EA;Lartigue C;Gibson DG;Assad-Garcia N;Chuang RY;Carrera W;Moodie M;Algire MA;Phan Q;Alperovich N;Vashee S;Merryman C;Venter JC;Smith HO;Glass JI;Hutchison CA 3rd
通讯作者：
Hutchison CA 3rd