Sequence-defined transposon mutant library of Burkholderia thailandensis.
Sequence-defined transposon mutant library of Burkholderia thailandensis.
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DOI：
10.1128/mbio.00604-13
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发表时间：
2013-11-05
期刊：
影响因子：
6.4
通讯作者：
Manoil C
中科院分区：
文献类型：
--
作者：
Gallagher LA;Ramage E;Patrapuvich R;Weiss E;Brittnacher M;Manoil C
我们构建了一个近饱和的转座子突变库Burkholderia thailandensis，一个低毒力的替代类鼻疽的病原体（类鼻疽伯克霍尔德氏菌）。使用转座子Tn 5衍生物产生了近42，000个独特突变体（约7.5个突变体/基因）的主要集合。这些菌株携带87%的预测蛋白编码基因的插入，对应于几乎所有在营养琼脂上生长所必需的基因。为了实现高的基因组覆盖率，我们开发了用于在DNA的极其富含GC的区域中的插入的有效序列鉴定的程序。为了促进菌株分布，我们创建了每个基因具有两个突变体的二级文库，其中大多数转座子位置已经通过重测序确认。双等位基因库中的突变图和获得菌株的程序可在和中找到。该文库应有助于全面的突变体筛选，并作为菌株的来源，以测试预测的基因型-表型关联。革兰氏阴性细菌类鼻疽伯克霍尔德氏菌是一种生物威胁剂，因为它具有气溶胶递送和内在抗生素耐药性的潜力，并且因为暴露会产生恶性感染。类鼻疽B氏菌的大规模研究受到限制，因为该微生物必须在生物安全性3级条件下操作。类鼻疽B.的近亲称为Burkholderia thailandensis，可以在限制性较低的条件下进行研究，已被验证为毒力、抗生素耐药性和其他性状研究中的低毒力替代物。为了便于对B. thailandensis的大规模研究，我们创建了该生物体的近饱和、序列定义的转座子突变体文库。该文库促进了鉴定类鼻疽B氏菌中保守的基因型-表型关联的遗传研究。
We constructed a near-saturation transposon mutant library for Burkholderia thailandensis, a low-virulence surrogate for the causative agent of melioidosis (Burkholderia pseudomallei). A primary set of nearly 42,000 unique mutants (~7.5 mutants/gene) was generated using transposon Tn5 derivatives. The strains carry insertions in 87% of the predicted protein-coding genes of the organism, corresponding to nearly all of those nonessential for growth on nutrient agar. To achieve high genome coverage, we developed procedures for efficient sequence identification of insertions in extremely GC-rich regions of DNA. To facilitate strain distribution, we created a secondary library with two mutants per gene for which most transposon locations had been confirmed by resequencing. A map of mutations in the two-allele library and procedures for obtaining strains can be found at and . The library should facilitate comprehensive mutant screens and serve as a source of strains to test predicted genotype-phenotype associations. The Gram-negative bacterium Burkholderia pseudomallei is a biothreat agent due to its potential for aerosol delivery and intrinsic antibiotic resistance and because exposure produces pernicious infections. Large-scale studies of B. pseudomallei are limited by the fact that the organism must be manipulated under biological safety level 3 conditions. A close relative of B. pseudomallei called Burkholderia thailandensis, which can be studied under less restrictive conditions, has been validated as a low-virulence surrogate in studies of virulence, antibiotic resistance and other traits. To facilitate large-scale studies of B. thailandensis, we created a near-saturation, sequence-defined transposon mutant library of the organism. The library facilitates genetic studies that identify genotype-phenotype associations conserved in B. pseudomallei.