Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis.
Comprehensive Essentiality Analysis of the Mycobacterium tuberculosis Genome via Saturating Transposon Mutagenesis.
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DOI：
10.1128/mbio.02133-16
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发表时间：
2017-01-17
期刊：
影响因子：
6.4
通讯作者：
Ioerger TR
中科院分区：
文献类型：
--
作者：
DeJesus MA;Gerrick ER;Xu W;Park SW;Long JE;Boutte CC;Rubin EJ;Schnappinger D;Ehrt S;Fortune SM;Sassetti CM;Ioerger TR
几十年来，鉴定细菌染色体上生存所必需的区域一直依赖于在随机转座子突变体库中定位整合位点，以找到无法维持插入的基因座。迄今为止，这些研究分析了亚饱和文库，需要应用统计方法来估计转座子覆盖率的缺口是生物选择而不是插入的随机性的结果的可能性。因此，许多基因组特征的重要性，特别是小的，不能可靠地评估。我们试图通过在结核分枝杆菌中创建完全饱和的转座子文库来克服这种限制。在通过深度测序评估该高度饱和文库的组成时，我们发现Himar 1元件的先前未知的序列偏差使得约9%的潜在TA二核苷酸插入位点不允许插入。我们使用了一个隐马尔可夫模型的重要性，占这种意想不到的偏见，使我们能够自信地评估的重要性的功能，包含少至2 TA网站，包括开放阅读框架（ORF），实验确定的非编码RNA，甲基化位点，和启动子。此外，确定了几个不对应于已知特征的重要区域，表明生长所必需的未表征功能。这项工作提供了结核分枝杆菌基因组的重要区域的权威目录和将饱和诱变应用于其他细菌的统计框架。转座子插入突变体文库测序已成为一种广泛使用的工具，用于探测基因在各种条件下的功能。Himar 1转座子通常被认为在所有TA二核苷酸处以相等的概率插入，因此其在突变体文库中的缺失被认为指示针对相应突变体的生物选择。通过对饱和的Himar 1文库进行测序，我们发现了TA二核苷酸不同样允许插入的证据。在多种原核生物中观察到插入偏倚，并影响转座子插入（TnSeq）数据的统计解释和基本基因组区域的表征。利用这些见解，我们分析了结核分枝杆菌完全饱和的TnSeq文库，使我们能够生成体外必要性的全面目录，包括比任何先前研究中发现的小的ORF，小（非编码）RNA（sRNA），启动子和其他基因组特征。
For decades, identifying the regions of a bacterial chromosome that are necessary for viability has relied on mapping integration sites in libraries of random transposon mutants to find loci that are unable to sustain insertion. To date, these studies have analyzed subsaturated libraries, necessitating the application of statistical methods to estimate the likelihood that a gap in transposon coverage is the result of biological selection and not the stochasticity of insertion. As a result, the essentiality of many genomic features, particularly small ones, could not be reliably assessed. We sought to overcome this limitation by creating a completely saturated transposon library in Mycobacterium tuberculosis. In assessing the composition of this highly saturated library by deep sequencing, we discovered that a previously unknown sequence bias of the Himar1 element rendered approximately 9% of potential TA dinucleotide insertion sites less permissible for insertion. We used a hidden Markov model of essentiality that accounted for this unanticipated bias, allowing us to confidently evaluate the essentiality of features that contained as few as 2 TA sites, including open reading frames (ORF), experimentally identified noncoding RNAs, methylation sites, and promoters. In addition, several essential regions that did not correspond to known features were identified, suggesting uncharacterized functions that are necessary for growth. This work provides an authoritative catalog of essential regions of the M. tuberculosis genome and a statistical framework for applying saturating mutagenesis to other bacteria. Sequencing of transposon-insertion mutant libraries has become a widely used tool for probing the functions of genes under various conditions. The Himar1 transposon is generally believed to insert with equal probabilities at all TA dinucleotides, and therefore its absence in a mutant library is taken to indicate biological selection against the corresponding mutant. Through sequencing of a saturated Himar1 library, we found evidence that TA dinucleotides are not equally permissive for insertion. The insertion bias was observed in multiple prokaryotes and influences the statistical interpretation of transposon insertion (TnSeq) data and characterization of essential genomic regions. Using these insights, we analyzed a fully saturated TnSeq library for M. tuberculosis, enabling us to generate a comprehensive catalog of in vitro essentiality, including ORFs smaller than those found in any previous study, small (noncoding) RNAs (sRNAs), promoters, and other genomic features.