Rapid mapping of insertional mutations to probe cell wall regulation in Cryptococcus neoformans.
Rapid mapping of insertional mutations to probe cell wall regulation in Cryptococcus neoformans.
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DOI：
10.1016/j.fgb.2015.06.003
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发表时间：
2015-09
期刊：
影响因子：
--
通讯作者：
Alspaugh JA
中科院分区：
文献类型：
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作者：
Esher SK;Granek JA;Alspaugh JA
随机插入诱变筛选是微生物遗传学研究的重要工具。真菌系统的研究人员利用植物病原体根癌农杆菌，通过独特的跨界细胞转接合过程，在其感兴趣的真菌物种中创建标记的随机突变，用于基因筛选。然而，识别插入位置传统上需要繁琐的基于 PCR 的方法，限制了该系统的有效通量。我们开发了一种高效的基因组测序和分析方法（AIM-Seq），以促进微生物中随机生成的基因组插入的识别。 AIM-Seq 结合了批量采样、全基因组测序和新型生物信息学流程 AIM-HII，可快速识别基因组插入位点。我们专门将该技术应用于人类真菌病原体新型隐球菌中农杆菌介导的转接合。通过这种方法，我们筛选了新型隐球菌细胞壁突变体文库，选择了 27 个感兴趣的突变体进行 AIM-Seq 分析。我们在这种病原真菌的细胞壁过程的已知和以前未知的调节因子中发现了 35 个假定的基因组插入。我们通过创建独立的突变株并分析所得的细胞壁表型来确认其中一部分的相关性。通过对这些突变进行基于序列的分析，我们观察到农杆菌转移 DNA 的“典型”插入以及非典型插入事件，包括大缺失和染色体重排。这种突变分析工具最初应用于新型隐球菌，可应用于广泛的实验系统和诱变方法，促进未来的微生物遗传筛选。
Random insertional mutagenesis screens are important tools in microbial genetics studies. Investigators in fungal systems have used the plant pathogen Agrobacterium tumefaciens to create tagged, random mutations for genetic screens in their fungal species of interest through a unique process of trans-kingdom cellular transconjugation. However, identifying the locations of insertion has traditionally required tedious PCR-based methods, limiting the effective throughput of this system. We have developed an efficient genomic sequencing and analysis method (AIM-Seq) to facilitate identification of randomly generated genomic insertions in microorganisms. AIM-Seq combines batch sampling, whole genome sequencing, and a novel bioinformatics pipeline, AIM-HII, to rapidly identify sites of genomic insertion. We have specifically applied this technique to Agrobacterium-mediated transconjugation in the human fungal pathogen Cryptococcus neoformans. With this approach, we have screened a library of C. neoformans cell wall mutants, selecting twenty-seven mutants of interest for analysis by AIM-Seq. We identified thirty-five putative genomic insertions in known and previously unknown regulators of cell wall processes in this pathogenic fungus. We confirmed the relevance of a subset of these by creating independent mutant strains and analyzing resulting cell wall phenotypes. Through our sequence-based analysis of these mutations, we observed “typical” insertions of the Agrobacterium transfer DNA as well as atypical insertion events, including large deletions and chromosomal rearrangements. Initially applied to C. neoformans, this mutant analysis tool can be applied to a wide range of experimental systems and methods of mutagenesis, facilitating future microbial genetic screens.