Perry Benjamin J, Yost Christopher K
Department of Biology, University of Regina, 3737 Wascana Parkway, Regina, SK, S4S 0A2, Canada.
BMC Microbiol. 2014 Nov 30;14:298. doi: 10.1186/s12866-014-0298-z.
The Rhizobiaceae family of Gram-negative bacteria often engage in symbiosis with plants of economic importance. Historically, genetic studies to identify the function of individual genes, and characterize the biology of these bacteria have relied on the use of classical transposon mutagenesis. To increase the rate of scientific discovery in the Rhizobiaceae there is a need to adapt high-throughput genetic screens like insertion sequencing for use in this family of bacteria. Here we describe a Rhizobiaceae compatible MmeI-adapted mariner transposon that can be used with insertion sequencing for high-throughput genetic screening.
The newly constructed mariner transposon pSAM_Rl mutagenized R. leguminosarum, S. meliloti, and A. tumefaciens at a high frequency. In R. leguminosarum, mutant pools were generated that saturated 88% of potential mariner insertions sites in the genome. Analysis of the R. leguminosarum transposon insertion sequencing data with a previously described hidden Markov model-based method resulted in assignment of the contribution of all annotated genes in the R. leguminosarum 3841 genome for growth on a complex medium. Good concordance was observed between genes observed to be required for growth on the complex medium, and previous studies.
The newly described Rhizobiaceaee compatible mariner transposon insertion sequencing vector pSAM_Rl has been shown to mutagenize at a high frequency and to be an effective tool for use in high-throughput genetic screening. The construction and validation of this transposon insertion sequencing tool for use in the Rhizobiziaceae will provide an opportunity for researchers in the Rhizobiaceae community to use high-throughput genetic screening, allowing for significant increase in the rate of genetic discovery, particularly given the recent release of genome sequences from many Rhizobiaceae strains.
革兰氏阴性菌根瘤菌科细菌常与具有经济重要性的植物形成共生关系。从历史上看，用于鉴定单个基因功能并表征这些细菌生物学特性的遗传学研究依赖于经典转座子诱变技术。为了提高根瘤菌科的科学发现率，需要采用适用于该细菌家族的高通量遗传筛选方法，如插入测序。在此，我们描述了一种与根瘤菌科兼容的经MmeI改造的水手转座子，可用于插入测序以进行高通量遗传筛选。
新构建的水手转座子pSAM_Rl能以高频率诱变豆科根瘤菌、苜蓿中华根瘤菌和根癌土壤杆菌。在豆科根瘤菌中，产生的突变体库覆盖了基因组中88%的潜在水手转座子插入位点。使用先前描述的基于隐马尔可夫模型的方法分析豆科根瘤菌转座子插入测序数据，确定了豆科根瘤菌3841基因组中所有注释基因对在复杂培养基上生长的贡献。在观察到的在复杂培养基上生长所需的基因与先前研究之间，观察到了良好的一致性。
新描述的与根瘤菌科兼容的水手转座子插入测序载体pSAM_Rl已被证明能以高频率诱变，是用于高通量遗传筛选的有效工具。这种用于根瘤菌科的转座子插入测序工具的构建和验证，将为根瘤菌科领域的研究人员提供使用高通量遗传筛选的机会，从而显著提高遗传发现率，特别是考虑到最近许多根瘤菌科菌株基因组序列的发布。
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