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首页> 外文期刊>Journal of bacteriology >Saturating Mutagenesis of an Essential Gene: a Majority of the Neisseria gonorrhoeae Major Outer Membrane Porin (PorB) Is Mutable
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Saturating Mutagenesis of an Essential Gene: a Majority of the Neisseria gonorrhoeae Major Outer Membrane Porin (PorB) Is Mutable

机译：必需基因的饱和诱变：淋病奈瑟氏球菌的主要外膜孔蛋白（PorB）的大多数是可变的。

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The major outer membrane porin (PorB) of Neisseria gonorrhoeae is an essential protein that mediates ion exchange between the organism and its environment and also plays multiple roles in human host pathogenesis. To facilitate structure-function studies of porin's multiple roles, we performed saturating mutagenesis at the porB locus and used deep sequencing to identify essential versus mutable residues. Random mutations in porB were generated in a plasmid vector, and mutant gene pools were transformed into N. gonorrhoeae to select for alleles that maintained bacterial viability. Deep sequencing of the input plasmid pools and the output N. gonorrhoeae genomic DNA pools identified mutations present in each, and the mutations in both pools were compared to determine which changes could be tolerated by the organism. We examined the mutability of 328 amino acids in the mature PorB protein and found that 308 of them were likely to be mutable and that 20 amino acids were likely to be nonmutable. A subset of these predictions was validated experimentally. This approach to identifying essential amino acids in a protein of interest introduces an additional application for next-generation sequencing technology and provides a template for future studies of both porin and other essential bacterial genes.
机译：淋病奈瑟氏球菌的主要外膜孔蛋白（PorB）是介导生物体与其环境之间离子交换的必需蛋白，并且在人宿主发病中也起着多种作用。为了促进孔蛋白的多重功能的结构功能研究，我们在 porB 位点进行了饱和诱变，并使用深度测序来鉴定必需残基和可变残基。在质粒载体中产生了 porB 中的随机突变，并将突变基因库转化到淋病奈瑟氏球菌中，以选择保持细菌活力的等位基因。对输入质粒库和输出淋病奈瑟氏球菌基因组DNA池的深度测序鉴定出每个突变体中都存在突变，然后比较这两个池中的突变以确定该生物体可以耐受哪些变化。我们检查了成熟的PorB蛋白中328个氨基酸的变异性，发现其中308个氨基酸可能是可变的，而20个氨基酸则是不可突变的。这些预测的子集已通过实验验证。这种鉴定目标蛋白中必需氨基酸的方法为下一代测序技术引入了另一项应用，并为孔蛋白和其他必需细菌基因的未来研究提供了模板。

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