Elucidating functions of commensal microbial genes in the mammalian gut is challenging because many commensals are recalcitrant to laboratory cultivation and genetic manipulation. We present Temporal FUnctional Metagenomics sequencing (TFUMseq), a platform to functionally mine bacterial genomes for genes that contribute to fitness of commensal bacteria in vivo. Our approach uses metagenomic DNA to construct large-scale heterologous expression libraries that are tracked over time in vivo by deep sequencing and computational methods. To demonstrate our approach, we built a TFUMseq plasmid library using the gut commensal Bacteroides thetaiotaomicron (Bt) and introduced Escherichia coli carrying this library into germfree mice. Population dynamics of library clones revealed Bt genes conferring significant fitness advantages in E. coli over time, including carbohydrate utilization genes, with a Bt galactokinase central to early colonization, and subsequent dominance by a Bt glycoside hydrolase enabling sucrose metabolism coupled with co-evolution of the plasmid library and E. coli genome driving increased galactose utilization. Our findings highlight the utility of functional metagenomics for engineering commensal bacteria with improved properties, including expanded colonization capabilities in vivo.

中文翻译：

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通过体内时间功能性宏基因组学改善哺乳动物肠道中的微生物适应性。

阐明共生微生物基因在哺乳动物肠道中的功能具有挑战性，因为许多共生微生物对实验室培养和基因操作都是顽固的。我们提出了时间功能性元基因组测序（TFUMseq），该平台可从功能上挖掘细菌基因组，寻找有助于体内共生细菌健康的基因。我们的方法使用宏基因组DNA来构建大规模的异源表达文库，并通过深度测序和计算方法在体内随时间进行追踪。为了证明我们的方法，我们使用肠道共生细菌拟杆菌（Bt）建立了TFUMseq质粒文库，并将携带该文库的大肠杆菌引入了无菌小鼠中。库克隆的种群动态显示，随着时间的推移，Bt基因在大肠杆菌中具有明显的适应性优势，包括碳水化合物利用基因，其中Bt半乳糖激酶在早期定居中处于中心地位，随后Bt糖苷水解酶占主导地位，使蔗糖代谢与质粒文库和大肠杆菌基因组共同进化共同驱动半乳糖利用增加。我们的发现突出了功能宏基因组学在工程共生细菌中的实用性，该细菌具有改善的特性，包括在体内扩展的定殖能力。