Lam Kathy N, Martens Eric C, Charles Trevor C
Department of Biology, University of Waterloo, Waterloo, Canada.
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
mSystems. 2018 Mar 27;3(3). doi: 10.1128/mSystems.00195-17. eCollection 2018 May-Jun.
Functional metagenomics is a powerful method that allows the isolation of genes whose role may not have been predicted from DNA sequence. In this approach, first, environmental DNA is cloned to generate metagenomic libraries that are maintained in Escherichia coli, and second, the cloned DNA is screened for activities of interest. Typically, functional screens are carried out using E. coli as a surrogate host, although there likely exist barriers to gene expression, such as lack of recognition of native promoters. Here, we describe efforts to develop Bacteroides thetaiotaomicron as a surrogate host for screening metagenomic DNA from the human gut. We construct a B. thetaiotaomicron-compatible fosmid cloning vector, generate a fosmid clone library using DNA from the human gut, and show successful functional complementation of a B. thetaiotaomicron glycan utilization mutant. Though we were unable to retrieve the physical fosmid after complementation, we used genome sequencing to identify the complementing genes derived from the human gut microbiome. Our results demonstrate that the use of B. thetaiotaomicron to express metagenomic DNA is promising, but they also exemplify the challenges that can be encountered in the development of new surrogate hosts for functional screening. Human gut microbiome research has been supported by advances in DNA sequencing that make it possible to obtain gigabases of sequence data from metagenomes but is limited by a lack of knowledge of gene function that leads to incomplete annotation of these data sets. There is a need for the development of methods that can provide experimental data regarding microbial gene function. Functional metagenomics is one such method, but functional screens are often carried out using hosts that may not be able to express the bulk of the environmental DNA being screened. We expand the range of current screening hosts and demonstrate that human gut-derived metagenomic libraries can be introduced into the gut microbe Bacteroides thetaiotaomicron to identify genes based on activity screening. Our results support the continuing development of genetically tractable systems to obtain information about gene function.
功能宏基因组学是一种强大的方法，可用于分离那些从DNA序列中无法预测其功能的基因。在这种方法中，首先，将环境DNA进行克隆以构建保存在大肠杆菌中的宏基因组文库，其次，对克隆的DNA进行感兴趣的活性筛选。通常，功能筛选是使用大肠杆菌作为替代宿主进行的，尽管可能存在基因表达障碍，例如对天然启动子缺乏识别。在此，我们描述了开发嗜热栖热放线菌作为筛选来自人类肠道宏基因组DNA的替代宿主的工作。我们构建了一种与嗜热栖热放线菌兼容的fosmid克隆载体，使用来自人类肠道的DNA生成了一个fosmid克隆文库，并展示了嗜热栖热放线菌聚糖利用突变体的成功功能互补。尽管互补后我们无法找回物理fosmid，但我们使用基因组测序来鉴定源自人类肠道微生物组的互补基因。我们的结果表明，使用嗜热栖热放线菌来表达宏基因组DNA是有前景的，但它们也例证了在开发用于功能筛选的新替代宿主时可能遇到的挑战。人类肠道微生物组研究得到了DNA测序进展的支持，这使得从宏基因组中获得数十亿碱基的序列数据成为可能，但受到基因功能知识的缺乏的限制，这导致这些数据集的注释不完整。需要开发能够提供有关微生物基因功能的实验数据的方法。功能宏基因组学就是这样一种方法，但功能筛选通常使用可能无法表达大部分被筛选环境DNA的宿主进行。我们扩大了当前筛选宿主的范围，并证明可以将源自人类肠道的宏基因组文库引入肠道微生物嗜热栖热放线菌中，以基于活性筛选来鉴定基因。我们的结果支持继续开发易于遗传操作的系统以获取有关基因功能的信息。
