High-Resolution Mapping of Bacterial Transcriptional Responses in Human-Associated Microbiota

项目摘要 微生物群落的功能图谱对于了解其对宿主健康的整体影响至关重要。大多数情况下， 微生物组样本的超基因组鸟枪法测序用于评估总的功能能力。然而，转录的 根据环境的不同，不同生物体的反应可能会有很大的不同，可能会产生很大的影响。许多 代谢功能只有在有机体敏锐地感觉到其体内存在特定底物后才能表现出来 环境。病原体只有在获得临界数量的病原体后才能表达毒力因子。总体来说，压力 在不断变化的非生物和生物条件下，反应对生存至关重要。能够全面规划 这些途径决定了微生物组的弹性、可塑性和病理功能，需要敏感、 强大的转录组学工具。 对细菌群落进行传统的RNAseq分析已经成为获得 转录信息，但由于需要技术解决办法，它提供了不完整的信息 关于转录景观的信息。核糖体RNA需要在测序前耗尽，它具有很差的 信噪比由不同的RNA衰减率引起，对非编码RNA的转录不敏感 具有二级结构或转录后修饰的基因。或者，RNA聚合酶(RNAP)的位置 可以进行评估，这提供了转录的实时读数。尽管所谓的新生记录测序已经 在大肠杆菌中进行，揭示了仅使用RNAseq时难以捉摸的转录暂停位点和其他现象， 这些方案依赖于RNAP的免疫沉淀，因此不适合复杂的微生物群落 RNAP可能非常多样化，需要物种特异性抗体。作为解决方案，Precision Run-On和 测序(PRO-SEQ)是一种最初为检查真核生物转录而创建的方法，它可能提供一个公正的 在培养细菌或复杂微生物群落中检测转录动力学的方法，如 人类微生物群。 我们的目标是测试PRO-SEQ应用于原核生物的可行性，并评估其捕获能力 与典型应激反应途径(热休克、氧气暴露和DNA)相关的转录动力学 损害)，使用一组定量指标。我们的目标是验证并在必要时修改协议，以便可以使用 在不同物种之间表现强劲。我们计划开发一种计算方法来测试转录现象的全部广度 用这种方法可以观察到的，例如转录暂停，双向转录，RNAP的差异 在物种间明显的功能，以及RNA衰减率等方面。如果成功，我们预计PRO-SEQ将 被用来研究人类相关微生物区系对宿主饮食、炎症信号、外源物质和 更直接地说，是人类转录电路。

Project Summary Functional profiling of microbial communities is critical to understanding their overall effects on host health. Most often, metagenomic shotgun sequencing of microbiome samples is used to assess total functional capacity. Yet, transcriptional responses may vary dramatically between organisms depending on the context, with potentially large effects. Many metabolic functions are only expressed after the organism acutely senses the presence of particular substrates in their environment. Pathogens may only express virulence factors after obtaining a critical quorum of pathogens. Overall, stress responses are critical for survival under changing abiotic and biotic conditions. Being able to comprehensively map out these pathways, which determine the resilience, plasticity, and patho-functions of the microbiome, requires sensitive, robust transcriptional –omics tools. Performing traditional RNAseq analyses on bacterial communities has been the predominant method to gain transcriptional information, but it is hampered by the need for technical workarounds and it provides incomplete information about the transcriptional landscape. Ribosomal RNA needs to be depleted prior to sequencing, it has a poor signal-to-noise ratio arising from varying RNA decay rates, and it is insensitive to the transcription of non-coding RNA that has secondary structure or post-transcriptional modifications. Alternatively, the position of RNA polymerase (RNAP) can be assessed, which provides a real-time readout of transcription. Although so-called nascent transcript sequencing has been performed in E. coli, revealing transcriptional pause sites and other phenomenon elusive when using RNAseq alone, these protocols rely on immunoprecipitation of RNAP and are therefore unsuitable for complex microbial communities where RNAP may be quite diverse and require species-specific antibodies. As a solution, Precision Run-On and SEQuencing (PRO-seq), a method originally created for examining transcription in eukaryotes, may provide an unbiased method to examine transcriptional dynamics on cultured bacteria or in complex microbial communities, such as the human microbiome. Our goal is to test the feasibility of PRO-seq when applied to prokaryotes and to evaluate its ability to capture transcriptional dynamics associated with canonical stress response pathways (heat-shock, oxygen exposure and DNA damage), using a set of quantitative metrics. We aim to validate, and if necessary, modify the protocol so it can be used robustly across species. We plan to develop a computational approach to test the full breadth of transcriptional phenomena that can be observed using this method, such as transcriptional pausing, bidirectional transcription, differences in RNAP function apparent across species, and RNA decay rates, among other aspects. If successful, we expect that PRO-seq will be adopted to study the responses of human-associated microbiota to host diet, inflammatory signals, xenobiotics and to human transcriptional circuitry, more directly.