Unraveling regulatory networks in biological nutrient removal (BNR) microbiomes

运营废水处理厂的很大一部分能源成本是为执行处理过程的微生物提供氧气。如果处理厂能够在低氧条件下运行，这些成本将会降低。目前尚不清楚微生物如何适应低氧条件，以及处理厂能否在最低限度的氧气曝气下有效运行。该项目将揭示废水处理系统中的关键微生物如何调节其内部基因网络，以应对工厂在最小曝气量下的运行。该项目的其他影响包括与指导高中生、本科生和研究生有关的教育活动，以及促进对自然和工程环境中基因调控的理解。如果成功，这个项目将提供变革性的信息，通过高级处理过程去除活性氮和磷，帮助保护国家的水安全。这项提议寻求开发和测试一个框架，以研究复杂微生物群的转录调控。具体地说，该框架将用于研究氨氧化细菌和聚磷酸盐积累生物中的转录调控网络(TRN)，这些微生物被确定为生物营养去除(BNR)微生物组的主要贡献者。该微生物群保持在一个高速的BNR生物反应器中，使用厌氧和微好氧条件的循环运行。建议的框架涉及：(I)使用元基因组数据来组装关键生物的基因组；时间序列元转录数据来确定感兴趣的生物中的基因表达模式；(Ii)染色质免疫沉淀(ChIP)和随后的基因组规模DNA测序(CHIP-SEQ)来识别特定转录因子(Tf)直接靶向的基因；以及(Iii)在电子系统基因组学中预测在组装的基因组中的一组共调控基因。该项目由四项任务组成。任务1的重点是通过使用新的工具改善基因组组装，利用新兴的测序技术获得长DNA序列读数，并创造感兴趣的生物的丰富。任务2涉及使用CHIP-SEQ对ANR靶向的亚硝酸单胞菌UW-LDO-1菌株的基因进行实验鉴定。任务3将使用CHIP-SEQ靶向RegA、ANR和DNR-三种假想的TF来控制Acumulibacter菌株UW-TNR-IC的好氧和缺氧呼吸。任务4包括对亚硝化单胞菌UW-LDO-1和Acumulibacter UW-TNR-IC的大规模TRN的电子重建。这种综合的系统生物学方法的有效使用具有变革性，因为它是首次尝试使用实验和计算方法相结合的方法在未培养的微生物中组装TRN。这个项目将提供两个关键组织如何在转录水平上调节呼吸的第一次分析。这个奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

A large fraction of the energy cost in operating a wastewater treatment plant is supplying oxygen to the microorganisms performing the treatment process. These costs would decrease if treatment plants could be operated under low-oxygen conditions. It remains unknown how microorganisms adapt to low-oxygen conditions and whether treatment plants can be effectively operated with minimal oxygen aeration. This project will reveal how key microorganisms in wastewater treatment systems regulate their internal gene networks in response to plant operations with minimal aeration. Other impacts of the project include educational activities related to mentoring high school, undergraduate, and graduate students, and advancing the understanding of gene regulation in natural and engineered environments. If successful, this project will provide transformative information that can help protect the Nation's water security through advanced treatment processes to remove both reactive nitrogen and phosphorus.This proposal seeks to develop and test a framework to study transcriptional regulation in complex microbiomes. Specifically, the framework will be used to investigate transcriptional regulatory networks (TRNs) in an ammonia-oxidizing bacterium and a polyphosphate accumulating organism identified as major contributors to a biological nutrient removal (BNR) microbiome. This microbiome is maintained in a high-rate BNR bioreactor operated using cycles of anaerobic and micro-aerobic conditions. The proposed framework involves: (i) the use of metagenomic data to assemble genomes of key organisms; time-series metatranscriptomic data to identify gene expression patterns in the organisms of interest; (ii) chromatin immunoprecipitation (ChIP) followed by genome-scale DNA sequencing (ChIP-seq) to identify genes directly targeted by a specific transcription factor (TF); and (iii) an in silico phylogenomic approach to predict sets of co-regulated genes in assembled genomes. The project consists of four Tasks. Task 1 focuses on improving genome assembly by using novel tools, obtaining long DNA sequence reads with an emerging sequencing technology, and creating enrichments of the organisms of interest. Task 2 relates to the use ChIP-seq to experimentally identify genes in Nitrosomonas strain UW-LDO-1 targeted by ANR. Task 3 will use ChIP-seq to target RegA, ANR, and DNR - three TFs hypothesized to control aerobic and anoxic respiration in Accumulibacter strain UW-TNR-IC. Task 4 consists of an in silico reconstruction of large-scale TRNs of Nitrosomonas UW-LDO-1 and Accumulibacter UW-TNR-IC. The effective use of this integrative systems biology methodology is transformational because it is the first attempt at assembling a TRN in uncultured microbes using a combination of experimental and computational approaches. This project will provide the first analysis of how respiration is regulated at the transcription level in two key organisms.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.