Jia Yangyang, Leung Marcus H Y, Tong Xinzhao, Wilkins David, Lee Patrick K H
School of Energy and Environment, City University of Hong Kong, Kowloon, Hong Kong SAR.
mSystems. 2019 Jan 22;4(1). doi: 10.1128/mSystems.00208-18. eCollection 2019 Jan-Feb.
Microbial communities are composed of populations with vastly different abundances and levels of metabolic and replicative activity, ranging from actively metabolizing and dividing to dormant or nonviable. The 16S rRNA/rDNA ratio is an emerging tool for evaluating cell-level metabolic activity independent of abundance. In this study, we used five long-term enriched model anaerobic digestion (AD) communities to investigate community composition, diversity, structure, and in particular activity based on the rRNA/rDNA ratio. We cross-validated the 16S amplicon-based results using two alternative operational taxonomic unit (OTU) formation methods (conventional 97% sequence similarity and 100% sequence similar zero-radius OTUs by UNOISE3) and compared these to metagenome-derived population genomes and metatranscriptomes. Significant positive correlations were observed between microbial total activity and abundance with both the amplicon- and omic-based methods. All three methods revealed disproportionately high transcription/abundance ratios for some rare taxa but lower ratios for most abundant taxa for all the communities, which was further corroborated by the high replication rate (iRep) of most low-abundance population genomes. Variation in microbial activity levels is increasingly being recognized as both an important dimension in community function and a complicating factor in sequencing-based survey methods. This study extends previous reports that rare taxa may contribute disproportionately to community activity in some natural environments, showing that this may also hold in artificially maintained model communities with well-described inputs, outputs, and biochemical functions. These results demonstrate that assessment of activity levels using the rRNA/rDNA ratio is robust across taxonomic unit formation methods and is independently corroborated by omics methods. The results also provide insight into the comparative advantages and disadvantages of different taxonomic unit formation methods in amplicon sequencing studies, showing that UNOISE3 provides comparable microbial diversity, structure, and activity information as the 97% sequence similarity method but potentially loses some phylogenetic diversity and creates more "phantom taxa" (which are present in the RNA pool but not the corresponding DNA pool).
微生物群落由丰度、代谢和复制活性水平差异极大的种群组成，范围从活跃代谢和分裂到休眠或无活力状态。16S rRNA/rDNA比率是一种新兴工具，用于独立于丰度评估细胞水平的代谢活性。在本研究中，我们使用了五个长期富集的模型厌氧消化（AD）群落，基于rRNA/rDNA比率研究群落组成、多样性、结构，特别是活性。我们使用两种替代的操作分类单元（OTU）形成方法（传统的97%序列相似性和通过UNOISE3的100%序列相似性零半径OTU）对基于16S扩增子的结果进行交叉验证，并将这些结果与宏基因组衍生的种群基因组和宏转录组进行比较。基于扩增子和组学的方法均观察到微生物总活性与丰度之间存在显著正相关。所有三种方法都显示，对于所有群落中的一些稀有分类群，转录/丰度比率过高，但对于大多数丰富分类群，该比率较低，这一点在大多数低丰度种群基因组的高复制率（iRep）中得到了进一步证实。微生物活性水平的变化越来越被认为是群落功能的一个重要维度，也是基于测序的调查方法中的一个复杂因素。本研究扩展了先前的报告，即稀有分类群在某些自然环境中可能对群落活性有不成比例的贡献，表明在具有明确输入、输出和生化功能的人工维持模型群落中也可能如此。这些结果表明，使用rRNA/rDNA比率评估活性水平在分类单元形成方法中是稳健的，并且得到了组学方法的独立证实。结果还深入了解了扩增子测序研究中不同分类单元形成方法的比较优势和劣势，表明UNOISE3提供了与97%序列相似性方法相当的微生物多样性、结构和活性信息，但可能会丢失一些系统发育多样性，并产生更多“幽灵分类群”（存在于RNA库中但不存在于相应DNA库中）。
