Department of Civil and Environmental Engineering, Colorado State University, Fort Collins, CO, USA.
College of Agricultural Sciences, Colorado State University, Fort Collins, CO, USA.
J Microbiol Methods. 2022 Jun;197:106481. doi: 10.1016/j.mimet.2022.106481. Epub 2022 May 6.
For many contaminants, biomarker genes are unknown or assays are unavailable, and most biomarker assays target the first pathway step. Herein, we obtained sequences for all of the genes in a previously hypothesized o-xylene degradation pathway based on similarities to analogous genes in a known toluene degradation pathway. Comparative metatranscriptomics resulted in sequences for genes annotated as bssA, bbsEF, bbsCD, and bbsB, while genes for bbsG and bbsH were notably missing. Prokaryotic Suppressive Subtractive Hybridization PCR cDNA Subtraction (Prokaryotic SSH-PCR cDNA Subtraction) was applied for the first time to a mixed-species microbiome to enrich abundances of genes up-regulated during o-xylene degradation prior to metatranscriptomic sequencing. The subtracted metatranscriptome was sequenced using the MinION; this approach was highly effective at retrieving sequences for biodegradation genes including the previously missing bbsG and bbsH. Reverse transcription quantitative PCR (RT-qPCR) analysis confirmed up-regulation. Thus, data reported herein lend credence to the previously hypothesized anaerobic o-xylene degradation pathway, and new biomarker assays are presented. A novel biomarker development tool for mixed species systems, Subtractive Community Metatranscriptomics (SCM), is demonstrated.
对于许多污染物，生物标志物基因是未知的或检测方法不可用的，而且大多数生物标志物检测方法针对的是第一个途径步骤。在此，我们根据与已知甲苯降解途径中的类似基因的相似性，获得了先前假设的邻二甲苯降解途径中所有基因的序列。比较宏转录组学导致了注释为 bssA、bbsEF、bbsCD 和 bbsB 的基因序列，而 bbsG 和 bbsH 的基因明显缺失。细菌抑制性消减杂交 PCR cDNA 消减（Prokaryotic SSH-PCR cDNA Subtraction）首次应用于混合物种微生物组，以富集邻二甲苯降解过程中上调的基因丰度，然后进行宏转录组测序。消减的宏转录组使用 MinION 进行测序；这种方法在检索包括先前缺失的 bbsG 和 bbsH 在内的生物降解基因序列方面非常有效。逆转录定量 PCR（RT-qPCR）分析证实了上调。因此，本文报告的数据为先前假设的厌氧邻二甲苯降解途径提供了依据，并提出了新的生物标志物检测方法。一种用于混合物种系统的新型生物标志物开发工具，即消减群落宏转录组学（SCM），得到了验证。
