University of Vienna, Centre for Microbiology and Environmental Systems Science, Division of Microbial Ecology, Vienna, Austria.
Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark.
Water Res. 2020 Nov 1;186:116372. doi: 10.1016/j.watres.2020.116372. Epub 2020 Sep 1.
Ammonia-oxidizing bacteria (AOB) of the betaproteobacterial genera Nitrosomonas and Nitrosospira are key nitrifying microorganisms in many natural and engineered ecosystems. Since many AOB remain uncultured, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been one of the most widely used approaches to study the community composition, abundance, and other features of AOB directly in environmental samples. However, the established and widely used AOB-specific 16S rRNA-targeted FISH probes were designed up to two decades ago, based on much smaller rRNA gene sequence datasets than available today. Several of these probes cover their target AOB lineages incompletely and suffer from a weak target specificity, which causes cross-hybridization of probes that should detect different AOB lineages. Here, a set of new highly specific 16S rRNA-targeted oligonucleotide probes was developed and experimentally evaluated that complements the existing probes and enables the specific detection and differentiation of the known, major phylogenetic clusters of betaproteobacterial AOB. The new probes were successfully applied to visualize and quantify AOB in activated sludge and biofilm samples from seven pilot- and full-scale wastewater treatment systems. Based on its improved target group coverage and specificity, the refined probe set will facilitate future in situ analyses of AOB.
氨氧化细菌（AOB）属于β变形菌纲的硝化单胞菌属和硝化螺旋菌属，是许多自然和工程生态系统中重要的硝化微生物。由于许多 AOB 仍然无法培养，因此使用 rRNA 靶向寡核苷酸探针的荧光原位杂交（FISH）一直是研究环境样品中 AOB 群落组成、丰度和其他特征的最广泛应用的方法之一。然而，现有的和广泛使用的 AOB 特异性 16S rRNA 靶向 FISH 探针是在大约二十年前设计的，当时可用的 rRNA 基因序列数据集比现在小得多。其中一些探针不能完全涵盖其目标 AOB 谱系，并且特异性较弱，导致应该检测不同 AOB 谱系的探针发生交叉杂交。在这里，开发并实验评估了一组新的高度特异性 16S rRNA 靶向寡核苷酸探针，这些探针补充了现有的探针，能够特异性检测和区分已知的β变形菌 AOB 的主要系统发育群。新探针成功地应用于可视化和量化来自七个中试和全规模废水处理系统的活性污泥和生物膜样品中的 AOB。基于其改进的目标群体覆盖范围和特异性，经过精细化的探针集将有助于未来对 AOB 的原位分析。
