Reichart Nicholas J, Steiger Andrea K, Van Fossen Elise M, McClure Ryan, Overkleeft Herman S, Wright Aaron T
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.
Leiden Institute of Chemistry, Leiden University, Leiden, The Netherlands.
ISME Commun. 2023 Sep 30;3(1):106. doi: 10.1038/s43705-023-00305-w.
Multi-omic analyses can provide information on the potential for activity within a microbial community but often lack specificity to link functions to cell, primarily offer potential for function or rely on annotated databases. Functional assays are necessary for understanding in situ microbial activity to better describe and improve microbiome biology. Targeting enzyme activity through activity-based protein profiling enhances the accuracy of functional studies. Here, we introduce a pipeline of coupling activity-based probing with fluorescence-activated cell sorting, culturing, and downstream activity assays to isolate and examine viable populations of cells expressing a function of interest. We applied our approach to a soil microbiome using two activity-based probes to enrich for communities with elevated activity for lignocellulose-degradation phenotypes as determined by four fluorogenic kinetic assays. Our approach efficiently separated and identified microbial members with heightened activity for glycosyl hydrolases, and by expanding this workflow to various probes for other function, this process can be applied to unique phenotype targets of interest.
多组学分析可以提供有关微生物群落内活性潜力的信息，但通常缺乏将功能与细胞联系起来的特异性，主要提供功能潜力或依赖于注释数据库。功能测定对于理解原位微生物活性以更好地描述和改善微生物组生物学是必要的。通过基于活性的蛋白质谱分析靶向酶活性可提高功能研究的准确性。在这里，我们介绍了一种将基于活性的探针与荧光激活细胞分选、培养和下游活性测定相结合的流程，以分离和检查表达感兴趣功能的活细胞群体。我们使用两种基于活性的探针将我们的方法应用于土壤微生物组，以富集具有由四种荧光动力学测定确定的木质纤维素降解表型的高活性群落。我们的方法有效地分离和鉴定了具有增强的糖基水解酶活性的微生物成员，并且通过将此工作流程扩展到针对其他功能的各种探针，该过程可以应用于感兴趣的独特表型靶点。
