Metagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.

元基因组学研究彻底改变了我们对各种生态系统中未培养微生物代谢潜力的理解。但是，许多这些基因组预测尚未经过实验测试，而基因组潜力的功能表达通常仍未解决。为了获得对细胞生理学的更彻底的了解，必须开发能够在接近原位条件下测试微生物代谢的新技术。在这里，我们提供了一项基准研究，以证明生物正交非规范性氨基酸标签（BONCAT）结合荧光激活细胞分选（FACS）和16S rRNA基因测序可用于鉴定在各种生长底物的存在或物理化学条件的变化。Thaumarchaeota，酸性细菌和Fervidibacteria。由于响应于底物修饰或顶空变化而发生的活动变化表明微生物对特定生长条件的偏爱，因此本次和未来的BONCAT-FACS研究的结果可能会为培养培养基的发展提供信息，以特别丰富未培养的微生物。最重要的是，BONCAT-FACS能够在尽可能接近原位条件的条件下提供未培养生物的生理信息。