Trexler Ryan V, Van Goethem Marc W, Goudeau Danielle, Nath Nandita, Malmstrom Rex R, Northen Trent R, Couradeau Estelle
Intercollege Graduate Degree Program in Ecology, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, United States.
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
Front Microbiol. 2023 Jun 26;14:1176751. doi: 10.3389/fmicb.2023.1176751. eCollection 2023.
Determining which microorganisms are active within soil communities remains a major technical endeavor in microbial ecology research. One promising method to accomplish this is coupling bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence activated cell sorting (FACS) which sorts cells based on whether or not they are producing new proteins. Combined with shotgun metagenomic sequencing (Seq), we apply this method to profile the diversity and potential functional capabilities of both active and inactive microorganisms in a biocrust community after being resuscitated by a simulated rain event. We find that BONCAT-FACS-Seq is capable of discerning the pools of active and inactive microorganisms, especially within hours of applying the BONCAT probe. The active and inactive components of the biocrust community differed in species richness and composition at both 4 and 21 h after the wetting event. The active fraction of the biocrust community is marked by taxa commonly observed in other biocrust communities, many of which play important roles in species interactions and nutrient transformations. Among these, 11 families within the Firmicutes are enriched in the active fraction, supporting previous reports indicating that the Firmicutes are key early responders to biocrust wetting. We highlight the apparent inactivity of many Actinobacteria and Proteobacteria through 21 h after wetting, and note that members of the Chitinophagaceae, enriched in the active fraction, may play important ecological roles following wetting. Based on the enrichment of COGs in the active fraction, predation by phage and other bacterial members, as well as scavenging and recycling of labile nutrients, appear to be important ecological processes soon after wetting. To our knowledge, this is the first time BONCAT-FACS-Seq has been applied to biocrust samples, and therefore we discuss the potential advantages and shortcomings of coupling metagenomics to BONCAT to intact soil communities such as biocrust. In all, by pairing BONCAT-FACS and metagenomics, we are capable of highlighting the taxa and potential functions that typifies the microbes actively responding to a rain event.
确定土壤群落中哪些微生物具有活性仍然是微生物生态学研究中的一项主要技术难题。实现这一目标的一种有前景的方法是将生物正交非天然氨基酸标记（BONCAT）与荧光激活细胞分选（FACS）相结合，后者根据细胞是否正在产生新蛋白质来对细胞进行分选。结合鸟枪法宏基因组测序（Seq），我们应用这种方法来描绘生物结皮群落中活性和非活性微生物在模拟降雨事件复苏后的多样性和潜在功能能力。我们发现BONCAT-FACS-Seq能够区分活性和非活性微生物库，尤其是在应用BONCAT探针后的数小时内。在湿润事件后的4小时和21小时，生物结皮群落的活性和非活性组分在物种丰富度和组成上均有所不同。生物结皮群落的活性部分以在其他生物结皮群落中常见的分类群为特征，其中许多在物种相互作用和养分转化中发挥重要作用。其中，厚壁菌门中的11个科在活性部分中富集，支持了先前的报告，表明厚壁菌门是生物结皮湿润的关键早期响应者。我们强调了许多放线菌和变形菌在湿润后21小时内明显不具有活性，并注意到在活性部分中富集的噬几丁质菌科成员在湿润后可能发挥重要的生态作用。基于活性部分中COG的富集情况，噬菌体和其他细菌成员的捕食以及不稳定养分的 scavenging和循环利用，似乎是湿润后不久的重要生态过程。据我们所知，这是BONCAT-FACS-Seq首次应用于生物结皮样本，因此我们讨论了将宏基因组学与BONCAT结合应用于完整土壤群落（如生物结皮）的潜在优势和缺点。总之，通过将BONCAT-FACS与宏基因组学相结合，我们能够突出那些对降雨事件有积极响应的典型微生物的分类群和潜在功能。
