Hatzenpichler Roland, Connon Stephanie A, Goudeau Danielle, Malmstrom Rex R, Woyke Tanja, Orphan Victoria J
Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA 91125;
Joint Genome Institute, Department of Energy, Walnut Creek, CA 94598.
Proc Natl Acad Sci U S A. 2016 Jul 12;113(28):E4069-78. doi: 10.1073/pnas.1603757113. Epub 2016 Jun 28.
To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.
为了解微生物在环境中的生物地球化学作用，确定它们在何时以及何种条件下具有代谢活性至关重要。生物正交非天然氨基酸标记（BONCAT）可以通过追踪合成氨基酸掺入新合成蛋白质的过程来揭示活性细胞。通过将BONCAT与靶向rRNA的荧光原位杂交（BONCAT-FISH）相结合，可以确定翻译活性细胞的系统发育身份。理论上，可以使用荧光激活细胞分选技术（BONCAT-FACS）分离BONCAT标记的细胞，以便进行后续的遗传分析。在此，据我们所知，在环境背景下首次应用BONCAT-FISH和BONCAT-FACS，我们探究了催化甲烷厌氧氧化（AOM）的微生物聚集体的翻译活性，AOM是海洋中甲烷的主要汇。这些聚集体通常由厌氧甲烷氧化古菌（ANME）和硫酸盐还原细菌组成，由于其原位生长速度缓慢，一直难以研究，关于它们的生态学以及ANME与相关伙伴之间相互作用的多样性仍存在基本问题。我们对超过16400个微生物聚集体进行的活性相关分析，据我们所知，首次提供了证据，表明隶属于所有五个主要ANME进化枝的AOM聚集体在受控条件下同时具有活性。令人惊讶的是，对单个BONCAT标记的聚集体进行分选，随后进行全基因组扩增和16S rRNA基因测序，揭示了ANME与了解较少的疣微菌门成员之间以前未被认识的相互作用。这一发现，连同我们观察到与ANME相关的疣微菌存在于各种地理上不同的甲烷渗漏环境中，表明AOM聚集体内的共生关系范围比以前认为的更广。
