National Science Review PERSPECTIVE 8: nwaa280, 2021 doi: 10.1093/nsr/nwaa280 Advance access publication 8 December 2020 MICROBIOLOGY Microbial dark matter coming to light: challenges and opportunities Jian-Yu Jiao 1, Lan Liu 1, Zheng-Shuang Hua 1,2, Bao-Zhu Fang 1,5, En-Min Zhou 1, Downloaded from https://academic.oup.com/nsr/article/8/3/nwaa280/6027462 by University of Nevada user on 30 June 2021 Nimaichand Salam1, Brian P. Hedlund 3,4 and Wen-Jun Li 1,5,∗ Microbes are the most abundant and of the major branches of the tree of microbiologists can readily design exper- diverse cellular life forms on Earth and life limits our understanding of the iments to probe in situ physiology and colonize a wide range of environmental microbial world. Until recent decades, design targeted cultivation approaches. niches. However, more than 99% of bac- microbiology research was mostly fo- terial and archaeal species have not been cused on pure cultures. Only with the MDM: A BAG FULL OF obtained in pure culture [1] and we have development of new advancements in only glimpsed the surface of this myste- DNA sequencing and computing has the SURPRISES rious microbial world. This is so-called great challenge of the genomic explo- Studies on SAGs and MAGs have un- Microbial Dark Matter (MDM): the ration of MDM directly from complex veiled some big surprises on the diver- enormous diversity of yet-uncultivated environmental samples been feasible. sity of microbes in nature and facilitated microbes that microbiologists can only Notably, single-amplified genomes major discoveries. During the last decade, study by using cultivation-independent (SAGs) and metagenome-assembled a number of MAGs or SAGs represent- techniques. Recently, a number of genomes (MAGs) obtained from ing major lineages have been retrieved international projects have dramatically single-cell genomic and metagenomic from environmental samples (Table 1). increased our understanding of the approaches, respectively, have become A few landmark papers on systematic and extent and distribution of microbial the most effective methods that enable evolutionary microbiology are as follows. diversity, including the Global Cata- organism-level genomic analysis of Hug et al. used more than 1000 MDM logue of Microorganisms (GCM), the complex microbial ecosystems without genomes, together with public genomic Genomic Encyclopedia of Bacteria and the need for cultivation, which brings data, to infer the tree of life and described Archaea (GEBA), the Earth Microbiome light to MDM. a hyper-diverse clade of MDM termed Project (EMP), the Genomic Encyclo- SAGs are typically accessed by se- the Candidate Phyla Radiation (CPR), pedia of Bacteria and Archaea-Microbial quencing amplified genomic DNA from subdividing the domain Bacteria [4]. Dark Matter (GEBA-MDM) and several individual cells using a variety of whole- Parks et al. recovered nearly 8000 MAGs primate microbiome projects; however, genome amplification (WGA) technolo- from more than 1500 metagenomic data the functional diversity of MDM is still gies. MAGs, on the other hand, are recov- sets to reconstruct an expanded ver- mysterious. This perspective addresses ered from metagenomic data sets using sion of the tree of life, and increased why MDM deserves scientific effort and computational binning tools (Fig. 1). the prokaryotic phylogenetic diversity by illustrates challenges and opportunities With high-quality SAGs or MAGs, the more than 30%, including 20 novel phyla in the future study of these enigmas. metabolicpotential of MDM canbe more of Bacteria and Archaea [5]. Recent re- reliably predicted compared to functions search has leaned on MAG and SAG TECHNOLOGY BRINGS LIGHT TO extrapolated from phylogenetic affilia- data sets to explore the Asgard archaea tion based on marker genes (e.g. through and propose new and exciting models on MDM 16S rRNA gene phylogeny) or from the origin of eukaryotic cellular complex- Our planet harbors a vast repository medium- or low-quality draft genomes ity [6]. Specifically, the entangle-engulf- of diverse microorganisms. More than [3]. However, even low-quality MAGs endogenizemodelforeukaryogenesishas 20 000 species (<1%) of bacterial and and SAGs can give researchers insight been proposed based on the study of archaeal species have been described and into three basic scientific questions: who the decade-long enrichment and isola- validly published [2], yet our nascent are they, where are they and what can tion of the Asgard archaeon ‘Candidatus understanding of the biology of some they do? Armed with this information, Prometheoarchaeum syntrophicum’ [7]. C The Author(s) 2020. Published by Oxford University Press on behalf of China Science Publishing & Media Ltd. This is an Open Access articleder distributedun the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Natl Sci Rev, 2021, Vol. 8, nwaa280 (a) (b) Downloaded from https://academic.oup.com/nsr/article/8/3/nwaa280/6027462 by University of Nevada user on 30 June 2021 What can they do Figure 1. Schematic of research approaches to answer the basic scientific questions about MDM. (a) Typical pipeline to produce SAGs or MAGs. Single- cell isolation requires specialized instrumentation, such as flow cytometry, microfluidics or micromanipulators, and clean rooms are also required for downstream handling. Genome binning tools are available, such as MetaBAT, MaxBin, GroopM, MetaWatt and CONCOCT. The quality of draft genomes could follow the minimum standards information about SAGs and MAGs, which was proposed by the Genome Standards Consortium to facilitate communication and more robust comparative genomic analyses of microbial diversity [3]. (b) Downstream analysis based on MDM genomes and a flowchart illustrating how genomic data can be integrated. Marker genes used for phylogenetic analysis are flexible enough toaccommodate changes over time. Combination of multi-omics, FISH, MAR, NanoSIMS, Raman and other techniques would provide compelling evidence for microbial function and the framework to design targeted cultivation strategies. These studies of MDM are changing the us a better understanding of the world in The first challenge is to communicate way we think about the origin and evo- which we live. better. Communication about MDM is lution of life. Another notable research currently difficult because the taxonomy and nomenclature are unstable. The cur- area that deserves scientific effort is the CHALLENGES AND metabolic versatility of MDM, which rent iteration of the International Code has uncovered significant surprises in re- OPPORTUNITIES of Nomenclature of Prokaryotes (ICNP), cent years. For example, genes encoding The current ease of DNA sequencing pro- which governs bacterial and archaeal enzymes for anaerobic methane oxida- vides ample genomic information for ex- nomenclature, does not cover unculti- tion and dissimilatory sulfate reduction ploring MDM, transporting MDM sci- vated microorganisms, making scientific were detected in Korarchaeota MAGs, ence from a data-poor past to a data-rich communication challenging. There is no suggesting these two functions, typically era. The volume of genomic data in public doubt that instability and synonymy in encoded by different partners of a syntro- databases will continue to increase, and microbial nomenclature lead to confu- phy, are coupled within a single organ- more and more MDM will be discovered sion in the scientific community and be- ism [8]. Additionally, genomic analysis of and probed. How to maximize the utility yond (e.g. agriculture, law, biotechnol- MDM has opened our eyes to new mi- of big data will continue to be at the fore- ogy and medicine). Therefore, a system crobial functions that can be applied for front of research in microbiology, and the of rules to name, compile and commu- advancements in biomedicine, bioenergy time is right to consider what we should nicate about MDM is urgently required. and biotechnology, and bioremediation. do in the post-genomic era. Here, we Toward this end, genome sequences have Functional versatility of MDM shows us outline three major challenges of MDM been proposed to replace or complement its ‘colorful’ side, surprises us and gives research. pure cultures to serve as type material for Page 2 of 5 Natl Sci Rev, 2021, Vol. 8, nwaa280 Table 1. Candidate new phyla revealed by using MAGs or SAGs. ∗ Candidate phylum MAG or SAG First description Reference Acetothermia (OP1/KB1 MAG, SAG Obsidian Pool, Yellowstone National Park Hugenholtz et al., 1998; Rinke et al., group) 2013 Aenigmarchaeota (DSEG)a SAG Homestake Mine Rinke et al., 2013 Aerophobetes (CD12) SAG Sakinaw Lake Rinke et al., 2013 Aigarchaeota (pSL4; MAG, SAG Geothermal water stream from a subsurface mine in Japan Takami et al., 2012; Rinke et al., 2013 HWCG-I)a Aminicenantes (OP8) SAG Obsidian Pool, Yellowstone National Park Hugenholtz et al., 1998; Rinke et al., 2013 Downloaded from https://academic.oup.com/nsr/article/8/3/nwaa280/6027462 by University of Nevada user on 30 June 2021 Atribacteria (OP9/JS1) MAG, SAG Obsidian Pool, Yellowstone National Park Hugenholtz et al., 1998; Dodsworth et al., 2013; Rinke et al., 2013 Bathyarchaeota (MCG)a MAG Marine sediment Lloyd et al., 2013 BD1–5 MAG Groundwater samples Wrighton et al., 2012 Berkelbacteria (ACD58) MAG Aquifer adjacent to the Colorado River Wrighton et al., 2014