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Bacteria work together to survive ’sdepths Sandeep Ravindran, Science Writer

Thousands of feet below the Earth’s surface seems an “We have long understood that exists in the unlikely cradle for life. Indeed, for a long , scien- deep subsurface, but we don’t actually know what tists wondered if any life could survive in such a hos- they’re doing in that environment,” says Maggie Lau, a tile, pitch black, -poor environment. geomicrobiologist at Princeton University and the study’s But about two decades ago, researchers found the first author. Lau and her colleagues identified and ana- first substantive evidence of living deep under- lyzed the metabolic networks used by deep-living bacte- ground. A Department of drilling project had rial communities. By capturing the “metabolic landscape” recovered rocks from more than a mile below the Earth’s of these underground environments, the authors showed surface in the Taylorsville Basin in Virginia, and scientists that the use a wider range of energy sources than were surprised to find that these rocks harbored bacteria. expected, and work together to survive (3). More recently, researchers went looking for micro- Studying these kinds of metabolic landscapes will scopic worms in deep mines, and discovered a variety of be crucial for understanding deep-living bacteria, says eukaryotic organisms living at these depths (1, 2). Most of Princeton University geomicrobiologist Tullis Onstott, these survive by eating deep-living bacteria. the study’s senior author. “It’s basically getting an im- But there’sstillalotwedon’t know about how the age of what is actually active down there,” he says. bacteria themselves survive in an environment without “It’s like catching them in the act.” or plentiful oxygen for energy. Recent work, based on samples painstakingly retrieved from deep gold Low-Energy Living mines, appears to provide a crucial clue: the bacteria work To examine deep-living bacterial communities, re- together in intricate networks, cooperating to survive. searchers either use drills to collect samples or travel

Working in the depths of a gold mine, Maggie Lau (Left) and Rachel L. Harris (Right) filter water to capture microbial cells onto a filter membrane. Image courtesy of Rachel L. Harris (Princeton University, Princeton).

788–790 | PNAS | January 31, 2017 | vol. 114 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1621079114 Downloaded by guest on September 25, 2021 phyllosillicates 632/31 69/83 PEP DSR 3/nd 258/100 URE APR + NifH organic-N NH3/NH4 25/16 DISPROPORTIONATION SAT 12/nd 74/nd PHS SULPHATE- ccNIR 146/43 HCP 90/nd REDUCTION PSR 5/nd HZS 6/nd - 2- 0 2- SO 2- SO 2- HZO/ S2 Sx S S2O3 3 4 28/nd HAO NirS SULPHUR 50/10 SuDH OXIDATION NO - NO - NO N ON 2/nd 482/38 2/nd 269/8 3 2 2 2 SQR SOX SOR SEC DENITRI- 491/2175 387/198 211/3 206/227 NIR NOR NOS 127/nd 97/nd FICATION NRA TET TAT 6/0 NRZ 30/nd 62/66 1511/899 127/89 15/33 NPD rDSR APR SAT NAP nitropropane

62/8 WL EMP or GLUCO 28/nd butyrate monosaccharides PRK CO2 CO2 RPE MEGEN or ANME 1/nd CO RuBP 100/11 41/nd 3/nd formate GCK 2 MT CO2 MTHFR 28/4 67/nd TKT 36/nd 4/nd G6PI 19/4 2/nd FWD 856/78 180/46 MTCH ME 143/157 180/46 1 CODH MTD 40/nd RuBisCo FBP 106/nd PFK FBP 219/94 12/8 CAD ECH 124/62 138/28 124/62 FDH FTFL 98/14 61/3 FBA PGK [CO-] [CH -] 12/6 3KAT 47/nd 203/139 FBA 3 MVH ACStase TPI 32/nd 203/139 3PG GAPDH 47/nd 5/nd TPI 63/7 PFL-AE GAPDH acetate 60/nd ADH ACDS 11/nd 138/28 MTR PFOR 104/3 PGK Acetyl 2/nd 194/147 2/nd -CoA 2/nd MCR PDH PGM PTA 32/21 37/7 Acetyl-CoA ACK 61/12 CH4 ENO MDH ACN acetate 76/2 74/6 pyruvate PK 57/34 5/nd PPDK 26/nd Acetyl-CoA ICDH HCO - 32/nd pyruvate FRD/ 3 Form- PEPC 81/nd HBD 26/5 QFR 69/nd 8/nd ACC aldehyde 3/nd 21/nd 13/nd 5/18 SHMT 10/nd MMO UbiX OOR/ CO hydro- SCS 2 HPCS HPR PGAM KOR 24/nd 32/nd 4/nd 2/nd - MCM 3PG HCO3 ADH ALDH DH CO2 Among the metabolic pathways expressed in the deep subsurface are those involving nitrogen (Upper Left), (Upper Right), and (Below). Black and gray solid lines indicate reactions mediated by ; black dotted lines in the indicate abiotic reactions. Reproduced from ref. 3.

to mines and caves that provide access to these learn about these organisms through traditional microbi- depths. Onstott studied deep life in several gold and ology approaches. Advances in DNA tech- diamond mines in South Africa. To collect samples for nologies have helped offer insights into the bacteria’s the current project, Lau ventured nearly a mile un- metabolic pathways. But such studies derground in South Africa’s Beatrix gold mine. aren’t sufficient to find out what these organisms actu- Getting to the study site is an odyssey in itself. Lau ally do in the depths. Researchers need to examine takes a mine cage down about a mile, and then walks the bacteria’s RNA and to determine when another half-mile or more, sometimes wading through and at what levels their metabolic and proteins flooded tunnels, all while carrying a heavy backpack. are expressed. She often relies on a headlamp to illuminate her work “When it comes down to constructing a model for how area in the pitch-black depths. the organisms that are present may or may not be active, To collect bacterial samples, Lau filters water from or how active they are, and what they contribute to the boreholes—tubes drilled into the ground to look for oil system, you really need to go beyond metagenomics and or minerals, which sometimes fill with underground get a proteomics and transcriptomics approach to address water—at her subterranean study site. Because this part that,” says Oregon State University geomicrobiologist of the Beatrix gold mine has no active mining, the Frederick Colwell, who was not involved in the study. borehole served as a fount of uncontaminated samples. The first step is to gather enough RNA and To collect enough samples for her analysis, Lau had to from deep-living microbes, a challenging feat in these filter nearly 23,000 gallons of water over 15 days. low- systems. Lau took advantage of her study “Getting good samples is always the biggest chal- site to collect large amounts of uncontaminated sam- lenge,” says Onstott. “We really need to sample large ples and devised methods of preserving the samples for volumes because the biomass concentration is so low.” her analysis (keeping them cold at all , for exam- Only a small fraction of deep-living bacteria have been ple). As a result, Lau was able to gather enough high- grown in laboratory cultures, limiting what researchers can quality DNA, RNA, and proteins for metagenomics,

Ravindran PNAS | January 31, 2017 | vol. 114 | no. 5 | 789 Downloaded by guest on September 25, 2021 metatranscriptomics, and metaproteomics assays, and complete the cycle. Another bacterial pair may offering a revealing look at the metabolic genes and oxidize and reduce . By pairing up, bacteria proteins expressed in these communities. can make use of a greater range of energy sources than Lau and Onstott found that different groups of would otherwise be feasible in these barren environ- deep-living bacteria paired up, with each eating sub- ments. “Cooperative functioning in the subsurface stances produced by the other and producing the seems to have benefits to enable the system to be more ” “ stable and more diverse than we thought, says Lau. Cooperative functioning in the subsurface seems to “It’s really a good way to think about it, as a bunch have benefits to enable the system to be more stable and of syntrophies, rather than competing reactions,” says more diverse than we thought.” Karen Lloyd a geomicrobiologist at the University of — Tennessee who was not involved in the study. The Maggie Lau study’s findings could expand the kinds of processes and organisms researchers look for when looking for other’s in return. This type of cross-feeding is life. “Their work is relevant to people working in very called “,” and may help bacteria survive different environments,” says Lloyd. these harsh environments. “In the subsurface envi- ronment, not many energy-producing processes are Deep Life Implications favorable,” says Lau. “In order to live here, organisms Those very different environments may include some on work with each other to make conditions favorable,” other worlds. The new study has implications for how life she says. “The organisms that they are detecting are not could survive not just in deep subsurface environments on new to science, but their collusion in this sort of sys- Earth, but also deep beneath the surface of other planets or “ tem, to me that’s pretty novel,” says Colwell. moons. On other planets where the surface is hostile and not habitable, the subsurface could be a refuge,” says Lau. Microbial Menu When searching for habitable environments on other When Lau and Onstott analyzed their data, they were in worlds, astronomers and astrobiologists generally look ’ for another surprise. The researchers had expected hy- for , methane, or . Lau s work sug- drogen, methane, and to be the major energy gests that could be another indicator of po- sources. “Because of the abiotic processes that can gen- tential life. And the syntrophic interactions she found erate these energy sources, people think reactions that suggest that networks of metabolic interactions could utilize these resources should be prevalent,” Lau explains. servetoincreasethechancesforlifetoexistin Instead, only a minority of the deep-living bacteria fed on extreme environments. ’ these chemicals. This minority produced sulfur and Looking for deep life on other planets isn t yet feasible. as metabolic byproducts, which then fueled the vast ma- But Lau and Onstott do plan to investigate whether these jority of the bacteria that live at these depths. same types of metabolic landscapes are found in other “ Previous geochemical work by Onstott suggested that deep environments on Earth. We are planning to extend —the reduction of nitrate by microbes— this analysis to multiple samples from different locations might be occurring in these underground communities (4). and also from different times,” says Lau. “Hopefully we will “Lo and behold, our data shows that nitrate is a very im- be able to resolve the metabolic landscape on a temporal portant source of energy in the deep subsurface,” says and spatial scale.” Lau initially plans to analyze weekly Lau. Nitrate is usually found at pretty low concentrations in and annually collected samples from the same borehole, these environments, and Lau suggests that bacteria may as as data from some other South African mines. be keeping nitrate levels low by feeding on it. Eventually the researchers hope to expand these Bacteria feeding on different substrates appeared efforts to other geographic locations. “In the next few to work in syntrophic pairs. For example, bacteria that years,” says Onstott, “developing new images from eat sulfate produce sulfide. This sulfide becomes the the molecular data from different sites to see what it’s food source for other bacteria, which produce sulfate showing us is going to be really terrific.”

1 Borgonie G, et al. (2011) Nematoda from the terrestrial deep subsurface of South Africa. 474(7349):79–82. 2 Borgonie G, et al. (2015) Eukaryotic opportunists dominate the deep-subsurface in South Africa. Nat Commun 6:8952. 3 Lau MCY, et al. (2016) An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers. Proc Natl Acad Sci USA 113(49):E7927–E7936. 4 BJ, et al. (2012) The origin of NO3− and N2 in deep subsurface fracture water of South Africa. Chem Geol 294-295:51–62.

790 | www.pnas.org/cgi/doi/10.1073/pnas.1621079114 Ravindran Downloaded by guest on September 25, 2021