Diverse syntrophic partnerships from deep-sea methane vents revealed by direct cell capture and metagenomics Annelie Pernthaler*†‡, Anne E. Dekas*, C. Titus Brown§, Shana K. Goffredi*, Tsegereda Embaye*, and Victoria J. Orphan*‡§ Divisions of *Geological and Planetary Sciences and §Biology, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125 Edited by David M. Karl, University of Hawaii, Honolulu, HI, and approved March 18, 2008 (received for review November 29, 2007) Microorganisms play a fundamental role in the cycling of nutrients of the metabolic potential of the entire community (4, 5); however, and energy on our planet. A common strategy for many microorgan- information regarding specific groups of microorganisms and in- isms mediating biogeochemical cycles in anoxic environments is terspecies associations is often lacking. Methods that enable the syntrophy, frequently necessitating close spatial proximity between selective extraction of individual microorganisms from native mi- microbial partners. We are only now beginning to fully appreciate the crobial communities before genome sequencing have shown prom- diversity and pervasiveness of microbial partnerships in nature, the ise for interpreting genome data in the context of specific micro- majority of which cannot be replicated in the laboratory. One notable organisms. Examples of these purification approaches for single example of such cooperation is the interspecies association between cells include microfluidics (6), optical trapping (7), immunomag- anaerobic methane oxidizing archaea (ANME) and sulfate-reducing netic capture (8), and flow cytometry (9, 10). The ability to identify bacteria. These consortia are globally distributed in the environment and characterize the metabolic potential of in situ syntrophic and provide a significant sink for methane by substantially reducing partnerships presents a greater challenge and requires new meth- the export of this potent greenhouse gas into the atmosphere. The odological strategies that are both compatible with metagenomic interdependence of these currently uncultured microbes renders analysis and can be applied in complex environmental samples, such them difficult to study, and our knowledge of their physiological as anoxic sediments, where these interspecies associations are likely capabilities in nature is limited. Here, we have developed a method to occur. to capture select microorganisms directly from the environment, In response to this need, we have developed a culture- using combined fluorescence in situ hybridization and immunomag- independent method called ‘‘magneto-FISH’’ that enables the netic cell capture. We used this method to purify syntrophic anaerobic capture and assessment of interspecies associations and corre- methane oxidizing ANME-2c archaea and physically associated mi- sponding metabolic properties of these organisms directly from croorganisms directly from deep-sea marine sediment. Metagenom- complex environments. Briefly, magneto-FISH allows for the tar- ics, PCR, and microscopy of these purified consortia revealed unex- geted magnetic capture of whole microorganisms and cell aggre- pected diversity of associated bacteria, including Betaproteobacteria gates, using 16S rRNA oligonucleotide probes to the exclusion of and a second sulfate-reducing Deltaproteobacterial partner. The de- other microorganisms within the environmental sample. In this tection of nitrogenase genes within the metagenome and subse- study, we applied magneto-FISH for targeted metagenomic inves- 15 tigations of in situ syntrophic assemblages, followed by genomics- quent demonstration of N2 incorporation in the biomass of these methane-oxidizing consortia suggest a possible role in new nitrogen enabled hypothesis development and subsequent hypothesis testing, inputs by these syntrophic assemblages. using a combination of pyrosequencing and isotope labeling ex- periments. The magneto-FISH method increases the probability of betaproteobacteria ͉ methanotrophy ͉ nitrogen fixation ͉ syntrophy identifying potentially hidden microbial associations and simplify- ing environmental genomics by specifically targeting the microor- ganism(s) of interest. ur understanding of the interplay between microorganisms Here, we investigate a globally significant but poorly understood Oand the environment has advanced tremendously in the past syntrophic association between as-yet uncultured groups of anaer- two decades through the application of environmental metagenom- obic methane-oxidizing archaea (ANME) and sulfate-reducing ics, microanalytical methodologies, novel cultivation methods, and bacteria; a microbial association responsible for consuming Ͼ80% coupling of stable and radiogenic isotopes with molecular analysis of the methane flux from the ocean (11). We can now begin to of organic biosignatures (i.e., DNA, RNA, protein, and lipids). The address key questions pertaining to the ecology and physiology of strict requirement for pure cultures has been alleviated through the these organisms, such as: How versatile is the syntrophic association refinement of these techniques, and the field of microbial ecology is now poised to fully illuminate the complex interrelationships within natural communities. Author contributions: A.P. and V.J.O. designed research; A.P., A.E.D., S.K.G., T.E., and V.J.O. The anaerobic cycling of carbon frequently relies on closely performed research; A.P. and C.T.B. contributed new reagents/analytic tools; A.P., A.E.D., coupled interdependent microbial associations (1). The prevalence C.T.B., S.K.G., and V.J.O. analyzed data; and A.P., C.T.B., and V.J.O. wrote the paper. and biogeochemical importance of these associations emphasizes The authors declare no conflict of interest. the need for effective methodologies that enable the study of novel This article is a PNAS Direct Submission. microbial processes and interspecies relationships in situ.Tothis Data deposition: The sequence reported in this paper has been deposited in the GenBank database [accession nos. EU622281–EU622312 (16S rRNA) and EU647340–EU647354 end, culture-independent environmental metagenomic studies (nifH)]. have expanded our understanding of the phylogenetic and meta- †Present address: Department of Environmental Microbiology, Centre for Environmental bolic diversity on Earth (2, 3). An important caveat of these studies Research, UFZ, Permoser Strasse 15, 04318 Leipzig, Germany. is the ability to interpret the metagenomic data in the context of ‡To whom correspondence may be addressed. E-mail: [email protected] or organism identity, which increases in difficulty with increasing [email protected]. community complexity. Direct sequencing of complex soils and This article contains supporting information online at www.pnas.org/cgi/content/full/ sediments, using high-throughput sequencing methods (shotgun 0711303105/DCSupplemental. cloning or clone-free pyrosequencing), yield important overviews © 2008 by The National Academy of Sciences of the USA 7052–7057 ͉ PNAS ͉ May 13, 2008 ͉ vol. 105 ͉ no. 19 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711303105 Downloaded by guest on September 25, 2021 Table 1. Comparison of 16S rRNA diversity recovered before and after capture of ANME-2c consortia, using Magneto-FISH 16S rRNA phylotypes, % Bulk sediment Bead capture clone library clone library Closest relative Archaea 26 92 ANME-2c 6 7 ANME-2a 21Methanococcoides burtonii 19 0 ANME-2b 44 0 Others (ANME-1, MBGD, MBGB) Bacteria Fig. 1. CARD-FISH of Eel River Basin sediment with probe ANME-2c࿝760 25 42 Deltaproteobacteria (Desulfobulbus (green) and a general DNA stain (DAPI, blue), before (a) and after (b) capture or Desulfosarcina) with paramagnetic beads (dark red, collage of six images). (Scale bars, 10 ␮m.) 032Betaproteobacteria (Burkholderiaceae) 09Alphaproteobacteria (Sphingomonas) 92Gammaproteobacteria (uncultured) between specific ANME groups and associated bacteria? What 16 7 Epsilonproteobacteria (uncultured) 0 4 CFB group* (AJ535255, AY768987) metabolic processes are available to them and how do they specif- 03Planctomyces (AF424488, EF424502) ically acquire essential nutrients in a carbon-dominated environ- 50 Ͻ1 Others (eight additional major lineages) ment? Do these properties vary between the different methane- oxidizing ANME ecotypes? Metagenomic studies of ANME Archaeal and bacterial 16S rRNA clone libraries were constructed by using archaea have primarily focused on methane and energy metabo- Ar20F-958r and Ba27F-1492r, respectively. Total number of clones in archaeal lism; however, the underlying nutrition and the intricacies of these library: 211 (bead capture) and 254 (bulk sediment). Total number of clones in bacterial library: 139 (bead capture) and 165 (bulk sediment). interspecies associations are still a mystery. Using the phylogenetic *CFB group: Cytophaga/Flexibacter/Bacterioides. selectiveness of the 16S rRNA targeted magneto-FISH assay, we broaden our understanding of the syntrophic lifestyle and specifi- cally nitrogen acquisition of a specific methane-oxidizing ecotype, ANME-2 subgroups represent unique ecotypes. Metagenomic se- known as the ANME-2c, and their coassociated bacterial partners quencing of the microbial community in methane seep sediment recovered from methane-rich deep-sea sediments. has shed light on the physiology and specific mechanism of AOM catalyzed by some of the ANME archaea (18, 19). However, details Results and Discussion of the metabolic regulation, environmental