Abstract
Recent discoveries suggest that photoheterotrophs (rhodopsin-containing bacteria (RBs) and aerobic anoxygenic phototrophs (AAPs)) and chemoautotrophs may be significant for marine and freshwater ecosystem productivity. However, their abundance and taxonomic identities remain largely unknown. We used a combination of single-cell and metagenomic DNA sequencing to study the predominant photoheterotrophs and chemoautotrophs inhabiting the euphotic zone of temperate, physicochemically diverse freshwater lakes. Multi-locus sequencing of 712 single amplified genomes, generated by fluorescence-activated cell sorting and whole genome multiple displacement amplification, showed that most of the cosmopolitan freshwater clusters contain photoheterotrophs. These comprised at least 10–23% of bacterioplankton, and RBs were the dominant fraction. Our data demonstrate that Actinobacteria, including clusters acI, Luna and acSTL, are the predominant freshwater RBs. We significantly broaden the known taxonomic range of freshwater RBs, to include Alpha-, Beta-, Gamma- and Deltaproteobacteria, Verrucomicrobia and Sphingobacteria. By sequencing single cells, we found evidence for inter-phyla horizontal gene transfer and recombination of rhodopsin genes and identified specific taxonomic groups involved in these evolutionary processes. Our data suggest that members of the ubiquitous betaproteobacteria Polynucleobacter spp. are the dominant AAPs in temperate freshwater lakes. Furthermore, the RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) gene was found in several single cells of Betaproteobacteria, Bacteroidetes and Gammaproteobacteria, suggesting that chemoautotrophs may be more prevalent among aerobic bacterioplankton than previously thought. This study demonstrates the power of single-cell DNA sequencing addressing previously unresolved questions about the metabolic potential and evolutionary histories of uncultured microorganisms, which dominate most natural environments.
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References
Allgaier M, Grossart H-P . (2006). Diversity and seasonal dynamics of actinobacteria populations in four Lakes in Northeastern Germany. Appl Environ Microbiol 72: 3489–3497.
Allgaier M, Uphoff H, Felske A, Wagner-Dobler I . (2003). Aerobic anoxygenic photosynthesis in Roseobacter clade bacteria from diverse marine habitats. Appl Environ Microbiol 69: 5051–5059.
Atamna-Ismaeel N, Sabehi G, Sharon I, Witzel K-P, Labrenz M, Jurgens K et al. (2008). Widespread distribution of proteorhodopsins in freshwater and brackish ecosystems. ISME J 2: 656–662.
Balashov SP, Imasheva ES, Boichenko VA, Anton J, Wang JM, Lanyi JK . (2005). Xanthorhodopsin: a proton pump with a light-harvesting carotenoid antenna. Science 309: 2061–2064.
Beja O, Suzuki MT, Heidelberg JF, Nelson WC, Preston CM, Hamada T et al. (2002). Unsuspected diversity among marine aerobic anoxygenic phototrophs. Nature 415: 630–633.
Béjà O, Aravind L, Koonin EV, Suzuki MT, Hadd A, Nguyen LP et al. (2000). Bacterial rhodopsin: evidence for a new type of phototrophy in the Sea. Science 289: 1902–1906.
Bouvier T, Del Giorgio PA, Gasol JM . (2007). A comparative study of the cytometric characteristics of high and low nucleic-acid bacterioplankton cells from different aquatic ecosystems. Environ Microbiol 9: 2050–2066.
Casamayor EO, García-Cantizano J, Pedrós-Alió C . (2008). Carbon dioxide fixation in the dark by photosynthetic bacteria in sulfide-rich stratified lakes with oxic–anoxic interfaces. Limnol Oceanogr 53: 1193–1203.
Cho J-C, Stapels MD, Morris RM, Vergin KL, Schwalbach MS, Givan SA et al. (2007). Polyphyletic photosynthetic reaction centre genes in oligotrophic marine Gammaproteobacteria. Environ Microbiol 9: 1456–1463.
Cleland D, Krader P, McCree C, Tang J, Emerson D . (2004). Glycine betaine as a cryoprotectant for prokaryotes. J Microbiol Methods 58: 31–38.
de la Torre JR, Christianson LM, Béjà O, Suzuki MT, Karl DM, Heidelberg J et al. (2003). Proteorhodopsin genes are distributed among divergent marine bacterial taxa. Proc Natl Acad Sci USA 100: 12830–12835.
Dean FB, Hosono S, Fang L, Wu X, Faruqi AF, Bray-Ward P et al. (2002). Comprehensive human genome amplification using multiple displacement amplification. Proc Natl Acad Sci USA 99: 5261–5266.
DeLong EF, Béjà O . (2010). The light-driven proton pump proteorhodopsin enhances bacterial survival during tough times. PLoS Biol 8: e1000359.
Eiler A, Beier S, Sawstrom C, Karlsson J, Bertilsson S . (2009). High ratio of bacteriochlorophyll biosynthesis genes to chlorophyll biosynthesis genes in bacteria of humic lakes. Appl Environ Microbiol 75: 7221–7228.
Fleming EJ, Langdon AE, Martinez-Garcia M, Stepanauskas R, Poulton N, Masland D et al. (2011). What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH. PLoS One 6: e17769.
García-Cantizano J, Casamayor E, Gasol J, Guerrero R, Pedrós-Alió C . (2005). Partitioning of CO2; incorporation among planktonic microbial guilds and estimation of in situ specific growth rates. Microb Ecol 50: 230–241.
Gasol JM, Del Giorgio PA . (2008). Physiological structure and single-cell activity in marine bacterioplankton. In: Kirchman DL (ed). Microbial Ecology of the Oceans. John Wiley & Sons Inc: Hoboken, NJ, pp 243–298.
Gich F, Overmann J . (2006). Sandarakinorhabdus limnophila gen. nov., sp. nov., a novel bacteriochlorophyll a-containing, obligately aerobic bacterium isolated from freshwater lakes. Int J Syst Evol Microbiol 56: 847–854.
Gomez-Alvarez V, Teal TK, Schmidt TM . (2009). Systematic artifacts in metagenomes from complex microbial communities. ISME J 3: 1314–1317.
Hess M, Sczyrba A, Egan R, Kim T-W, Chokhawala H, Schroth G et al. (2011). Metagenomic discovery of biomass-degrading genes and genomes from cow rumen. Science 331: 463–467.
Heywood JL, Sieracki ME, Bellows W, Poulton NJ, Stepanauskas R . (2011). Capturing diversity of marine heterotrophic protists: one cell at a time. ISME J 5: 674–684.
Hill PG, Zubkov MV, Purdie DA . (2010). Differential responses of Prochlorococcus and SAR11-dominated bacterioplankton groups to atmospheric dust inputs in the tropical Northeast Atlantic Ocean. FEMS Microbiol Lett 306: 82–89.
Jezberová J, Jezbera J, Brandt U, Lindström ES, Langenheder S, Hahn MW . (2010). Ubiquity of Polynucleobacter necessarius ssp. asymbioticus in lentic freshwater habitats of a heterogenous 2000 km2 area. Environ Microbiol 12: 658–669.
Jiao N, Zhang Y, Zeng Y, Hong N, Liu R, Chen F et al. (2007). Distinct distribution pattern of abundance and diversity of aerobic anoxygenic phototrophic bacteria in the global ocean. Environ Microbiol 9: 3091–3099.
Jiao N, Zhang F, Hong N . (2010). Significant roles of bacteriochlorophyll a supplemental to chlorophyll a in the ocean. ISME J 4: 595–597.
Jochem FJ, Lavrentyev PJ, First MR . (2004). Growth and grazing rates of bacteria groups with different apparent DNA content in the Gulf of Mexico. Mar Biol 145: 1213–1225.
Kvist T, Ahring B, Lasken R, Westermann P . (2007). Specific single-cell isolation and genomic amplification of uncultured microorganisms. Appl Microbiol Biotechnol 74: 926–935.
Li W . (2009). Analysis and comparison of very large metagenomes with fast clustering and functional annotation. BMC Bioinform 10: 359.
Longnecker K, Homen DS, Sherr EB, Sherr BF . (2006). Similar community structure of biosynthetically active prokaryotes across a range of ecosystem trophic states. Aquat Microb Ecol 42: 265–276.
Longnecker K, Sherr BF, Sherr EB . (2005). Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem. Appl Environ Microbiol 71: 7737–7749.
Ludwig W, Strunk O, Westram R, Richter L, Meier H, Yadhukumar et al. (2004). ARB: a software environment for sequence data. Nucl Acids Res 32: 1363–1371.
Marcy Y, Ouverney C, Bik EM, Lösekann T, Ivanova N, Martin HG et al. (2007). Dissecting biological ‘dark matter’ with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth. Proc Natl Acad Sci USA 104: 11889–11894.
Mašín M, Nedoma J, Pechar L, Koblížek M . (2008). Distribution of aerobic anoxygenic phototrophs in temperate freshwater systems. Environ Microbiol 10: 1988–1996.
McCarren J, DeLong EF . (2007). Proteorhodopsin photosystem gene clusters exhibit co-evolutionary trends and shared ancestry among diverse marine microbial phyla. Environ Microbiol 9: 846–858.
McKinlay JB, Harwood CS . (2010). Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. Proc Natl Acad Sci USA 107: 11669–11675.
Meyer F, Paarmann D, D’Souza M, Olson R, Glass E, Kubal M et al. (2008). The metagenomics RAST server—a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinform 9: 386.
Minin VN, Dorman KS, Fang F, Suchard MA . (2005). Dual multiple change-point model leads to more accurate recombination detection. Bioinformatics 21: 3034–3042.
Morgan JL, Darling AE, Eisen JA . (2010). Metagenomic sequencing of an in vitro-simulated microbial community. PLoS One 5: e10209.
Page KA, Connon SA, Giovannoni SJ . (2004). Representative freshwater bacterioplankton isolated from Crater Lake, Oregon. Appl Environ Microbiol 70: 6542–6550.
Potvin M, Lovejoy C . (2009). PCR-based diversity estimates of artificial and environmental 18S rRNA gene libraries. J Eukaryot Microbiol 56: 174–181.
Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J et al. (2007). SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucl Acids Res 35: 7188–7196.
Raghunathan A, Ferguson Jr HR, Bornarth CJ, Song W, Driscoll M, Lasken RS . (2005). Genomic DNA amplification from a single bacterium. Appl Environ Microbiol 71: 3342–3347.
Rappé MS, Giovannoni SJ . (2003). The uncultured microbial majority. Annu Rev Microbiol 57: 369–394.
Rusch DB, Halpern AL, Sutton G, Heidelberg KB, Williamson S, Yooseph S et al. (2007). The Sorcerer II global ocean sampling expedition: Northwest Atlantic through Eastern Tropical Pacific. PLoS Biol 5: e77.
Sabehi G, Loy A, Jung K-H, Partha R, Spudich JL, Isaacson T et al. (2005). New insights into metabolic properties of marine bacteria encoding proteorhodopsins. PLoS Biol 3: e273.
Sharma AK, Sommerfeld K, Bullerjahn GS, Matteson AR, Wilhelm SW, Jezbera J et al. (2009). Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria. ISME J 3: 726–737.
Sharma AK, Zhaxybayeva O, Papke RT, Doolittle WF . (2008). Actinorhodopsins: proteorhodopsin-like gene sequences found predominantly in non-marine environments. Environ Microbiol 10: 1039–1056.
Stamatakis A . (2006). RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690.
Stepanauskas R, Sieracki ME . (2007). Matching phylogeny and metabolism in the uncultured marine bacteria, one cell at a time. Proc Natl Acad Sci USA 104: 9052–9057.
Suyama T, Shigematsu T, Takaichi S, Nodasaka Y, Fujikawa S, Hosoya H et al. (1999). Roseateles depolymerans gen. nov., sp. nov., a new bacteriochlorophyll a-containing obligate aerobe belonging to the beta-subclass of the Proteobacteria. Int J Syst Bacteriol 49: 449–457.
Tabita FR, Hanson TE, Satagopan S, Witte BH, Kreel NE . (2008). Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms. Philos Trans R Soc Ser B 363: 2629–2640.
Tillett D, Neilan BA . (2000). Xanthogenate nucleic acid isolation from cultured and environmental cyanobacteria. J Phycol 36: 251–258.
Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM . (2007). Primer3Plus, an enhanced web interface to Primer3. Nucl Acids Res 35: W71–W74.
Wagner-Döbler I, Biebl H . (2006). Environmental biology of the marine Roseobacter lineage. Annu Rev Microbiol 60: 255–280.
Waidner LA, Kirchman DL . (2005). Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in the Delaware River. Environ Microbiol 7: 1896–1908.
Wang Y, Hammes F, Boon N, Chami M, Egli T . (2009). Isolation and characterization of low nucleic acid (LNA)-content bacteria. ISME J 3: 889–902.
Warnecke F, Sommaruga R, Sekar R, Hofer JS, Pernthaler J . (2005). Abundances, identity, and growth state of Actinobacteria in mountain lakes of different UV transparency. Appl Environ Microbiol 71: 5551–5559.
Wernersson R, Pedersen AG . (2003). RevTrans: multiple alignment of coding DNA from aligned amino acid sequences. Nucl Acids Res 31: 3537–3539.
Woyke T, Xie G, Copeland A, González JM, Han C, Kiss H et al. (2009). Assembling the marine metagenome, one cell at a time. PLoS One 4: e5299.
Yurkov VV, Beatty JT . (1998). Aerobic anoxygenic phototrophic bacteria. Microbiol Mol Biol Rev 62: 695–724.
Yutin N, Suzuki MT, Beja O . (2005). Novel primers reveal wider diversity among marine aerobic anoxygenic phototrophs. Appl Environ Microbiol 71: 8958–8962.
Zhang K, Martiny AC, Reppas NB, Barry KW, Malek J, Chisholm SW et al. (2006). Sequencing genomes from single cells by polymerase cloning. Nat Biotechnol 24: 680–686.
Zubkov MV, Allen JI, Fuchs BM . (2004). Coexistence of dominant groups in marine bacterioplankton community; a combination of experimental and modelling approaches. J Mar Biol Assoc UK 84: 519–529.
Zubkov MV . (2009). Photoheterotrophy in marine prokaryotes. J Plankton Res 31: 933–938.
Zwart G, Crump BC, Agterveld MPK-v, Hagen F, Han S-K . (2002). Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers. Aquat Microb Ecol 28: 141–155.
Acknowledgements
We thank Wendy Korjeff-Bellows and Jane Heywood for fieldwork assistance, Stefan Bertilsson and Siv Andersson for access to their metagenomic data, Katherine McMahon and Todd Miller for Wisconsin lake sampling and DNA extraction, and Vladimir M Minin for his advice on recombination event analysis. This research was supported by the NSF Grants DEB-841933 and OCE-821374 to RS and by a Maine Technology Institute research infrastructure grant to the Bigelow Laboratory.
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Martinez-Garcia, M., Swan, B., Poulton, N. et al. High-throughput single-cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton. ISME J 6, 113–123 (2012). https://doi.org/10.1038/ismej.2011.84
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DOI: https://doi.org/10.1038/ismej.2011.84
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