The actinobacterial acI lineage is among the most successful and ubiquitous freshwater bacterioplankton found on all continents, often representing more than half of all microbial cells in the lacustrine environment and constituting multiple ecotypes. However, stably growing pure cultures of the acI lineage have not been established despite various cultivation efforts based on ecological and genomic studies on the lineage, which is in contrast to the ocean from which abundant microorganisms such as Prochlorococcus, Pelagibacter, and Nitrosopumilus have been isolated. Here, we report the first two pure cultures of the acI lineage successfully maintained by supplementing the growth media with catalase. Catalase was critical for stabilizing the growth of acI strains irrespective of the genomic presence of the catalase-peroxidase (katG) gene. The two strains, representing two novel species, displayed differential phenotypes and distinct preferences for reduced sulfurs and carbohydrates, some of which were difficult to predict based on genomic information. Our results suggest that culture of previously uncultured freshwater bacteria can be facilitated by a simple catalase-supplement method and indicate that genome-based metabolic prediction can be complemented by physiological analyses.
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The complete genome sequences of strains IMCC25003 and IMCC26103 obtained from cell pellets using the Illumina MiSeq platform have been deposited in GenBank with the accession numbers CP029557 for IMCC25003 and CP029558 for IMCC26103.
Glöckner FO, Zaichikov E, Belkova N, Denissova L, Pernthaler J, Pernthaler A, et al. Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria. Appl Environ Microbiol. 2000;66:5053–65.
Zwart G, Crump BC, Agterveld MPK-v, Hagen F, Han S-K. Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers. Aquat Microb Ecol. 2002;28:141–55.
Warnecke F, Amann R, Pernthaler J. Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages. Environ Microbiol. 2004;6:242–53.
Newton RJ, Jones SE, Eiler A, McMahon KD, Bertilsson S. A guide to the natural history of freshwater lake bacteria. Microbiol Mol Biol Rev. 2011;75:14–49.
Parfenova VV, Gladkikh AS, Belykh OI. Comparative analysis of biodiversity in the planktonic and biofilm bacterial communities in Lake Baikal. Microbiology. 2013;82:91–101.
Newton RJ, McLellan SL. A unique assemblage of cosmopolitan freshwater bacteria and higher community diversity differentiate an urbanized estuary from oligotrophic Lake Michigan. Front Microbiol. 2015;6:1028.
Li J, Zhang J, Liu L, Fan Y, Li L, Yang Y, et al. Annual periodicity in planktonic bacterial and archaeal community composition of eutrophic Lake Taihu. Sci Rep. 2015;5:15488.
Henson M, Jordan H, Greg S, Patrick F, Markus P, Frederick S, et al. Nutrient dynamics and stream order influence microbial community patterns along a 2914 kilometer transect of the Mississippi River. Limnol Oceanogr. 2018;63:1837–55.
Satinsky B, Fortunato C, Doherty M, Smith C, Sharma S, Ward N, et al. Metagenomic and metatranscriptomic inventories of the lower Amazon River, May 2011. Microbiome. 2015;3:39.
Savio D, Sinclair L, Ijaz UZ, Parajka J, Reischer GH, Stadler P, et al. Bacterial diversity along a 2600 km river continuum. Environ Microbiol. 2015;17:4994–5007.
Allgaier M, Grossart H-P. Diversity and seasonal dynamics of Actinobacteria populations in four lakes in northeastern Germany. Appl Environ Microbiol. 2006;72:3489–97.
Allgaier M, Brückner S, Jaspers E, Grossart H-P. Intra- and inter-lake variability of free-living and particle-associated Actinobacteria communities. Environ Microbiol. 2007;9:2728–41.
Newton RJ, Jones SE, Helmus MR, McMahon KD. Phylogenetic ecology of the freshwater Actinobacteria acI lineage. Appl Environ Microbiol. 2007;73:7169–76.
Eckert EM, Salcher MM, Posch T, Eugster B, Pernthaler J. Rapid successions affect microbial n-acetyl-glucosamine uptake patterns during a lacustrine spring phytoplankton bloom. Environ Microbiol. 2012;14:794–806.
Salcher MM, Posch T, Pernthaler J. In situ substrate preferences of abundant bacterioplankton populations in a prealpine freshwater lake. ISME J. 2013;7:896–907.
Pérez MT, Hörtnagl P, Sommaruga R. Contrasting ability to take up leucine and thymidine among freshwater bacterial groups: implications for bacterial production measurements. Environ Microbiol. 2010;12:74–82.
Buck U, Grossart H-P, Amann R, Pernthaler J. Substrate incorporation patterns of bacterioplankton populations in stratified and mixed waters of a humic lake. Environ Microbiol. 2009;11:1854–65.
Ghai R, Rodriguez-Valera F, McMahon KD, Toyama D, Rinke R, Cristina Souza de Oliveira T, et al. Metagenomics of the water column in the rristine upper course of the Amazon River. PLoS ONE. 2011;6:e23785.
Ghai R, McMahon KD, Rodriguez-Valera F. Breaking a paradigm: cosmopolitan and abundant freshwater actinobacteria are low GC. Environ Microbiol Rep. 2012;4:29–35.
Sharma AK, Sommerfeld K, Bullerjahn GS, Matteson AR, Wilhelm SW, Jezbera J, et al. Actinorhodopsin genes discovered in diverse freshwater habitats and among cultivated freshwater Actinobacteria. ISME J. 2009;3:726–37.
Martinez-Garcia M, Swan BK, Poulton NJ, Gomez ML, Masland D, Sieracki ME, et al. High-throughput single-cell sequencing identifies photoheterotrophs and chemoautotrophs in freshwater bacterioplankton. ISME J. 2012;6:113–23.
Garcia SL, McMahon KD, Martinez-Garcia M, Srivastava A, Sczyrba A, Stepanauskas R, et al. Metabolic potential of a single cell belonging to one of the most abundant lineages in freshwater bacterioplankton. ISME J. 2013;7:137–47.
Ghylin TW, Garcia SL, Moya F, Oyserman BO, Schwientek P, Forest KT, et al. Comparative single-cell genomics reveals potential ecological niches for the freshwater acl Actinobacteria lineage. ISME J. 2014;8:2503–16.
Ghai R, Mizuno CM, Picazo A, Camacho A, Rodriguez-Valera F. Key roles for freshwater Actinobacteria revealed by deep metagenomic sequencing. Mol Ecol. 2014;23:6073–90.
Salcher MM, Šimek K. Isolation and cultivation of planktonic freshwater microbes is essential for a comprehensive understanding of their ecology. Aquat Microb Ecol. 2016;77:183–96.
Rappé MS. Stabilizing the foundation of the house that ‘omics builds: the evolving value of cultured isolates to marine microbiology. Curr Opin Microbiol. 2013;16:618–24.
Jezbera J, Sharma AK, Brandt U, Doolittle WF, Hahn MW. ‘Candidatus Planktophila limnetica’, an actinobacterium representing one of the most numerically important taxa in freshwater bacterioplankton. Int J Syst Evol Microbiol. 2009;59:2864–9.
Garcia SL, McMahon KD, Grossart HP, Warnecke F. Successful enrichment of the ubiquitous freshwater acI Actinobacteria. Environ Microbiol Rep. 2014;6:21–7.
Garcia SL, Buck M, McMahon KD, Grossart H-P, Eiler A, Warnecke F. Auxotrophy and intrapopulation complementary in the ‘interactome’ of a cultivated freshwater model community. Mol Ecol. 2015;24:4449–59.
Garcia SL, Stevens SLR, Crary B, Martinez-Garcia M, Stepanauskas R, Woyke T, et al. Contrasting patterns of genome-level diversity across distinct co-occurring bacterial populations. ISME J. 2018;12:742–55.
Kang I, Kim S, Islam MR, Cho J-C. The first complete genome sequences of the acI lineage, the most abundant freshwater Actinobacteria, obtained by whole-genome-amplification of dilution-to-extinction cultures. Sci Rep. 2017;7:42252.
Neuenschwander SM, Ghai R, Pernthaler J, Salcher MM. Microdiversification in genome-streamlined ubiquitous freshwater Actinobacteria. ISME J. 2018;12:185–98.
Giovannoni SJ, Cameron Thrash J, Temperton B. Implications of streamlining theory for microbial ecology. ISME J. 2014;8:1553–65.
Moore LR, Rocap G, Chisholm SW. Physiology and molecular phylogeny of coexisting Prochlorococcus ecotypes. Nature. 1998;393:464–7.
Rappe MS, Connon SA, Vergin KL, Giovannoni SJ. Cultivation of the ubiquitous SAR11 marine bacterioplankton clade. Nature. 2002;418:630–3.
Konneke M, Bernhard AE, de la Torre JR, Walker CB, Waterbury JB, Stahl DA. Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature. 2005;437:543–6.
Konstantinidis KT, Tiedje JM. Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci USA. 2005;102:2567–72.
Kim S, Kang I, Cho JC. Genomic analysis of a freshwater actinobacterium, “Candidatus Limnosphaera aquatica” strain IMCC26207, isolated from Lake Soyang. J Microbiol Biotechnol. 2017;27:825–33.
Morris JJ, Kirkegaard R, Szul MJ, Johnson ZI, Zinser ER. Facilitation of robust growth of Prochlorococcus colonies and dilute liquid cultures by “Helper” heterotrophic bacteria. Appl Environ Microbiol. 2008;74:4530–4.
Morris JJ, Johnson ZI, Szul MJ, Keller M, Zinser ER. Dependence of the cyanobacterium Prochlorococcus on hydrogen peroxide scavenging microbes for growth at the ocean’s surface. PLoS One. 2011;6:e16805.
Kim JG, Park SJ, Damste JSS, Schouten S, Rijpstra WIC, Jung MY, et al. Hydrogen peroxide detoxification is a key mechanism for growth of ammonia-oxidizing archaea. Proc Natl Acad Sci USA. 2016;113:7888–93.
Desagher S, Glowinski J, Premont J. Pyruvate protects neurons against hydrogen peroxide-induced toxicity. J Neurosci. 1997;17:9060–7.
Varma SD, Hegde KR. Lens thiol depletion by peroxynitrite. Protective effect of pyruvate. Mol Cell Biochem. 2007;298:199–204.
Glorieux C, Calderon PB. Catalase, a remarkable enzyme: targeting the oldest antioxidant enzyme to find a new cancer treatment approach. Biol Chem. 2017;398:1095–108.
Cory RM, Davis TW, Dick GJ, Johengen T, Denef VJ, Berry MA, et al. Seasonal Dynamics in Dissolved Organic Matter, Hydrogen Peroxide, and Cyanobacterial Blooms in Lake Erie. Front Mar Sci. 2016;3:54.
Rusak SA, Richard LE, Peake BM, Cooper WJ, Bodeker GE. The influence of solar radiation on hydrogen peroxide concentrations in freshwater. Mar Freshw Res. 2010;61:1147–53.
Berry MA, Davis TW, Cory RM, Duhaime MB, Johengen TH, Kling GW, et al. Cyanobacterial harmful algal blooms are a biological disturbance to Western Lake Erie bacterial communities. Environ Microbiol. 2017;19:1149–62.
Morris JJ, Lenski RE, Zinser ER. The Black Queen Hypothesis: evolution of dependencies through adaptive gene loss. mBio. 2012;3:e00036–12.
Zamocky M, Gasselhuber B, Furtmuller PG, Obinger C. Molecular evolution of hydrogen peroxide degrading enzymes. Arch Biochem Biophys. 2012;525:131–44.
Okazaki Y, Nakano S-I. Vertical partitioning of freshwater bacterioplankton community in a deep mesotrophic lake with a fully oxygenated hypolimnion (Lake Biwa, Japan). Environ Microbiol Rep. 2016;8:789–788.
Carini P, Steindler L, Beszteri S, Giovannoni SJ. Nutrient requirements for growth of the extreme oligotroph ‘Candidatus Pelagibacter ubique’ HTCC1062 on a defined medium. ISME J. 2013;7:592–602.
Davis HC, Guillard RRL. Relative value of ten genera of micro-organisms as food for oyster and clam larvae. Fish Bull. 1958;58:293–304.
Warnecke F, Sommaruga R, Sekar R, Hofer JS, Pernthaler J. Abundances, identity, and growth state of actinobacteria in mountain lakes of different UV transparency. Appl Environ Microbiol. 2005;71:5551–9.
Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–77.
King DW, Cooper WJ, Rusak SA, Peake BM, Kiddle JJ, O’Sullivan DW, et al. Flow injection analysis of H2O2 in natural waters using acridinium ester chemiluminescence: Method development and optimization using a kinetic model. Anal Chem. 2007;79:4169–76.
Sasser M. Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101. Newark, DE: MIDI Inc 1990.
The authors are grateful to Dr. Sung-Keun Rhee and Dr. Jong-Geol Kim for their help in measuring H2O2 concentration. This study was supported by the Mid-Career Research Program (to J-CC, No. NRF-2016R1A2B2015142) and Science Research Center grant (to J-CC, No. NRF-2018R1A5A1025077) through the National Research Foundation (NRF) funded by the Ministry of Sciences and ICT, and by the Basic Science Research Program funded by the Ministry of Education, Republic of Korea (to IK, No. NRF-2016R1A6A3A11934789; to J-HS, No. NRF-2016R1A6A3A11935361).
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Kim, S., Kang, I., Seo, J. et al. Culturing the ubiquitous freshwater actinobacterial acI lineage by supplying a biochemical ‘helper’ catalase. ISME J 13, 2252–2263 (2019). https://doi.org/10.1038/s41396-019-0432-x
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