Abstract
Redox sensor green (RSG), a novel fluorescent dye from Invitrogen was employed as a tool for real-time detection of microbes metabolically active in situ, in combination with flow cytometry and cell sorting. Lake Washington sediment, an environment known for high rates of methane oxidation, was used as a model, and methylotrophs were targeted as a functional group. We first tested and optimized the performance of the dye with pure methylotroph cultures. Most cells in actively growing cultures were positive for staining, whereas in starved cultures, few cells fluoresced. However, starved cells could be activated by addition of substrate. High numbers of fluorescing cells were observed in a Lake Washington sediment sample, and activation of subpopulations of cells was demonstrated in response to methane, methanol, methylamine and formaldehyde. The fraction of the population activated by methane was investigated in more detail, by phylogenetic profiling. This approach showed that the major responding species were the Methylomonas species, previously isolated from the site, and Methylobacter species that have not yet been cultivated from Lake Washington. In addition, from the methane-stimulated fraction, uncultivated bacterial sequences were obtained that belonged to unclassified Deltaproteobacteria, unclassified Verrucomicrobiles and unclassified Acidobacteria, suggesting that these microbes may also be involved in methane metabolism. The approach was further tested for its utility in facilitating enrichment for functional types that possess specific metabolic activities but resist cultivation. It was demonstrated that in enrichment cultures inoculated with cells that were sorted after stimulation with methane, Methylobacter sequences could be detected, whereas in enrichment cultures inoculated by randomly sorted cells, Methylomonas species quickly outcompeted all other types.
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References
Anesti V, McDonald IR, Ramaswamy M, Wade WG, Kelly DP, Wood AP . (2005). Isolation and molecular detection of methylotrophic bacteria occurring in the human mouth. Environ Microbiol 7: 1227–1238.
Auman AJ, Stolyar S, Costello AM, Lidstrom ME . (2000). Molecular characterization of methanotrophic isolates from freshwater lake sediment. Appl Environ Microbiol 66: 5259–5266.
Borneman J . (1999). Culture-independent identification of microorganisms that respond to specified stimuli. Appl Environ Microbiol 65: 3398–3400.
Costello AM, Lidstrom ME . (1999). Molecular characterization of functional and phylogenetic genes from natural populations of methanotrophs in lake sediments. Appl Environ Microbiol 65: 5066–5074.
Dunfield PF, Yuryev A, Senin P, Smirnova AV, Stott MB, Hou S et al. (2007). Methane oxidation by an extremely acidophilic bacterium of the phylum Verrucomicrobia. Nature 450: 879–882.
Eek KM, Sessions AL, Lies PD . (2007). Carbon-isotopic analysis of microbial cells sorted by flow cytometry. Geobiol 5: 89–95.
Gray D, Yue RS, Chueng CY, Godfrey W . (2005). Bacterial vitality detected by a novel fluorogenic redox dye using flow cytometry. In: Abstracts of the American Society of Microbiology Meeting. American Society for Microbiology, Washington, DC, USA, http://probes.invitrogen.com/servlets/posters/.
Green BD, Keller M . (2006). Capturing the uncultivated majority. Curr Opin Biotechnol 17: 236–240.
Gruden CL, Khijniak A, Ariaens P . (2003). Activity assessment of microorganisms eluted from sediments using 5-cyano-2,3-ditolyl tetrazolium chloride: a quantitative comparison of flow cytometry to epifluorescent microscopy. J Microbiol Methods 55: 865–874.
Harder W, Attwood M, Quayele JR . (1973). Methanol assimilation by Hyphomicrobium spp. J Gen Microbiol 78: 155–163.
Helenius A, Simons K . (1975). Solubilization of membranes by detergents. Biochim Biophys Acta 415: 29–79.
Islam T, Jensen S, Reigstad LJ, Larsen O, Birkeland NK . (2008). Methane oxidation at 55 degrees C and pH 2 by a thermoacidophilic bacterium belonging to the Verrucomicrobia phylum. Proc Natl Acad Sci USA 105: 300–304.
Kalyuzhnaya MG, Bowerman S, Lara JC, Lidstrom ME, Chistoserdova L . (2006a). Methylotenera gen. nov., a new genus in the family Methylophilaceae, and description of Methylotenera mobila, an obligate methylamine utilizing bacterium. Int J Syst Evol Microbiol 56: 2819–2823.
Kalyuzhnaya MG, Bowerman S, Nercessian O, Lidstrom ME, Chistoserdova L . (2005b). Highly divergent genes for methanopterin-linked C1 transfer reactions in Lake Washington, assessed via metagenomic analysis and mRNA detection. Appl Envir Microbiol 71: 8846–8854.
Kalyuzhnaya MG, Nercessian O, Lidstrom ME, Chistoserdova L . (2005c). Development and application of polymerase chain reaction primers based on fhcD for environmental detection of methanopterin-linked C1 metabolism in bacteria. Environ Microbiol 7: 1269–1274.
Kalyuzhnaya MG, Stolyar SM, Auman AJ, Lara JC, Lidstrom ME, Chistoserdova L . (2005a). Methylosarcina lacus sp. nov., a methanotroph from Lake Washington, Seattle, USA, and emended description of the genus Methylosarcina. Int J Syst Evol Microbiol 55: 2345–2350.
Kalyuzhnaya MG, Zabinsky R, Bowerman S, Baker DR, Lidstrom ME, Chistoserdova L . (2006b). Fluorescence in situ hybridization-flow cytometry-cell sorting-based method for separation and enrichment of type I and type II methanotroph populations. Appl Environ Microbiol 72: 4293–4301.
Lane DJ . (1991). 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds.). Nucleic Acid Techniques in Bacterial Systematics. Wiley: New York, pp 115–175.
Manefield M, Whiteley AS, Griffiths RI, Bailey MJ . (2002). RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl Environ Microbiol 68: 5367–5373.
Miller JA, Kalyuzhnaya MG, Noyes E, Lara JC, Lidstrom ME, Chistoserdova L . (2005). Labrys methylaminiphilus, sp. nov., a new facultatively methylotrophic bacterium from a freshwater lake sediment. Int J Syst Evol Microbiol 55: 1247–1253.
Moosvi SA, Pacheco CC, McDonald IR, De Marco P, Pearce DA, Kelly DP et al. (2005). Isolation and properties of methanesulfonate-degrading Afipia felis from Antarctica and comparison with other strains of A. Environ Microbiol 7: 22–33.
Muyzer G, de Waal EC, Uitterlinden A . (1993). Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59: 695–700.
Nakatsu CH, Hristova K, Hanada S, Meng XY, Hanson JR, Scow KM et al. (2006). Methylibium petroleiphilum gen. nov., sp. nov., a novel methyl tert-butyl ether-degrading methylotroph of the Betaproteobacteria. Int J Syst Evol Microbiol 56: 983–989.
Nemec T, Jernejc K . (2002). Influence of Tween 80 on lipid metabolism of an Aspergillus niger strain. Appl Biochem Biotech 101: 229–238.
Nercessian O, Noyes E, Kalyuzhnaya MG, Lidstrom ME, Chistoserdova L . (2005). Bacterial populations active in metabolism of C1 compounds in the sediment of Lake Washington, a freshwater lake. Appl Envir Microbiol 71: 6885–6899.
Orphan VJ, House CH, Hinrichs K-U, McKeegan KD, DeLong EF . (2001). Methane-consuming archaea revealed by directly coupled isotopic and phylogenetic analysis. Science 293: 484–487.
Pol A, Heijmans K, Harhangi HR, Tedesco D, Jetten MSM, Op den Camp HJM . (2007). Methanotrophy below pH 1 by a new Verrucomicrobia species. Nature 450: 874–878.
Radajewski S, Ineson P, Parekh NR, Murrell JC . (2000). Stable-isotope probing as a tool in microbial ecology. Nature 403: 646–649.
Rappe MS, Giovanoni SJ . (2003). The uncultured microbial majority. Annu Rev Microbiol 57: 369–394.
Schloss PD, Handelsman J . (2005). Metagenomics for studying unculturable microorganisms: cutting the Gordian knot. Genome Biology 6: 229.
Sekiguchi H, Tomioka N, Nakahara T, Uchiyama H . (2001). A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis. Biotechnol Lett 23: 1205–1208.
Ullrich S, Karrasch B, Hoppe H, Jeskulke K, Mehrens M . (1996). Toxic effects on bacterial metabolism of the Redox Dye 5-cyano-2,3-ditolyl tetrazolium chloride. Appl Environ Microbiol 62: 4587–4593.
Van Mooy BAS, Devol AH, Keil RG . (2004). Quantifying 3H-thymidine incorporation rates by a phylogenetically defined group of marine planktonic bacteria (Bacteriodetes phylum). Environ Microbiol 6: 1061–1069.
Whittenbury R, Phillips KC, Wilkinson JF . (1970). Enrichment, isolation and some properties of methane-utilizing bacteria. J Gen Microbiol 61: 205–218.
Zeng G, Fu H, Zhong H, Yuan X, Fu M, Wang W et al. (2007). Co-degradation with glucose of four surfactants, CTAB, Triton X-100, SDS and Rhamnolipid, in liquid culture media and compost matrix. Biodegrad 18: 303–310.
Acknowledgements
We are grateful to SR Levine and E Latypova (University of Washington) for assistance in sequencing and to V Karpiak (Molecular Probes) for helpful suggestions on RSG use. We acknowledge the UW Immunology/Cell Analysis Facility team for assistance in cell-sorting experiments. This work was funded by the National Science Foundation as part of the Microbial Observatories Program (MCB-0131957).
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Kalyuzhnaya, M., Lidstrom, M. & Chistoserdova, L. Real-time detection of actively metabolizing microbes by redox sensing as applied to methylotroph populations in Lake Washington. ISME J 2, 696–706 (2008). https://doi.org/10.1038/ismej.2008.32
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DOI: https://doi.org/10.1038/ismej.2008.32
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