Abstract
Carbon cycling in the hypersaline microbial mats from Chiprana Lake, Spain is primarily dependent on phototrophic microorganisms with the ability to fix CO2 into organics that can be further utilized by aerobic as well as anaerobic heterotrophic bacteria. Here, mat pieces were incubated in seawater amended with 14C sodium bicarbonate and the incorporation of the radiocarbon in the small subunit ribosomal RNA (SSU rRNA) of mat organisms was followed using scintillation counter and autoradiography. Different domains of SSU rRNA were separated from the total RNA by means of streptavidin-coated magnetic beads and biotin-labeled oligonucleotide probes. The 14C label was detected in isolated RNA by both scintillation counter and autoradiography, however the latter technique was less sensitive. Using scintillation counter, the radiolabel incorporation increased with time with a maximum rate of 0.18 Bq ng−1 detected after 25 days. The bacterial SSU rRNA could be captured using the magnetic beads, however the hybridization efficiency was around 20%. The captured RNA was radioactively labeled, which could be mainly due to the fixation of radiocarbon by phototrophic organisms. In conclusion, the incubation of microbial mats in the presence of radiolabeled bicarbonate leads to the incorporation of the 14C label into RNA molecules through photosynthesis and this label can be detected using scintillation counter. The used approach could be useful in studying the fate of fixed carbon and its uptake by other microorganisms in complex microbial mats, particularly when species-specific probes are used and the hybridization efficiency and RNA yield are further optimized.
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References
Stal LJ (1995) Physiological ecology of cyanobacteria in microbial mats and other communities. New Phytol 131:1–32
Bateson MM, Ward DM (1988) Photoexcretion and fate of glycolate in a hot spring cyanobacterial mat. Appl Environ Microbiol 54:1738–1743
Anderson KL, Tayne TA, Ward DM (1987) Formation and fate of fermentation products in hot-spring cyanobacterial mats. Appl Environ Microbiol 53:2343–2352
Jonkers HM, Abed RMM (2003) Identification of aerobic heterotrophic bacteria from the photic zone of a hypersaline microbial mat. Aquat Microb Ecol 30:127–133
Nold SC, Ward DM (1996) Photosynthate partitioning and fermentation in hot spring microbial mat communities. Appl Environ Microbiol 62:4598–4607
Abed RMM, Safi NMD, Köster J, de Beer D, Rullkötter J, Garcia-Pichel F (2002) Microbial diversity of a heavily polluted microbial mat and its community changes following degradation of petroleum compounds. Appl Environ Microbiol 68:1674–1683
Radajewski S, Ineson P, Parekh NR, Murrel JC (2000) Stable-isotope probing as a tool in microbial ecology. Nature 403:646–649
Whiteley AS, Manefield M, Lueders T (2006) Unlocking the ‘microbial black box’ using RNA-based stable isotope probing technologies. Curr Opin Biotechnol 17:67–71
Lueders T, Manefield M, Friedrich MW (2004) Enhanced sensitivity of DNA-and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation. Environ Microbiol 6:73–78
MacGregor BJ, Boshker HTS, Amann R (2006) Comparison of rRNA and polar lipid-derived fatty acid biomarkers for assessment of 13C-substrate incorporation by microorganisms in marine sediments. Appl Environ Microbiol 72:5246–5253
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
Li T, Wu T-D, Mazeas L, Toffin L, Guerquin-Kern J-L, Leblon G, Bouchez T (2008) Simultaneous analysis of microbial identity and function using NanoSIMS. Environ Microbiol 10:580–588
Musat N, Halm H, Winterholler B, Hoppe P, Peduzzi S, Hillion F, Horreard F, Amann R, Jorgensen BB, Kuypers MMM (2008) A single-cell view on the ecophysiology of anaerobic phototrophic bacteria. Proc Natl Acad Sci U S A 18:17861–17866
Lee N, Nielsen PH, Andreasen KH, Juretschko S, Nielsen JL, Schleifer KH, Wagner M (1999) Combination of fluorescently in situ hybridization and microautoradiography—a new tool for structure-function analyses in microbial ecology. Appl Environ Microbiol 65:1289–1297
Ogiyama S, Takeda H, Ishii N, Uchida S (2010) Migration of 14C in the paddy soil-to-rice plant system after 14C-acetic acid breakdown by microorganisms below the plow layer. J Environ Radiol 101:177–184
MacGregor BJ, Brüchert V, Fleisher S, Amann R (2002) Isolation of small subunit rRNA for stable isotopic characterization. Environ Microbiol 4:451–464
Jonkers HM, Ludwig R, de Wit R, Pringault O, Muyzer G, Niemann H, Finke N, de Beer D (2003) Structural and functional analysis of a microbial mat ecosystem from a unique permanent hypersaline inland lake: ‘La Salada de Chiprana’ (NE Spain). FEMS Microbiol Ecol 44:175–189
Stahl DA, Flesher B, Mansfield HR, Montgomery L (1988) Use of phlogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol 54:1079–1084
Alm EW, Stahl DA (2000) Critical factors influencing the recovery and integrity of rRNA extracted from environmental samples: use of an optimized protocol to measure depth-related biomass distribution in freshwater sediments. J Microbiol Methods 40:153–162
Mastrangeli R, Micangeli E, Donini S (1996) Cloning of murine LAG-3 by magnetic bead bound homologous probes and PCR (GENE-CAPTURE PCR). Anal Biochem 241:93–102
Zheng DD, Alm EW, Stahl DA, Raskin L (1996) Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies. Appl Environ Microbiol 62:4504–4513
Amann RI, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S ribosomal RNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925
Hicks RE, Amann RI, Stahl DA (1992) Dual staining of natural bacterioplankton with 4′, 6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S ribosomal RNA sequences. Appl Environ Microbiol 58:2158–2163
Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic and environmental studies in microbiology. J Bacteriol 172:762–770
Hunt WL, Foote RH (1967) Efficiency of liquid scintillation counting and autoradiography for detecting tritium in spermatozoa. Radiat Res 31:63–73
Sawada S, Asakura S, Daimon H, Furihata C (1995) Comparison of autoradiography, liquid scintillation counting and immunoenzymatic staining of 5-bromo-2’-deoxyuridine for measurement of unscheduled DNA synthesis and replicative DNA synthesis in rat liver. Mutat Res 344:109–116
Acknowledgments
I would like to thank the Max-Planck Institute for supporting this research and for allowing me to work in their radioactivity laboratories. Special thanks to Dirk de Beer for his support and to Barbara MacGregor for teaching me RNA techniques.
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Abed, R.M.M. Detection and Capturing of 14C Radioactively-Labeled Small Subunit rRNA from Mixed Microbial Communities of a Microbial Mat Using Magnetic Beads. Indian J Microbiol 52, 88–93 (2012). https://doi.org/10.1007/s12088-011-0239-6
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DOI: https://doi.org/10.1007/s12088-011-0239-6