Chapter 15 : FISH-Microautoradiography and Isotope Arrays for Monitoring the Ecophysiology of Microbes Within Their Natural Environment

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Preview this chapter:
Zoom in

FISH-Microautoradiography and Isotope Arrays for Monitoring the Ecophysiology of Microbes Within Their Natural Environment, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555816896/9781555815370_Chap15-1.gif /docserver/preview/fulltext/10.1128/9781555816896/9781555815370_Chap15-2.gif


The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR), as well as the isotope array, which are described in this chapter, are both based on the use of radioactive isotopes for revealing a specific ecophysiology and rRNA for identifying the respective organisms and provide the direct means to identify microbes that catalyze a defined process in an ecosystem. In the combination of both approaches, DNA-stable isotope probing (SIP) serves to rapidly narrow down the number of 16S rRNA genes, which might originate from substrate-consuming microbes, and FISH-MAR is then used to prove the actual functional involvement of these bacteria. Isotope arrays benefit from the fact that rRNA is labeled faster than DNA after exposure of a cell to a suitable labeled substrate, but are less sensitive than FISH-MAR because a single biomarker and not all cellular compounds contribute to the radioactive signal. If used with CO as the activity marker, isotope arrays are able to detect metabolic activity of community members that comprise not more than 1% of all cells in the investigated sample. NanoSIMS-based techniques are even more sensitive than FISH-MAR and offer a reliable quantification of the incorporated radiotracer per cell, but do not provide information about the labeled compound classes in the cell, are much more timeconsuming, and can only be performed in a few laboratories worldwide.

Citation: Wagner M. 2011. FISH-Microautoradiography and Isotope Arrays for Monitoring the Ecophysiology of Microbes Within Their Natural Environment, p 305-316. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch15
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of FIGURE 1

FISH-MAR analyses of a novel member of the in an ammonia-oxidizing enrichment culture. The left column displays the fluorescent signals after hybridization of the enrichment with a specific probe for the betaproteobacterium. The middle column shows the corresponding MAR signals, and the right column presents an overlay of both figures. (Top row) Dead control. The cells in the enrichment were killed prior to incubation with CO and 1 mM NH for 16 h. No incorporation of CO is detectable, excluding chemography or labeling of the cells by the radiotracer in the absence of metabolic activity. (Middle row) The enrichment was incubated with 0.1 mM ammonium in the presence of CO for 16 h. The cells of the betaproteobacterium are MAR positive, indicating that the bacterium is an autotrophic ammonia oxidizer. (Bottom row) Same experiment as in the middle panel but in the presence of 1 mM ammonium. The cells of the betaproteobacterium are strongly labeled, demonstrating that this organism fixes more CO in the presence of 1 mM than in the presence of 0.1 mM. Scale bar corresponds to 10 μM. It should be noted that other microbes than the betaproteobacterium were present in the sample. These microbes are not visible in the FISH pictures, but they could have caused silver grain formation at spots where no cells of the betaproteobacterium were located. Photos by Roland Hatzenpichler.

Citation: Wagner M. 2011. FISH-Microautoradiography and Isotope Arrays for Monitoring the Ecophysiology of Microbes Within Their Natural Environment, p 305-316. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch15
Permissions and Reprints Request Permissions
Download as Powerpoint


1. Adamczyk, J.,, M. Hesselsoe,, N. Iversen,, M. Horn,, A. Lehner,, P. H. Nielsen,, M. Schloter,, P. Roslev, and, M. Wagner. 2003. The isotope array, a new tool that employs substrate-mediated labeling of rRNA for determination of microbial community structure and function. Appl. Environ. Microbiol. 69: 68756887.
2. Alonso, C., and, J. Pernthaler. 2005. Incorporation of glucose under anoxic conditions by bacterioplankton from coastal North Sea surface waters. Appl. Environ. Microbiol. 71: 17091716.
3. Amann, R., and, B. M. Fuchs. 2008. Single-cell Identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat. Rev. Microbiol. 6: 339348.
4. Amann, R.,, J. Snaidr,, M. Wagner,, W. Ludwig, and, K. H. Schleifer. 1996. In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol. 178: 34963500.
5. Barker, H. A. 1941. The chemistry and metabolism of bacteria. Annu. Rev. Biochem. 10: 553580.
6. Behrens, S.,, T. Losekann,, J. Pett-Ridge,, P. K. Weber,, W. O. Ng,, B. S. Stevenson,, I. D. Hutcheon,, D. A. Relman, and, A. M. Spormann. 2008. Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS. Appl. Environ. Microbiol. 74: 31433150.
7. Brock, T. D., and, M. L. Brock. 1966. Autoradiography as a tool in microbial ecology. Nature 209: 734736.
8. Caro, L. G., and, M. Schnos. 1965. Tritium and phos-phorus-32 in high-resolution autoradiography. Science 149: 6062.
9. Chen, Y., and, J. C. Murrell. 2010. When metagenomics meets stable-isotope probing: progress and perspectives. Trends Microbiol. 18: 157163.
10. Cottrell, M. T., and, D. L. Kirchman. 2000. Natural assemblages of marine proteobacteria and members of the Cytophaga-Flavobacter cluster consuming low- and high-molecular-weight dissolved organic matter. Appl. Environ. Microbiol. 66: 16921697.
11. Cottrell, M. T., and, D. L. Kirchman. 2003. Contribution of major bacterial groups to bacterial biomass production (thymidine and leucine incorporation) in the Delaware estuary. Limnol. Oceanogr. 48: 168178.
12. Daims, H.,, A. Bruehl,, R. Amann,, K. H. Schleifer, and, M. Wagner. 1999. The domain-specific probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22: 434444.
13. Daims, H.,, S. Luecker, and, M. Wagner. 2006. daime, a novel image analysis program for microbial ecology and biofilm research. Environ. Microbiol. 8: 200213.
14. DeLong, E. F. 2005. Microbial community genomics in the ocean. Nat. Rev. Microbiol. 3: 459469.
15. DeLong, E. F.,, G. S. Wickham, and, N. R. Pace. 1989. Phylogenetic stains: ribosomal RNA-based probes for the Identification of single cells. Science 243: 13601363.
16. Dijkhuizen, L., and, W. Harder. 1985. Microbial metabolism of carbon dioxide, p. 409423. In H. Dalton (ed.), Comprehensive Biotechnology: the Principles of Biotechnology. Pergamon Press, Oxford, United Kingdom.
17. Fiencke, C., and, E. Bock. 2004. Genera-specific immunofluorescence labeling of ammonia oxidizers with polyclonal antibodies recognizing both sub-units of the ammonia monooxygenase. Microb. Ecol. 47: 374384.
18. Fliermans, C. B., and, E. L. Schmidt. 1975. Autoradiography and immunofluorescence combined for au-tecological study of single cell activity with Nitrobacter as a model system. Appl. Microbiol. 30: 676684.
19. Frias-Lopez, J.,, Y. Shi,, G. W. Tyson,, M. L. Coleman,, S. C. Schuster,, S. W. Chisholm, and, E. F. DeLong. 2008. Microbial community gene expression in ocean surface waters. Proc. Natl. Acad. Sci. USA 105: 38053810.
20. Furnham, N.,, J. S. Garavelli,, R. Apweiler, and, J. M. Thornton. 2009. Missing in action: enzyme functional annotations in biological databases. Nat. Chem. Biol. 5: 521525.
21. Ginige, M. P.,, P. Hugenholtz,, H. Daims,, M. Wagner,, J. Keller, and, L. L. Blackall. 2004. Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community. Appl. Environ. Microbiol. 70: 588596.
22. Hackman, C. R., and, H. Vapaatalo. 1972. Reproducible chemography in autoradiographs of rat brain. Experientia 28: 492493.
23. Handelsman, J. 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68: 669685.
24. Hesselsoe, M.,, S. Fureder,, M. Schloter,, L. Bodrossy,, N. Iversen,, P. Roslev,, P. H. Nielsen,, M. Wagner, and, A. Loy. 2009. Isotope array analysis of Rhodocy-clales uncovers functional redundancy and versatility in an activated sludge. ISME J. 3: 13491364.
25. Hesselsoe, M.,, J. L. Nielsen,, P. Roslev, and, P. H. Nielsen. 2005. Isotope labeling and microautora-diography of active heterotrophic bacteria on the basis of assimilation of 14CO2. Appl. Environ. Microbiol. 71: 646655.
26. Hoshino, T., and, A. Schramm. 2010. Detection of denitrification genes by in situ rolling circle amplification-fluorescence in situ hybridization to link metabolic potential with identity inside bacterial cells. Environ. Microbiol. 12: 25082517( 10)
27. Hoshino, T.,, L. S. Yilmaz,, D. R. Noguera,, H. Daims, and, M. Wagner. 2008. Quantification of target molecules needed to detect microorganisms by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition-FISH. Appl. Environ. Microbiol. 74: 50685077.
28. Huang, W. E.,, K. Stoecker,, R. Griffiths,, L. New-Bold,, H. Daims,, A. S. Whiteley, and, M. Wagner. 2007. Raman-FISH: combining stable-isotope Raman spectroscopy and fluorescence in situ hybridization for the single cell analysis of identity and function. Environ. Microbiol. 9: 18781889.
29. Jeffery, C. J. 2003. Moonlighting proteins: old proteins learning new tricks. Trends Genet. 19: 415417.
30. Karp, P. D.,, I. M. Keseler,, A. Shearer,, M. Latendresse,, M. Krummenacker,, S. M. Paley,, I. Paulsen,, J. Collado-Vides,, S. Gama-Castro,, M. Peralta-Gil,, A. Santos-Zavaleta,, M. I. Penaloza-Spinola,, C. Bonavides-Martinez, and, J. Ingraham. 2007. Multidimensional annotation of the Escherichia coli K-12 genome. Nucleic Acids Res. 35: 75777590.
31. Knoechel, R., and, J. Kalff. 1976. The applicability of grain density autoradiography to the quantitative determination of algal species production: a critique. Limnol. Oceanogr. 21: 583590.
32. Kragelund, C.,, Y. Kong,, J. Van Der Waarde,, K. Thelen,, D. Eikelboom,, V. Tandoi,, T. R. Thomsen, and, P. H. Nielsen. 2006. Ecophysiology of different filamentous Alphaproteobacteria in industrial wastewater treatment plants. Microbiology 152: 30033012.
33. Lee, N.,, P. H. Nielsen,, K. H. Andreasen,, S. Juretschko,, J. L. Nielsen,, K. H. Schleifer, and, M. Wagner. 1999. Combination of fluorescent in situ hybridization and microautoradiography—a new tool for structure-function analyses in microbial ecology. Appl. Environ. Microbiol. 65: 12891297.
34. Li, T.,, T. D. Wu,, L. Mazeas,, L. Toffin,, J. L. Guerquin-Kern,, G. Leblon, and, T. Bouchez. 2008. Simultaneous analysis of microbial identity and function using NanoSIMS. Environ. Microbiol. 10: 580588.
35. Loy, A.,, F. Maixner,, M. Wagner, and, M. Horn. 2007. probeBase—an online resource for rRNA-targeted oligonucleotide probes: new features 2007. Nucleic Acids Res. 35: D800804.
36. Maixner, F.,, D. R. Noguera,, B. Anneser,, K. Stoecker,, G. Wegl,, M. Wagner, and, H. Daims. 2006. Nitrite concentration influences the population structure of Nitrospira-like bacteria. Environ. Microbiol. 8: 14871495.
37. Malmstrom, R. R.,, R. P. Kiene, and, D. L. Kirchman. 2004. Identification and enumeration of bacteria assimilating dimethylsulfoniopropionate (DMSP) in the North Atlantic and Gulf of Mexico. Limnol. Oceanogr. 49: 597606.
38. Musat, N.,, H. Halm,, B. Winterholler,, P. Hoppe,, S. Peduzzi,, F. Hillion,, F. Horreard,, R. Amann,, B. B. Jorgensen, and, M. M. Kuypers. 2008. A single-cell view on the ecophysiology of anaerobic phototrophic bacteria. Proc. Natl. Acad. Sci. USA 105: 1786117866.
39. Neufeld, J. D.,, M. Wagner, and, J. C. Murrell. 2007. Who eats what, where and when? Isotope-labelling experiments are coming of age. ISME J. 1: 103110.
40. Nielsen, J. L., and, P. H. Nielsen. 2002a. Enumeration of acetate-consuming bacteria by microautoradiography under oxygen and nitrate respiring conditions in activated sludge. Water Res. 36: 421428.
41. Nielsen, J. L., and, P. H. Nielsen. 2002b. Quantification of functional groups in activated sludge by microautoradiography. Water Sci. Technol. 46: 389395.
42. Nielsen, J. L., and, P. H. Nielsen. 2005. Advances in microscopy: microautoradiography of single cells. Methods Enzymol. 397: 237256.
43. Nielsen, J. L.,, D. Christensen,, M. Kloppenborg, and, P. H. Nielsen. 2003. Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization. Environ. Microbiol. 5: 202211.
44. Ouverney, C. C., and, J. A. Fuhrman. 1999. Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ. Appl. Environ. Microbiol. 65: 17461752.
45. Pearl, H. W., and, E. A. Stull. 1979. In defence of grain density autoradiography. Limnol. Oceanogr. 24: 11661169.
46. Pernthaler, A., and, R. Amann. 2004. Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Appl. Environ. Microbiol. 70: 54265433.
47. Pernthaler, J., and, R. Amann. 2005. Fate of heterotrophic microbes in pelagic habitats: focus on populations. Microbiol. Mol. Biol. Rev. 69: 440461.
48. Pernthaler, A.,, J. Pernthaler, and, R. Amann. 2002. Fluorescence in situ hybridization and catalyzed reporter deposition for the Identification of marine bacteria. Appl. Environ. Microbiol. 68: 30943101.
49. Power, M. E.,, D. Tilman,, J. A. Estes,, B. A. Menge,, W. J. Bond,, L. S. Mills,, G. Daily,, J. C. Castilla,, J. Lubchenco, and, R. T. Paine. 1996. Challenges in the quest for keystones. Bioscience 46: 609620.
50. Ram, R. J.,, N. C. Verberkmoes,, M. P. Thelen,, G. W. Tyson,, B. J. Baker,, R. C. Blake, 2nd,, M. Shah,, R. L. Hettich, and, J. F. Banfield. 2005. Community proteomics of a natural microbial biofilm. Science 308: 19151920.
51. Rogers, A. W. 1979. Techniques of Autoradiography. Elsevier/North Holland Biomedical Press, Amsterdam, The Netherlands.
52. Salpeter, M. M.,, L. Bachmann, and, E. E. Salpeter. 1969. Resolution in electron microscope radioautography. J. Cell Biol. 41: 132.
53. Shi, Y.,, G. W. Tyson, and, E. F. DeLong. 2009. Meta-transcriptomics reveals unique microbial small RNAs in the ocean’s water column. Nature 459: 266269.
54. Stahl, D. A., and, R. Amann. 1991. Development and application of nucleic acid probes, p. 205248. In E. Stackebrandt and, M. Goodfellow (ed.), Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, New York, NY.
55. Staley, J. T., and, A. Konopka. 1985. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu. Rev. Microbiol. 39: 321346.
56. Stoecker, K.,, C. Dorninger,, H. Daims, and, M. Wagner. 2010. Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) improves signal intensity and increases rRNA accessibility. Appl. Environ. Microbiol. 76: 922926.
57. Strous, M.,, E. Pelletier,, S. Mangenot,, T. Rattei,, A. Lehner,, M. W. Taylor,, M. Horn,, H. Daims,, D. Bartol-Mavel,, P. Wincker,, V. Barbe,, N. Fonknechten,, D. Vallenet,, B. Segurens,, C. Schenowitz-Truong,, C. Medigue,, A. Collingro,, B. Snel,, B. E. Dutilh,, H. J. Op Den Camp,, C. Van Der Drift,, I. Cirpus,, K. T. van de Pas-Schoonen,, H. R. Harhangi,, L. Van Niftrik,, M. Schmid,, J. Keltjens,, J. Van De Vossenberg,, B. Kartal,, H. Meier,, D. Frishman,, M. A. Huynen,, H. W. Mewes,, J. Weissenbach,, M. S. Jetten,, M. Wagner, and, D. Le Paslier. 2006. Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature 440: 790794.
58. Teira, E.,, T. Reinthaler,, A. Pernthaler,, J. Pernthaler, and, G. J. Herndl. 2004. Combining catalyzed reporter deposition-fluorescence in situ hybridization and microautoradiography to detect substrate utilization by Bacteria and Archaea in the deep ocean. Appl. Environ. Microbiol. 70: 44114414.
59. Urich, T.,, A. Lanzen,, J. Qi,, D. H. Huson,, C. Schleper, and, S. C. Schuster. 2008. Simultaneous assessment of soil microbial community structure and function through analysis of the meta-transcriptome. PLoS One 3: e2527.
60. Vaniman, D. A.,, S. Furlano,, J. Chipera,, K. Thompson, and, I. Triay. 1996. Microautoradiography in studies of Pu(V) sorption by trace and fracture minerals in tuff. Mat. Res. Soc. Symp. Proc. 412: 639646.
61. Verberkmoes, N. C.,, A. L. Russell,, M. Shah,, A. Godzik,, M. Rosenquist,, J. Halfvarson,, M. G. Lefsrud,, J. Apajalahti,, C. Tysk,, R. L. Hettich, and, J. K. Jansson. 2009. Shotgun metaproteomics of the human distal gut microbiota. ISME J. 3: 179189.
62. Vila, M.,, R. Simo,, R. P. Kiene,, J. Pinhassi,, J. M. Gonzalez,, M. A. Moran, and, C. Pedros-Alio. 2004. Use of microautoradiography combined with fluorescence in situ hybridization to determine dimethylsulfoniopropionate incorporation by marine bacterioplankton taxa. Appl. Environ. Microbiol. 70: 46484657.
63. Wagner, M. 2009. Single-cell ecophysiology of microbes as revealed by Raman microspectroscopy or secondary ion mass spectrometry imaging. Annu. Rev. Microbiol. 63: 411429.
64. Wagner, M.,, M. Horn, and, H. Daims. 2003. Fluorescence in situ hybridisation for the Identification and characterisation of prokaryotes. Curr. Opin. Microbiol. 6: 302309.
65. Wagner, M.,, P. H. Nielsen,, A. Loy,, J. L. Nielsen, and, H. Daims. 2006. Linking microbial community structure with function: fluorescence in situ hybridization-microautoradiography and isotope arrays. Curr. Opin. Biotechnol. 17: 8391.
66. Wagner, M.,, M. Schmid,, S. Juretschko,, K. H. Trebesius,, A. Bubert,, W. Goebel, and, K. H. Schleifer. 1998. In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes. FEMS Microbiol. Lett. 160: 159168.
67. Wagner, M.,, H. Smidt,, A. Loy, and, J. Zhou. 2007. Unravelling microbial communities with DNA-microarrays: challenges and future directions. Microb. Ecol. 53: 498506.
68. Wilmes, P., and, P. L. Bond. 2006. Metaproteomics: studying functional gene expression in microbial ecosystems. Trends Microbiol. 14: 9297.
69. Zhou, J. 2003. Microarrays for bacterial detection and microbial community analysis. Curr. Opin. Microbiol. 6: 288294.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error