, Volume 7, Issue 5, pp 409–413 | Cite as

Substrate uptake in extremely halophilic microbial communities revealed by microautoradiography and fluorescence in situ hybridization

  • Ramon Rosselló-Mora
  • Natuschka Lee
  • Josefa Antón
  • Michael Wagner
Original Paper


The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp. and the conspicuous square Archaea had been detected. Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such brines. The results indicated that hitherto uncultured 'square Archaea' do actively incorporate amino acids and acetate. However, Salinibacter spp. only showed amino acid incorporation in pure culture, but no evidence of such activity in their natural environment could be demonstrated. No glycerol incorporation was observed for any component of the microbial community.


Fluorescence in situ hybridization Halophiles In situ physiology Microautoradiography Salinibacter Square archaea Uncultured microorganisms 



We are grateful to the Zaforteza-Dezcallar family for allowing access to the S'Avall salterns, and to Annatina Zarda, who encouraged us to undergo this study. This work was supported by the following institutions' research projects: the Spanish Ministry of Science and Technology with grants BOS2000-1123C02-01 and BOS2000-1123C02-02 (to R.R.M. and J.A.). R.R.M. acknowledges the grant of a Marina Bueno Fellowship (CSIC-DFG), which covered travel expenses to perform the current project.


  1. Amann R, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990) Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56:1919–1925PubMedGoogle Scholar
  2. Amann R, Ludwig W, Schleifer K-H (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedGoogle Scholar
  3. Antón J, Llobet-Brossa E, Rodríguez-Valera F, Amann R (1999) Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds. Environ Microbiol 1:517–523PubMedGoogle Scholar
  4. Antón J, Rosselló-Mora R, Rodríguez-Valera F, Amann R (2000) Extremely halophilic bacteria in crystallizer ponds from solar salterns. Appl Environ Microbiol 66:3052–3057PubMedGoogle Scholar
  5. Antón J, Oren A, Benlloch S, Rodríguez-Valera F, Amann R, Rosselló-Mora R (2002) Salinibacter ruber gen. nov., sp. nov., a novel extremely halophilic member of the Bacteria from saltern crystallizer ponds. Int J Syst Evol Microbiol 52:485–491PubMedGoogle Scholar
  6. Benlloch S, López-López A, Casamayor EO, Øvreas L, Goddard V, Daae FL, Smerdon G, Massana R, Joint l, Thingstad F, Pedrós-Alió C, Rodríguez-Valera F (2002) Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern. Environ Microbiol 4:349–360CrossRefPubMedGoogle Scholar
  7. Brock TD, Brock L (1966) Autoradiography as a tool in microbial ecology. Nature 208:734–736Google Scholar
  8. Casamayor EO, Massana R, Benlloch S, Øvreas L, Díez B, Goddard VJ, Gasol JM, Joint I, Rodríguez-Valera F, Pedrós-Alió C (2002) Changes in archaeal, bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern. Environ Microbiol 4:338–348CrossRefPubMedGoogle Scholar
  9. Cottrell MT, Kirchman DL (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 66:1692–1697CrossRefPubMedGoogle Scholar
  10. Daims H, Nielsen JL, Nielsen PH, Schleifer K-H, Wagner M (2001) In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in waste water treatment plants. Appl Environ Microbiol 67:5273–5284CrossRefPubMedGoogle Scholar
  11. Gray ND, Head IM (2001) Linking genetic identity and function in communities of uncultured bacteria. Environ Microbiol 3:481–492CrossRefPubMedGoogle Scholar
  12. Gray ND, Howarth R, Pickup RW, Jones JG, Head IM (2000) Use of combined microautoradiography and fluorescence in situ hybridization to determine carbon metabolism in mixed natural communities of uncultured bacteria from the genus Achromatium. Appl Environ Microbiol 66:4518–4522CrossRefPubMedGoogle Scholar
  13. Lee N, Halkjaer P, Andreasen KH, Juretschko S, Nielsen JL, Wagner M (1999) Combination of fluorescent in situ hybridization and microautoradiography: a new tool for structure-function analyses in microbial ecology. Appl Environ Microbiol 65:1289–1297PubMedGoogle Scholar
  14. Nielsen PH, Andreasen K, Wagner M, Blackall LL, Lemmer H, Seviour RJ (1998) Variability of type 021N in activated sludge as determined by in situ substrate uptake pattern and in situ hybridization with fluorescent rRNA targeted probes. Water Sci Technol 37:423–430CrossRefGoogle Scholar
  15. Nielsen PH, Andreasen K, Lee N, Wagner M (1999) Use of microautoradiography and fluorescent in situ hybridization for characterization of microbial activity in activated sludge. Water Sci Technol 39:1-9CrossRefGoogle Scholar
  16. Oren A (1990a) Estimation of the contribution of halobacteria to the bacterial biomass and activity in solar salterns by the use of bile salts. FEMS Microbiol Ecol 73:41–48CrossRefGoogle Scholar
  17. Oren A (1990b) The use of protein synthesis inhibitors in the estimation of the contribution of halophilic archaebacteria to bacterial activity in hypersaline environments. FEMS Microbiol Ecol 73:187–192CrossRefGoogle Scholar
  18. Oren A (1990c) Thymidine incorporation in saltern ponds of different salinities: estimation of in situ growth rates of halophilic Archaeobacteria and Eubacteria. Microb Ecol 19:43–51Google Scholar
  19. Oren A (1993) Availability, uptake and turnover of glycerol in hypersaline environments. FEMS Microbiol Lett 12:15–23CrossRefGoogle Scholar
  20. Oren A (1994) The ecology of extremely halophilic archaea. FEMS Microbiol Rev 13:415–440Google Scholar
  21. Oren A (2002) Molecular ecology of extremely halophilic Archaea and Bacteria. FEMS Microbiol Ecol 39:1-7Google Scholar
  22. Oren A, Duker S, Ritter S (1996) The polar lipid composition of Walsby's square bacterium. FEMS Microbiol Lett 138:135–140Google Scholar
  23. Ouverney CC, Fuhrman JA (1999) Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ. Appl Environ Microbiol 65:1746–1752PubMedGoogle Scholar
  24. Ouverney CC, Fuhrman JA (2000) Marine planktonic Archaea take up aminoacids. Appl Environ Microbiol 66:4829–4833PubMedGoogle Scholar
  25. Pedrós-Alió C, Calderón-Paz JI, MacLean MH, Medina G, Marrasé C, Gasol JM, Guixa-Boixereu N (2000) The microbial food web along salinity gradients. FEMS Microbiol Ecol 32:143–155CrossRefPubMedGoogle Scholar
  26. Raskin L, Stromley JM, Rittmann BE, Stahl DA (1994) Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens. Appl Environ Microbiol 60:1232–1240PubMedGoogle Scholar
  27. Rodríguez-Valera F, Acinas SG, Antón J (1999) Contribution of molecular techniques to the study of microbial diversity in hypersaline environments. In: Oren A (ed) Microbiology and biogeochemistry of hypersaline environments. CRC, Boca Raton, Fla., pp 27–38Google Scholar
  28. Rosselló-Mora R, Thamdrup B, Schäfer H, Weller R, Amann R (1999) The response of the microbial community of marine sediments to organic carbon input under anaerobic conditions. Syst Appl Microbiol 22:237–248PubMedGoogle Scholar
  29. Stahl DA, Amann R (1991) Development and application of nucleic acid probes. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, Chichester, pp 205–248Google Scholar
  30. Stoeckenius W (1981) Walsby's square bacterium: fine structure of an orthogonal prokaryote. J Bacteriol 148:352–360PubMedGoogle Scholar
  31. Wagner M, Rath G, Amann R, Koops H-P, Schleifer K-H (1995) In situ identification of ammonia-oxidizing bacteria. Syst Appl Microbiol 17:251–264Google Scholar
  32. Walsby AE (1980) A square bacterium. Nature 283:69–71Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • Ramon Rosselló-Mora
    • 1
  • Natuschka Lee
    • 2
  • Josefa Antón
    • 3
  • Michael Wagner
    • 4
  1. 1.Grup d'Oceanografia InterdisciplinarInstitut Mediterrani d'Estudis Avançats (CSIC-UIB)EsporlesSpain
  2. 2.Lehrstuhl für MikrobiologieTechnische Universität MünchenMunichGermany
  3. 3.División de Microbiología, Departamento de Fisiología, Genética y MicrobiologíaUniversidad de AlicanteSan Vicente del RaspeigSpain
  4. 4.Department of Microbial Ecology, Institute of Ecology and Conservation BiologyVienna UniversityViennaAustria

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