Deciphering Functions of Uncultured Microorganisms

난배양성 미생물의 기능 분석 방법

  • Published : 2009.03.31

Abstract

Microbes within complex communities show quite different physiology from pure cultured microbes. However, historically the study of microbes has focused on single species in pure culture and most of microbes are unculturable in our labs, so understanding of complex communities lags behind understanding of pure cultured cells. Methodologies including stable isotope probing (SIP), a combination of fluorescence in situ hybridization (FISH) and microautoradiography (MAR), isotope micrarray, and metagenomics have given insights into the uncultivated majority to link phylogenetic and functional information. Here, we review some of the most recent literatures, with an emphasis on methodological improvements to the sensitivity and utilities of these methods to link phylogeny and function in complex microbial communities.

미생물 군집 내의 미생물은 순수하게 배양된 미생물과는 다른 생리적 특징을 갖는다. 전통적으로 미생물 연구는 순수배양에 초점을 맞추어 이루어져 왔고 실제 생태계에 존재하는 대부분의 미생물들이 난배양성 미생물로 알려져 있다. 따라서 복잡한 미생물 군집에서 미생물의 기능에 대한 연구는 실질적으로 미진한 실정이다. 그러나 stable isotope probing (SIP), fluorescence in situ hybridization (FISH)와 microautoradiography (MAR)의 조합, isotope micrarray, 메타게노믹스 등의 새로운 분석방법들은 미생물 군집 내에서 난배양성 미생물의 기능 분석을 어느 정도 가능하게 해 주었다. 본 논문에서는 이들 방법 등에 대해 간단히 설명하고 좀 더 정확한 결과를 얻기 위한 최신 연구 동향을 소개하고자 한다.

Keywords

References

  1. Adamczyk, J., M. Hesselsoe, N. Inversion, M. Horn, A. Lehner, P.H. Nielsen, M. Schloter, P. Roslev, and M. Wagner. 2003. The isotope array: a new tool to determine microbial community structure and function using substrate mediated labeling of ribosomal RNA. Appl. Environ. Microbiol. 69, 6875-6887 https://doi.org/10.1128/AEM.69.11.6875-6887.2003
  2. Borneman, J. 1999. Culture-independent identification of microorganisms that respond to specified stimuli. Appl. Environ. Microbiol. 65, 3398-3400
  3. Boschker, J.T., S.C. Nold, P. Wellsbury, D. Bos, W. de Graaf, R. Pel, R.J. Parkes, and T.E. Cappenberg. 1998. Direct linking of microbial populations to specific biogeochemical processes by 1$^13$C-labelling of biomarkers. Nature 392, 801-805 https://doi.org/10.1038/33900
  4. Committee on metagenomics: challenges and functional applications, National research council. 2007. Parallels with traditional microbial genome sequencing, pp. 60-63. In The new science of metagenomics: revealing the secrets of our microbial planet. The national academies, USA
  5. DeLong, E.F., G.S. Wickham, and N.R. Pace. 1989. Phylogenetic stains: Ribosomal RNA-based probes for the identification of single microbial cells. Science 243, 1360-1363 https://doi.org/10.1126/science.2466341
  6. Dumont, M.G. and J.C. Murrell. 2005. Stable isotope probing -linking microbial identity to function. Nature Rev. Microbiol. 3, 499-504 https://doi.org/10.1038/nrmicro1162
  7. Green, B.D. and M. Keller. 2006. Capturing the uncultivated majority. Curr. Opin. Biotechnol. 17, 236-240 https://doi.org/10.1016/j.copbio.2006.05.004
  8. Handelsman, J., M.R. Rondon, S.F. Brady, J. Clardy, and R.M. Goodman. 1998. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 5, R245-R249 https://doi.org/10.1016/S1074-5521(98)90108-9
  9. Huang, W.E., K. Stoecker, R. Griffiths, L. Newbold, 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, 1878-1889 https://doi.org/10.1111/j.1462-2920.2007.01352.x
  10. Hugenholtz, P. 2002. Exploring prokaryotic diversity in the genomic era. Genome Biology 3, 1-8
  11. Jeon, C.O., W. Park, P. Padmanabhan, C. DeRito, J.R. Snape, and E.L. Madsen. 2003. Discovery of a previously undescribed bacterium with distinctive dioxygenase that is responsible for in situ biodegradation in contaminated sediment. Proc. Nat. Acad. Sci. USA 100, 13591-13596 https://doi.org/10.1073/pnas.1735529100
  12. Kitano, H. 2002. System biology : a brief overview. Science 295, 1662-1664 https://doi.org/10.1126/science.1069492
  13. Kong, Y.H., J.L. Nielsen, and P.H. Nielsen. 2005. Identity and ecophysiology of uncultured actinobacterial polyphosphateaccumulating organisms in full-scale enhanced biological phosphorus removal plants. Appl. Environ. Microbiol. 71, 4076-4085 https://doi.org/10.1128/AEM.71.7.4076-4085.2005
  14. Kuypers, M.M.M. and B.B. J$\o$rgensen. 2007. The future of single cell environmental microbiology. Environ. Microbiol. 9, 6-7 https://doi.org/10.1111/j.1462-2920.2006.01222_5.x
  15. 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, 1289-1297
  16. Manefield, M., A.S. Whiteley, R.I. Griffiths, and M.J. Bailey. 2002. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl. Environ. Microbiol. 68, 5367-5373 https://doi.org/10.1128/AEM.68.11.5367-5373.2002
  17. Morgenroth, E., A. Obermayer, E. Arnold, A. Bruhl, M. Wagner, and P.A. Wilderer. 2000. Effect of long-term idle periods on the performance of sequencing batch reactors. Water Sci. Technol. 41, 105-113
  18. Munch, R. 2003. Robert Koch. Microbes Infect. 5, 69-74 https://doi.org/10.1016/S1286-4579(02)00053-9
  19. Nielsen, J.L., D. Christensen, M. Kloppenborg, and P.H. Nielsen. 2003. Quantification of cell-specific substrate uptake by probedefined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization. Environ. Microbiol. 5, 202-211 https://doi.org/10.1046/j.1462-2920.2003.00402.x
  20. Orphan, V.J., C.H. House, K.U. Hinrichs, K.D. McKeegan, and E.F. DeLong. 2001. Methane-consuming Archaea revealed by directly coupled isotopic and phylogenetic analysis. Science 293, 484-487 https://doi.org/10.1126/science.1061338
  21. Pace, N.R. 1997. A molecular view of microbial diversity and the biosphere. Science 276, 734-740 https://doi.org/10.1126/science.276.5313.734
  22. Park, J., S. Congeevaram, D. Ki, and J.M. Tiedje. 2006. Use of stable isotope probing in selectively isolating target microbial community genomes from environmental samples for enhancing resolution in ecotoxicological assessment. Mol. Cell. Toxicol. 2, 11-14
  23. Pernthaler, A. and R. Amann. 2004. Simultaneous fluorescence in situ hybridization of mRNA and rRNA in environmental bacteria. Appl. Environ. Microbiol. 70, 5426-5433 https://doi.org/10.1128/AEM.70.9.5426-5433.2004
  24. Radajewski, S., P. Ineson, N. Parekh, and J. Murrell. 2000. Stableisotope probing as a tool in microbial ecology. Nature 403, 646-649 https://doi.org/10.1038/35001054
  25. Schmid, M.C., S. Schmitz-Esser, M. Jetten, and M. Wagner. 2001. 16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium oxidizers: implications for phylogeny and in situ detection. Environ. Microbiol. 3, 450-459 https://doi.org/10.1046/j.1462-2920.2001.00211.x
  26. Stanley, J.T. and A. Konopka. 1985. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Ann. Rev. Microbiol. 39, 321-346 https://doi.org/10.1146/annurev.mi.39.100185.001541
  27. Streit, W.R. and R.A. Schmitz. 2004. Metagenomics - the key to the uncultured microbes. Curr. Opin. Microbiol. 7, 492-498 https://doi.org/10.1016/j.mib.2004.08.002
  28. Streit, W.R., R. Daniel, and K.E. Jaeger. 2004. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms. Curr. Opin. Biotechnol. 15, 1-6 https://doi.org/10.1016/j.copbio.2003.12.005
  29. Taniguchi, A. and K. Hamasaki. 2008. Community structures of actively growing bacteria shift along a north-south transect in the western North Pacific. Environ. Microbiol. 10, 1007-1017 https://doi.org/10.1111/j.1462-2920.2007.01521.x
  30. 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, 4411-4414 https://doi.org/10.1128/AEM.70.7.4411-4414.2004
  31. Vo, N.X.Q., H. Kang, and J. Park. 2007. Functional metagenomics using stable isotope probing: a review. Environ. Eng. Res. 12, 231-237 https://doi.org/10.4491/eer.2007.12.5.231
  32. Wang, R.F., M.L. Beggs, L.H. Robertson, and C.E. Cerniglia. 2002. Design and evaluation of oligonucleotide-microarray method for the detection of human intestinal bacteria in fecal samples. FEMS Microbiol. Lett. 213, 175-182 https://doi.org/10.1111/j.1574-6968.2002.tb11302.x