Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Dec;63(12):4907–4913. doi: 10.1128/aem.63.12.4907-4913.1997

In Situ Reverse Transcription, an Approach To Characterize Genetic Diversity and Activities of Prokaryotes

F Chen, J M Gonzalez, W A Dustman, M A Moran, R E Hodson
PMCID: PMC1389309  PMID: 16535753

Abstract

Reverse transcription of RNA molecules inside intact bacterial cells was carried out by using reverse transcriptase with a single oligonucleotide complementary to specific 16S rRNA or mRNA sequences. Fluorescently labeled nucleotides were incorporated into each transcribed cDNA inside cells. This protocol is termed in situ reverse transcription (ISRT). In this study, by using species-specific primers targeting unique regions of the 16S rRNA sequences, ISRT was used successfully to detect and enumerate the two lignin-degrading bacteria Microbulbifer hydrolyticus IRE-31 and Sagittula stellata E-37 in culture mixtures and complex enrichment communities selected for lignin degradation. Image analysis revealed that M. hydrolyticus IRE-31 and S. stellata E-37 accounted for approximately 30 and 2%, respectively, of the total bacterial cells in lignin enrichment communities. Populations estimated by ISRT were comparable to those estimated by in situ hybridization (ISH) techniques and to those estimated by hybridization against extracted community DNA. ISRT was also successfully used to detect Pseudomonas putida F1 expressing the todC1 gene in seawater exposed to toluene vapor. ISRT provided a higher signal intensity than ISH, especially when targeting mRNA. The calculated pixel intensities resulting from ISRT were up to 4.2 times greater than those from ISH. This suggests that multiple incorporation of fluorescently labeled nucleotides into cDNA provides a high sensitivity for phylogenetic identification of bacterial populations as well as detection of cells expressing a specific functional gene within complex bacterial communities.

Full Text

The Full Text of this article is available as a PDF (636.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alfreider A., Pernthaler J., Amann R., Sattler B., Glockner F., Wille A., Psenner R. Community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake by in situ hybridization. Appl Environ Microbiol. 1996 Jun;62(6):2138–2144. doi: 10.1128/aem.62.6.2138-2144.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amann R. I., Binder B. J., Olson R. J., Chisholm S. W., Devereux R., Stahl D. A. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol. 1990 Jun;56(6):1919–1925. doi: 10.1128/aem.56.6.1919-1925.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amann R. I., Ludwig W., Schleifer K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995 Mar;59(1):143–169. doi: 10.1128/mr.59.1.143-169.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DeLong E. F., Wickham G. S., Pace N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989 Mar 10;243(4896):1360–1363. doi: 10.1126/science.2466341. [DOI] [PubMed] [Google Scholar]
  5. Gibson D. T., Hensley M., Yoshioka H., Mabry T. J. Formation of (+)-cis-2,3-dihydroxy-1-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida. Biochemistry. 1970 Mar 31;9(7):1626–1630. doi: 10.1021/bi00809a023. [DOI] [PubMed] [Google Scholar]
  6. Gibson D. T., Koch J. R., Kallio R. E. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene. Biochemistry. 1968 Jul;7(7):2653–2662. doi: 10.1021/bi00847a031. [DOI] [PubMed] [Google Scholar]
  7. Giovannoni S. J., DeLong E. F., Olsen G. J., Pace N. R. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol. 1988 Feb;170(2):720–726. doi: 10.1128/jb.170.2.720-726.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. González J. M., Whitman W. B., Hodson R. E., Moran M. A. Identifying numerically abundant culturable bacteria from complex communities: an example from a lignin enrichment culture. Appl Environ Microbiol. 1996 Dec;62(12):4433–4440. doi: 10.1128/aem.62.12.4433-4440.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gosden J. R., Lawson D. Oligonucleotide PRINS DNA synthesis. Methods Mol Biol. 1997;71:1–6. doi: 10.1385/0-89603-395-3:1. [DOI] [PubMed] [Google Scholar]
  10. Hahn D., Amann R. I., Ludwig W., Akkermans A. D., Schleifer K. H. Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides. J Gen Microbiol. 1992 May;138(5):879–887. doi: 10.1099/00221287-138-5-879. [DOI] [PubMed] [Google Scholar]
  11. Hahn D., Amann R. I., Zeyer J. Detection of mRNA in streptomyces cells by whole-cell hybridization with digoxigenin-labeled probes. Appl Environ Microbiol. 1993 Aug;59(8):2753–2757. doi: 10.1128/aem.59.8.2753-2757.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hicks R. E., Amann R. I., Stahl D. A. Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences. Appl Environ Microbiol. 1992 Jul;58(7):2158–2163. doi: 10.1128/aem.58.7.2158-2163.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hodson R. E., Dustman W. A., Garg R. P., Moran M. A. In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities. Appl Environ Microbiol. 1995 Nov;61(11):4074–4082. doi: 10.1128/aem.61.11.4074-4082.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Karner M., Fuhrman J. A. Determination of Active Marine Bacterioplankton: a Comparison of Universal 16S rRNA Probes, Autoradiography, and Nucleoid Staining. Appl Environ Microbiol. 1997 Apr;63(4):1208–1213. doi: 10.1128/aem.63.4.1208-1213.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Mobarry B. K., Wagner M., Urbain V., Rittmann B. E., Stahl D. A. Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ Microbiol. 1996 Jun;62(6):2156–2162. doi: 10.1128/aem.62.6.2156-2162.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Møller S., Pedersen A. R., Poulsen L. K., Arvin E., Molin S. Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy. Appl Environ Microbiol. 1996 Dec;62(12):4632–4640. doi: 10.1128/aem.62.12.4632-4640.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Patterson B. K., Till M., Otto P., Goolsby C., Furtado M. R., McBride L. J., Wolinsky S. M. Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science. 1993 May 14;260(5110):976–979. doi: 10.1126/science.8493534. [DOI] [PubMed] [Google Scholar]
  18. Ramsing N. B., Fossing H., Ferdelman T. G., Andersen F., Thamdrup B. Distribution of bacterial populations in a stratified fjord (Mariager Fjord, Denmark) quantified by in situ hybridization and related to chemical gradients in the water column. Appl Environ Microbiol. 1996 Apr;62(4):1391–1404. doi: 10.1128/aem.62.4.1391-1404.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ramsing N. B., Kühl M., Jørgensen B. B. Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes. Appl Environ Microbiol. 1993 Nov;59(11):3840–3849. doi: 10.1128/aem.59.11.3840-3849.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schramm A., Larsen L. H., Revsbech N. P., Ramsing N. B., Amann R., Schleifer K. H. Structure and function of a nitrifying biofilm as determined by in situ hybridization and the use of microelectrodes. Appl Environ Microbiol. 1996 Dec;62(12):4641–4647. doi: 10.1128/aem.62.12.4641-4647.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Trebesius K., Amann R., Ludwig W., Mühlegger K., Schleifer K. H. Identification of Whole Fixed Bacterial Cells with Nonradioactive 23S rRNA-Targeted Polynucleotide Probes. Appl Environ Microbiol. 1994 Sep;60(9):3228–3235. doi: 10.1128/aem.60.9.3228-3235.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wagner M., Amann R., Lemmer H., Schleifer K. H. Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure. Appl Environ Microbiol. 1993 May;59(5):1520–1525. doi: 10.1128/aem.59.5.1520-1525.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Weiss P., Schweitzer B., Amann R., Simon M. Identification in situ and dynamics of bacteria on limnetic organic aggregates (lake snow). Appl Environ Microbiol. 1996 Jun;62(6):1998–2005. doi: 10.1128/aem.62.6.1998-2005.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yamaguchi N., Inaoka S., Tani K., Kenzaka T., Nasu M. Detection of specific bacterial cells with 2-hydroxy-3-naphthoic acid-2'-phenylanilide phosphate and fast red TR in situ hybridization. Appl Environ Microbiol. 1996 Jan;62(1):275–278. doi: 10.1128/aem.62.1.275-278.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Zylstra G. J., Gibson D. T. Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli. J Biol Chem. 1989 Sep 5;264(25):14940–14946. [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES