In situ reverse transcription, an approach to characterize genetic diversity and activities of prokaryotes.

Applied and Environmental Microbiology
F ChenR E Hodson

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, especiall...Continue Reading

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Citations

Nov 5, 2003·Proceedings of the National Academy of Sciences of the United States of America·C O JeonE L Madsen
Jul 27, 2001·FEMS Microbiology Letters·D S BachoonR E Hodson
May 29, 1999·Letters in Applied Microbiology·K KurokawaM Nasu
Jun 1, 2000·Applied and Environmental Microbiology·A S Whiteley, M J Bailey
Nov 15, 2000·FEMS Microbiology Reviews·A BruneH Cypionka
Jan 16, 2013·Chromosome Research : an International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology·Alla KrasikovaElena Vasilevskaya
Feb 24, 2001·Environmental Microbiology·M Espinosa-Urgel, R Kolter
Jun 5, 2015·Frontiers in Microbiology·Grace PoldKristen M DeAngelis
Jan 5, 2002·Applied and Environmental Microbiology·Katsuji TaniMasao Nasu
Sep 5, 2008·Current Protocols in Cytometry·Jonathan Porter
Jul 2, 1998·Applied and Environmental Microbiology·S StrettonA E Goodman
Sep 23, 2000·Journal of Microbiological Methods·J Porter, R W Pickup
Jun 11, 1999·Current Opinion in Biotechnology·B J MacGregor

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