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

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.