Chen, F., J.M. González, W.A. Dustman, M. A. Moran, and R. E.
Hodson. 1997. In situ reverse transcriptase: an approach to
characterize genetic diversity and activity of prokaryotes. Appl.
Environ. Microbiol. 63:4907-4913.
Reverse transcription of RNA molecules inside intact bacterial cells
was carried out using reverse transcriptase with a single oligonucleotide
complementary to specific 16S rRNA or mRNA sequences. Fluorescently-labeled
nucleotides were incoporated into each transcribed cDNA inside cells. This
protocol is termed in situ reverse transcription (ISRT). In this
study, 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
with those estimated by in situ hybridization (ISH) techniques and
with those estimated by hybridizations 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 compared to ISH, especially when targeting mRNA. The
calculated pixel intensities resulted from ISRT were up to 4.2 times more
intensive than those from ISH. It suggests that multiple incorporation of
fluorescently-labeled nucleotides into cDNA provided a high sensitivity for
phylogenetic identification of bacterial populations as well as detection of
cells expressing a specific gene within complex bacterial communities.