In situ reverse transcription-PCR and detection of nirS genes by Fluorescent In Situ Hybridization ( FISH ) to study denitrifying bacteria

Continental margin sediments constitute only about 10% of the total sediment surface area in the world’s oceans, nevertheless they are the dominant sites of nitrogen (N) cycling. Recent studies suggest that the oceanic nitrogen budget is unbalanced, primarily due to a higher nitrogen removal rate in contrast to the fixation rate, and it has been suggested that denitrification activity contributes significantly to this imbalance. Although denitrification in marine environments has been studied intensively at the process level, little is known about the species abundance, composition, distribution, and functional differences of the denitrifying population. Understanding the diversity of microbial populations in marine environments, their responses to various environmental factors such as NO3 , and how this impact the rate of denitrification is critical to predict global N dynamics. Environmental Microbiology has the prompt to study the influence of each microbial population on a biogeochemical process within a given ecosystem. Culture-dependent and -independent techniques using nucleic acid probes can access the identity and activity of cultured and uncultured microorganisms. Nucleic acid probes can target distintict genes which set phylogenetic relationships, such as rDNA 16S, DNA gyrase (gyrB) and RNA polymerase sigma 70 factor (rpoD). In the other hand, the genetic capabilities and their expression could be tracked using probes that target several functional genes, such as nirS, nirK, nosZ, and nifH, which are genes involved in denitrification. Selective detection of cells actively expressing functional genes within a community using In Situ Reverse Transcription-PCR (ISRT-PCR) could become a powerful culture-independent technique in microbial ecology. Here we describe an approach to study the expression of nirS genes in denitrifying bacteria. Pure cultures of Pseudomonas stutzeri and Paracoccus denitrificans, as well as co-cultures with nondenitrifying populations were used to optimize the ISRT-PCR protocol. Cells grown on nitrate broth were harvested and fixed at both logarithmic (24-48 h) and stationary phase (7 days). Fixed and RNA protectedcc cells were spotted on microscope slides to optimize cell wall permeabilization conditions with lyzozyme and proteinase K. Subsequently, ISRT-PCR was performed with NirS 1F and NirS 6R primers using the QIAGEN OneStep RT-PCR Kit. Amplification products within the cell were detected by Fluorescent In Situ Hybridization (FISH) at 40oC overnight using a Cy3 labeled internal probe, specifically designed to detect the nirS gene. After hybridization, the cells were counterstained with DAPI and examined by confocal fluorescence microscopy. P. stutzeri cells treated with RNase and Pseudomonas G179 (a nirK denitrifying strain) were used as negative controls. Optimal cell permeabilization was achieved using 1 mg ml lyzozyme for 30 min and 2 μg ml Proteinase K. RNase treated cells did not fluoresce after FISH, but were detectable by DAPI. Only nirS-type denitrifying cells in log phase (80-95% of total direct cell counts) were detected by this approach while fewer cells (5-10%) were detectable after 7 days in stationary phase. Co-cultures of P. denitrificans with a non-denitrifying isolate resulted in selective identification of target cells, thus supporting the potential use of this approach for gene expression analysis at the community level.