Microscopic detection of the toluene dioxygenase gene and its expression inside bacterial cells in seawater using prokaryotic in situ PCR

The toluene dioxygenase (todC1) gene and its mRNA transcripts were amplified by in situ PCR and in situ RT-PCR, respectively, in intact cells of the bacterium Pseudomonas putida F1. In situ amplicons of DNA and mRNA were then detected by hybridizing to a fluorescently labeled oligonucleotide. In situ PCR protocols were developed to distinguish between cells of P. putida F1 (possessing the todC1 gene) and P. putida AC10R (lacking the todC1 gene); the method was sensitive enough to detect amplified products from a single copy of the todC1 gene. P. putida F1 cells were also introduced into seawater with toluene addition. Cells expressing todC1 and total cells were detectable by in situ RT-PCR and Yo-Pro 1 counterstaining, respectively. Nearly 90% of cells expressing the todC1 gene were detected in seawater amended with toluene at day 3, but no cells expressing todC1 were detected in seawater not exposed to toluene. Our results suggest that in situ PCR amplification can be a useful technique for studying presence or absence of a specific gene and gene expression of bioremediative bacteria at the individual cell level following release into natural environments.