In situ detection of denitrifying bacteria by mRNA-targeted nucleic acid probes and catalyzed reporter deposition Michael V.W. Kofoed, Peter Stief, Morten Poulsen, and Andreas Schramm Department of Biological Sciences, Microbiology, University of Aarhus, Denmark Denitrification, the sequential reduction of nitrate to dinitrogen gas, is essential for the removal of fixed nitrogen from natural and engineered ecosystems. However, community structure and activity dynamics of denitrifying bacteria in most systems are poorly understood, partially due to difficulties in identifying and quantifying (active) denitrifiers. The goal of this study was therefore to develop a protocol for the in situ detection of denitrifying bacterial cells by targeting the mRNA of denitrification genes, hereby linking denitrification activity directly to the single-cell level. Target genes were narG (encoding nitrate reductase) and nosZ (encoding nitrous oxide reductase), to detect nitrate-reducing and completely denitrifying bacteria, respectively. Enzyme-labelled oligonucleotide probes and digoxygenin-labelled polynucleotide probes were evaluated for in situ hybridization in combination with immunochemical detection and catalyzed fluorescent reporter deposition (CARD-FISH). The general feasibility of the approach was first tested with pure cultures of Pseudomonas stutzeri and various denitrifying and nitrate-reducing isolates. Detailed studies of probe specificity and hybridization conditions using Clone-FISH of narG and nosZ libraries prepared from freshwater sediment revealed a sequence similarity threshold of about 80% for detectable hybridization with polynucleotide probes. Consequently, polynucleotide probes need to be designed based on habitat-specific sequence information. In contrast, oligonucleotide probes can be designed to target a broader range of denitrifying bacteria; however, they require two-pass CARD-FISH, which may result in (too) high background fluorescence. In a first application example, habitat-specific polynucleotide probes were used to quantify bacteria expressing narG and nosZ in freshwater sediments and the guts of benthic invertebrates.
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Annual Conference of the Association for General and Applied Microbiology (VAAM), 2007