Development of molecular marker techniques for detection of denitrification in marine environments, with using denitrification-related genes.

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I tried to develop new techniques for estimating heterotrophic denitrification and anaerobic ammonium oxidation (anammox) in coastal environments, to reconstruct the new nitrogen cycling model. First of all, I determined three genes relative to enzymes for denitrification metabolism, that is nitrate reductase (nirS), nitrite reductase (norB) and nitrous oxide reductase (nosZ), from strains of Marinobacter spp. that was isolated from a seawater environment. After that I established quantative PCR system for RNA of these genes. I applied this system for investigation of expression properties of the Marinobacter spp. and elucidated that the two closely related strains of Marinobacter have different expression patterns of the denitrification genes. This suggests that it is difficult to estimate denitrification activities in situ by quantification of the denitrification genes. I also develop a selective PCR technique for detecting anammox bacteria-related 16S rRNA genes from sediment and seawater samples. With using this protocol, I discovered the area where anammox bacteria inhabit and potentially produced dinitrogen gas from ammonium with nitrite. Results from the ^<15>N tracer experiment with GC-MS and the newly developed molecular technique suggested that anammox bacterial population was present in freshwater, estuary and seawater sediment however no anammox bacterial population existed in the sediment of hypoxia (seawater mass in which scarcely or no oxygen contains). From the clone library analysis of anammox bacteria-related 16S rRNA genes amplified, I propose the compartmentalize of anammox bacterial population along with salinity gradient, because in freshwater environments, Candidatus "Kuennenia"-like bacteria and Ca. "Brocadia"-like bacteria dominantly inhabit while Ca. "Scalindua sorokini"-like bacteria were present abundantly in seawater environments.