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

本研究试图开发新的技术手段来评估海岸带环境中的异养反硝化和厌氧氨氧化，以重建新的氮循环模式。首先，从海蛞蝓菌株中确定了硝酸还原酶（nirS）、亚硝酸还原酶（norB）和一氧化二氮还原酶（nosZ）三个与反硝化代谢相关的基因。是从海水环境中分离出来的。在此基础上，建立了这些基因的RNA定量PCR体系。我应用这个系统的表达特性的调查marinspp。并阐明了两株关系密切的海洋细菌具有不同的反硝化基因表达模式。这表明，这是很难估计反硝化活性在原位通过定量的反硝化基因。我还开发了一种选择性PCR技术，用于检测厌氧氨氧化细菌相关的16 S rRNA基因从沉积物和海水样品。通过使用该协议，我发现了厌氧氨氧化细菌栖息的区域，并可能从亚硝酸铵中产生二氮气体。利用<15>GC-MS和新发展的分子生物学技术进行的^ N示踪实验结果表明，淡水、河口和海水沉积物中存在厌氧氨氧化菌群，而缺氧沉积物中不存在厌氧氨氧化菌群。通过对厌氧氨氧化细菌相关16 SrRNA基因克隆文库的分析，提出了厌氧氨氧化细菌种群沿着盐度梯度的划分，因为在淡水环境中，类Kuennenia细菌和Ca.“Brocadia”样细菌占主导地位，而Ca.“Scalindua sorokini”类细菌在海水环境中大量存在。

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.