Murdock Sheryl A, Juniper S Kim
School of Earth and Ocean Sciences, University of Victoria, Victoria, British Columbia, Canada
School of Earth and Ocean Sciences, University of Victoria, Victoria, British Columbia, Canada.
Appl Environ Microbiol. 2017 Mar 2;83(6). doi: 10.1128/AEM.02753-16. Print 2017 Mar 15.
Denitrifying may dominate nitrogen loss processes in marine habitats with intense redox gradients, but assessment of their importance is limited by the currently available primers for nitrite reductase genes. Nine new primers targeting the gene of denitrifying were designed and tested for use in sequencing and quantitative PCR on two microbial mat samples (vent 2 and vent 4) from the Calypso hydrothermal vent field, Bay of Plenty, New Zealand. Commonly used and primer sets nirS1F/nirS6R, cd3aF/R3cd, nirK1F/nirK5R, and F1aCu/R3Cu were also tested to determine what may be missed by the common single-primer approach to assessing denitrifier diversity. The relative importance of in these samples was evaluated by 16S rRNA gene sequencing. represented up to 75.6% of 16S rRNA libraries, but genes from this group were not found with commonly used primers. Pairing of the new primer EPSnirS511F with either EPSnirS1100R or EPSnirS1105R recovered sequences from members of the genera , , and The new quantitative PCR primers EPSnirS103F/EPSnirS530R showed dominance of denitrifying in vent 4 compared to vent 2, which had greater representation by "standard" denitrifiers measured with the cd3aF/R3cd primers. Limited results from commonly used primers suggest biased amplification between primers. Future application of multiple and primers, including the new epsilonproteobacterial primers, will improve the detection of denitrifier diversity and the capability to identify changes in dominant denitrifying communities. Estimating the potential for increasing nitrogen limitation in the changing global ocean is reliant on understanding the microbial community that removes nitrogen through the process of denitrification. This process is favored under oxygen limitation, which is a growing global-ocean phenomenon. Current methods use the nitrite reductase genes and to assess denitrifier diversity and abundance using primers that target only a few known denitrifiers and systematically exclude denitrifying , a group known to dominate in reducing environments, such as hydrothermal vents and anoxic basins. As oxygen depletion expands in the oceans, it is important to study denitrifier community dynamics within those areas to predict future global ocean changes. This study explores the design and testing of new primers that target epsilonproteobacterial and reveals the varied success of existing primers, leading to the recommendation of a multiple-primer approach to assessing denitrifier diversity.
在具有强烈氧化还原梯度的海洋生境中，反硝化作用可能主导着氮损失过程，但目前用于亚硝酸还原酶基因的引物限制了对其重要性的评估。设计并测试了9种针对反硝化细菌基因的新引物，用于对来自新西兰丰盛湾卡吕普索热液喷口场的两个微生物垫样本（喷口2和喷口4）进行测序和定量PCR。还测试了常用的nirS和nirK引物组nirS1F/nirS6R、cd3aF/R3cd、nirK1F/nirK5R以及F1aCu/R3Cu，以确定在评估反硝化细菌多样性的常见单引物方法中可能遗漏的信息。通过16S rRNA基因测序评估了这些样本中ε-变形菌纲的相对重要性。ε-变形菌纲在16S rRNA文库中占比高达75.6%，但使用常用引物未发现该类群的基因。新引物EPSnirS511F与EPSnirS1100R或EPSnirS1105R配对，从硫单胞菌属、硫还原菌属和硫化还原菌属成员中获得了ε-变形菌纲序列。新的定量PCR引物EPSnirS103F/EPSnirS530R显示，与喷口2相比，喷口4中反硝化ε-变形菌纲占主导地位，喷口2中用cd3aF/R3cd引物检测到的“标准”反硝化细菌占比更高。常用nirS引物的有限结果表明引物之间存在扩增偏差。包括新的ε-变形菌纲反硝化细菌引物在内的多种nirS和nirK引物的未来应用，将提高反硝化细菌多样性的检测能力以及识别优势反硝化细菌群落变化的能力。估计在不断变化的全球海洋中氮限制增加的可能性，依赖于了解通过反硝化过程去除氮的微生物群落。这个过程在氧气限制条件下更有利，而氧气限制是一种日益普遍的全球海洋现象。目前的方法使用亚硝酸还原酶基因nirS和nirK，通过仅针对少数已知反硝化细菌的引物来评估反硝化细菌的多样性和丰度，并系统性地排除了反硝化ε-变形菌纲，这是一个已知在还原环境（如热液喷口和缺氧盆地）中占主导地位的类群。随着海洋中氧气消耗的扩大，研究这些区域内反硝化细菌群落动态对于预测未来全球海洋变化很重要。本研究探索了针对ε-变形菌纲反硝化细菌的新引物的设计和测试，并揭示了现有引物的不同成功情况，从而推荐采用多引物方法来评估反硝化细菌多样性。
