AmoA-Targeted Polymerase Chain Reaction Primers for the Specific Detection and Quantification of Comammox Nitrospira in the Environment.
AmoA-Targeted Polymerase Chain Reaction Primers for the Specific Detection and Quantification of Comammox Nitrospira in the Environment.
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DOI：
10.3389/fmicb.2017.01508
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发表时间：
2017
影响因子：
5.2
通讯作者：
Daims H
中科院分区：
文献类型：
--
作者：
Pjevac P;Schauberger C;Poghosyan L;Herbold CW;van Kessel MAHJ;Daebeler A;Steinberger M;Jetten MSM;Lücker S;Wagner M;Daims H
硝化作用，即氨通过亚硝酸盐氧化为硝酸盐，一直被认为是由氨氧化微生物和亚硝酸盐氧化微生物协同作用催化的。直到最近，完全氨氧化剂（“comammox”）才在硝化螺旋菌属细菌中被发现，它们自己将氨氧化成硝酸盐，以前认为它们只含有典型的亚硝酸盐氧化剂。硝基螺旋菌在自然界中分布广泛，但为了评估其在生态系统中的分布和功能重要性，需要独立于栽培的工具来区分硝基螺旋菌和严格的亚硝酸盐氧化硝基螺旋菌。在这里，我们开发了新的PCR引物集，专门针对编码氨单加氧酶亚基A的amoA基因。虽然现有引物只捕获了已知comammox amoA多样性的一小部分，但在包含326个comammox amoA基因的参考数据库中，新的引物集覆盖了高达95%的comammox amoA进化支a序列和92%的进化支B序列，并在引物结合位点上提供了序列信息。将引物应用于来自工程系统（地下水井、饮用水处理和废水处理厂）和其他生境（稻田和森林土壤、水稻根际、咸淡湖沉积物和淡水生物膜）的13个样品中，检测到所有样品中的comammox Nitrospira，并揭示了大多数生境中comammox的相当大的多样性。通过避免使用高度简并的引物制剂，并使用与现有的comammox amoA基因匹配的寡核苷酸等摩尔混合物，实现了对comammox amoA极好的引物特异性。这些等摩尔引物混合物的定量PCR具有高度的敏感性和特异性，能够有效地定量环境样品中A支和B支comammox amoA基因拷贝数。与典型氨氧化剂相比，测量到的comammox硝基螺旋菌的相对丰度在不同环境下变化很大。新的comammox amoa靶向引物使未来在不同栖息地的硝化微生物的研究更加全面。例如，它们可用于监测在不断变化的环境条件下未培养的共生菌的种群动态，以及对工程和农业生态系统中改变处理的反应。
Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be catalyzed by the concerted activity of ammonia- and nitrite-oxidizing microorganisms. Only recently, complete ammonia oxidizers (“comammox”), which oxidize ammonia to nitrate on their own, were identified in the bacterial genus Nitrospira, previously assumed to contain only canonical nitrite oxidizers. Nitrospira are widespread in nature, but for assessments of the distribution and functional importance of comammox Nitrospira in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite-oxidizing Nitrospira are required. Here we developed new PCR primer sets that specifically target the amoA genes coding for subunit A of the distinct ammonia monooxygenase of comammox Nitrospira. While existing primers capture only a fraction of the known comammox amoA diversity, the new primer sets cover as much as 95% of the comammox amoA clade A and 92% of the clade B sequences in a reference database containing 326 comammox amoA genes with sequence information at the primer binding sites. Application of the primers to 13 samples from engineered systems (a groundwater well, drinking water treatment and wastewater treatment plants) and other habitats (rice paddy and forest soils, rice rhizosphere, brackish lake sediment and freshwater biofilm) detected comammox Nitrospira in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox amoA was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox amoA genes. Quantitative PCR with these equimolar primer mixtures was highly sensitive and specific, and enabled the efficient quantification of clade A and clade B comammox amoA gene copy numbers in environmental samples. The measured relative abundances of comammox Nitrospira, compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox amoA-targeted primers enable more encompassing future studies of nitrifying microorganisms in diverse habitats. For example, they may be used to monitor the population dynamics of uncultured comammox organisms under changing environmental conditions and in response to altered treatments in engineered and agricultural ecosystems.
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影响因子：
5.2
作者：
Daebeler A;Abell GC;Bodelier PL;Bodrossy L;Frampton DM;Hefting MM;Laanbroek HJ
通讯作者：
Laanbroek HJ
影响因子：
14.9
作者：
Ludwig, W;Strunk, O;Schleifer, KH
通讯作者：
Schleifer, KH
影响因子：
4.4
作者：
Daims, H;Nielsen, JL;Wagner, M
通讯作者：
Wagner, M
DOI：
10.1073/pnas.0506625102
发表时间：
2005-10-11
影响因子：
11.1
作者：
Francis, CA;Roberts, KJ;Oakley, BB
通讯作者：
Oakley, BB
影响因子：
11
作者：
Angel, Roey;Claus, Peter;Conrad, Ralf
通讯作者：
Conrad, Ralf