RT Journal Article
SR Electronic
T1 <em>AmoA</em>-targeted polymerase chain reaction primers for the specific detection and quantification of comammox <em>Nitrospira</em> in the environment
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 096891
DO 10.1101/096891
A1 Petra Pjevac
A1 Clemens Schauberger
A1 Lianna Poghosyan
A1 Craig W. Herbold
A1 Maartje A.H.J. van Kessel
A1 Anne Daebeler
A1 Michaela Steinberger
A1 Mike S. M. Jetten
A1 Lücker Sebastian
A1 Wagner Michael
A1 Daims Holger
YR 2017
UL http://biorxiv.org/content/early/2017/02/06/096891.abstract
AB 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 known 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. Thus, the new comammox amoA-targeted primers will enable more encompassing studies of nitrifying microorganisms in diverse ecosystems.