AmoA-Targeted Polymerase Chain Reaction Primers for the Specific Detection and Quantification of Comammox Nitrospira in the Environment

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 <i>Nitrospira</i>, previously assumed to contain only canonical nitrite oxidizers. <i>Nitrospira</i> are widespread in nature, but for assessments of the distribution and functional importance of comammox <i>Nitrospira</i> in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite-oxidizing <i>Nitrospira</i> are required. Here we developed new PCR primer sets that specifically target the <i>amoA</i> genes coding for subunit A of the distinct ammonia monooxygenase of comammox <i>Nitrospira</i>. While existing primers capture only a fraction of the known comammox <i>amoA</i> diversity, the new primer sets cover as much as 95% of the comammox <i>amoA</i> clade A and 92% of the clade B sequences in a reference database containing 326 comammox <i>amoA</i> 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 <i>Nitrospira</i> in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox <i>amoA</i> was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox <i>amoA</i> 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 <i>amoA</i> gene copy numbers in environmental samples. The measured relative abundances of comammox <i>Nitrospira</i>, compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox <i>amoA</i>-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.