AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammoxNitrospirain the environment

Abstract

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 Pjevac et al.Comammox amoA-Targeted PCR Primers 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.

CO2assimilation strategies in stratified lakes: Diversity and distribution patterns of chemolithoautotrophs

“…). However, the differentiation between comammox and strict NOB is also not possible with other potential marker genes, such as nxrB and 16S rRNA genes (Pinto et al ., ; Pjevac et al ., ). Therefore, the functional role of Nitrospira populations in the nitrogen cycle at our study sites remains unresolved.…”

Comammox Nitrospira are abundant ammonia oxidizers in diverse groundwater‐fed rapid sand filter communities

“…While this method has a broad coverage for CuMMOs, it does not have good coverage of comammox amoA , and due to its two‐step nature, is not amenable to quantification. Another set of primers has high coverage and specificity, but requires separate primer sets for clades A and B (Pjevac et al ., ). A third group has combined a previously designed pmoA primer with a newly designed comammox specific primer.…”

“…As all source waters are freshwater, sulfate was extremely low in all environments (< 25 mg/l), so it was interesting to find that it was an important explanatory variable in shaping the comammox guild, even at such low concentrations. Notably, while comammox Nitrospira have been detected in brackish lakes and marine sediments (van Kessel et al ., ; Pjevac et al ., ), they have mainly been described in freshwater environments (Pinto et al ., ; Palomo et al ., ; Y. Wang et al ., ) and have not been detected in marine environments (Daims et al ., ). Thus, it is possible that sensitivity to sulfate varies across the comammox Nitrospira , even at the low concentrations observed in freshwater environments.…”

“…The simultaneous discovery of comammox Nitrospira by a number of groups (Daims et al ., ; van Kessel et al ., ; Pinto et al ., ; Palomo et al ., ) disrupts our long‐held understanding of nitrification. The low prevalence of comammox Nitrospira in most environments examined to date (Pjevac et al ., ), combined with their resistance to cultivation, explains how these organisms were overlooked for so long. The sole enrichments of comammox bacteria come from environments where they made up relatively small fractions of the original communities (Daims et al ., ; van Kessel et al ., ).…”

The pH dependency of N‐converting enzymatic processes, pathways and microbes: effect on net N2O production

“…NirK , the gene for Cu‐NIR, is also present in comammox, but no evidence of NO accumulation in comammox enrichments cultures suggests that nitrite reduction may not be particularly robust (Daims et al ., ). Comammox have been found in different water engineered systems, including WWTPs (Chao et al ., ; Gonzalez‐Martinez et al ., ; Pjevac et al ., ). However, their abundance in wastewater treatment plants is lower than AOB, which is likely due to the competitive advantage of AOB in systems with high substrate concentrations (Costa et al ., ; Kits et al ., ).…”