Use of Newly Designed Primers for Quantification of Complete Ammonia-Oxidizing (Comammox) Bacterial Clades and Strict Nitrite Oxidizers in the Genus Nitrospira

Abstract

Complete ammonia oxidizing (comammox) bacteria play key roles in environmental nitrogen cycling and all belong to the genus Nitrospira, which was originally believed to include only strict nitrite-oxidizing bacteria (sNOB). Thus, differential estimation of sNOB abundance from comammox Nitrospira has become problematic since both contain nitrite oxidoreductase genes that serve as common targets for sNOB detection. Herein, we developed novel comammox Nitrospira clades A- and B-specific primer sets targeting the α-subunit of ammonia monooxygenase gene (amoA) and a sNOB-specific primer set targeting cyanase gene (cynS) for quantitative PCR (qPCR). The high coverage and specificity of these primers had been checked by metagenome and metatranscriptome datasets. Efficient and specific amplification with these primers were demonstrated using various environmental samples. Using the newly designed primers, we successfully estimated the abundances of comammox Nitrospira and sNOB in samples from two chloramination-treated drinking water systems and found that, in most samples, comammox Nitrospira clade A was the dominant Nitrospira, and also served as the primary ammonia oxidizer. Compared with other ammonia oxidizers, comammox Nitrospira took advantage in process water samples in these two drinking water systems. We also demonstrated sNOB can be readily misrepresented with the previous method, calculated by subtracting the comammox abundance from the total Nitrospira, especially when the comammox Nitrospira proportion is relatively high. The new primer sets were successfully applied for the quantification of comammox Nitrospira and sNOB, which may prove useful in understanding the roles of Nitrospira in nitrification in various ecosystems. Importance Nitrospira is a dominant nitrite-oxidizing bacteria in many artificial and natural environments. The discovery of complete ammonia oxidizers in Nitrospira prevents the use of previously identified primers targeting Nitrospira 16S rRNA gene or nitrite oxidoreductase (nxr) gene for differential determination of strict nitrite-oxidizing bacteria in Nitrospira (sNOB) and comammox Nitrospira. We designed three novel primer sets that enabled quantification of comammox Nitrospira clades A and B and sNOB with high coverage, specificity, and accuracy in various environments. With designed primer sets, sNOB and comammox Nitrospira were differentially estimated in drinking water systems, and we found that comammox clade A was overcome sNOB and other ammonia oxidizers in process water samples. Accurate quantification of comammox Nitrospira and sNOB using the newly designed primers will provide essential information for evaluating the contribution of Nitrospira to nitrification in various ecosystems.

Niche differentiation of comammox Nitrospira in sediments of the Three Gorges Reservoir typical tributaries, China

“…The qRT-PCR was performed to determine the amoA gene abundance of comammox Nitrospira clade A, comammox Nitrospira clade B, AOA, and AOB using QuantStudio™ 6 Flex quantitative PCR instrument (Thermo Fisher Scientific, Singapore). Four primer pairs, namely, CA377f/C576r 36 , CB377f/C576r 36 , Arch- amoA F/Arch- amoA R 37 , and amoA –1Fmod/GenAOBR 38 were used for the quantification of the aforementioned four amoA genes, respectively. Quantitative PCR was carried out in 10 μL amplification system containing 5.0 μL of T5 Fast qPCR Mix (2 ×), 0.4 μL of each Primer (10 μM), 0.2 μL of ROX Reference Dye II (50 ×), 1 μL of template DNA, and 3.0 μL of distilled deionized water (ddH 2 O).…”

Abundance and Niche Differentiation of Comammox in the Sludges of Wastewater Treatment Plants That Use the Anaerobic–Anoxic–Aerobic Process

“…The reaction volumes and procedures of partial nested PCR were performed as previously described [ 12 ]. Genomic DNA was diluted 10-fold to avoid interference from environmental factors [ 37 ]. qPCR primers used in this study are listed in Table S1 .…”

“…The results were analyzed using MxPro qPCR software (version 3.0) (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer’s instructions. The construction of standard plasmids and standard curves was performed as previously reported [ 12 , 37 ].…”

Plant Species–Driven Distribution of Individual Clades of Comammox Nitrospira in a Subtropical Estuarine Wetland

“…Quantitative PCR was performed on a CFX96 Optical Real-Time Detection System (Bio-Rad Laboratories Inc., Hercules, CA, USA). The primer sets CA377f/C576r and CB377f/C576r [37] were used for the amplification of comammox Nitrospira clade A and clade B, respectively. Each 20-μL reaction mixture contained 10 μL of SYBR qPCR mix (Vazyme, Nanjing, China), 0.2 μL of forward and reverse primers (20 μM), 1 μL of DNA template and 8.6 μL of sterilized water.…”