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

Abstract

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