Nitrite Reductase (nirK) as an Alternative Molecular Marker for Ammonia-oxidizing Archaea: Quantification and Characterization of Thaumarchaeal nirK Genes in Estuarine Sediments from San Francisco Bay

Nitrification, the microbially-mediated oxidation of ammonia to nitrate via nitrite, is a key component of the biogeochemical nitrogen cycle. The first and rate-limiting step of nitrification is the aerobic oxidation of ammonia to nitrite. Recent molecular ecological studies targeting ammonia monooxygenase (amoA) genes have identified ammonia-oxidizing archaea (AOA) as key players in the process. In addition to the ammonia oxidation machinery that includes the amoA gene, AOA also possess a gene for copper-containing nitrite reductase (nirK). Despite the notably high abundance and expression of archaeal nirK in various environments, the distribution patterns and functional role of this gene in AOA remain mostly unknown. In the present study, the diversity and abundance of nirK genes in estuarine sediments were investigated using quantitative polymerase chain reaction, cloning and sequencing approaches. The abundance and phylogeny were compared with that of the conventional marker gene, amoA. Overall, nirK was observed to be significantly more abundant than amoA in the sediment samples, with variation in copy numbers spanning several orders of magnitude between sampling sites. The substantial difference in relative abundance suggests that the conventionally used amoA primers could be underestimating AOA diversity in marine/estuarine sediments. Phylogenetic analysis targeting archaeal nirK revealed a number of unique sequence types, as well as many that clustered with sequences from previous estuarine studies and cultured AOA isolates, including Nitrosopumilus maritimus and Nitrosoarchaeaum limnia. The nirK phylogeny was mostly congruent with that of archaeal amoA. Overall, this study provides new insights into the diversity and abundance of archaeal nirK genes in estuarine sediments, and highlights the importance of further investigating the physiological role of this gene in AOA, as well as its suitability as a marker gene for studying AOA in the environment.