Archaea are widespread in extreme and temperate environments, and cultured representatives cover a broad spectrum of metabolic capacities, which sets them up for potentially major roles in the biogeochemistry of their ecosystems. The detection, characterization, and quantification of archaeal functions in mixed communities require Archaea-specific primers or probes for the corresponding metabolic genes. Five pairs of degenerate primers were designed to target archaeal genes encoding key enzymes of nitrogen cycling: nitrite reductases NirA and NirB, nitrous oxide reductase (NosZ), nitrogenase reductase (NifH), and nitrate reductases NapA/NarG. Sensitivity towards their archaeal target gene, phylogenetic specificity, and gene specificity were evaluated in silico and in vitro. Owing to their moderate sensitivity/coverage, the novel nirB-targeted primers are suitable for pure culture studies only. The nirA-targeted primers showed sufficient sensitivity and phylogenetic specificity, but poor gene specificity. The primers designed for amplification of archaeal nosZ performed well in all 3 criteria; their discrimination against bacterial homologs appears to be weakened when Archaea are strongly outnumbered by bacteria in a mixed community. The novel nifH-targeted primers showed high sensitivity and gene specificity, but failed to discriminate against bacterial homologs. Despite limitations, 4 of the new primer pairs are suitable tools in several molecular methods applied in archaeal ecology. 1. Introduction Archaea have been detected in virtually all types of extreme and moderate environments. They play multiple ecological roles, colonizing certain newly emerging habitats [1, 2], interacting with animals such as corals [3, 4], sponges [5, 6], termites , or ruminants, forming part of microbe-microbe symbioses [8–10], and driving numerous processes in the biogeochemical C, N, S, and Fe cycles. In addition to relatively well-studied isolates of extremophilic or methanogenic Archaea, uncultured representatives have been detected by their 16S rRNA genes or by metabolic genes that classify their owners into the guilds of sulfate reducers, diazotrophs, ammonia oxidizers, or methanogens. Despite their widespread occurrence, a mere handful of nonmethanogenic Archaea has been isolated from moderate habitats [11–13]. While such isolates are indispensable for insight into archaeal ecophysiology, they have been recalcitrant to cultivation efforts, so that our current ecological research on mesophilic Archaea largely depends on cultivation-independent methods. Molecular
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