Homologues of nitrite reductases in ammonia-oxidizing archaea: diversity and genomic context Rita Bartossek, 1,2 Graeme W. Nicol, 3 Anders Lanzen, 1 Hans-Peter Klenk 4 and Christa Schleper 1,2 * 1 Department of Biology, Centre for Geobiology, University of Bergen, PO Box 7803, 5020 Bergen, Norway. 2 Department of Genetics in Ecology, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria. 3 Institute of Biological & Environmental Sciences, Cruickshank Building, University of Aberdeen, St Machar Drive, Aberdeen AB24 3UU, UK. 4 DSMZ, German Collection of Microorganisms and Cell Cultures, 38124 Braunschweig, Inhoffenstraße 7B, Germany. Summary Ammonia-oxidizing archaea are frequent and ubiqui- tous inhabitants of terrestrial and marine environ- ments. As they have only recently been detected, most aspects of their metabolism are yet unknown. Here we report on the occurrence of genes encoding potential homologues of copper-dependent nitrite reductases (NirK) in ammonia-oxidizing archaea of soils and other environments using metagenomic approaches and PCR amplification. Two pairs of highly overlapping 40 kb genome fragments, each containing nirK genes of archaea, were isolated from a metagenomic soil library. Between 68% and 85% of the open reading frames on these genome fragments had homologues in the genomes of the marine archaeal ammonia oxidizers Nitrosopumilus mariti- mus and Cenarchaeum symbiosum. Extensions of NirK homologues with C-terminal fused amicyanin domains were deduced from two of the four fosmids indicating structural variation of these multicopper proteins in archaea. Phylogenetic analyses including all major groups of currently known NirK homologues revealed that the deduced protein sequences of marine and soil archaea were separated into two highly divergent lineages that did not contain bacte- rial homologues. In contrast, another separated lineage contained potential multicopper oxidases of both domains, archaea and bacteria. More nirK gene variants directly amplified by PCR from several envi- ronments indicated further diversity of the gene and a widespread occurrence in archaea. Transcription of the potential archaeal nirK in soil was demonstrated at different water contents, but no significant increase in transcript copy number was observed with increased denitrifying activity. Introduction Nitrification, the oxidation of ammonia to nitrate via nitrite, is an important step in the biogeochemical nitrogen cycle. Until recently, in most environments this activity was thought to be restricted to specific microbial groups within the gamma and betaproteobacteria (Purkhold et al., 2000; Kowalchuk and Stephen, 2001). However, there is now increasing evidence that archaea make an important con- tribution to this process. Specific lineages of archaea from marine and soil environments possess the genes for ammonia monooxygenase (AMO), the key enzyme for ammonia oxidation (Schleper et al., 2005; Treusch et al., 2005; Hallam et al., 2006a) and a few archaeal strains capable of autotrophic growth by ammonia oxidation have recently been isolated (Könneke et al., 2005) or obtained in enrichment cultures (Hatzenpichler et al., 2008; de la Torre et al., 2008). Like their bacterial counterparts these organisms appear to be chemolithoautotrophs, who produce their vital energy through the oxidation of ammonia to nitrite (Könneke et al., 2005). amoA genes of archaea encoding subunit A of ammonia monooxygenase have now been isolated from many environments ranging from pristine and agricultural soils, marine plankton and sediments, to the tissues of invertebrates and hot springs (Francis et al., 2005; Leininger et al., 2006; Wuchter et al., 2006; Hallam et al., 2006b; Beman et al., 2007; He et al., 2007; Reigstad et al., 2008; de la Torre et al., 2008). In addition, quantitative molecular studies indicate that archaea with the potential to oxidize ammonia occur in high numbers and often exceed numbers of ammonia- oxidizing bacteria by orders of magnitude (Leininger et al., 2006; Wuchter et al., 2006). Received 3 September, 2009; accepted 4 December, 2009. *For correspondence. E-mail christa.schleper@univie.ac.at; Tel. (+43) 1427757800; Fax (+43) 142779578. Environmental Microbiology (2010) doi:10.1111/j.1462-2920.2010.02153.x © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd