Homologues of nitrite reductases in ammonia-oxidizing archaea: diversity and genomic context
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Homologues of nitrite reductases in ammonia-oxidizing archaea: diversity and genomic contextRita 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 [email protected]; 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
Transcript of Homologues of nitrite reductases in ammonia-oxidizing archaea: diversity and genomic context
Homologues of nitrite reductases inammonia-oxidizing archaea: diversity andgenomic contextemi_2153 1..14
Rita Bartossek,1,2 Graeme W. Nicol,3 Anders Lanzen,1
Hans-Peter Klenk4 and Christa Schleper1,2*1Department of Biology, Centre for Geobiology,University of Bergen, PO Box 7803, 5020 Bergen,Norway.2Department of Genetics in Ecology, University ofVienna, Althanstrasse 14, A-1090 Vienna, Austria.3Institute of Biological & Environmental Sciences,Cruickshank Building, University of Aberdeen, StMachar Drive, Aberdeen AB24 3UU, UK.4DSMZ, German Collection of Microorganisms and CellCultures, 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 encodingpotential homologues of copper-dependent nitritereductases (NirK) in ammonia-oxidizing archaea ofsoils and other environments using metagenomicapproaches and PCR amplification. Two pairs ofhighly overlapping 40 kb genome fragments, eachcontaining nirK genes of archaea, were isolated froma metagenomic soil library. Between 68% and 85% ofthe open reading frames on these genome fragmentshad homologues in the genomes of the marinearchaeal ammonia oxidizers Nitrosopumilus mariti-mus and Cenarchaeum symbiosum. Extensions ofNirK homologues with C-terminal fused amicyanindomains were deduced from two of the four fosmidsindicating structural variation of these multicopperproteins in archaea. Phylogenetic analyses includingall major groups of currently known NirK homologuesrevealed that the deduced protein sequences ofmarine and soil archaea were separated into twohighly divergent lineages that did not contain bacte-
rial homologues. In contrast, another separatedlineage contained potential multicopper oxidases ofboth domains, archaea and bacteria. More nirK genevariants directly amplified by PCR from several envi-ronments indicated further diversity of the gene and awidespread occurrence in archaea. Transcription ofthe potential archaeal nirK in soil was demonstratedat different water contents, but no significant increasein transcript copy number was observed withincreased 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 wasthought to be restricted to specific microbial groups withinthe gamma and betaproteobacteria (Purkhold et al., 2000;Kowalchuk and Stephen, 2001). However, there is nowincreasing evidence that archaea make an important con-tribution to this process. Specific lineages of archaea frommarine and soil environments possess the genes forammonia monooxygenase (AMO), the key enzyme forammonia oxidation (Schleper et al., 2005; Treusch et al.,2005; Hallam et al., 2006a) and a few archaeal strainscapable of autotrophic growth by ammonia oxidation haverecently been isolated (Könneke et al., 2005) or obtainedin enrichment cultures (Hatzenpichler et al., 2008; de laTorre et al., 2008). Like their bacterial counterparts theseorganisms appear to be chemolithoautotrophs, whoproduce their vital energy through the oxidation ofammonia to nitrite (Könneke et al., 2005). amoA genes ofarchaea encoding subunit A of ammonia monooxygenasehave now been isolated from many environments rangingfrom pristine and agricultural soils, marine plankton andsediments, 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). Inaddition, quantitative molecular studies indicate thatarchaea with the potential to oxidize ammonia occur inhigh 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. *Forcorrespondence. E-mail [email protected]; 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
The first evidence of ammonia monooxygenase homo-logues associated with archaea was found through thegenomic DNA fragment 54d9 retrieved from a sandyecosystem in Darmstadt, Germany. Interestingly, this frag-ment also encoded a gene for a homologue of a copper-dependent nitrite reductase (NirK) (Treusch et al., 2005).NirK proteins are a key component of denitrifying metabo-lisms that reduce nitrite to nitric oxide (NO). Unlike nitrifi-cation, the metabolic capacity of denitrification (reductionof NO2
- to NOx compounds and N2) is widely distributedamong many different lineages of heterotrophic, oftenfacultative anaerobic bacteria and also in some archaea(Zumft, 1997; Cabello et al., 2004). In addition to thecopper-containing enzyme NirK, which contains at leastone type 1 and one type 2 copper centre, a secondnon-homologous haem-cytochrome cd1 type nitritereductase (NirS) is found in several denitrifying bacteriaand in a few hyperthermophilic archaea (Philippot, 2002).This protein, however, is less broadly distributed thanNirK.
NirK is not only associated with heterotrophic bacteria,but also with nitrifying autotrophic bacteria. It was firstdescribed in Nitrosomonas europaea (Ritchie and Nicho-las, 1974) but has now also been found in most otherammonia-oxidizing bacteria studied to date (Cantera andStein, 2007a). In these organisms, NirK seems to beinvolved in denitrification activity. This process of ‘nitrifierdenitrification’ is thought to produce substantial quantitiesof the greenhouse gas nitrous oxide (N2O), especially atthe interface between aerobic and anaerobic habitats(Goreau et al., 1980; Lipschultz et al., 1981; Yoshidaet al., 1989). The NO2 reduction by autotrophic nitrifyingorganisms has even been assumed to contribute as muchor even more N2O to the atmosphere than denitrificationby heterotrophs in terrestrial and marine ecosystems(Webster and Hopkins, 1996; Dore et al., 1998).
Although the role of NirK has not been elucidated for allammonia-oxidizing bacteria, the work of Beaumont andcolleagues (2002), Schmidt and colleagues (2004),Schmidt (2009) and Cantera and Stein (2007b) togethershows that NirK in N. europaea is required for nitritereduction from oxic to anoxic conditions and furthermore,the protein supports growth of the organism by supportingthe oxidation of ammonia to nitrite (Cantera and Stein,2007b).
The deduced archaeal NirK protein sequence encodedon the soil fosmid 54d9 was only about 30% similar toknown bacterial nitrite reductases (Treusch et al., 2005).An alignment with known NirK proteins revealed that allresidues known to be involved in copper coordination(type 1 and type 2) (Kukimoto et al., 1994) were con-served in 54d9 NirK (Treusch et al., 2005). Furthermore, itencoded a potential transmembrane domain at itsN-terminus probably constituting a signal peptide. Unlike
its homologues in bacteria and other archaea, the NirK of54d9 contained an extension of 180 amino acids at theC-terminus which encoded an amicyanin with the con-served residues for copper type 1 coordination and a motifcharacteristic for these blue copper proteins (Canterset al., 2000). In analogy to the role of blue copper proteinsin electron transfer, e.g. in methylotrophic bacteria(usually coupled with methylamine dehydrogenase)(Tobari and Harada, 1981), it is probably involved in anintramolecular electron transfer to the catalytic domain(Treusch et al., 2005).
In this article we describe novel variants of potentialarchaeal nirK genes, isolated from a large insert metage-nomic library and from other environments. We presentthe genomic context of three novel archaeal nirK homo-logues from soil, a phylogenetic analysis of multicopperproteins from marine and terrestrial environmentstogether with their bacterial counterparts and initial tran-scriptional characterizations. Together our data indicatethat these archaeal proteins are widespread and arepotentially as important for ammonia-oxidizing archaea asthey are for their bacterial counterparts.
Results
Amplification and isolation of archaeal nirK homologuesfrom metagenomic libraries and environmental samples
Several sets of primers for the amplification of nirK-relatedgenes from archaea were designed based on an align-ment of deduced amino acid sequences of various NirKsfrom bacteria and two known (at that time) archaealhomologues; one from the halophilic archaeon Haloarculamarismortui and the second from soil fosmid 54d9 whichis affiliated to Group 1.1b (Treusch et al., 2005). Fourprimers (AnirK2F, 3F, 4R and 5R) (Table 1) were designedsuch that a bacterial consensus would be avoided inconserved regions as much as possible and enable pref-erential amplification of archaeal genes. Three additionalprimers (sAnirK1F, 2R and 3R) were then designed basedon nirK sequences retrieved from the first screeninground. A total of 17 different environmental samplesincluding various soils, freshwater and associated sedi-ments, compost, chicken manure and invertebrates werescreened for the presence of nirK genes (Table S1). Allsamples had tested positive earlier for the presence ofcrenarchaeal 16S rRNA genes after PCR amplificationwith primers 20F and 958R (Ochsenreiter et al., 2003)(data not shown). Of the 17 samples, six gave putativenirK PCR products of the expected size [Rotböll meadowsoil (ROB); asparagus agricultural field (SPA); chickenmanure (CM); Darmbach stream biofilm (DVII) and GrubePrinz von Hessen lake sediment/water (GPvHS/W)].These amplicons were then used for constructing clone
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libraries. Inserts that exhibited different restriction pat-terns were sequenced. For all six libraries putative nirKgene sequences were retrieved. However, no individualprimer set retrieved exclusively nirK sequences. Of 89sequenced inserts, 52 (58%) were identified as nirKhomologues as determined from sequence alignmentsand phylogenetic analysis (see below) with the rest pre-sumably being the product of unspecific amplification. Inaddition, different primer pairs varied in their success inamplifying nirK gene sequences from individual DNAextracts. For example, nirK-related gene sequences fromchicken manure were only obtained with AnirK3F–AnirK4R, which did not work for amplifications fromRotböll soil. A new specific primer set (sAnirK1F–sAnirK3R) needed to be designed for the screening ofrecombinant DNA libraries.
To obtain additional metagenomic fragments, twofosmid libraries from soil were screened for the presenceof archaeal nirK genes. Libraries RUD003 (Treuschet al., 2005) and RUD001 (Quaiser et al., 2002) con-tained 30 366 and 25 344 fosmid clones, respectively,with an average insert length of approximately 40 kb. Inearlier screenings, one fosmid was found in each librarythat contained a 16S rRNA gene of archaea, indicatinglow coverage of archaeal genomes in both libraries. Thelibraries were screened with primers sAnirK1F andsAnirK3R (Table 1) and three additional fosmidscontaining a nirK homologue (29d5, 57a5 and 76h13)were identified from library RUD003 (Treusch et al.,2005) from which fosmid 54d9 had previously beenobtained. The inserts of the three fosmids (c. 40 kb)were fully sequenced and analysis revealed that most ofthe genes exhibited either highest similarity to knownarchaeal homologues or were even exclusively found inthe archaeal domain (see below). From this, it was con-cluded that all three fosmids stemmed from archaea and
not bacteria. The deduced protein sequences of all threenirK genes exhibited the conserved amino acid residuesfor coordination of a type 1 and type 2 copper centrerespectively (Godden et al., 1991; Kukimoto et al., 1994;Dodd et al., 1997) (see Fig. 1). However, only one of thethree new fosmid sequences contained the fused ami-cyanin observed with the NirK of 54d9.
Phylogenetic analysis of NirK proteins from bacteriaand archaea
Phylogenetic analysis of the NirK protein sequencesdeduced from the four soil fosmid clones was performedtogether with reference multicopper protein sequencesfrom bacteria and archaea using three different treereconstruction methods (Fig. 2). The four deducedarchaeal soil protein sequences formed a monophyleticgroup distinct from all other multicopper proteins (includ-ing those from other archaea) with 100% bootstrapsupport. Overall, all sequences could be placed into oneof two major groupings. The first group contained the tworecognized lineages of bacterial NirK sequences plus alineage associated with archaea within the Halobacteri-ales. The second major group included a lineage with thefour soil NirK-like sequences, a lineage with homologoussequences from the genomes of the ammonia-oxidizingarchaea Nitrosopumilus maritimus and Cenarchaeumsymbiosum, plus additional sequences from bacteria(including ammonia and nitrite-oxidizing bacteria) andfrom environmental surveys.
The intra-cluster identity between the four soil NirKsequences ranged between 74% and 94% at the aminoacid level (Table 2). Interestingly, this is comparable tothe high level of identity found between two NirK proteinsof the marine ammonia-oxidizing archaeon N. maritimus(Nmar_1259 and Nmar_1667, 91% at the amino acid
Table 1. Primers used to amplify gene fragments from archaeal nitrite reductases.
Primera Sequence 5′→3′ bp Positionb
AnirK2F GAACCWGGWCARTSWAARAC 20 370–389AnirK3F CCKGGYGYKTTTWTKTATCATTGT 24 412–435AnirK4R KGMKTCKWSGTTKGGKCC 18 781–798AnirK5R ACKYYKGAKAKKGCGTGRTC 20 964–983sAnirK1F GGTGCKGACGGWCTKAAYGG 20 436–455sAnirK2R ACRTATTTGTGRGMCATWCCGTT 23 658–680sAnirK3R ATRTACCAWCKWGTCAATTCACC 23 748–77020961Fc AACGAAGCTGTGTCATGCTCAC 22 Upstream of nirK23567Rc CGGATAATGAGGGACCTAAATG 22 Downstream of nirKqAnirK1F TTCAAGGTAAAACCAGGTG 19 733–751qAnirK1R CTGGAGGWATCCACCATGTTTC 22 886–907
a. Primer name prefixes: A for archaea, s for specific and q for quantitative PCR. Forward and reverse primers are indicated by the last letters Fand R respectively.b. Positions in the nirK gene of Fosmid 54d9.c. Primers homologous to positions outside nirK, used for generating standard template for quantitative PCR. Numbering representing positionson fosmid 54d9.
Nitrite reductase of soil archaea 3
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
level). However, these two sequence groups are rela-tively divergent, with the NirK sequences of N. maritimusshowing much greater similarity (� 66%) to sequencesobtained from the metagenomic study of the SargassoSea compared to those from soil (Venter et al., 2004).Hallam and colleagues (2006a) reported the presence ofa NirK in the genome sequence of C. symbiosum, thesymbiont of a marine sponge, which is related to N. mar-
itimus within the 16S rRNA-defined lineage Group 1.1a.This protein showed high similarity with proteins fromseveral bacteria as well as two additional proteins fromN. maritimus (Nmar_1131 and Nmar_1663). However,analysis of conserved metal-binding residues indicatesthat this particular group of proteins do not contain themethionine residue normally found in the copper type 1centre of nitrite reductases, but possess a leucine typical
10 20 30 40 50 70 80 90 100 110 |....|....|....|....|....|....|....|....| |....|....|....|....|....|....|....|....|. 29d5 NGTIPGPVISIDQGDTLNITLKNEG--QTIHSLDFHAGFGP KTWSLTGEFPGVFMYHCGADGLNGVWEHISNGMYGGIVVHPT 54d9 NGTIPGPVISIDQGDTLNITLKNEG--QTIHSLDFHAGFGP KTWSLTGEFPGVFLYHCGADGLNGVWEHISNGMYGGIVVHPT 57a5 NGTIPGPVISIDQGDTLNITLKNEG--KTIHSLDFHAGYGP KTWSLTGEFPGVFIYHCGADGLNGIWEHISNGMYGGIVVHPK 76h13 NGSIPGPAISIDQGDTLNITLKNEG--QTIHSLDFHAGYGP KTWSLKADYPGAFLYHCGADGLNGVWEHISNGMYGGIIVHPA N.europ DGKVPGPVVRVTEGDTVEFTLINDKNSKNSHSMDFHAARLD KKYTFTADNPGVFFYHCGSDP---MIQHIARGMYGVIIVDPK R.sphae DGSIPGPLMIVHEGDYVELTLINPPENTMPHNIDFHAATGA VVLRFKATRAGAFVYHCAPGGP-MIPWHVVSGMAGCIMVLPR N.multi NNRVPGPLLRVKVGDTITINLHNSASSTNIHSIDFHAVTGP KSFTFKSLHPGLFVYHCATPM---VAHHIANGMYGMVLVEPE A.cyclo NGSVPGPLMVVHENDYVELRLINPDTNTLLHNIDFHAATGA TTLRFKATKPGVFVYHCAPEG--MVPWHVTSGMNGAIMVLPR A.faeca NGTVPGPLMVVHQDDYLELTLINPETNTLMHNIDFHAATGA TILRFKATKPGVFVYHCAPPG--MVPWHVVSGMNGAIMVLPR Nmar_1259 SEQVPGPTLRVTQGDVVKMTLTIPDDEVTGHGNDMHASQMS SQYCYIAESAGMFKYHCSGVKLIGMDQHVLSGMYGITIVDPA Nmar_1667 NQQVPGPTLRVTQGDVVKMTLTIPDDEVTGHGNDMHASQIS AQYCYIAEVAGTFKYHCSGVKLIGMDQHVLSGMYGIAIVDPA Nmar_1131 NGTVPGPTIRATEGDVVRIKFINNG--EKEHTMHFHGIHPA FVYEFEAGPVGVHPYHCHVMP---LEEHIVHGLYGVFIVDPK Nmar_1663 NGTVPGPTIRATEGDLLRINFINNG--DKPHTMHFHGEHPA FTYEFEAGPVGVHPYHCHVMP---LEEHIAHGLYGVFIVDPK CENSYa_1582 NGTVPGPTIRATQGDLVRIHFINNG--EKEHTMHFHGIHRP FTYEWRAEPVGVHPYHCHVMP---LEEHIVHGLYGVYIVDPK Frankia sp. NGTVPGPVIRATEGDLLRVRFTNGS--MHPHTIHFHGIHPA FVYNFPATPYGMHMYHCHATP---LKKHIAKGLYGALIIDPP R.xylan NGQVPGPTLRAREGDRLRVAFKNGD--THPHTIHFHGFHPA FVYEFEAGPFGCHLYHCHTMP---LKKHIEKGLYGAFIVDPR N.farci NARVPGPTLRCREGELLRVHFRNGS--EHPHTLHFHGIHPA TTYEFDATPFGLHLFHCHVGP---LAEHIARGMYGTFIVDPP R.eryth NSRIPGPALRCSEGDRLRVKFVNGS--AHPHTMHFHGIHPA FTYEFDATPFGLHLYHCHVSP---LAEHIARGMYGTFIVDPA 260 270 280 290 300 310 320 330 340 |....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|. 29d5 GPNDGVSFHFISGILSVKDGSNTANNGFGTQDIND-ETWWIPPGSGSVIESTFPEGGVYVGVDHAM-ADVVKGGAFAVLAVE 54d9 GPNDGVSFHFISGMLSVRDGSNTANNGFGTQDIND-ETWWIPPGSGSVIESTFPQEGVYVGVDHAM-SDVVKGGAFAVLAVE 57a5 GPNDGVSFHFIGGMLSVRDGTNQANNGLGTQDIND-ETWWIPPAAGSVIESTFPEAGLYVGVDHAM-ADVVKGAAFAVVAME 76h13 GPNDGVSFHFISGILSVKDGSNAANNGYGTQDMND-ETWWIPPGSGSVIESTFPQPGVYVGVDHAM-SDVVKGGAFAVVAME N.europ GPNELSSLHPIAGIW---DRVYPSGNPKNVQYAL--QSYLIGAGDAATLDLISPVEGANAIVDHSM-RHAHSGAIAVIMFTN R.sphae QPNRDSRPHLIGGHG---DLVWETGKFHNAPE-RDLETWFIRGGSAGAALYKFLQPGVYAYVNHNLIEAVHKGATAHVLVEG N.multi GPNLTSSFHIIGEVF---DKVYDQASLTSPPL-TDVQTTLVPPGGATIVEFKVDYPGRYILVDHAL-SRMEKGLAGYLTVRG A.cyclo QANRDTRPHLIGGHG---DYVWATGKFRNPPD-LDQETWLIPGGTAGAAFYTFRQPGVYAYVNHNLIEAFELGAAGHFKVTG A.faeca QANRDTRPHLIGGHG---DYVWATGKFNTPPD-VDQETWFIPGGAAGAAFYTFQQPGIYAYVNHNLIEAFELGAAAHFKVTG Nmar_1259 QGNEPVFFHIVGEIL---DRVTQ-GN-RVQSAAT--ETWLLGGSQGMIVDLVFDEPGAYAAVNHDY-AAIYTGAATVFVAGD Nmar_1667 NGNEPVFFHIVGEIL---DRVVQ-GN-RVQSAGT--ETWNIGGSQGAIIDLVFDEPGVYAAVNHDY-AAIYTGAASVFVAGD Nmar_1131 EFDPINNLHLHGNLY---KYY-PTGTDIVPSFYTD--MITLSQTERGIMEFEYEYPGKYLFHAHKV-EFSEKGWVGIFMVNE Nmar_1663 EFDPINNLHLHGNLY---QYY-PTGTDIVPSEFTD--MITLSQTERGIMEFKYQYPGKYLFHAHKV-EFSEKGWVGVFLVKD CENSYa_1582 EFDPINNFHLHGNLF---HYY-PTGTDTVPSFYTD--MITLSQTERGIMEFSYDFPGKYLFHAHKV-EFSEKGWSGIFLAEG Frankia sp. EFDLINSFHLHGEFF---RYY-PTGSGDQY-EYTD--TVMLCQGQRGIIEIEFHYPGRFMFHAHQS-EFAELGWMGFFEVTN R.xylan EFDLVNSFHIHANFF---DYY-PTGTRLEPSEFTD--TKMFAQGERGIMEFRYRFPGRFMFHAHVS-EFAELGWMGLFEVEG N.farci EYDPINSFHVHGNFF---DYY-PTGTRLQSSEFTD--TIVQGQGQRGICEMRFPHPGKYMFHAHKT-EFAELGWMGFFEVSD R.eryth EYDPINSFHIHGNFF---DYY-PTGTRLEPSEFTD--TIVQAQGQRGICELRFPHPGKYMFHAHKT-EFADLGWMGFFEVTE Copper centre type 1 Copper centre type 2 Putative trinuclear type 2/type 3 copper centre
Nir
K
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Nir
K
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Fig. 1. Alignment of nitrite reductases (NirK) and multicopper oxidases (MCO) from a selection of archaea and bacteria with copper centretype 1, 2 and 3 coordinating amino acid residues. Dark grey background shading: identical residues in all compared sequences; light greybackground shading: similar residues in all compared sequences. Copper coordinating residues are highlighted in bold with solid grey, solidblack and striped grey arrows indicating copper centre type 1, copper centre type 2 and putative trinuclear type 2/type 3 copper centre,respectively. Nitrite reductases are from soil fosmids 54d9, 57a5, 29d5, 76h13, Nitrosomonas europaea (N. europ), Rhodobacter sphaeroides(R. sphae), Nitrosospira multiformis (N. multi), Achromobacter cycloclastes (A. cyclo), Alcaligenes faecalis (A. faeca) and Nitrosopumilusmaritimus (Nmar_1259 and Nmar_1667). Multicopper oxidases are from N. maritimus (Nmar_1131 and Nmar_1663), Cenarchaeumsymbiosum (CENSYa_1582), a Frankia species, Rubrobacter xylanophilus (R. xylan), Nocardia farcinica (N. farci) and Rhodococcuserythropolis (R. eryth).
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of type 1 copper centre of multicopper oxidases such asfungal laccases (Fig. 1, position 102) (Sakurai andKataoka, 2007). In addition, all four histidine residuesrequired for coordinating a trinuclear type 2/type 3copper centre typical of multicopper oxidases arepresent (Fig. 1, positions 43, 87, 270, 321) (MacPherson
and Murphy, 2007). These findings strongly suggest thatproteins of this group are multicopper oxidases and notnitrite reductases.
Phylogenetic analyses were also performed on derivedNirK sequences from cloned PCR products from six dif-ferent clone libraries retrieved with five different primer
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11
A
Nit
rob
acte
rw
ino
gra
dskyi
Nitrosom
onas e
uropaea
ATCC 1
9718
Nitr
osom
onas e
uropae
a ATC
C 2
5978
Nitrosomonas eutro
pha C91
0.20
Nitrosopumilus maritimus 1667
Nitrosopumilus maritimus 1259
Rubro
bacte
rxyla
nophilu
s
Cenarchaeum symbiosum A
Halophiles
Soil fosmids
Nitrosopumilus& marine metagenome
Ha
lofe
rax
lu
ce
nte
ns
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Halo
arc
ula
mari
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ui
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fera
xd
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itri
fican
s
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100 100
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100
75
Nitro
sopum
ilus m
aritim
us 1
131
Nitrosopum
ilus maritim
us 1663
Fig. 2. Phylogenetic analysis of archaeal and bacterial derived NirK and multicopper oxidase protein sequences. Sequences from Archaea(putative ammonia oxidizers plus halophiles) are highlighted in blue and sequences from nitrifying bacteria are highlighted in green. Thedistance tree was constructed using the minimum evolution criterion with JTT correction with 157 unambiguously aligned positions. Boot-strap values represent the most conservative value from three treeing methods (distance, parsimony and maximum likelihood with 1000,1000 and 100 replicates respectively). Circles represent multifurcating branches of major clades as the relative branching order could notbe supported in the majority of bootstrap replicates using any method. Grey branches represent aligned sequences (nine in total) whichwere shorter than the 157 derived amino acid positions used in the alignment, and deletion of these missing sites was performed in pair-wise comparisons.
Nitrite reductase of soil archaea 5
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
pairs (Fig. 3). With the exception of sequences fromchicken dung DNA, sequences from all habitats wereclosely related to the four soil NirK sequences (� 87%and 84% identity at nucleotide and amino acid levelrespectively). The six sequences obtained from chickendung were placed within two other highly divergent lin-eages and were obtained only with primer set AnirK3F–AnirK4R, reflecting a substantial difference in specificitycompared with the other primer combinations used. Fivesequences were associated with Geobacillus-like NirKand one clone insert exhibited greatest similarity withsequences associated with Halobacteriales. Due to theirhigh divergence from the soil NirK sequences recovered,it was not possible to determine whether these latter pro-teins (Fig. 3, b and c) are associated with bacteria orarchaea.
Genomic context of nirK-like genes from a metagenomicsoil library
Of the four nirK-containing fosmids, two pairs werehighly similar and syntenic to each other (Fig. 4). Fosmid54d9 and 57a5 overlapped in 22 genes whose orienta-tion and synteny was conserved, except that 54d9 con-tained a small insertion of three additional genes.Fosmids 29d5 and 76h13 showed even greater similaritywith an overlap of 29 genes and 29d5 containing oneregion with an additional 10 genes. In line with thisfinding, the nirK genes of the two fosmid pairs showed asignificant difference with 29d5 and 76h13 possessing aputative nitrite reductase of 452 amino acids in bothcases, and the predicted NirK of 57a5 containing anextension of 98 amino acids that showed high similarity
Table 2. Pairwise sequence identity comparison of the four nirK genes from soil (nucleic acid/amino acid).
Nucleic/amino acids (%)a 54d9 57a5 29d5 57a5+AMI
54d9 – – – –57a5 78.2/73.6 – – –29d5 94.5/93.4 79.6/75.8 – –76h13 86.0/83.3 79.4/75.1 89.3/86.3 –54d9+AMI – – – 78.3/74.4
a. Comparisons were made using the EMBOSS comparison tool.AMI, amicyanin domain.
Fig. 3. Phylogenetic analysis of partialNirK-like sequences retrieved from differentenvironmental samples (ROB, Rotböllmeadow soil; SPA, asparagus agriculturalfield; CM, chicken manure; DVII, Darmbachstream biofilm; GPvHS/W, Grube Prinz vonHessen lake sediment/water). Numbers afterthe name of the environmental sourcedescribe which of five primer sets was usedfor generating that cloned sequence [1:AnirK2F-AnirK4R; 2: AnirK3F-AnirK4R; 3:AnirK2F-AnirK5R; 4: AnirK3F-AnirK5R; 5:sAnirK1F-sAnirK3R (Table 1)]. Retrievedsequences were placed within one of threedivergent groups and showed highestsimilarity to (a) soil fosmid, (b) Geobacillus sp.or (c) Halobacteriales NirK sequences. Due tothe relatively short sequences obtained andthe relatively large divergence between thethree group of sequences, phylogeneticanalyses were performed separately tomaximize the amount of unambiguouslyaligned amino acid positions included (97, 111and 126 positions for trees a, b and crespectively). The scale bar represents 0.05changes per amino acid position for all trees.
Haloarcula marismortuiHaloferax lucentense
Haloferax denitrificans
CM2_24
Geobacillus denitrificansGeobacillus klaustophilus
CM2_21CM2_11 & 1 other
CM2_22CM2_18
54d9GPvHS5_45
GPvHW5_48GPvHS5_41
29d5
SPA5_23
SPA3_01 & 1 otherSPA5_21
57a5
ROB1_08 & 14 others
SPA5_24
GPvHS5_42DVII5_28
GPvHW5_46
SPA5_25SPA3_02DVII5_26
ROB3_03 & 8 othersROBe3_22 & 2 others
ROB3_14 & 3 others
SPA5_2276h13
ROB4_23
0.05
a) Soil fosmid-like
b) Geobacillus sp.-like
c) Halobacteriales-like
6 R. Bartossek et al.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
to an amicyanin, as was found in 54d9 (Treusch et al.,2005). Unexpectedly however, the greatest similarityobserved between any two of the nirK genes (excludingthe amicyanin domain) was between those of 54d9 and29d5 (94.5%, on nucleic acid level, Table 2). The nitritereductase gene in 54d9/57a5 was surrounded by a puta-tive adenylosuccinate lyase gene and a chloride channelprotein gene, whereas it was flanked by uncharacterizedconserved protein genes in 29d5/76h13. The overallG+C content of the four fosmids varied between 33.7%(57a5) and 36.4% (54d9) (Table 3) and was similar tothat found earlier for archaeal fosmids from environmen-
tal soil and marine samples (Quaiser et al., 2002;Treusch et al., 2004; Martin-Cuadrado et al., 2007). TheG+C content of the four nirK genes was slightly higherthan average, i.e. between 41.2% and 42.6%, but still inthe range found for other genes on the genome frag-ments.
The four fosmids shared many genes with the genomesof N. maritimus and C. symbiosum. Between 68% (fosmid57a5) and 85% (fosmid 76h13) of the predicted ORFs hadhomologues in the two genomes and the vast majority ofthe predicted proteins exhibited highest similarity to theircounterparts in the two marine organisms in BLASTP
57a5
2 3 4 5 6 7 8 11 12 15 21 22 23 24 25 27 30 36 40 41 43
2 4 6 7 8 10 11 13 16 21 22 24 25 27 30 34 35 36 37 38 39 40
29d5
76h13
54d9
2 3 4 6 7 8 11 12 15 17 20 24 25 36 39 40 42 43 44
CENSYa
Nmar
nirK 16S 23S
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amoC
amoA
amoB
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Nmar
1 2 5 6 9 11 14 18 19 22 25 26 28 29 30 33 34 38 39 40 41
03
58
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06
79
06
80
Thaumarchaeota
Archaea
Bacteria
no homologues
only in RUD003
only in Thaumarchaeota
nirK
4323164
22 33 37
Fig. 4. Schematic representation of genome fragment 54d9 (Treusch et al., 2005) and the three fosmid inserts containing NirK discussed inthis study. Homologous genes are connected by blue shadings. Colour code refers to the group of organisms to which a respective gene ismost similar as determined by BLAST searches. Regions of synteny to the genomes of Cenarchaeum symbiosum (CENSYa) andNitrosopumilus maritimus (Nmar) are indicated above and below. For annotation of genes see text and Table S2.
Table 3. General characteristics of the four archaeal genome fragments.
Fosmid 54d9 57a5 29d5 76h13
Length (bp) 43 377 35 462 37 794 36 567Length nirK (bp) 1 614 1 653 1 359 1 359Length NirK (aa) 537 550 452 452Fused amicyanin domain Yes Yes No NoG+C content (%) 36.5 33.7 35.5 35.9Coding density (%) 83 80 84 88Number of ORFs 43 41 45 44Hypothetical proteins 06 06 10 07Uncharacterized conserved proteins 19 15 13 14Proteins with assigned function 18 20 22 23Proteins with most similar orthologue in ammonia-oxidizing archaea 34 24 27 33Proteins with orthologue only in ammonia-oxidizing archaea 12 9 9 11
Nitrite reductase of soil archaea 7
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
searches (red and orange shading in Fig. 4). Further-more, syntenic gene clusters were found between the soilfosmids and the marine genomes, most extensivelybetween fosmids 29d5/76h13 and N. maritimus (Fig. 4,lower part). The syntenic regions were mainly comprisedof genes involved in amino acid or nucleotide biosynthesisand translation, as well as the AmoAB cluster.
No gene synteny was found between the two fosmidpairs, whereas a remarkable level of conservation wasobserved between 54d9 and 57a5 as well as 29d5 and76h13. In both cases perfect synteny is maintained exceptfor specific insertions of a few genes (8–10 in 54d9, 26–35in 29d5, 20 and 21 in 76h13). With one exception (anhsp20 homologue) these insertions encode hypotheticalprotein genes, perhaps indicating that they stem frommobile genetic elements or viruses. A total of 23 proteinencoding genes had exclusive similarity to genes ofammonia-oxidizing archaea (including the metagenomicclones), but were not found in any other archaea(Table S2). None of these proteins (except topoisomeraseIB, see below) could be assigned a specific function.Potentially, some of these proteins might be involved inspecific energy metabolism of ammonia-oxidizingarchaea. Additionally, a number of genes with assignablefunction that might be directly or indirectly involved inspecific energy metabolisms have homologues in otherarchaeal and/or bacterial genomes. Those include genesfor ferredoxins, Fe-S cluster formation, ubiquionone/menaquinone biosynthesis, nitroreductase, sulfite/nitritereductase, cation transporters and diverse oxidoreduc-tases (Table S2). Other genes on the fosmids are involvedin protein synthesis, transcription and regulation, proteinsecretion, DNA replication and cofactor biosynthesis.
Interestingly, fosmids 29d5/76h13 contained a gene for atopoisomerase IB, homologues of which have onlyrecently been recognized in archaea (Brochier-Armanetet al., 2008a). While topoisomerase IA is found wide-spread in archaea, topoisomerase IB seems to berestricted to the proposed phylum Thaumarchaeota(including ammonia-oxidizing archaea) and might havebeen present in the last common ancestor of eukaryotesand archaea (Brochier-Armanet et al., 2008a,b).
Highly conserved promoter regions upstream ofnirK genes
Comparison of the upstream region of all four nirK ORFsrevealed a region of c. 90 nucleotides upstream of thetranslational start codon that was almost identical in thefour fosmids (Fig. 5), whereas other intergenic regionswere less conserved. Around position -27 (with respect tothe translation start), a sequence that matched the con-sensus of archaeal promoters (the so-called TATA-box)and an adjacent BR element could be dissected. Thesemotifs were also found in the same distance upstream ofthe ORF in the more distantly related N. maritimus, whichindicates that it represents the basic promoter of nirK. In asignificant fraction of genes in archaea, transcriptionaland translational start are found on the same nucleotideor codon, as would be predicted for nirK in this case, dueto the location of the TATA box (Torarinsson et al., 2005).The highly conserved region upstream of the nirK geneson the soil fosmids is unexpected and perhaps indicates aspecific and conserved mechanism of regulation. Thepresence of translational stop codons upstream of thepredicted nirK start rules out the possibility that this high
10 20 30 40 50 60 70 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| 54d9 TTAGTTTTTGATAAAAAAAT-TCTAATTAATATAGAGCTAGTGTATAATTTCAACAAAATTCTAA-TCTA 57a5 TTACATTTTGGTATAAAAAT-TCCAATTAATATTTAGCTAGCATATAGTTTCAGCAAATTTATGA-GCTA 76h13 TCTTGATTAGATTAAAAAATATATATATTCTTTAAAATAATTGTATAATTTCAACAAAATTATAA-TCTA 29d5 TCCTGATTAGGTTAAAAAATATGCATATTTTTTAAAATTAATGTATAATTTCAACAAAATTCTAAATCTA Nmar_1667 CAAAAAACTTCTATTTTATACAAGTCCTATAAACGATCAATGAAAAAAATCTGGTTTTATTTTTAAAAAT Nmar_1259 TCTTGATCTAATCTAAATTTCATACAAAATAGCCTCAACTACCCATTATTCATTCTTTTTCAGCATCATA 80 90 100 110 120 130 140 ....|....|....|....|....|....|....|....|....|....|....|....|....|....| 54d9 AATTAAAATATTTTATTCTTATCTCAGCATAATATTTATAGTGGAAACCTTTACAATCCGCTAATGAATC 57a5 AATTAAAATATTTTATTCTTATCTTAGAATAATATTTATAGTGGAATACTATACAATGCGATAATGAATC 76h13 AATTAAAATATTTTATTCTTATCTCAGAATAATATTTATAGTGGAAAACTTTACAATCCGCTAATGAATC 29d5 AATTAAAATATTTTATTCTTATCTCAGCATAATATTTATAGTGGAAACCTTTACAATCCGCTAATGAATC Nmar_1667 GTGGTGAAAATGCACTCTAGGCAAGGGGAATTATTATATAGGA-TTCGCAGTCACGGTGCTTAATGAATA Nmar_1259 TCACATTTTCATAATTCCCAAATTGGGAAATTGTTATATAGGAACTGGGTACCATCGTGCATAATGAATA BRE TRATS xoB-ATAT
Fig. 5. Alignment of start and upstream regions of nirK sequences from four soil fosmids and two from the genome of N. maritimus. The ATGstart codon and TATA box/BR element are highlighted (Bell et al., 1999). Identical positions conserved in 66% of the sequences are shadedgrey.
8 R. Bartossek et al.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
conservation is due to an encoded protein or anN-terminal extension of NirK (not shown). Analysis ofupstream regions of other genes potentially involved innitrogen transformations (e.g. amoA, amoB, homologuesto orf 38 on fosmid 54d9) does not exhibit similarupstream conservation either within individual genomesor between different organisms.
Amicyanin domains are fused to various proteins inammonia-oxidizing archaea
A comparison of the deduced amino acid sequences ofthe potential amicyanins encoded by fosmids 54d9 and57a5 with other proteins in the databases revealedhighest similarities to proteins of unknown function inother ammonia-oxidizing archaea and to amicyanins ofmethylotrophs and a few other bacteria. Similarities toother small blue copper proteins (plastocyanin, halocya-nin, azurin, etc.) were much lower. Furthermore, a signa-ture recently described to be specific for amicyanins anddistinguishing those from other blue copper proteins (Giriet al., 2004) was also found in the archaeal homologuesof oceanic and terrestrial origin (Fig. S1). Interestingly,most amicyanins identified in this way formed parts oflarger potential proteins in ammonia-oxidizing archaea.
Archaeal nirK expression under denitrifying conditions
In order to determine whether archaeal nirK expressioncould be detected in soil and whether expression levelswere related to overall denitrifying activity, a series ofmicrocosms was established. Nitrous oxide productionrates were measured in Rotböll soil microcosms as ameasure of overall denitrifying activity at four differentwater concentrations and nirK transcripts were measuredby qPCR from randomly reverse transcribed total RNAusing primers that were specifically designed for nirKgenes of archaea from Rotböll soil (Table 1). Rates ofnitrous oxide production (and denitrification activity)(Table S3) were lowest at 15% water content and highestat 25% water content with 0.3 and 6.1 nmol N2O drysoil g-1 h-1 respectively. Conversely, the highest archaealnirK expression occurred at 15% water content with14.7 ¥ 105 copies dry soil g-1 and the lowest expressionlevel at 25% water content with 5.8 ¥ 105 copies drysoil g-1. While this preliminary experiment clearly demon-strates expression of the archaeal nirK gene in the envi-ronment, transcriptional activity did not seem to becorrelated to denitrification.
Discussion
Molecular studies in microbial ecology often rely on theamplification or quantification of key metabolic genes
from environmental samples. However, metagenomicanalyses have demonstrated in the past few years thatmany if not all protein families cover a much broadersequence space than that usually captured with primersin PCR-based environmental surveys. Some of the mostintriguing examples are the finding of an unexpectedlyhigh diversity of proteorhodopsin genes in the SargassoSea (Venter et al., 2004) and the discovery of novelammonia monooxygenase gene variants in archaea(Treusch et al., 2005). Similarly, we demonstrate herethat the sequence diversity of nitrite reductases andrelated proteins is broader than previously observed. Thenovel gene variants would not have been amplified byprimer sets previously used in PCR-based studies of nirKdiversity (Hallin and Lindgren, 1999; Braker et al., 2000;Prieme et al., 2002; Heylen et al., 2006). Using theexpanded sequence space shown here, it should be pos-sible to design novel primer sets suitable for the ampli-fication of the expanded group of nitrite reductases andmulticopper proteins.
Unlike most bacterial copper-dependent nitrite reduc-tases that contain one type 1 and one type 2 coppercentre per monomer, an additional C-terminal amicyanindomain is encoded in the genes of fosmids 54d9 and57a5. Further extensions of NirK by electron transferprotein domains have also recently been recognized inbacteria. For example, the NirK of the methylotrophicdenitrifying bacterium Hyphomicrobium denitrificans con-tains two type 1 Cu centres and one type 2 Cu centre(Yamaguchi et al., 2004), as predicted for the proteinsencoded on the archaeal fosmids. However, in H. denitri-ficans, the additional cupredoxin domain is fused to theN-terminus of the protein and not to the C-terminus. It isthought that the additional copper centre type 1 of thecupredoxin domain is involved in electron transferbetween the other copper centres in the NirK protein(Nojiri et al., 2007). However, the cupredoxin domain isnot essential for the function of the NirK as its removalincreased the activity of the nitrite reductase (Yamaguchiet al., 2003, 2004). Interestingly this NirK forms ahexamer in solution (Nojiri et al., 2007) and not a trimerlike the canonical CuNIRs. The structural analysis dem-onstrated that the enzyme retains the well-characterizedcoupled copper type 1 and copper type 2 centres, in whichthe catalytic type 2 Cu core is also built with ligands fromneighbouring monomers (Nojiri et al., 2007). Interestingly,another extended NirK has been found in the bacterialammonia oxidizer, Nitrosospira multiformis, which alsocontains a gene with a predicted blue copper protein at itsN-terminus (Norton et al., 2008). More variants of NirKwith additional cupredoxin domains at the N-terminus(and with additional class I cytochrome c domains at theC-terminus) have also recently been predicted by Ellisand colleagues (2007) based on genomic searches. It will
Nitrite reductase of soil archaea 9
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
be most interesting to determine the three-dimensionalstructures of the novel bacterial and archaeal NirK fami-lies in order to understand electron transport in theseenzymes and to shed light on their evolutionary descentfrom a functional perspective.
Our analysis on the occurrence of amicyanins inammonia-oxidizing archaea indicates that fusion of thesesmall copper proteins to other proteins is a commontheme in these organisms, whereas this seems to be arare event in bacteria (Fig. S1).
Similar to earlier studies on NirK diversity, our analysesconfirm that the distribution of nitrite reductases in deni-trifying and nitrifying organisms does not follow their 16SrRNA gene-defined phylogenetic placement (Jones et al.,2008). Cantera and Stein (2007a) reported that the NirK,associated with nitrifying bacteria, can be highly diver-gent, including those associated with organisms withinone genus such as Nitrosomonas. The divergence in NirKassociated with ammonia-oxidizing archaea appears tobe similar, with sequences clearly placed within at leasttwo distinct lineages.
However, it is noteworthy that there is a deep separa-tion between archaeal NirK from soil and marine environ-ments, both of which represent a separate and deeplybranching lineage without known homologues from bac-teria. This may indicate that NirK emerged early in theevolution of ammonia-oxidizing archaea and was notrecently transferred from other organisms. In contrast, afurther lineage that probably encompasses a distinct typeof multicopper oxidases can be dissected, which containshomologues from C. symbiosum, N. maritimus as well asa diverse range of bacteria including Actinobacteria, Pro-teobacteria and Cyanobacteria (Fig. 1). In these multicop-per proteins, the residues Asp98, Ala137, Ile257 andVal304 (numbering according to the protein of Achromo-bacter cycloclastes) are exchanged to histidine residueswithout exception. According to predictions by MacPher-son and Murphy (2007), this indicates that the proteinscontain a trinuclear copper centre and represent multicop-per oxidases rather than nitrite reductases. Interestingly,this would indicate that all ammonia-oxidizing archaeamight contain such a multicopper oxidase, whereas anitrite reductase is present in N. maritimus and marineand soil metagenomes, but not in C. symbiosum. Further-more, different from archaeal NirK, the multicopper oxi-dases might have been transferred horizontally recentlyamong bacteria and archaea as proteins of both domainsform a distinct and separate lineage in the phylogeneticanalysis.
We have not been able to amplify archaeal nirK genesfrom all environments in which potential archaealammonia oxidizers have been detected. However, webelieve that this is due to the primers used in this study.Based on the novel dataset it should be possible to design
novel nirK-specific primers with broader specificity, whichwould allow amplification of further variants. It will beinteresting to see in future studies if ammonia-oxidizingarchaea all contain nitrite reductases, similar to their bac-terial counterparts, in which this enzyme seems to occurbroadly (Cantera and Stein, 2007a).
In our initial studies on nirK transcription in soil micro-cosms, we could clearly demonstrate that the archaealgene is actively expressed in the environment. Relativeexpression was, however, greatest under conditions withlowest nitrous oxide production.
Further biochemical and physiological studies of thisprotein will be needed to elucidate its function. In the lightof the fact that the energy metabolism of ammonia-oxidizing archaea has not been elucidated, it might wellbe that this multicopper protein exhibits other or additionalactivities beside nitrite reduction.
While the genomic content of the characterized soilfragments gives some interesting hints to specific metabo-lisms of ammonia oxidizers, it may also serve to clarify theevolutionary descent of ammonia-oxidizing archaea.Recently, Brochier-Armanet and colleagues (2008b) haveproposed that C. symbiosum and its relatives constitute aseparate phylum within the domain of Archaea based onr-protein phylogenies and genome content. Initial studieson further genome fragments and genomes of ammonia-oxidizing archaea (A. Spang, R. Hatzenpichler, T. Rattei,C. Brochier-Armanet, C. Schleper, M. Wagner et al., inpreparation) and the detection of many ORFs in our soilfosmids that are exclusively found in other ammonia-oxidizing archaea appear to confirm this finding.
Since their discovery in 2005, much progress has beenmade in the characterization of ammonia-oxidizingarchaea, involving extensive studies on their occurrencein diverse ecosystems, metagenomic analyses and somegrowth characterizations of the cultivated isolate, N. mar-itimus. However, the physiology of ammonia-oxidizingarchaea remains largely unknown and future studies onthe biochemistry and genetics of these archaea will becrucial in order to shed light on their metabolic pathwaysand their ecological importance.
Experimental procedures
NirK diversity studies
To examine the diversity of archaeal nirK sequences in envi-ronmental samples, DNA was isolated from 0.5 g of sampleusing the FastDNA SPIN Kit for soil (MP Biomedicals, Heidel-berg, Germany) according to manufacturer’s instructions.Sets of degenerated primers (Table 1) specific for archaealnirK were designed based on an alignment of the two avail-able (at that time) archaeal nirK sequences from fosmid 54d9and H. marismortui, avoiding conserved regions from bacte-rial nirK genes. Primer combinations AnirK2F–AnirK4R,
10 R. Bartossek et al.
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology
AnirK3F–AnirK4R, AnirK2F–AnirK5R and AnirK3F–AnirK5Rwere used to amplify fragments of approximately 429, 387,614 and 572 bp in length respectively.
Successful amplification of nirK genes varied between dif-ferent environmental samples for each primer set thus requir-ing the use of a range of different primer combinations for allsamples. All PCR amplifications were performed in 50 mlreaction volumes using a TGradient thermocycler (Biometra,Goettingen, Germany) with DAP Goldstar DNA polymerase(Eurogentec, Köln, Germany) at 1 unit per reaction, 1¥ Opti-buffer, 2 mM MgCl2, 200 mM of each dNTP, 40 pmol of everyprimer and 10–100 ng of genomic DNA. Gene fragmentswere amplified using a thermocycling protocol of 5 min ofinitial denaturation of DNA at 95°C, followed by 35 cycles of95°C for 60 s, annealing at 48°C for 60 s and elongation at72°C for 50 s. A final elongation step at 72°C for 10 mincompleted the amplification.
Cloning of PCR products and sequencing
PCR products of the expected size were ligated into the pCR4-TOPO vector (Invitrogen, Karlsruhe, Germany) and trans-formed into One Shot TOP10 chemically competent Escheri-chia coli cells (Invitrogen). Transformants were screened forinserts of the correct size by colony PCR. Restriction digestswith EcoRI were used to analyse the diversity of gene frag-ments prior to sequencing. Inserts with unique restrictionpattern were sequenced using Big Dye Chemistry (ABI,Applied Biosystems, Oslo, Norway) with M13 forward andreverse vector primers on an ABI PRISM 377 capillarysequencer (ABI).
Screening for nirK
Two fosmid libraries, Rud003 (Treusch et al., 2005) andRud001 (Quaiser et al., 2002), were screened for the pres-ence of archaeal nirK genes by PCR using primers sAnirK1Fand sAnirK3R (Table 1) in 25 ml of reactions as describedpreviously. DNA-pools of every 384-well plate were firstscreened for identification of nirK fosmids. Pools 29, 57and 76 of Rud003 produced positive amplifications of theexpected size. Cells from these three plates were grown and24 individual pools (representing one column of 16 clones)were produced from each plate by mixing samples of cells in25 ml of H2O. Cells were lysed by heat (99°C for 10 min) andafter subsequent centrifugation 1 ml of the supernatant wasused for PCR. To identify the final coordinate, 1 ml of crudecell extract was used in the PCR, without prior lysis.
Sequence analysis of three archaeal fosmid inserts
To sequence fosmids 29d5, 57a5 and 76h13, subclone librar-ies for shotgun sequencing were prepared from extractedfosmid DNA by mechanical shearing and cloning of 2–3 kbfragments into pGEM-T Easy vector (Promega, Mannheim,Germany). Subclone libraries were sequenced until at leasteight times coverage of the fosmid sequence was achieved.Resulting reads were assembled and finished using thePHRED/PHRAP/CONSED package (Ewing et al., 1998;Gordon et al., 1998).
Sequence annotation
Protein coding sequences were identified using Glimmer v3(Delcher et al., 2007), BLASTX searches of public protein data-bases (GenBank nr and env_nr) and the IMG/M metagenomeanalysis system (Markowitz et al., 2008). Annotations weremanually curated based on BLASTP alignments to the samedatabases and amino acid correlation scores using Artemis.Only hits with an E-value less than 10-5 were consideredsignificant. tRNA genes were identified using tRNAScan-SEv1.23 (Lowe and Eddy, 1997). COGNITOR was used fordetermining COG assignment and functional categories(Tatusov et al., 2001). Putative protein family assignments,domains, trans-membrane regions and signal peptideswere predicted using the InterProScan Web Service at theEuropean Bioinformatics Centre (Quevillon et al., 2005)including all available applications (BlastProDom, FPrint-Scan, HMMPIR, HMMPfam, HMMSmart, HMMTigr,HMMPanther, PatternScan, ProfileScan, ScanRegExp,Gene3D, SuperFamily, SignalP and TMHMM).
Phylogenetic analysis of the archaeal nirK genes
Derived amino acid sequences from the four soil fosmidswere aligned with reference bacterial and archaeal NirKsequences plus multicopper proteins from N. maritimus andC. symbiosum. Alignments were performed using CLUSTALW
implemented in BioEdit and manually adjusted, takinginto consideration alignment of conserved metal-bindingdomains. Phylogenetic analysis of derived NirK sequenceswas performed using MEGA (Tamura et al., 2007) withminimum evolution and the JTT substitution model. To allowthe inclusion of nine shorter NirK sequences from ammonia-oxidizing bacteria, the pairwise comparison tool was imple-mented. Bootstrap support was calculated for minimumevolution, parsimony (MEGA) and maximum-likelihood analy-ses with site variation (invariable sites and eight variablegamma rates) using PHYML (Guindon and Gascuel, 2003)with 1000, 1000 and 100 replicates respectively. Environmen-tal NirK sequences were shorter than those of the fourretrieved fosmid sequences. Therefore, only closely relatedsequences were used in phylogenetic analyses to allow theinclusion of as many alignable positions as possible.
Microcosm set-up and N2O measurements
Rotböll soil (10 g dry weight) was added to 44 ml serum vialbottles. Each microcosm was then individually supplementedwith 50 mg NH4NO3-N soil g-1 and the water content adjusted(from an original concentration of 9.2%) to a final concentra-tion of 15%, 20%, 25% or 30% (w/w) with three microcosmsestablished for each water concentration. Microcosms wereincubated at 25°C with loose fitting caps to allow airexchange. After 5 days, nitrous oxide concentrations weredetermined using the method of Garcia-Ruiz and Baggs(2007). Before gas sampling, microcosms were sealed with arubber septum for 1 h prior to the removal of 1 ml of head-space gas and direct injection into an Agilent 6890 gas chro-matograph calibrated with 1 and 10 ppm standards. After gasmeasurements, microcosm soil was removed and frozenimmediately at -80°C for analysis of nirK mRNA transcripts.
Nitrite reductase of soil archaea 11
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Reverse transcription and qPCR
Total nucleic acids were extracted from microcosm soil usingthe method of Griffiths and colleagues (2000). Briefly, 0.5 g ofsoil (wet weight) was added to a 2 ml microcentrifuge tubecontaining a silica and ceramic bead mixture (Hybaid,Ashford, Middlesex, UK) with 0.5 ml of CTAB/phosphateextraction buffer (pH 7.8) and 0.5 ml of phenol : chloro-form : isoamyl alcohol (25:24:1). Tubes were then shaken for30 s in a Hybaid Ribolyser (Hybaid) at a speed of 4 m s-1
before centrifugation at 16 000 g for 5 min. The top aqueousphase was removed and extracted with a further 0.25 ml ofchloroform : isoamyl alcohol (24:1) followed by centrifuga-tion. The aqueous phase was then removed and nucleicacids precipitated by adding 2 vols 30% (w/v) PEG 6000 in1.6 M NaCl and leaving on ice for 2 h. Nucleic acids werethen pelleted by centrifugation at 16 000 g for 20 min andwashed in 1 ml of ice-cold 70% ethanol (v/v) before furthercentrifugation at 16 000 g for 20 min. The ethanol wash solu-tion was poured off before removing residual liquid with usinga micropipette. Pellets were dried by heating in a dry-block at55°C for ~1 min and the nucleic acid pellets dissolved in 50 mlof sterile H2O.
cDNA was produced from extracted RNA as describedpreviously (Nicol et al., 2008). Briefly, DNA was digestedusing RQ1 DNase (Promega) before producing cDNA usingSuperscript II reverse transcriptase (Invitrogen) with randomhexamer primers (Invitrogen) at a concentration of 8 mM.Negative controls included water only (no RNA) and RNA butno RT enzyme in the final step.
qPCR was performed using a Bio-Rad MyIQ Single-ColorReal-Time PCR Detection System (Biorad, Hertfordshire,UK) with Qiagen QuantiFast SYBR Green PCR Master Mix(Qiagen, West Sussex, UK) according to manufacturer’sinstructions with both primers at 1.0 mM. For generating aqPCR standard, a 2648 bp PCR product was amplified fromsoil fosmid clone 54d9 (using primers homologous to posi-tions 20940–20961 and 23567–23588) (Table 1) andincluded the entire nirK gene (orf 26). For the qPCR assay, 24nirK sequences were aligned using BioEdit Sequence Align-ment Editor (Hall, 1999) and a primer set resulting in a 174 bpproduct was designed specific to all nirK sequences obtainedfrom Rötboll soil (qAnirK1F, qAnirK2R) (Table 1). After purifi-cation and quantification, a dilution series of template stan-dard was produced from 108 to 102 copies. Amplificationefficiencies were greater than 95% with an r2 value of > 0.99.Determined quantities were then adjusted to copies per gramof dry soil.
Accession numbers
All sequences have been deposited in the GenBank data-base with Accession No. GU059106 to GU059162.
Acknowledgements
The authors would like to thank Anja Spang and Tim Urich forhelpful discussions. G.W.N. is funded by a NERC AdvancedFellowship (NE/D010195/1). A Royal Society InternationalJoint Project grant awarded to G.W.N. and C.S. is also grate-fully acknowledged.
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Supporting information
Additional Supporting Information may be found in the onlineversion of this article:
Fig. S1. Schematic representation (drawn to scale) display-ing the location of an identified 12-nucleotide signature rep-resentative of amicyanin domains (in orange) present ingenes of archaea and bacteria (in blue). The amino acidmotifs in green represent the permitted variation of amicyanindomains as proposed by Giri and colleagues (2004). Aminoacid positions which vary from the proposed signatures arehighlighted in bold and underlined. Sequences shown wereretrieved from the NCBI database after BLASTP searches andinclude genes found in the archaea N. maritimus (Nmar),HF4000 (ALOHA marine fosmids) and C. symbiosum(CENSYa) and bacteria.Table S1. Environmental samples screened for archaeal16S rRNA and nirK genes.Table S2. Predicted RNA and protein encoding genes in cre-narchaeal fosmids 29d5, 76h13, 57a5 and 54d9 (a to drespectively).Table S3. Nitrous oxide production rates and nirK transcriptcopies measured in Rotböll meadow soil microcosms estab-lished at different water contents. Values represent the mean(and standard error) of triplicate microcosms.
Please note: Wiley-Blackwell are not responsible for thecontent or functionality of any supporting materials suppliedby the authors. Any queries (other than missing material)should be directed to the corresponding author for the article.
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