refined nrfa phylogeny improves pcr-based nrfa gene detection · ing a lax hmm coverage of 11% as...

Refined NrfA Phylogeny Improves PCR-Based nrfA Gene Detection

Allana Welsh,a Joanne C. Chee-Sanford,a,b Lynn M. Connor,b Frank E. Löffler,c,d,e,f Robert A. Sanforda

University of Illinois at Urbana-Champaign, Urbana, Illinois, USAa; USDA-ARS, Urbana, Illinois, USAb; Department of Microbiology, University of Tennessee, Knoxville,Tennessee, USAc; Center for Environmental Biotechnology, University of Tennessee, Knoxville, Tennessee, USAd; Department of Civil and Environmental Engineering,University of Tennessee, Knoxville, Tennessee, USAe; University of Tennessee and Oak Ridge National Laboratory (UT-ORNL) Joint Institute for Biological Sciences (JIBS)and Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USAf

Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrialnitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous prod-ucts). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite am-monification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme hadnot been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA cladeswith robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the firstheme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH orKXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfAproteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new nrfA-containing isolates. nrfA amplicons obtainedwith template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providingsupport for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populationsand will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.

Understanding the mechanisms controlling nitrogen (N) cycledynamics is crucial for improving models that predict thefluxes of nitrate, nitrite, N2O, and associated carbon turnover (1).Conceptually, the N cycle consists of two types of processes, thosethat drive N loss (denitrification and anammox) and those thatpromote N retention (N fixation and dissimilatory nitrate reduc-tion to ammonium [DNRA]) (Fig. 1). Comprehensive under-standing of the identities and activities of microorganisms in-volved in N cycling, particularly in soils, is still limited, becausemany key players have not been identified and cultured, and theapplication of molecular tools has been hampered by the limitednumber of target gene sequences.

Fertilizer use is increasing globally and now contributes abouthalf of the biologically available N to the global N cycle (2). Thefate of N in soil is of paramount concern in agriculture, whereefficient use of fertilizer remains a challenge and managementpractices can drive the extent of either N loss or N retention in soil(3). Denitrification leads to N loss from agroecosystems throughemissions of N2 and the potent greenhouse gas N2O (4). Anam-mox could also contribute to N loss from soils through the con-version of ammonium and nitrite to N2 gas (5). The relative con-tribution of DNRA to N cycling may have been overlooked, as anumber of studies suggested that DNRA plays a greater role innitrite turnover than previously thought (6). In contrast to deni-trification, DNRA, or, more precisely, respiratory nitrite-ammon-ification (7) would promote N retention in soils (8), because am-monium electrostatically interacts with the soil matrix and hasminimal volatilization potential at circumneutral soil pH (9). Un-like denitrification, the contribution of respiratory nitrite am-monification to N cycle processes has received little attention insoil ecosystems (6).

Recent studies using N isotopes suggested that DNRA is morerelevant for nitrate/nitrite turnover than previously thought. Forexample, in estuarine sediments, DNRA was shown to co-occur

with denitrification (10, 11), and in oceanic oxygen minimumzones/coastal shelf waters, DNRA has been reported to provideammonia that is oxidized to dinitrogen gas by anammox bacteria(12, 13). [15N]nitrate additions to soil from an old growth forestshowed that DNRA exceeded denitrification with little overall Nloss from the system (14). Templer et al. (15) showed that DNRAaccounted for 25% of the reduction of added [15N]nitrate andcontributed significantly to N retention in a tropical forest soil.Although agricultural soils differ from mature forest soil ecosys-tems in terms of nutrient inputs, plant communities, and landmanagement practices, respiratory nitrite ammonification inagroecosystems may be significant over temporal and spatialscales.

Another consideration for understanding N retention versus Nloss processes in ecosystems is the fact that nitrite, and not nitrate,is the key branching point in the N cycle. The term DNRA isactually a misnomer consisting of two independent reductionsteps (7). Nitrate is first reduced to nitrite by a reductase and isnot necessarily linked with any downstream process, such asnitrite reduction to N2 or ammonium (16). Depending on theenvironmental conditions, nitrite may undergo dissimilatoryreduction via denitrification, respiratory nitrite ammonification(i.e., DNRA), or anammox. None of these processes depends on

Received 17 October 2013 Accepted 16 January 2014

Published ahead of print 24 January 2014

Editor: C. R. Lovell

Address correspondence to Allana Welsh, [email protected].

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.03443-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.03443-13

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the presence of nitrate. In addition, if the nitrite generated fromnitrate reduction diffuses to oxic zones, it may be subject to oxi-dation to nitrate via nitrification (Fig. 1) (7). Many studies havecharacterized the diversity of the microbial community associatedwith denitrification by targeting the genes encoding the nitritereductase NirK or NirS (17, 18, 19, 20). The nitrite reductase NrfA,associated with respiratory nitrite ammonification, provides ananalogous target to monitor organisms potentially mediating thisunderstudied N retention process. In contrast to decades of stud-ies focused on denitrification, nrfA-containing organisms havereceived less attention, and their contributions to N retention areunclear.

Reduction of nitrite to ammonium catalyzed by NrfA has beenextensively studied in model organisms like Escherichia coli andWolinella succinogenes (21, 22). NrfA shares evolutionary historywith octaheme c-type cytochrome proteins, like the hydroxyl-amine oxidoreductases and octaheme nitrite reductases (23, 24).What distinguishes NrfA from these other c-type cytochromes is apentaheme structural core with an unusual CXXCK motif (whereX can be any amino acid) in the first heme-binding motif (25, 26).A small number of NrfA proteins have been noted to have aCXXCH motif in this domain, suggesting it is not a universaldistinguishing feature (22, 23). Despite the known lack of motifconservation, previous molecular studies evaluating NrfA diver-sity focused on the CXXCK motif; therefore, they were not com-prehensive (27).

Although many bacterial populations harbor the nrfA gene inthe environment, particularly in soil ecosystems (6, 28), little isknown about the diversity of NrfA in these habitats. Common soilbacterial taxa, such as Anaeromyxobacter dehalogenans and Desul-fitobacterium spp., reduce nitrate to ammonium via nitrite (29, 30,31), yet studies to explore their contributions and of other poten-tial DNRA populations to nitrate and/or nitrite turnover in soilecosystems are absent. PCR primers targeting nrfA genes in envi-ronmental samples were reported, but the design was based ononly six sequences available at the time (27). Since then, many

more nrfA sequences have become available, but a comprehensiveNrfA phylogenetic analysis has not been performed.

To advance understanding of microbes contributing to DNRA,we performed a comprehensive analysis of NrfA sequences andidentified novel diagnostic features shared among all known NrfA.The NrfA phylogenetic analysis extended the evolutionary diver-sity of this protein and revealed a universal diagnostic motif. Theexpanded phylogeny guided the design of a new PCR primer setthat captures the known nrfA gene diversity, which will be usefulfor assessing the prevalence and activity of potential respiratorynitrite-ammonifying populations in natural and engineered eco-systems.

MATERIALS AND METHODSPhylogenetic analyses. Gene sequences of putative nrfA genes wereevaluated initially in the Functional Gene Pipeline and Repository(FUNGENE) (http://fungene.cme.msu.edu/) database, version 6.5 (Sep-tember 2011). Sequences that were initially annotated as nrfA inFUNGENE were further pared by evaluating a range of filter thresholds(7% to 19% hidden Markov model [HMM] coverage) in the FUNGENEsearch and establishing an optimal filter to maximize inclusion of diver-gent nrfA genes while reducing the inclusion of non-nrfA sequences. Us-ing a lax HMM coverage of 11% as the filter threshold resulted in 684putative nrfA sequences that ranged in size from �120 bp to �1,600 bpand included genes from whole genomes, pure cultures, and unculturedcloned sequences from the environment. An alignment of the 684 se-quences was made using a ClustalW algorithm (32) and the TranslationAlign feature in GENEIOUS 5.3.4 (Biomatters Ltd., Auckland, New Zea-land). The data set was further sorted as either nrfA or non-nrfA genesequences by hand by simultaneous inspection of the nucleotides and theencoded amino acid residues. Using criteria based on the presence ofcatalytically important amino acid residues (22), 474 nrfA sequences wereretained from the initial set of 684 sequences. The alignment of 474 nrfAsequences was used for the design of a new PCR primer set described later.The 210 non-nrfA gene sequences were retained for subsequent in silicoanalyses.

Prior to conducting a comprehensive phylogenetic analysis of NrfA,we updated the sequence data set in April 2012 by adding an additional 35

FIG 1 Major pathways of nitrogen (N) metabolism mediated by distinct functional microbial populations in the environment. Denitrification and DNRA arehighlighted as dissimilatory reduction pathways involving nitrite, depicted as a central intermediate of both oxidative and reductive processes. Under anoxicconditions, consumption of nitrite can lead to either N loss (NO, N2O, N2) via denitrification and anammox or N retention (NH4

�) via DNRA. Numbers at thebottom of the figure indicate the oxidation state of the N compound directly above. N compounds involved are shown relative to their presence as gases or liquids.

Nitrite Reductase (NrfA) Phylogeny and Detection

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nrfA sequences from the FUNGENE and GenBank (33) databases. Thisalignment file was curated by removing partial nrfA gene sequences andredundant sequences. Sequences were translated into amino acid residuesusing GENEIOUS, and redundant sequences were removed on the basisof �98% amino acid similarity calculated using a simple distance matrixgenerated using MEGA v4 (34). The remaining 272 NrfA amino acidsequences were aligned using a MAFFT algorithm (35) through theGUIDANCE web server (http://guidance.tau.ac.il) (36). Bayesian phylo-genetic analyses for 272 NrfA amino acid sequences (1,086 aligned aminoacid residues) was completed using MRBAYES, version 3.0, in three inde-pendent runs and included Metropolis-coupled Markov chain MonteCarlo (MCMCMC) sampling, a mixed amino acid model, 2 million gen-erations, and sampling every 1,000 trees (37). The outgroup was the oc-taheme nitrite reductase (ONR) from Thioalkalivibrio nitratireducens(38). A 50% majority-rule consensus tree for the Bayesian output of pos-terior probabilities (PP) was created with 50% of the trees removed asburn-in to ensure that log likelihoods had reached an asymptote (37).MEGA was used to display treefiles and was used to create a sequencesimilarity matrix to aid in defining nrfA gene clade assignments. For pre-sentation purposes, a similar Bayesian analysis was performed on a subsetof 67 NrfA sequences representative of the diversity of the full data set. Togauge whether a small fragment of NrfA carried phylogenetic informationsimilar to that of the full gene, a similar Bayesian phylogenetic analysis wasperformed on the subset of 67 NrfA sequences using only the amino acidresidues between the third and fourth heme-binding motifs.

Design of a new PCR primer set to target nrfA and in silico analyses.Using the alignment of 474 nrfA gene sequences resulting from the Sep-tember 2011 FUNGENE search, new PCR primers targeting nrfA geneswere designed by examining nucleotide regions that were conserved in atleast 75% of the nrfA sequences. A new forward primer, nrfAF2aw(5=-CAR TGY CAY GTB GAR TA), which includes a portion of the thirdheme-binding motif, was paired with the previously published reverseprimer nrfAR1 (5=-TWN GGC ATR TGR CAR TC) (27), which encodesthe fourth heme-binding motif. The forward and reverse primer sequenceregions correspond to positions 637 to 654 and 889 to 906, respectively, inthe nrfA gene sequence of Wolinella succinogenes and generate a 269-bpamplicon. In silico analyses using the Primer Test feature in GENEIOUSquantified the number of mismatches occurring with nrfA and non-nrfAsequences using primer set nrfAF2aw and nrfAR1.

PCR conditions and validation of nrfA amplification from pure cul-tures. PCR amplification of the nrfA gene was conducted in 25-�l reactionmixtures with the following reagent concentrations: 1� PCR buffer(Clontech Laboratories Inc., Mountain View, CA, USA), 2.5 mM MgCl2,25 �g/ml T4 gene 32 protein (Roche Applied Science, Indianapolis, IN,USA), 0.2 mM each deoxynucleoside triphosphate (dNTP), 0.8 �M eachprimer (nrfAF2aw and nrfAR1), 0.025 U/�l TaKaRa Ex Taq DNA poly-merase (Clontech), and �100 ng template DNA. Thermal cycling condi-tions included an initial denaturation step at 95°C for 5 min, followed by30 cycles of 95°C for 30 s, 53°C for 30 s, and 72°C for 20 s and a finalextension step at 72°C for 10 min. DNA from eight pure cultures known tohave nrfA (Escherichia coli strain K-12 ATCC 15222, Shewanella loihicastrain PV-4, Anaeromyxobacter dehalogenans strain 2CP-1, Desulfitobac-terium hafniense strain DCB-1, Desulfovibrio vulgaris strain Hildenbor-ough, Geobacter bemidjiensis strain Bem, and Wolinella succinogenes) wereused as positive test cultures. Each pure culture used has a fully sequencedgenome containing nrfA genes to validate the specificity of the new nrfAprimer set, nrfAF2aw and nrfAR1, and to optimize conditions for PCRamplification. Negative PCR controls were DNA from Pseudomonas fluo-rescens strain Pf-5 NRRL B-23932 and water. Restriction fragment lengthpolymorphisms (RFLP) resulting from actual restriction enzyme diges-tion of the amplified product of the nrfA genes with each reference strainmatched the expected fragment sizes generated using in silico analysis andwere used as putative confirmation of nrfA gene amplification.

Analysis of nrfA gene clones from new bacterial isolates and soil.DNA from 40 new bacterial cultures isolated under nitrite selection (de-

scribed below) was PCR amplified using primer set nrfAF2aw and nrfAR1.The amplicons from samples yielding an �250-bp product were gel pu-rified (Ultra Clean 15 DNA purification kit; MoBio, Carlsbad, CA), ligatedinto pGEM-T Easy vector (Promega), and transformed into E. coli JM109competent cells (Promega). One nrfA gene clone from each isolate wassequenced using the vector primer T7 (W. M. Keck Center for Compara-tive and Functional Genomics, Urbana, IL).

The primer set nrfAF2aw and nrfAR1 was used to amplify nrfA genesin samples of pooled DNA extracted from soil taken as cored depths fromthe surface to 30 cm at two contrasting agricultural field sites (Havana andUrbana, IL). For comparison, we also tested primers nrfAF1 and nrfAR1(27). Briefly, the field sites were sampled in November 2011 as part oflong-term soil monitoring experiments being conducted at two agricul-tural field sites, both with long histories of commercial corn and soybeancrop production. The soil texture at the Urbana site is clay (10 to 20%), silt(60 to 70%), and loam (10 to 20%), while the Havana site is primarily sand(93%). PCR amplification was conducted in triplicate using conditionsdescribed above, and the products were gel purified and pooled into onesample per soil source. The PCR products were cloned, and 48 clonescorresponding to each soil site (95 in total) were selected at random forsequencing.

A separate phylogenetic analysis included the addition of 110 clonedsequences obtained following PCR amplification of nrfA genes using theprimer set nrfAF2aw and nrfAR1 (described below) from soil and newnitrite-reducing bacterial isolates. Each cloned sequence (�250 bp) wastranslated into amino acid residues using GENEIOUS. The alignment ofthe 272 NrfA sequences described above was trimmed to include only theregion amplified by the nrfAF2aw and nrfAR1 PCR primer set and thenmerged with the 110 cloned sequences and realigned using the MAFFTalgorithm in GUIDANCE (n � 382; 188 aligned amino acid residues).Maximum likelihood (ML) analyses were completed using the RAxML-HPC Blackbox (v 7.2.6) program (39) on the computer cluster of theCyberInfrastructure for Phylogenetic RESearch project (CIPRES; www.phylo.org) from the online servers at the San Diego SupercomputingCenter. Settings included GTR�CAT approximation for rate heterogene-ity (40), invariant sites, empirical base frequencies, estimation of the boot-strap (BS) replicates necessary for this data set, and input of the Bayesiantree generated using full-length NrfA sequences as a constraint tree.MEGA was used to create similarity matrices to group nrfA sequencesamplified from soil DNA and soil isolates obtained in this study withsequences from pure cultures.

Cultivation of bacterial isolates. Isolation of new nitrite-reducingbacteria was accomplished with 1-g samples of Havana or Urbana soil,each diluted 10-fold serially (10�6 final concentration) in 9-ml anaerobi-cally prepared phosphate buffer (2 mM, pH 7.0). Samples (0.1 ml) of eachdilution were inoculated onto anaerobically prepared agar medium usinga basal medium formulation (41) with the following amendments: so-dium nitrite (1 mM), sodium acetate or sodium succinate (5 mM), yeastextract (0.01%, wt/vol) (BD, Franklin Lakes, NJ, USA), and Bacto agar(1.5%); some formulations also included ammonium chloride (4 mM).All cultures were incubated at 25°C in an anaerobic chamber (Coy Labo-ratory Products Inc., Grasslake, MI, USA) containing a primary head-space of N2 with 5% CO2 and 2% H2. Individual colonies were selectedperiodically over 1 month of incubation and restreaked onto fresh me-dium at least twice until purity was achieved.

DNA extraction. DNA was extracted from pure cultures for evaluat-ing the presence of nrfA genes using the following protocol: liquid cultureswere centrifuged, the pelleted cells were resuspended in TE (10 mM Tris,1 mM EDTA, pH 8.0), cells were lysed by subjecting them to three cyclesof freeze/thaw (�80°C freezer to 56°C water bath), and then they wereincubated overnight at 37°C in 1% SDS with 1.0 mg/ml Proteinase K(Promega, Madison, WI, USA). DNA was precipitated with isopropanol(42), and concentrations were estimated based on quantitative DNA stan-dards analyzed using gel electrophoresis. DNA from W. succinogenes was

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provided by Jim Hemp (Department of Biochemistry, University of Illi-nois at Urbana-Champaign).

To obtain soil DNA, individual aliquots (�0.5 g) of Urbana (U) andHavana (H) soil were used for extraction using a phenol-chloroform pro-tocol (43). Additional purification of DNA was made with Sepharose 4B(Sigma-Aldrich, St. Louis, MO, USA) gel exclusion, modified from Jack-son et al. (44) prior to PCR amplification. To obtain a single compositeDNA sample for use in amplification of the nrfA gene, 10-�l aliquots ofDNA were pooled from individual soil extracts. All DNA was stored at�20°C for use in this study.

Nucleotide sequence accession numbers. Sequences of the new nrfAgene fragments obtained in this study were deposited in the GenBankdatabase under accession numbers JX293721 to JX293830.

RESULTSNrfA amino acid alignment and phylogeny. A total of 474 pen-taheme proteins with NrfA-characteristic conserved amino acidresidues (45) were identified in an alignment of 684 proteins. Theywere putatively identified as NrfA and retrieved from theFUNGENE database. All 474 NrfA proteins harbored a KXRH orKXQH motif between the third and fourth heme-binding motifs(Fig. 2).

Of those sequences, a CXXCK motif in the first heme-bindingsite was found in 420 NrfA sequences, while the remaining 54sequences had a CXXCH motif in this region. Of the original 684aligned protein sequences, 210 did not match the NrfA criteriasensu stricto (i.e., were not pentaheme proteins and/or lacked thekey KXRH or KXQH motif between the third and fourth heme-binding motifs). These 210 sequences represented proteins classi-fied as octaheme nitrite reductases (ONR), hydroxylamine oxi-doreductases (HAO), tetraheme cytochrome c proteins, andmultiheme cytochrome c proteins.

After removal of redundant sequences (i.e., sequences with�98% amino acid identity), the NrfA alignment resulted in 272distinct NrfA sequences (401 to 623 amino acids in length) with amean pairwise amino acid residue identity of 48%. Using at least a30% mean amino acid sequence divergence to any neighboringclade and �90% Bayesian posterior probability support, NrfAproteins were partitioned into 18 distinct clades (A to R) (see Fig.S1 and S3 in the supplemental material).

The NrfA phylogeny did not necessarily correspond with or-ganismal taxonomic delineations. For example, NrfA sequences ofa variety of taxa belonging to the Firmicutes were distributed inclades I, K, M, N, P, Q, and R, while Bacteroidetes NrfA occurredonly in clade H (Fig. 3). While Gammaproteobacteria NrfA pro-teins were spread over clades A, B, C, D, and E, the diversity withinthis Proteobacteria subphylum originated from a single branch,indicating a monophyletic origin. The NrfA phylogeny revealedthat nrfA was only found in bacterial genomes, and no NrfA se-quences were encoded in the available archaeal genomes (see Fig.S1 in the supplemental material). All NrfA sequences in clades J, K,and L shared a CXXCH motif in the first heme-binding motif,while members of all other clades possessed a correspondingCXXCK motif (Fig. 2).

The majority of the analyzed genomes possess a single nrfAgene copy; however, some had multiple copies. Bacillus selenitire-ducens had four nrfA genes encoding NrfA with 87 to 100% aminoacid identity, all of which belong to clade P. The nrfA genes withinone organism could encode proteins as divergent from each otheras the NrfA proteins of separate genera. For example, the twopredicted NrfA proteins of A. dehalogenans strain 2CP-1 (clades J

and K) shared 64% amino acid similarity, as did the NrfA from E.coli (clade A) and Actinobacillus ureae (clade E). Shewanella putre-faciens strain 200 had three identical NrfA proteins (clade D) andone NrfA protein that belonged to clade C that shared 48% aminoacid identity with the other NrfA of clade D.

Identification of NrfA diagnostic regions and design of nrfA-targeted PCR primers. The alignment of 474 NrfA sequencesidentified conserved regions suitable for the design of PCR prim-ers (Fig. 2). Specifically, the third and fourth heme-binding motifsthat flanked the region containing the conserved KXRH or KXQHmotif present in all NrfA sequences appeared suitable to target thebreadth of the currently known nrfA diversity. Phylogenetic anal-ysis of NrfA amino acid sequences between the third and fourthheme-binding motifs were found to be phylogenetically coherentwith the NrfA alignment of full-length sequences, and similarclade divisions were supported even with the partial NrfA se-quences (see Fig. S2 in the supplemental material). The PCRprimer pair nrfAF2aw and nrfAR1, designed to target this regionbetween the third and fourth heme-binding motifs, was evaluatedfor its ability to amplify nrfA fragments (Fig. 2). An in silico anal-ysis predicted that these primers would amplify 403 of the 474(85%) nrfA sequences. Of the 15% nrfA genes not predicted to beamplified, most were associated with only one or two representa-tive genera; thus, most of the NrfA diversity is covered with thenew primer set. Primers nrfAF2aw and nrfAR1 had few (i.e., 3)mismatches with all nrfA sequences among the clades, and thecomputational analysis predicted amplification of nrfA sequencesrepresenting all NrfA clades.

Using the new primer set nrfAF2aw/nrfAR1, the nrfA gene wasamplified using DNA from eight pure cultures, representing 10NrfA clades, except for G. bemidjiensis and the negative-control P.fluorescens. Restriction fragment length polymorphism (RFLP)analyses of nrfA gene amplicons (�250 bp) showed the corre-sponding predicted fragment sizes, indicating that nrfA was am-plified (data not shown). The nrfA genes associated with G. bemi-djiensis (clade I) and one copy of the clade D nrfA gene of S. loihicastrain PV-4 (Fig. 3) were not amplified with the new primer set.This was consistent with in silico analysis of predicted mismatches(3 mismatches in G. bemidjiensis and 7 mismatches in S. loihica)between the primers and these specific nrfA sequences (see Fig. S3in the supplemental material).

Amplification of nrfA from soil bacterial isolates and soilDNA. Using the primer set nrfAF2aw and nrfAR1 with templateDNA from nitrite-reducing bacterial isolates from two contrast-ing agricultural soils from Urbana (clay/silt/loam) and Havana(sand), IL, yielded PCR amplicons of the expected size with 16 ofthe 40 isolates tested (Table 1; also see Fig. S4 in the supplementalmaterial). The NrfA amino acid sequences predicted from theamplified fragments from Urbana soil isolates shared 99.5%amino acid identity with each other and were 90% identical to theNrfA of Bacillus sp. strain INLA3E in clade P (Table 1). Bacterialisolates from the Havana soil harbored NrfA sequences thatshared 99% amino acid identity with each other and were 85%identical to the NrfA of Providencia species in clade A.

Application of primer set nrfAF2aw and nrfAR1 to DNA ex-tracted from Havana and Urbana agricultural soils yielded nrfAsequences with fragment sizes ranging from 215 bp to 251 bp (themean amplicon size was 239 bp). Of the 95 cloned sequences, 94were confirmed as nrfA fragments based on sequence similarityand the presence of the characteristic NrfA motif KXRH or KXQH




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FIG 2 Partial alignment of amino acid residues of 11 representative NrfA proteins harbored by nine bacterial genera. Conserved and catalytically importantresidues are shaded in gray. Heme-binding motifs (CXXCX) and distal heme-ligating histidines (H) are over- and underlined in black. The five heme-bindingmotifs of NrfA are labeled 1 to 5. The first heme-binding motif with either a CXXCK or a CXXCH motif is highlighted in red. Black triangles indicate residuesassociated with the enzyme active site. Open triangles represent residues purportedly involved in forming the substrate/product channel (45). The highlyconserved KXRH or KXQH motif between the 3rd and 4th heme-binding motifs is highlighted in green. Strain abbreviations are the following: E. coli, Escherichiacoli strain HS; S. loih-D, Shewanella loihica strain PV-4 clade D (Fig. 3); S. loih-C, S. loihica strain PV-4 clade C (Fig. 3); W. succ, Wolinella succinogenes strain DSM1740; P. gingiv, Porphyromonas gingivalis strain W83; B. selen, Bacillus selenitireducens strain MLS10; D. vulga, Desulfovibrio vulgaris subsp. vulgaris strainHildenborough; G. metal, Geobacter metallireducens strain GS 15; A. deh-K, Anaeromyxobacter dehalogenans strain 2CP-1 clade K (Fig. 3); A. deh-J, A. dehalo-genans strain 2CP-1 clade J (Fig. 3); C. jejuni, Campylobacter jejuni subsp. jejuni 414.



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in the translated amino acid sequences (Fig. 2). A comparativeamino acid analysis of the cloned nrfA gene fragments amplifiedfrom the soil samples showed a mean pairwise amino acid identityof 68%. The majority (82) of the NrfA sequences clustered inclades J and K (Table 1; also see Fig. S5 in the supplemental mate-rial) and were most similar to the NrfA proteins associated withthe Myxococcales and the genus Opitutus. Interestingly, similar

NrfA proteins from clades J and K were obtained from both Ha-vana and Urbana soil DNA samples, even though these soils havecontrasting properties.

DISCUSSION

NrfA has been characterized as a pentaheme nitrite reductase witha unique CXXCK motif in the first heme-binding (catalytic) motif

FIG 3 Bayesian phylogenetic tree based on analysis of 1,086 amino acid residues encoded by nrfA for a representative subset of taxa (n � 67). Numbers indicateBayesian posterior probabilities for that branch. Clade assignments are designated A through R and are based on at least 30% mean pairwise amino acid sequencedivergence to a neighboring clade and �90% Bayesian posterior probabilities. NrfA electron donor redox partners (either NrfB or NrfH) are indicated on the left.NrfA with a CXXCH motif in the first heme-binding motif are indicated on the right. All other sequences have a CXXCK motif in this region. The octaheme nitritereductase (ONR) of Thioalkalivibrio nitratireducens was used as an outgroup. The scale bar represents 0.2 amino acid replacements per site. Shaded taxa have beendemonstrated to reduce nitrate or nitrite to ammonium. References describing the physiological activity of each organism are indicated in boldface, italicizednumbers following the name except for those marked by asterisks: *, S. Yoon, personal communication; **, M. W. Fields, personal communication.




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(25, 26). This enzyme system shares considerable similarity withthe octaheme cytochrome c (OCC) family of proteins, making itsidentification challenging. It has even been suggested that a pen-taheme nitrite reductase similar to NrfA gave rise to related OCCproteins (23). Because of the evolutionary similarities in proteinstructure and function, identification of key diagnostic features iscritical for distinguishing NrfA from other closely related pro-teins. The misidentification of 210 of the 684 sequences as NrfA inthe FUNGENE database at the time of our analysis (September2011) exemplified the challenge to clearly delineate NrfA. A sub-sequent examination of an updated FUNGENE database did showimprovement in what genes were identified as NrfA and, moreimportantly, did not show any new diversity (data not shown).

Comprehensive analysis of all known NrfA sequences has ex-panded our understanding of the characteristic features of thisprotein. In the alignment of 474 NrfA sequences, 11% (54 se-quences), including all NrfA in clades J, K, and L, possess aCXXCH motif in the first heme-binding motif. This modified firstheme-binding domain had previously been noted in the NrfA inCampylobacter jejuni, which has been shown to reduce nitrite toammonium (66, 67). Anaeromyxobacter dehalogenans strains re-duce nitrite to ammonium (29) and also have the CXXCH NrfAmotif. Thus, functional NrfA sequences possess either the CXXCKor CXXCH motif in the first heme-binding domain of respiratorynitrite ammonifiers.

Catalytically important residues, such as the KXRH or KXQHmotif between the third and fourth heme-binding domains, arefound in all NrfA sequences. Prior analysis of NrfA identified theKXQH motif, and the protein structure from eight organisms sug-gested the involvement of this motif in the formation of the sub-strate-product channel (45, 65, 68). Our sequence analysis alsoidentified a similar sequence, KXRH, in the same location in asubset of the NrfA sequences, suggesting a similar role.

The phylogenetic analysis of NrfA sequences delineated 18clades and is the most comprehensive analysis to date. PreviousNrfA phylogenies used representative sequences representingonly 6 to 12 of the 18 clades of NrfA delineated in this study (23,27, 69). The clade classification proposed here facilitates the phy-logenetic placement of new nrfA genes obtained from environ-mental samples. Such a robust classification can easily be ex-panded to include additional clades should future studies discoveran even greater NrfA diversity.

In some cases, the assignment of NrfA to distinct clades fol-lowed the 16S rRNA gene phylogeny. For example, the NrfA pro-teins of the Gammaproteobacteria originated from a single ances-tral gene that subsequently branched into clades A, B, C, D, and E(Fig. 3). Monophyly of the Gammaproteobacteria NrfA may berelated to structural changes in the protein found among mem-bers of this subdivision, allowing for interaction with the electrontransfer protein NrfB (70). The tetraheme protein NrfH purport-edly acts, in a fashion similar to that of NrfB, as an electron trans-fer redox partner with NrfA in all other clades (Fig. 3) (45, 71). Incontrast to the Gammaproteobacteria, NrfA proteins of clades J, K,and L are populated with taxa from six different phyla (i.e., Pro-teobacteria, Verrucomicrobia, Acidobacteria, Planctomycetes, Fir-micutes, and Chloroflexi) (Fig. 3), suggesting that the respiratorynitrite ammonification genetic elements are mobile and were ac-quired independently by members of these taxonomic groups (72,73). Similar observations have been made with nirK in some deni-trifiers, where the evolutionary histories of the nirK genes weredistinct from the phylogeny of the 16S rRNA genes from the sameorganisms (74). Therefore, environmental nrfA sequences can beascribed to a clade (Fig. 3), but assignments to specific genera orspecies are not possible (with the possible exception of some nrfAsequences of the Gammaproteobacteria).

Further complicating assignment of NrfA sequences to specifictaxonomic groups is the presence of multiple, divergent nrfA cop-ies on a single genome. Of the 228 organisms representing thediversity of NrfA in the alignment of 272 proteins, 30 organismsrepresenting 14 genera had more than one nrfA copy (see Fig. S1 inthe supplemental material). This is in contrast to patterns revealedin the phylogenies of NO-forming nitrite reductases among deni-trifiers, where only 3 of 101 bacterial strains with the nirS gene andnone of 147 strains with nirK had multiple copies (74). The func-tion of different NrfA proteins within one organism is unknown.Physiological studies of a Thauera sp. with two different nirS cop-ies encoding denitrifying nitrite reductases revealed that one copywas expressed constitutively and the other was positively regulatedby nitrate (75). Similar differences in gene expression may explainthe role of multiple NrfA proteins in diverse genera; however, thisobservation clearly warrants further exploration.

The identified diagnostic features common to all known NrfAproteins enabled the design of a new PCR primer set that amplifiesnrfA genes belonging to all clades and is applicable to environ-mental samples. The amplified region corresponds to the diagnos-tic motifs between the third and fourth heme-binding domainsand retains sufficient phylogenetic information for assignment toNrfA clades. For example, the nrfA gene fragments from bacterialisolates and environmental clone sequences could be confidentlyassigned to the different NrfA clades (Table 1; also see Fig. S5 inthe supplemental material). Efforts to use a previously publishednrfA-targeted PCR primer set (27) failed to yield any ampliconsfrom the soil DNA samples (data not shown) and generated false-

TABLE 1 NrfA amino acid residue identity deduced from nrfA geneclones retrieved from soil and nitrite-reducing soil isolates from Urbanaand Havana soilsc

CladeaNo. ofclones/isolates

Mean groupidentityb(%)

Representative soilclone/strain

UrbanaG 1 UCC1L 3 88.6 UCE2J 9 77.9 UCC2K 27 74.5 UCA1M 8 89 UCA11

HavanaJ 6 77.9 HCA6K 40 74.5 HCB2

Urbana isolatesP 10 99.5 UAAc-1

Havana isolatesA 6 99.5 HAc-4

a As delineated in Fig. 3.b Mean pairwise similarity for NrfA amino acid residues in the isolate or clone groupwithin a clade.c Clade assignments were based on phylogenetic analyses of 188 aligned amino acidresidues deduced from the soil clones (n � 94) and isolates (n � 16) using thephylogeny of the full-length NrfA proteins (1,086 amino acid residues) as a constrainttree to guide assignment of these short fragments to the correct clade.

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negative results. This finding emphasizes that the updated NrfAphylogeny and improved primer design enables a more compre-hensive tool to evaluate nrfA gene presence and diversity in natu-ral and engineered environments.

Even though the clone library generated from each soil DNAsample was not exhaustive (47 clones per sample), the dominanceof nrfA gene sequences belonging to clades J and K suggests thatmembers of these clades contribute to DNRA activity in thesesoils. The nrfAF2AW and nrfAR1 primer set effectively covers thebreadth of the currently known nrfA gene diversity; thus, findingonly certain predominant clades in Urbana and Havana soilscould reflect the diversity of nrfA-harboring bacterial communi-ties in these soils. The nrfA gene fragments cloned from both soilsare similar to the genes harbored by the soil-dwelling A. dehaloge-nans, a genus known for DNRA activity (29) and previously de-tected in the Urbana and Havana soils (30).

In summary, this study presents an updated and comprehen-sive description of the currently known NrfA diversity and a usefulframework to categorize NrfA. The finding of a diverse range ofnrfA genes amplified with DNA from soil and bacterial isolatesprovides a basis for testing the contributions of DNRA to N cy-cling (Fig. 1). The combined application of the new nrfA PCRprimer set and process-level measures of respiratory nitrite am-monification (e.g., reverse transcriptase quantitative real-timePCR, 15N isotope fate studies) promises to generate new insightsinto the ecological significance of DNRA for N retention in natu-ral and engineered ecosystems.

ACKNOWLEDGMENTS

This research was supported by the U.S. Department of Energy, Office ofBiological and Environmental Research, Genomic Science Program, awardDE-SC0006662, and USDA-ARS CRIS Project 3611-12220-008-00D.

REFERENCES1. Canfield DE, Glazer AN, Falkowski PG. 2010. The evolution and future

of Earth’s nitrogen cycle. Science 330:192–196. http://dx.doi.org/10.1126/science.1186120.

2. Gruber N, Galloway JN. 2008. An Earth-system perspective of the globalnitrogen cycle. Nature 451:293–296. http://dx.doi.org/10.1038/nature06592.

3. Robertson GP, Vitousek PM. 2009. Nitrogen in agriculture: balancingthe cost of an essential resource. Annu. Rev. Environ. Resour. 34:97–125.http://dx.doi.org/10.1146/annurev.environ.032108.105046.

4. Conrad R. 1996. Soil microorganisms as controllers of atmospheric tracegases (H2, CO, CH4, OCS, N2O, and NO). Microbiol. Rev. 60:609 – 640.

5. Hu B-I, Shen L-D, Xu X-Y, Zheng P. 2011. Anaerobic ammoniumoxidation (anammox) in different natural ecosystems. Biochem. Soc.Trans. 39:1811–1816. http://dx.doi.org/10.1042/BST20110711.

6. Rütting T, Boeckx P, Müller C, Klemedtsson L. 2011. Assessment of theimportance of dissimilatory nitrate reduction to ammonium for the ter-restrial nitrogen cycle. Biogeosciences 8:1169 –1196. http://dx.doi.org/10.5194/bg-8-1169-2011.

7. Simon J, Klotz MG. 2013. Diversity and evolution of bioenergetic systemsinvolved in microbial nitrogen compound transformations. Biochim. Bio-phys. Acta 1827:114–135. http://dx.doi.org/10.1016/j.bbabio.2012.07.005.

8. Silver WL, Thompson AW, Reich A, Ewel JJ, Firestone MK. 2005.Nitrogen cycling in tropical plantation forests: potential controls on ni-trogen retention. Ecol. Appl. 15:1604 –1614. http://dx.doi.org/10.1890/04-1322.

9. Myrold DD. 2005. Transformations of nitrogen, p 333–372. In Sylvia DM,Fuhrmann JJ, Hartel PG, Zuberer DA (ed), Principles and applications ofsoil microbiology, 2nd ed. Pearson Prentice Hall, Upper Saddle River, NJ.

10. Gardner WS, McCarthy MJ, An S, Sobolev D, Sell KS, Brock D. 2006.Nitrogen fixation and dissimilatory nitrate reduction to ammonium(DNRA) support nitrogen dynamics in Texas estuaries. Limnol. Ocean-ogr. 51:558 –568. http://dx.doi.org/10.4319/lo.2006.51.1_part_2.0558.

11. Koop-Jakobsen K, Giblin AE. 2010. The effect of increased nitrate load-ing on nitrate reduction via denitrification and DNRA in salt marsh sedi-ments. Limnol. Oceanogr. 55:789 – 802. http://dx.doi.org/10.4319/lo.2009.55.2.0789.

12. Lam P, Lavik G, Jensen MM, van de Vossenberg J, Schmid M, WoebkenD, Gutiérrez D, Amann R, Jetten MSM, Kuypers MMM. 2009. Revisingthe nitrogen cycle in the Peruvian oxygen minimum zone. Proc. Natl.Acad. Sci. U. S. A. 106:4752– 4757. http://dx.doi.org/10.1073/pnas.0812444106.

13. Jensen MM, Lam P, Revsbech NP, Nagel B, Gaye B, Jetten MSM,Kuypers MMM. 2011. Intensive nitrogen loss over the Omani Shelf due toanammox coupled with dissimilatory nitrite reduction to ammonium.ISME J. 5:1660 –1670. http://dx.doi.org/10.1038/ismej.2011.44.

14. Rütting T, Huygens D, Müller C, Van Cleemput O, Godoy R, BoeckxP. 2008. Functional role of DNRA and nitrite reduction in a pristine southChilean Nothofagus forest. Biogeochemistry 90:243–258. http://dx.doi.org/10.1007/s10533-008-9250-3.

15. Templer PH, Silver WL, Pett-Ridge J, DeAngelis KM, Firestone MK.2008. Plant and microbial controls on nitrogen retention and loss in ahumid tropical forest. Ecology 89:3030 –3040. http://dx.doi.org/10.1890/07-1631.1.

16. Klotz MG, Stein LY. 2008. Nitrifier genomics and evolution of the nitro-gen cycle. FEMS Microbiol. Lett. 278:146 –156. http://dx.doi.org/10.1111/j.1574-6968.2007.00970.x.

17. Wallenstein MD, Myrold DD, Firestone MK, Voytek M. 2006. Envi-ronmental controls on denitrifiying communities and denitrificationrates: insights from molecular methods. Ecol. Appl. 16:2143–2152. http://dx.doi.org/10.1890/1051-0761(2006)016[2143:ECODCA]2.0.CO;2.

18. Oakley BB, Francis CA, Roberts KJ, Fuchsman CA, Srinivasan S, StaleyJT. 2007. Analysis of nitrite reductase (nirK and nirS) genes and cultiva-tion reveal depauperate community of denitrifying bacteria in the BlackSea suboxic zone. Environ. Microbiol. 9:118 –130. http://dx.doi.org/10.1111/j.1462-2920.2006.01121.x.

19. Smith JM, Ogram A. 2008. Genetic and functional variation in denitrifierpopulations along a short-term restoration chronosequence. Appl. Envi-ron. Microbiol. 74:5615–5620. http://dx.doi.org/10.1128/AEM.00349-08.

20. Santoro AE, Boehm AB, Francis CA. 2006. Denitrifier community com-position along a nitrate and salinity gradient in a coastal aquifer. Appl.Environ. Microbiol. 72:2102–2109. http://dx.doi.org/10.1128/AEM.72.3.2102-2109.2006.

21. Einsle O. 2011. Structure and function of formate-dependent cytochromec nitrite reductase, NrfA. Methods Enzymol. 496:399 – 422. http://dx.doi.org/10.1016/B978-0-12-386489-5.00016-6.

22. Simon J. 2002. Enzymology and bioenergetics of respiratory nitrite am-monification. FEMS Microbiol. Rev. 26:285–309. http://dx.doi.org/10.1111/j.1574-6976.2002.tb00616.x.

23. Klotz MG, Schmid MC, Strous M, Op den Camp HJM, Jetten MSM,Hooper AB. 2008. Evolution of an octahaem cytochrome c protein familythat is key to aerobic and anaerobic ammonia oxidation by bacteria. En-viron. Microbiol. 10:3150 –3163. http://dx.doi.org/10.1111/j.1462-2920.2008.01733.x.

24. Kern M, Klotz MG, Simon J. 2011. The Wolinella succinogenes mcc genecluster encodes an unconventional respiratory sulphite reduction system.Mol. Microbiol. 82:1515–1530. http://dx.doi.org/10.1111/j.1365-2958.2011.07906.x.

25. Eaves DJ, Grove J, Staudenmann W, James P, Poole RK, White SA,Griffiths I, Cole JA. 1998. Involvement of products of the nrfEFG genes inthe covalent attachment of haem c to a novel cysteine-lysine motif in thecytochrome c552 nitrite reductase from Escherichia coli. Mol. Microbiol.28:205–216.

26. Stach P, Einsle O, Schumaker W, Kurun E, Kroneck PMH. 2000.Bacterial cytochrome c nitrite reductase: new structural and functionalaspects. J. Inorg. Biochem. 79:381–385. http://dx.doi.org/10.1016/S0162-0134(99)00248-2.

27. Mohan SB, Schmid M, Jetten MSM, Cole JR. 2004. Detection andwidespread distribution of the nrfA gene encoding nitrite reduction toammonia, a short circuit in the biological nitrogen cycle that competeswith denitrification. FEMS Microbiol. Ecol. 49:433– 443. http://dx.doi.org/10.1016/j.femsec.2004.04.012.

28. Kraft B, Strous M, Tegetmeyer HE. 2011. Microbial nitrate respiration—genes, enzymes and environmental distribution. J. Biotechnol. 155:104 –117. http://dx.doi.org/10.1016/j.jbiotec.2010.12.025.

29. Sanford RA, Cole JR, Tiedje JM. 2002. Characterization and description




http://aem.asm

.org/D

ownloaded from

http://dx.doi.org/10.1126/science.1186120http://dx.doi.org/10.1126/science.1186120http://dx.doi.org/10.1038/nature06592http://dx.doi.org/10.1038/nature06592http://dx.doi.org/10.1146/annurev.environ.032108.105046http://dx.doi.org/10.1042/BST20110711http://dx.doi.org/10.5194/bg-8-1169-2011http://dx.doi.org/10.5194/bg-8-1169-2011http://dx.doi.org/10.1016/j.bbabio.2012.07.005http://dx.doi.org/10.1890/04-1322http://dx.doi.org/10.1890/04-1322http://dx.doi.org/10.4319/lo.2006.51.1_part_2.0558http://dx.doi.org/10.4319/lo.2009.55.2.0789http://dx.doi.org/10.4319/lo.2009.55.2.0789http://dx.doi.org/10.1073/pnas.0812444106http://dx.doi.org/10.1073/pnas.0812444106http://dx.doi.org/10.1038/ismej.2011.44http://dx.doi.org/10.1007/s10533-008-9250-3http://dx.doi.org/10.1007/s10533-008-9250-3http://dx.doi.org/10.1890/07-1631.1http://dx.doi.org/10.1890/07-1631.1http://dx.doi.org/10.1111/j.1574-6968.2007.00970.xhttp://dx.doi.org/10.1111/j.1574-6968.2007.00970.xhttp://dx.doi.org/10.1890/1051-0761(2006)016[2143:ECODCA]2.0.CO%3B2http://dx.doi.org/10.1890/1051-0761(2006)016[2143:ECODCA]2.0.CO%3B2http://dx.doi.org/10.1111/j.1462-2920.2006.01121.xhttp://dx.doi.org/10.1111/j.1462-2920.2006.01121.xhttp://dx.doi.org/10.1128/AEM.00349-08http://dx.doi.org/10.1128/AEM.72.3.2102-2109.2006http://dx.doi.org/10.1128/AEM.72.3.2102-2109.2006http://dx.doi.org/10.1016/B978-0-12-386489-5.00016-6http://dx.doi.org/10.1016/B978-0-12-386489-5.00016-6http://dx.doi.org/10.1111/j.1574-6976.2002.tb00616.xhttp://dx.doi.org/10.1111/j.1574-6976.2002.tb00616.xhttp://dx.doi.org/10.1111/j.1462-2920.2008.01733.xhttp://dx.doi.org/10.1111/j.1462-2920.2008.01733.xhttp://dx.doi.org/10.1111/j.1365-2958.2011.07906.xhttp://dx.doi.org/10.1111/j.1365-2958.2011.07906.xhttp://dx.doi.org/10.1016/S0162-0134(99)00248-2http://dx.doi.org/10.1016/S0162-0134(99)00248-2http://dx.doi.org/10.1016/j.femsec.2004.04.012http://dx.doi.org/10.1016/j.femsec.2004.04.012http://dx.doi.org/10.1016/j.jbiotec.2010.12.025http://aem.asm.orghttp://aem.asm.org/

of Anaeromyxobacter dehalogenans gen. nov., sp. nov., an aryl-halorespiring facultative anaerobic myxobacterium. Appl. Environ. Mi-crobiol. 68:893–900. http://dx.doi.org/10.1128/AEM.68.2.893-900.2002.

30. Sanford RA, Wagner DD, Wu Q, Chee-Sanford JC, Thomas SH, Cruz-Garcia C, Rodriguez G, Massol-Deya A, Krishnani KK, Ritalahti KM,Nissen S, Konstantinidis KT, Löffler FE. 2012. Unexpected nondenitri-fier nitrous oxide reductase gene diversity and abundance in soils. Proc.Natl. Acad. Sci. U. S. A. 109:19709 –19714. http://dx.doi.org/10.1073/pnas.1211238109.

31. Madsen T, Licht D. 1992. Isolation and characterization of an anaerobicchlorophenol-transforming bacterium. Appl. Environ. Microbiol. 58:2874 –2878.

32. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG.1997. The CLUSTAL_X Windows interface: flexible strategies for multiplesequence alignment aided by quality analysis tools. Nucleic Acids Res.25:4876 – 4882. http://dx.doi.org/10.1093/nar/25.24.4876.

33. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Sayers EW. 2011.GenBank. Nucleic Acids Res. 39:D32–D37. http://dx.doi.org/10.1093/nar/gkq1079.

34. Tamura K, Dudley J, Nei M, Kumar S. 2007. Molecular evolutionarygenetics analysis (MEGA) software version 4. Mol. Biol. Evol. 24:1596 –1599. http://dx.doi.org/10.1093/molbev/msm092.

35. Katoh K, Kuma K, Toh H, Miyata T. 2005. MAFFT version 5: improve-ment in accuracy of multiple sequence alignment. Nucleic Acids Res. 33:511–518. http://dx.doi.org/10.1093/nar/gki198.

36. Penn O, Privman E, Ashkenazy H, Landan G, Graur D, Pupko T. 2010.GUIDANCE: a web server for assessing alignment confidence scores. Nu-cleic Acids Res. 2010:W23–W28. http://dx.doi.org/10.1093/nar/gkq443.

37. Huelsenbeck JP, Ronquist F. 2001. MRBAYES: Bayesian inference ofphylogenetic trees. Bioinformatics 17:754 –755. http://dx.doi.org/10.1093/bioinformatics/17.8.754.

38. Tikhonova T, Tikhonov A, Trofimov A, Polyakov K, Boyko K,Cherkashin E, Rakitina T, Sorokin D, Popov V. 2012. Comparativestructural and functional analysis of two octaheme nitrite reductases fromclosely related Thioalkalivibrio species. FEBS J. 279:4052– 4061. http://dx.doi.org/10.1111/j.1742-4658.2012.08811.x.

39. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phy-logenetic analyses with thousands of taxa and mixed models. Bioinformat-ics 22:2688 –2690. http://dx.doi.org/10.1093/bioinformatics/btl446.

40. Stamatakis A. 2006. Phylogenetic models of rate heterogeneity: a highperformance computing perspective, p 1– 8. In Proceedings of the IEEEInternational Parallel and Distributed Processing Symposium 2006, Rho-des, Greece. IEEE, New York, NY.

41. Sanford RA, Cole JA, Löffler FE, Tiedje JM. 1996. Characterization ofDesulfitobacterium chlororespirans sp. nov., which grows by coupling theoxidation of lactate to the reductive dechlorination of 3-chloro-4-hydroybenzoate. Appl. Environ. Microbiol. 62:3800 –3808.

42. Sambrook J, Russell D. 2001. Molecular cloning: a laboratory manual,3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

43. Tsai Y-L, Olsen BH. 1991. Rapid method for direct extraction of DNAfrom soil and sediment. Appl. Environ. Microbiol. 57:1070 –1074.

44. Jackson CR, Harper JP, Willoughby D, Roden EE, Churchill PF. 1997.A simple, efficient method for the separation of humic substances andDNA from environmental samples. Appl. Environ. Microbiol. 63:4993–4995.

45. Einsle O, Stach P, Messerschmidt A, Simon J, Kröger A, Huber R,Kroneck PM. 2000. Cytochrome c nitrite reductase from Wolinella succi-nogenes. J. Biol. Chem. 275:39608 –39616. http://dx.doi.org/10.1074/jbc.M006188200.

46. Cole JA. 1978. The rapid accumulation of large quantities of ammoniaduring nitrite reduction by Escherichia coli. FEMS Microbiol. Lett. 4:327–329. http://dx.doi.org/10.1111/j.1574-6968.1978.tb02891.x.

47. Liu MC, Bakel BW, Liu M-Y, Dao TN. 1988. Purification of Vibrio fisherinitrite reductase and its characterization as a hexaheme c-type cyto-chrome. Arch. Biochem. Biophys. 262:259 –265. http://dx.doi.org/10.1016/0003-9861(88)90187-7.

48. Cooper DC, Picardal FW, Schmmelmann A, Coby AJ. 2003. Chemicaland biological interactions during nitrate and goethite reduction by She-wanella putrefaciens 200. Appl. Environ. Microbiol. 69:3517–3525. http://dx.doi.org/10.1128/AEM.69.6.3517-3525.2003.

49. Cruz-García C, Murray AE, Klappenbach JA, Stewart V, Tiedje JM.2007. Respiratory nitrate ammonification by Shewanella oneidensis MR-1.J. Bacteriol. 189:656 – 662. http://dx.doi.org/10.1128/JB.01194-06.

50. Forsythe SJ, Dolby JM, Webster ADB, Cole JA. 1988. Nitrate- andnitrite-reducing bacteria in the achlorhydric stomach. J. Med. Microbiol.25:253–259. http://dx.doi.org/10.1099/00222615-25-4-253.

51. Wolin MJ, Wolin EA, Jacobs NJ. 1961. Cytochrome-producing anaero-bic vibrio, Vibrio succinogenes, sp. n. J. Bacteriol. 81:911–917.

52. Schumaker W, Kroneck PM. 1992. Anaerobic energy metabolism of thesulfur-reducing bacterium “Spirillum” 5175 during dissimilatory nitratereduction to ammonia. Arch. Microbiol. 157:464 – 470. http://dx.doi.org/10.1007/BF00249106.

53. Socransky SS, Holt SC, Leadbetter ER, Tanner ACR, Savitt E, Ham-mond BF. 1979. Capnocytophaga: new genus of Gram-negative glidingbacteria. III. Physiological characterization. Arch. Microbiol. 122:29 –33.

54. Takahashi N, Saito K, Schachtele CF, Yamada T. 1997. Acid toleranceand acid-neutralizing activity of Porphyromonas gingivalis, Prevotella in-termedia and Fusobacterium nucleatum. Oral Microbiol. Immunol. 12:323–328. http://dx.doi.org/10.1111/j.1399-302X.1997.tb00733.x.

55. Sung Y, Fletcher KE, Ritalahti KM, Apkarian RP, Ramos-Hernandez N,Sanford RA, Mesbah NM, Löffler FE. 2006. Geobacter lovleyi sp. nov.strain SZ, a novel metal-reducing and tetrachlorothene-dechlorinatingbacterium. Appl. Environ. Microbiol. 72:2775–2782. http://dx.doi.org/10.1128/AEM.72.4.2775-2782.2006.

56. Lovley DR, Giovannoni SJ, White DC, Champine JE, Phillips EJP,Gorby YA, Goodwin S. 1993. Geobacter metallireducens gen. nov. sp. nov.,a microorganism capable of coupling the complete oxidation of organiccompounds to the reduction of iron and other metals. Arch. Microbiol.159:336 –344. http://dx.doi.org/10.1007/BF00290916.

57. Huber R, Rossnagel P, Woese CR, Rachel R, Langworthy TA, StetterKO. 1996. Formation of ammonium from nitrate during chemolithoau-totrophic growth of the extremely thermophilic bacterium Ammonifexdegensii gen. nov. sp. nov. Syst. Appl. Microbiol. 9:40 – 49.

58. Sellars MJ, Hall SJ, Kelly DJ. 2002. Growth of Campylobacter jejunisupported by respiration of fumarate, nitrate, nitrite, trimethylamine-N-oxide, or dimethyl sulfoxide requires oxygen. J. Bacteriol. 184:4187– 4196.http://dx.doi.org/10.1128/JB.184.15.4187-4196.2002.

59. Henstra AM, Stams AJM. 2004. Novel physiological features of Carboxy-dothermus hydrogenoformans and Thermoterrabacterium ferrireducens.Appl. Environ. Microbiol. 70:7236 –7240. http://dx.doi.org/10.1128/AEM.70.12.7236-7240.2004.

60. Havemann SA, Greene EA, Stilwell CP, Voordouw JK, Voordouw G.2004. Physiological and gene expression analysis of inhibition of Desulfo-vibrio vulgaris Hildenborough by nitrite. J. Bacteriol. 186:7944 –7950.http://dx.doi.org/10.1128/JB.186.23.7944-7950.2004.

61. Liu MC, Peck HD, Jr. 1981. The isolation of a hexaheme cytochromefrom Desulfovibrio desulfuricans and its identification as a new type ofnitrite reductase. J. Biol. Chem. 256:13159 –13164.

62. Rauschenbach I, Narasingarao P, Häggblom MM. 2011. Desulfurispiril-lum indicum sp. nov., a selenate- and selenite-respiring bacterium isolatedfrom an estuarine canal. Int. J. Syst. Evol. Microbiol. 61:654 – 658. http://dx.doi.org/10.1099/ijs.0.022392-0.

63. Blum JS, Bindi AB, Buzzelli J, Stolz JF, Oremland RS. 1998. Bacillusarsenocoselenatis, sp. nov., and Bacillus selenitrireducens, sp. nov.: two ha-loalkipliles from Mono Lake, California that respire oxyanions of sele-nium and arsenic. Arch. Microbiol. 171:19 –30. http://dx.doi.org/10.1007/s002030050673.

64. Lanigan GW. 1976. Peptococcus heliotrinreducens, sp. nov., a cytochrome-producing anaerobe which metabolizes pyrrolizidine alkaloids. J. Gen.Microbiol. 94:1–10. http://dx.doi.org/10.1099/00221287-94-1-1.

65. Lockwood CWJ, Clarke TA, Butt JN, Hemmings AM, Richardson DJ.2011. Characterization of the active site and calcium binding in cyto-chrome c nitrite reductases. Biochem. Soc. Trans. 39:1871–1875. http://dx.doi.org/10.1042/BST20110731.

66. Pittman MS, Elvers KT, Lee L, Jones MA, Poole RK, Park SF, Kelly DJ.2007. Growth of Campylobacter jejuni on nitrate and nitrite: electrontransport to NapA and NrfA via NrfH and distinct roles for NrfA and theglobin Cgb in protection against nitrosative stress. Mol. Microbiol. 63:575–590. http://dx.doi.org/10.1111/j.1365-2958.2006.05532.x.

67. Kern M, Eisel F, Scheithauer J, Kranz RG, Simon J. 2010. Substratespecificity of three cytochrome c haem lyase isoenzymes from Wolinellasuccinogenes: unconventional haem c binding motifs are not sufficient forhaem c attachment by NrfI and CcsA1. Mol. Microbiol. 75:122–137. http://dx.doi.org/10.1111/j.1365-2958.2009.06965.x.

68. Einsle O, Messerschmidt A, Stach P, Bourenkov GP, Bartunik HD,

Welsh et al.



http://aem.asm

.org/D

ownloaded from

http://dx.doi.org/10.1128/AEM.68.2.893-900.2002http://dx.doi.org/10.1073/pnas.1211238109http://dx.doi.org/10.1073/pnas.1211238109http://dx.doi.org/10.1093/nar/25.24.4876http://dx.doi.org/10.1093/nar/gkq1079http://dx.doi.org/10.1093/nar/gkq1079http://dx.doi.org/10.1093/molbev/msm092http://dx.doi.org/10.1093/nar/gki198http://dx.doi.org/10.1093/nar/gkq443http://dx.doi.org/10.1093/bioinformatics/17.8.754http://dx.doi.org/10.1093/bioinformatics/17.8.754http://dx.doi.org/10.1111/j.1742-4658.2012.08811.xhttp://dx.doi.org/10.1111/j.1742-4658.2012.08811.xhttp://dx.doi.org/10.1093/bioinformatics/btl446http://dx.doi.org/10.1074/jbc.M006188200http://dx.doi.org/10.1074/jbc.M006188200http://dx.doi.org/10.1111/j.1574-6968.1978.tb02891.xhttp://dx.doi.org/10.1016/0003-9861(88)90187-7http://dx.doi.org/10.1016/0003-9861(88)90187-7http://dx.doi.org/10.1128/AEM.69.6.3517-3525.2003http://dx.doi.org/10.1128/AEM.69.6.3517-3525.2003http://dx.doi.org/10.1128/JB.01194-06http://dx.doi.org/10.1099/00222615-25-4-253http://dx.doi.org/10.1007/BF00249106http://dx.doi.org/10.1007/BF00249106http://dx.doi.org/10.1111/j.1399-302X.1997.tb00733.xhttp://dx.doi.org/10.1128/AEM.72.4.2775-2782.2006http://dx.doi.org/10.1128/AEM.72.4.2775-2782.2006http://dx.doi.org/10.1007/BF00290916http://dx.doi.org/10.1128/JB.184.15.4187-4196.2002http://dx.doi.org/10.1128/AEM.70.12.7236-7240.2004http://dx.doi.org/10.1128/AEM.70.12.7236-7240.2004http://dx.doi.org/10.1128/JB.186.23.7944-7950.2004http://dx.doi.org/10.1099/ijs.0.022392-0http://dx.doi.org/10.1099/ijs.0.022392-0http://dx.doi.org/10.1007/s002030050673http://dx.doi.org/10.1007/s002030050673http://dx.doi.org/10.1099/00221287-94-1-1http://dx.doi.org/10.1042/BST20110731http://dx.doi.org/10.1042/BST20110731http://dx.doi.org/10.1111/j.1365-2958.2006.05532.xhttp://dx.doi.org/10.1111/j.1365-2958.2009.06965.xhttp://dx.doi.org/10.1111/j.1365-2958.2009.06965.xhttp://aem.asm.orghttp://aem.asm.org/

Huber R, Kroneck PM. 1999. Structure of cytochrome c nitrite reductase.Nature 400:476 – 480. http://dx.doi.org/10.1038/22802.

69. Takeuchi J. 2006. Habitat segregation of a functional gene encoding ni-trate ammonification in estuarine sediments. Geomicrobiol. J. 23:75– 87.http://dx.doi.org/10.1080/01490450500533866.

70. Clarke TA, Cole JA, Richardson DJ, Hemmings AM. 2007. The crystalstructure of the pentahaem c-type cytochrome NrfB and characterizationof its solution-state interaction with the pentahaem nitrite reductaseNrfA. Biochem. J. 406:19 –30. http://dx.doi.org/10.1042/BJ20070321.

71. Simon J, Gross R, Einsle O, Kroneck PM, Kröger A, Klimmek O. 2000.A NapC/NirT-type cytochrome c (NrfH) is the mediator between the qui-none pool and the cytochrome c nitrite reductase of Wolinella succino-genes. Mol. Microbiol. 35:686 – 696.

72. Alvarez L, Bricio C, Gómez MJ, Berenguer J. 2011. Lateral transfer of the

denitrification pathway genes among Thermus thermophilus strains. Appl.Environ. Microbiol. 77:1352–1358. http://dx.doi.org/10.1128/AEM.02048-10.

73. Gogarten JP, Doolittle WF, Lawrence JG. 2002. Prokaryotic evolution inlight of gene transfer. Mol. Biol. Evol. 19:2226 –2238. http://dx.doi.org/10.1093/oxfordjournals.molbev.a004046.

74. Jones CM, Stres B, Rosenquist M, Hallin S. 2008. Phylogenetic analysisof nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal acomplex evolutionary history for denitrification. Mol. Biol. Evol. 25:1955–1966. http://dx.doi.org/10.1093/molbev/msn146.

75. Etchebehere C, Tiedje JM. 2005. Presence of two different active nirSnitrite reductase genes in a denitrifying Thauera sp. from a high-nitrate-removal-rate reactor. Appl. Environ. Microbiol. 71:5642–5645. http://dx.doi.org/10.1128/AEM.71.9.5642-5645.2005.




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Refined NrfA Phylogeny Improves PCR-Based nrfA Gene DetectionMATERIALS AND METHODSPhylogenetic analyses.Design of a new PCR primer set to target nrfA and in silico analyses.PCR conditions and validation of nrfA amplification from pure cultures.Analysis of nrfA gene clones from new bacterial isolates and soil.Cultivation of bacterial isolates.DNA extraction.Nucleotide sequence accession numbers.

RESULTSNrfA amino acid alignment and phylogeny.Identification of NrfA diagnostic regions and design of nrfA-targeted PCR primers.Amplification of nrfA from soil bacterial isolates and soil DNA.

DISCUSSIONACKNOWLEDGMENTSREFERENCES

refined nrfa phylogeny improves pcr-based nrfa gene detection · ing a lax hmm coverage of 11% as...

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