Unexpected nondenitrifier nitrous oxide reductase gene diversity and abundance in soils Robert A. Sanforda, Darlene D. Wagnerb, Qingzhong Wuc, Joanne C. Chee-Sanfordd, Sara H. Thomasc, Claribel Cruz-Garcíac, Gina Rodríguezc,e, Arturo Massol-Deyáe, Kishore K. Krishnanif, Kirsti M. Ritalahtig,h, Silke Nisseng,h, Konstantinos T. Konstantinidisb,c, and Frank E. Löfflerg,h,i,1 aDepartment of Geology, University of Illinois, Urbana, IL 61801; bSchool of Biology and cSchool of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332; dUS Department of Agriculture—Agricultural Research Service, Urbana, IL 61801; eDepartment of Biology, University of Puerto Rico, Mayagüez, Puerto Rico 00681; fNational Institute of Abiotic Stress Management, Indian Council of Agricultural Research, Pune 413115, India; gDepartment of Microbiology, University of Tennessee, Knoxville, TN 37996; hBiosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831; and iDepartment of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN 37996 Edited by Edward F. DeLong, Massachusetts Institute of Technology, Cambridge, MA, and approved October 19, 2012 (received for review July 2, 2012) − Agricultural and industrial practices more than doubled the intrin- dissimilatory nitrate reduction to ammonium (DNRA)] (NO3 / fi − + + − sic rate of terrestrial N xation over the past century with drastic NO2 → NH4 ), and nitrosifyers (NH4 → NO2 ) contribute to consequences, including increased atmospheric nitrous oxide (N2O) N2O release (15–21), and additional N2O emissions result from concentrations. N2O is a potent greenhouse gas and contributor to coupled microbial–abiotic processes (chemodenitrification). For ozone layer destruction, and its release from fixed N is almost en- example, ferric iron-reducing bacteria generate ferrous iron, which tirely controlled by microbial activities. Mitigation of N2O emissions reacts chemically with nitrite to produce N2O(22–24). Contem- fi to the atmosphere has been attributed exclusively to denitri ers porary greenhouse gas models presume that N2O-to-N2 reduction possessing NosZ, the enzyme system catalyzing N2OtoN2 reduction. (i.e., the final step of the denitrification pathway) is the major We demonstrate that diverse microbial taxa possess divergent nos attenuation process controlling N2O flux to the atmosphere. clusters with genes that are related yet evolutionarily distinct from Denitrification sensu stricto (i.e., complete denitrification) is the typical nos genes of denitirifers. nos clusters with atypical nosZ a process catalyzed by microorganisms that possess the enzymatic occur in Bacteria and Archaea that denitrify (44% of genomes), do − − − machinery including Nar and/or Nap (NO3 → NO2 ), Nir (NO2 not possess other denitrification genes (56%), or perform dissimila- → NO), Nor (NO → N2O), and Nos (N2O → N2)forthestepwise tory nitrate reduction to ammonium (DNRA; (31%). Experiments with − reduction of NO3 to N2 (21). Attempts to predict N2Oemissions the DNRA soil bacterium Anaeromyxobacter dehalogenans demon- based on denitrifier nosZ gene abundance and expression revealed strated that the atypical NosZ is an effective N O reductase, and PCR- 2 an incongruity between the predicted and the actual N2Oemis- based surveys suggested that atypical nosZ are abundant in terres- sions, suggesting the existence of an unaccounted N2Osink(25– trial environments. Bioinformatic analyses revealed that atypical nos 28). To explore the basis of this discrepancy, we screened 126 clusters possess distinctive regulatory and functional components bacterial and 7 archaeal genomes containing nosZ and found (e.g., Sec vs. Tat secretion pathway in typical nos), and that previous a broad distribution of nosZ genes across 16 taxonomic groups of nosZ-targeted PCR primers do not capture the atypical nosZ diversity. Bacteria and the Archaea. This analysis revealed uncharacterized Collectively, our results suggest that nondenitrifying populations (novel) nosZ genes encoding functional NosZ (N Oreductionto with a broad range of metabolisms and habitats are potentially sig- 2 fi N2) in diverse taxonomic groups, far exceeding the known nosZ ni cant contributors to N2O consumption. Apparently, a large, pre- diversity of complete denitrifiers. Thus, our study expands the viously unrecognized group of environmental nosZ has not been current understanding of the nitrogen cycle and provides enhanced accounted for, and characterizing their contributions to N2O con- means to monitor and model N2O emissions into the atmosphere. sumption will advance understanding of the ecological controls on SCIENCES fi fl N2O emissions and lead to re ned greenhouse gas ux models. Results and Discussion ENVIRONMENTAL Expanded NosZ Diversity and Prevalence in Taxonomically Diverse nitrogen cycle | climate change Populations. To date, N2O-to-N2 reduction in the environment has been attributed exclusively to denitrifying microorganisms ossil fuel combustion, agricultural practices (e.g., the cultiva- expressing the typical Z-type NosZ (29, 30). Typical nosZ genes fi Ftion of legumes promoting microbial N2 xation, manure ap- (77 sequences) were found on 75 genomes belonging to the plication), and the conversion of nonreactive N2 to ammonium in Alpha-, Beta-, and Gammaproteobacteria harboring complete – the Haber Bosch process are the main causes of the increased sets of denitrification genes. Ten genomes harboring a typical fi input of xed (reactive) nitrogen (N) into the environment (1). nosZ lacked nirS or nirK homologs, corroborating previous obser- This anthropogenic contribution to the N imbalance has a series of vations that some microorganisms with a typical nosZ may not environmental consequences, and particularly troubling are cli- denitrify (21, 30). Interestingly, bioinformatic sequence analyses mate change concerns stemming from the release of N2O gas into the atmosphere. N2O is a greenhouse gas with a global warming potential 310 times greater than that of the equivalent amount of – Author contributions: R.A.S. and F.E.L. designed research; R.A.S., Q.W., J.C.C.-S., S.H.T., G.R., CO2 (2 4) and promotes ozone destruction in the stratosphere K.K.K., and F.E.L. performed research; C.C.-G., A.M.-D., K.M.R., and S.N. contributed new (5–8). Global measurements demonstrate that atmospheric N2O reagents/analytic tools; R.A.S., D.D.W., Q.W., S.N., K.T.K., and F.E.L. analyzed data; and R.A.S., has increased steadily over the past 250 y (9), indicating that cur- D.D.W., J.C.C.-S., K.M.R., and F.E.L. wrote the paper. rent global sources exceed global sinks [(10); World Meteorolog- The authors declare no conflict of interest. ical Organization, www.wmo.int/pages/mediacentre/press_releases/ This article is a PNAS Direct Submission. documents/GHG_bull_6_en.pdf]. The major sources for atmo- Freely available online through the PNAS open access option. spheric N2O are biotic and coupled abiotic processes occurring in Data deposition: The sequences reported in this paper have been deposited in the Gen- soil, sediment, and subsurface ecosystems (11–14). Diverse mi- Bank database (accession nos. GU185838.1–GU185857.1). − − crobial populations of complete denitrifiers (NO3 /NO2 → N2), 1To whom correspondence should be addressed. E-mail: frank.loeffl[email protected]. − − incomplete denitrifiers (NO3 /NO2 → N2O), nitrate reducers − − This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (NO3 → NO2 ), ammonifiers [i.e., microorganisms performing 1073/pnas.1211238109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1211238109 PNAS | November 27, 2012 | vol. 109 | no. 48 | 19709–19714 Downloaded by guest on September 29, 2021 Denitrification DNRA 0.1 NirK NirS NrfA Anaeromyxobacter dehalogenans 2CP-1 Anaeromyxobacter dehalogenans K Deltaproteobacteria Anaeromyxobacter dehalogenans 2CP-C Anaeromyxobacter sp. Fw109-5 Verrucomicrobia Desulfomonile tiedjei DCB-1 Opitutus terrae PB90-1 Bacteroidetes Opitutaceae bacterium TAV5 Salinibacter ruber strain MS and DSM 13855 Gemmatimonadetes Marivirga tractuosa DSM 4126 Spirochaetes Cellulophaga algicola DSM 14237 Maribacter sp. HTCC2501 Chlorobi Robiginitalea biformata HTCC2510 Gramella forsetii KT0803 Aquificae Gemmatimonas aurantiaca T-27 Leptospira biflexa (Patoc) Firmicutes Ignavibacterium album JCM 16511 Hydrogenobacter thermophilus TK-6 Atypical NosZ Chloroflexi Persephonella marina EX-H1 Prevotella denticola F0289 Alphaproteobacteria Dyadobacter fermentans DSM 18053 Haliscomenobacter hydrossis DSM 1100 Betaproteobacteria Pedobacter saltans DSM 12145 Flavobacteriaceae bacterium 3519-10 Deferribacteres Riemerella anatipestifer DSM 15868 Desulfitobacterium dehalogenans, Epsilonproteobacteria D. dichloroeliminans, D. hafniense Y51 and DCB-2 Desulfotomaculum ruminis DSM 2154 Gammaproteobacteria Desulfosporosinus meridiei DSM 13257 Geobacillus thermodenitrificans NG80-2 Euryarchaeota Sphaerobacter thermophilus DSM 20745 Caldilinea aerophila DSM 14535 Crenarchaeota Thermomicrobium roseum DSM 5159 Rhodothermus marinus DSM 4252 Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense C. Accumulibacter phosphatis cIIA st. UW-1, Dechloromonas aromatica RCB, and Dechlorosoma suillum PS Denitrovibrio acetiphilus DSM 12809 Wolinella succinogenes DSM 1740 Campylobacter fetus subsp. fetus 82-40 Campylobacter concisus and C. curvus 525.92 Sulfurimonas denitrificans, S. autotrophica, Nitratiruptor sp. SB155-2, Sulfurovum sp. NBC37-1, and Nitratifractor salsuginis Ferroglobus placidus