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A nitrogenase-like enzyme system catalyzesmethionine, ethylene, and methane biogenesisJustin A. North1, Adrienne B. Narrowe2, Weili Xiong3*, Kathryn M. Byerly1, Guanqi Zhao1,Sarah J. Young1, Srividya Murali1, John A. Wildenthal1†, William R. Cannon4,5, Kelly C. Wrighton2,Robert L. Hettich3, F. Robert Tabita1‡

Bacterial production of gaseous hydrocarbons such as ethylene and methane affects soil environmentsand atmospheric climate. We demonstrate that biogenic methane and ethylene from terrestrial andfreshwater bacteria are directly produced by a previously unknown methionine biosynthesis pathway.This pathway, present in numerous species, uses a nitrogenase-like reductase that is distinct fromknown nitrogenases and nitrogenase-like reductases and specifically functions in C–S bond breakage toreduce ubiquitous and appreciable volatile organic sulfur compounds such as dimethyl sulfide and(2-methylthio)ethanol. Liberated methanethiol serves as the immediate precursor to methionine,while ethylene or methane is released into the environment. Anaerobic ethylene production by thispathway apparently explains the long-standing observation of ethylene accumulation in oxygen-depletedsoils. Methane production reveals an additional bacterial pathway distinct from archaealmethanogenesis.

Nitrogenases are an ancient group of en-zymes, existing ~3.2 billion years ago,before the evolution of oxygenic photo-synthesis and subsequent widespreadoxygenation (1, 2). Their essential func-

tion is reduction of dinitrogen gas into am-monia, which contributes over half of theannual global nitrogen fixation required forthe synthesis of nucleic and amino acids byall life on Earth (3). Ancestors to nitrogenasein anaerobic prokaryotes also gave rise to dis-tinct nitrogenase-like reductases for bacterialphotosynthesis and archaeal methanogenesiscofactor metabolism (4–7). These include thedark-operative protochlorophyllide oxidoreduc-tase (DPOR) and chlorophyllide a oxidoreduc-tase (COR) of bacteriochlorophyll biosynthesis,as well as Ni2+-sirohydrochlorin a,c-diamidereductive cyclase for biosynthesis of the ar-chaeal methyl coenzyme-M reductase cofactorF430 (4–7). However, the evolutionary historyof nitrogen fixation revealed overlooked ni-trogen fixation–like (NFL) sequences in thegenomes of anaerobic bacteria with entirelyunknown function. Some of these sequenceswere associated with sulfur metabolism andtransport genes (8, 9), suggesting that the

nitrogenase family evolved to tackle elemen-tal processes beyond nitrogen and carbonassimilation.We previously observed production of eth-

ylene gas (>1 mmol h−1 g−1 dry cell weight) byphotosynthetic Alphaproteobacteria such asRhodospirillumrubrumandRhodopseudomonaspalustris when growing anaerobically underthe low sulfate concentrations (<200 mM) com-monly encountered in their freshwater andsoil habitats (fig. S1) (10). We traced the pre-cursor of ethylene to (2-methylthio)ethanol(MT-EtOH). This volatile organic sulfur com-pound (VOSC) was produced from by-productsof S-adenosyl-L-methionine (SAM) utilizationto regenerate methionine [Fig. 1A, dihydroxy-acetone phosphate (DHAP) shunt] (10). SAMis a key cellular cofactor synthesized directlyfrom methionine and is required by all or-ganisms for diverse processes including DNA,RNA, and protein methylations; polyamine andneurotransmitter synthesis; quorum sensing;and 5′-deoxyadenosyl radical generation by rad-ical SAM enzymes (11). However, the enzymesresponsible for the liberation of sulfur fromMT-EtOH formethionine regeneration and con-comitant ethylene formation were unresolved(10). Therefore, we grewR. rubrum under condi-tions for ethylene induction (50 mM limitingsulfate or 1 mM MT-EtOH as the sole sulfursource) and ethylene repression (1mMsulfate)(fig. S1) (10). Differential abundance analysisidentified multiple proteins that increased bymore than 20-fold in proteomes from inducedcells compared with those from repressedcells (Fig. 1B). Among these were enzymes in-volved in cysteine and methionine metabo-lism: homoserine/serine O-acetyltransferase,

O-acetylhomoserine sulfhydrylase, cystathioninebeta-synthase, and cystathionine gamma-lyase(Fig. 1, reactions 2, 3, 6, and 7, respectively).Unexpectedly, several proteins previously

identified as NFL sequences of unknown func-tion (8, 9) showed some of the highest increasesin abundance under ethylene-inducing con-ditions (Fig. 1B, Rru_A0772-Rru_A0773 andRru_A0793–Rru_A0796; see fig. S2 and dataS1 for gene organization and sequences). Inaddition, there was also a large increase inabundance of two proteins likely involvedin iron-sulfur cluster metabolism, NifS cysteinedesulfurase and a putative [4Fe-4S] scaffoldprotein (Fig. 1B, Rru_A1068-Rru_A1069).Rru_A1068-Rru_A1069 appear analogous tothe Azotobacter vinelandii NifUS system forsynthesis of nitrogenase-destined iron-sulfurclusters from cysteine (12). However, the preciseiron-sulfur cluster assembly pathway inR. rubrumis unknown. The involvement of the nitrogenase-like system in ethylene production was fur-ther bolstered by the R. rubrum transposonmutant strain WRdht-66B3, having an in-activated gene encoding a putative nitro-genase reductase–like iron protein (Fig. 1B,Rru_A0795). This and other mutants iden-tified in a random mutagenesis screen wereunable to grow anaerobically with MT-EtOHas the sole sulfur source but could still growusing sulfate, indicating a defect in the ethylene-producing pathway (fig. S3). Consistent withthe transposon Tn5 mutagenesis results, spe-cific deletion of NFL gene cluster Rru_A0793–Rru_A0796 rendered R. rubrum incapableof growth or production of ethylene abovebasal levels with MT-EtOH as the sole sul-fur source (Fig. 2, A and B, and fig. S4C). Thisresult confirmed that the putative nitrogenase-like system encoded by NFL gene clusterRru_A0793–Rru_A0796 was essential for as-similating sulfur from MT-EtOH to produceethylene and methionine.We then tested whether other biologically

relevant VOSCs could be used by this putativenitrogenase-like enzyme system (Fig. 2, A andB, and fig. S5A). In addition to MT-EtOH, VOSCutilization with concomitant hydrocarbon pro-duction was specific to dimethyl sulfide (DMS),the most abundant environmental VOSC, andethyl methyl sulfide (EMS) (Fig. 2, A and B).Analogous to MT-EtOH (10), use of DMS orEMS resulted in methane or ethane produc-tion, respectively, in a 1:1 stoichiometry (Fig. 3,A and B). Specific deletion of the other twoNFL genes, Rru_A0772-Rru_A0773, did not af-fect growth or hydrocarbon production (Fig. 2,A and B, and fig. S4B). Thus, we designateR. rubrum genes Rru_A0793–Rru_A0796, pre-viously identified as NFL genes nflBHDK ofunknown function (8, 9), as methylthio-alkanereductase genes marBHDK. This designationis based on corresponding amino acid sim-ilarity to R. rubrummolybdenum nitrogenase

RESEARCH

North et al., Science 369, 1094–1098 (2020) 28 August 2020 1 of 5

1Department of Microbiology, The Ohio State University,Columbus, OH 43210, USA. 2Department of Soil and CropSciences, Colorado State University, Fort Collins, CO 80523,USA. 3Chemical Sciences Division, Oak Ridge NationalLaboratory, Oak Ridge, TN 37830, USA. 4Pacific NorthwestNational Laboratory, Richland, WA 99352, USA. 5Departmentof Mathematics, University of California, Riverside, Riverside,CA 92507, USA.*Present address: Center for Food and Safety and Applied Nutrition,U.S. Food and Drug Administration, College Park, MD 20740, USA.†Present address: Washington University in St. Louis School ofMedicine, St. Louis, MO 63110, USA.‡Corresponding author. Email: tabita.1@osu.edu

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gene products NifB (synthesis of the NifB-cofactor precursor to the nitrogenase catalyticcofactor), NifH (nitrogenase-reductase ironprotein), NifD (nitrogenase catalytic subunita), and NifK (nitrogenase catalytic subunit b)(figs. S6 to S9). NFL genes Rru_A0772-Rru_A0773remain designated nflDK genes of unknownfunction (8, 9).When all R. rubrum NFL genes were de-

leted (strain D0772:3/D0793:6) and specific genecombinations were reintroduced via expres-sion from a plasmid, expression of marBHDKwas necessary and sufficient to restore growthand hydrocarbon metabolism from VOSCs(Fig. 2, B and C, and fig. S5, B and C). The NFLgenes of unknown function, nflDK, could notreplacemarDK in complementing for growth.Upon feeding cells expressing marBH andnflDK with VOSCs, ethylene and ethane pro-ductionwas poorly catalyzed at three- to fourfoldabove basal levels, and no methane enhance-

ment was observed (Fig. 2, B and C, and fig. S5,B and C). This revealed that R. rubrum NflDKcould only weakly catalyze methylthio-alkanereduction, indicating a different primary func-tion. Given that nflDK is expressed not just inthe presence of MT-EtOH but also in responseto general sulfate limitation (Fig. 1, B and C),NflDK may catalyze sulfur liberation fromalternate, albeit unknown, compounds. Alter-nately, given gene proximity and amino acidsimilarity (40%) to MarDK, NflDK may serveas accessory proteins for MarDK assemblyanalogous to NifEN (13), as detailed in the sup-plementary text section of the supplementarymaterials. These results demonstrated the re-quirement of the MarBHDK nitrogenase-likesystem for the anaerobic assimilation of sulfurfrom common environmental VOSCs such asDMS and MT-EtOH to support growth andmethionine metabolism. Moreover, these ob-servations revealed a previously unknown

mechanism for the bacterial production ofmethane and ethylene.Wemetabolically observed the link between

VOSC utilization andmethionine synthesis viathe marBHDK gene products by feeding ex-periments with (2-methyl[14C]thio)ethanol,which enabled us to follow the methylthiogroup of MT-EtOH. Upon feeding the wild-type strain, MT-EtOH was consumed. La-beledmethanethiol (14CH3-SH) andmethionine(methyl-14C) were concomitantly producedand observed at low levels (~2% of MT-ETOHconcentration) until MT-EtOH was depleted(Fig. 2D). These low levels, as previously ob-served for methanethiol metabolism from 5′-methylthioadenosine in R. rubrum (12), arelikely due to the flux ofmethanethiol tomethi-onine and subsequent utilization thereof forprotein synthesis andSAM-dependent processes(11). This flux is substantiated by 14C incor-poration from MT-EtOH into insoluble cell

North et al., Science 369, 1094–1098 (2020) 28 August 2020 2 of 5

A B C

Fig. 1. Nitrogenase-like proteins linked to VOSC utilization. (A) Methylthio-alkane reductase (1, blue), the gene product of marBHDK (proposed), convertsVOSCs to ethylene, methane, and methanethiol for methionine biosynthesis (redbox). MT-EtOH is produced by the widespread DHAP shunt (gray shaded box)(10, 25, 26). (B) R. rubrum proteins with increased abundance whenmethylthio-alkane reductase activity is induced (“MT-EtOH” or “Lo”

50 mM sulfate) versus repressed (“Hi” 1 mM sulfate). Isolated Tn5transposon mutants (fig. S3), which could not utilize MT-EtOH for growth,are marked with an X. (C) Changes in gene transcript abundance ofR. rubrum parent strain (WRdht) and SalR deletion strain (0785::Tn5)under “Hi” and “Lo” sulfate. Asterisk symbol indicates no significantchange. P > 0.25, two-tailed.

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material (fig. S10). Conversely, in themarBHDKdeletion strain there was no detectable metab-olism of MT-EtOH and thus no methanethiolor methionine production (Fig. 2E and fig. S10).Given that ethylene, ethane, and methane are

produced from MT-EtOH, EMS, and DMS, re-spectively, the observedmethanethiol is con-sistent with C–S single bond reduction andrelease of the methylthio group by the methyl-thio-alkane reductase (Figs. 1A, reaction 1,

and 2F). Each process is thermodynamicallyfavored for the substrates and products ob-served (Fig. 2F and fig. S11). Themethanethiol,along with O-acetyl-L-homoserine, then servesas a substrate for O-acetylhomoserine sulf-hydrylase, which catalyzes the synthesis ofmethionine (Fig. 1A, reaction 3) (14). This de-fines an anaerobic methylthio-alkane reductasemethionine synthesis pathway and establishesthe role of a nitrogenase-like enzyme systemin sulfur metabolism (Fig. 1A, red box).Sulfur metabolism is evidently the primary

function of these nitrogenase-like methylthio-alkane reductases, as opposed to nitrogenfixation by nitrogenase. R. rubrum has molyb-denum nitrogenase (NifHDK), which is thedefault nitrogenase, and iron-only nitrogenase(AnfHDGK), which is synthesized in the ab-sence of molybdenum (9). In in vivo activityassays, theR. rubrummolybdenumnitrogenasecould not perform methylthio-alkane reduc-tion, and methylthio-alkane reductase didnot exhibit nitrogenase activity (Fig. 3D; gluta-mate as nitrogen source and 50 mM sulfate).Indeed, nitrogenase and methylthio-alkanereductase activities were independent and sepa-rately regulated, and both systems could be ex-pressed simultaneously (Fig. 3D). R. rubrumnitrogenase gene expression (nifHDK) isregulated by the transcriptional regulatorNifA in response to nitrogen availability (13).Methylthio-alkane reductase activity in thepresence of 1 mM MT-EtOH or DMS wasregulated by sulfate availability, with amedianeffective concentration (EC50) of ~150 mM sul-fate for 50% repression of activity (Fig. 3C).Our random mutagenesis screen identifiedthe specific regulatory gene in the vicinity ofmarBHDK (Rru_A0785; Fig. 1B and figs. S2and S3).We designate this LysR family regulatoras SalR (sulfur salvage regulator) (data S1).Inactivation of salR rendered strains incapableof growth or hydrocarbon production usingMT-EtOH, DMS, or EMS as the sole sulfursource (Fig. 2, A and B, and fig. S4E; strain0785::Tn5). Transcriptomics and differentialexpression analysis of the parent (WRdht) andsalR deletion strain (0785::Tn5) growing undermarBHDK inducing and repressing conditionsrevealed that marBHDK and the rest of themethylthio-alkane reductase methionine syn-thesis pathway are under transcriptional con-trol of SalR (Fig. 1C). Thus, when sufficientsulfur is available (>150 mM), expression ap-pears repressed, but when sulfate becomeslimiting, marBHDK and O-acetylhomoserinesulfhydrylase gene transcription is specifi-cally up-regulated via SalR to use VOSCs formethionine metabolism (Fig. 1A, reactions1 and 3).The nitrogenase superfamily is composed of

the bona fide nitrogenase sequences (groups Ito III) and nitrogen fixation–like sequences(NFL; groups IV to VI) (Fig. 4) (9). Phylogenetic

North et al., Science 369, 1094–1098 (2020) 28 August 2020 3 of 5

Fig. 2. Genes marBHDK are required for anaerobic methionine metabolism from VOSCs. (A) Growthand average total hydrocarbon production of strains using sulfate or VOSCs (see figs. S4 and S15).Asterisk symbol, not applicable. (B) Total amount of hydrocarbons produced when cells were fed with theindicated VOSC. (C) Plasmid-based complementation studies of NFL genes for growth of R. rubrumNFL gene deletion strain. O.D. 660 nm, optical density measured at 660 nm. Error bars in (A) to (C) arestandard deviation for N = 3 independent biological replicates. (D and E) Identification of methionine[retention time (RT) = 8.5 min] and methanethiol (RT = 28.3 min) upon feeding R. rubrum strainswith (2-methyl[14C]thio)ethanol (RT = 22.8 min). (F) Change in Gibbs free energy under standardconditions for the conversion of VOSCs to methanethiol and the corresponding hydrocarbon. H2

represents 2H+ and 2e− from an electron donor, as detailed in the materials and methods section ofthe supplementary materials.

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analysis places methylthio-alkane reductasehomologs in their own clade within group IV,which we denote as group IV-C (Fig. 4 and fig.S12). In contrast, the R. rubrum NflD proteinresides in a separate clade with other NflD se-quences of unknown function (Fig. 4), consistentwith the poor methylthio-alkane reductaseactivity exhibited by NflDK (Fig. 2B). Bacteriahaving MarBHDK sequence homologs of thispreviously uncharacterized group IV-C cladeinclude members of the Fibrobacter and Bac-teroidetes phyla, Rhodospirillales and Rhizo-biales within the Proteobacteria phylum, and

Selenomonadales and Clostridium specieswithin the Firmicutes phylum (fig. S13). Toverify our phylogeny results for the Proteobac-teria, we tested R. palustris and Blastochlorisviridis, which have group IV-C marBHDKhomologs. We also tested closely related speciesRhodobacter capsulatus, which have nitroge-nase andnflBHDKbut nomarBHDK (Fig. 4 andfigs. S12 and S14; Rp, Bv, Rc). Both R. palustrisand B. viridiswere able to growwithMT-EtOH,EMS, or DMS as the sole sulfur source andcorrespondingly produced ethylene, ethane,or methane (Fig. 2A and fig. S15), demonstrat-

ing that methylthio-alkane reductase homo-logs from these organisms catalyze the sameprocess. Conversely,R. capsulatus could not useany of these VOSCs as the sole sulfur source forgrowth (Fig. 2A and fig. S15), like R. rubrumexpressing nflDK but notmarDK (Fig. 2, B andC), indicating that group IV NFL proteins ofunknown function catalyze different processesthan methylthio-alkane reductase.There are several noteworthy similarities

and differences betweenMarBHDK and othernitrogenase and nitrogenase-like sequences(figs. S6 to S9), as detailed in the supplementary

North et al., Science 369, 1094–1098 (2020) 28 August 2020 4 of 5

Fig. 4. Methylthio-alkane reductases are phylogeneticallydistinct. Phylogenetic tree of NifD superfamily homologs.The scale bar represents the number of substitutionsper site. Nodes with UFBoot support values ≥ 95% areindicated with black circles. Clade labeling: Group IV-A(NfaD, nitrogen fixation IV-A) (27); Group IV-B (CfbD,Ni2+-sirohydrochlorin a,c-diamide reductive cyclase) (4, 5);Group IV-C (MarD, putative methylthio-alkane reductase);Groups IV and VI (NflD, nitrogen fixation–like of unknownfunction); Group V (ChlN, DPOR; and BchY, COR).Clade labels and colors are per Raymond et al. (9)and Méheust et al. (28). Av, Azotobacter vinelandii;Bv, Blastochloris viridis; Ep, Endomicrobium proavitum;Rc, Rhodobacter capsulatus; Rp, Rhodopseudomonas palustris;Ru, Rhodospirillum rubrum.

Fig. 3. Methylthio-alkane reductase and nitroge-nase are independent. (A and B) Stoichiometricproduction of methane and ethane by cells fed withDMS and EMS. R2, coefficient of determination.(C) Competition assays for methylthio-alkane reduc-tase repression in cells grown with 1 mM MT-EtOHor DMS plus the indicated amount of sulfate.Nonlinear fit to the Hill equation gives EC50DMS/sulfate =140 mM sulfate and EC50MT-EtOH/sulfate = 110 mMsulfate for 50% activity with DMS and MT-EtOH assubstrate, respectively. (D) Whole-cell methylthio-alkane reductase (Mar) and molybdenum nitrogenase(NifHDK) activities for wild type (WT) andD0772:3/D0793:6 deletion (DD) strains undermethylthio-alkane reductase inducing (50 mM sulfate)or repressing (1 mM sulfate) and NifHDK inducing(Glu, glutamate) or repressing (NH4

+, ammonium)conditions. Standard deviations are [(A) to (C)]the error bars or (D) are <10% for N =3 biological replicates.

CEthylene from1 mM MT-EtOHMethane from1 mM DMS

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text. MarDK have all the requisite cysteines forP-cluster coordination found in the bonafide nitrogenases but not in the nitrogenase-like systems for bacteriochlorophyll and F430biosynthesis (figs. S6 and S7) (9). The nitroge-nase P-cluster shuttles electrons originatingfrom the NifH [4Fe-4S] cluster to the catalyticFeMo cofactor (13). In addition, MarH has theconserved NifH [4Fe-4S] cluster coordinationandMgATP hydrolysis domains for transferringelectrons to a P-cluster (fig. S8) (9). Methylthio-alkane reductases also appear similar to nitro-genases owing to thepresence ofNifBhomologs.MarB has all of the conserved motifs found innitrogenase NifB sequences, particularly theradical SAM domain for carbide insertion intothe NifB-cofactor precursor of the FeMo co-factor (fig. S9) (12). However, the conservedNifD histidine responsible for coordinating theFeMo-cofactor molybdenum and homocitratein nitrogenase is replaced with acidic aminoacids in MarD (fig. S6) (9, 15, 16), which is sug-gestive of an analogous but distinct catalyticcofactor used by methylthio-alkane reductase.Together, this indicates that methylthio-alkanereductase proceeds via a mechanism, albeitcurrently unknown, similar to that of nitro-genase (17).Methane release fromDMSby themethylthio-

alkane reductases is separate and distinct fromthe other known nonarchaeal methanogenicprocesses, including photosynthesis-linkedmeth-ane production by cyanobacteria (18), meth-ane release frommethylphosphonates bymarinebacteria (19), and direct reduction of carbondioxide to methane by iron-only nitrogenase(AnfDHGK) (20). In waterlogged soils, strictlyanaerobic microbial processes produce ethyl-ene that can accumulate to levels inhibitoryto plant root growth, causing crop damage(21, 22). Early attempts at identifying ethylene-producing organisms unexpectedly isolatedoxygen-dependent soil bacteria and fungi

(23, 24). The organisms andmethylthio-alkanereductases we have identified here functionanaerobically and could contribute to this soil-ethylene paradox (10).

REFERENCES AND NOTES

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BMC Genomics 13, 162 (2012).9. J. Raymond, J. L. Siefert, C. R. Staples, R. E. Blankenship,

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11. N. Parveen, K. A. Cornell, Mol. Microbiol. 79, 7–20(2011).

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13. Y. Zhang, E. L. Pohlmann, P. W. Ludden, G. P. Roberts,J. Bacteriol. 182, 983–992 (2000).

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(2017).16. L.-M. Zhang, C. N. Morrison, J. T. Kaiser, D. C. Rees, Acta

Crystallogr. D 71, 274–282 (2015).17. D. Sippel et al., Science 359, 1484–1489 (2018).18. M. Bižić et al., Sci. Adv. 6, eaax5343 (2020).19. D. Repeta et al., Nat. Geosci. 9, 884–887 (2016).20. Y. Zheng et al., Nat. Microbiol. 3, 281–286 (2018).21. K. A. Smith, R. S. Russell, Nature 222, 769–771 (1969).22. S. M. N. Manik et al., Front. Plant. Sci. 10, 140 (2019).23. J. M. Lynch, Nature 240, 45–46 (1972).24. J. M. Lynch, Nature 256, 576–577 (1975).25. J. A. North et al., Mol. Microbiol. 113, 923–937 (2020).26. G. A. W. Beaudoin et al., Nat. Commun. 9, 3105 (2018).27. H. Zheng, C. Dietrich, R. Radek, A. Brune, Environ. Microbiol.

18, 191–204 (2016).28. R. Méheust et al., ISME J. 10.1038/s41396-020-0716-1

(2020).

ACKNOWLEDGMENTS

We thank D. Canniffe (University of Liverpool) for providingBlastochloris viridis. Funding: Electronic structure calculationswere performed at the Environmental Molecular Sciences Laboratory

(EMSL), a National Scientific User Facility sponsored by the U.S.Department of Energy (DOE), Office of Science, Biological andEnvironmental Research (BER) and located at Pacific NorthwestNational Laboratory, which is operated by Battelle, for the U.S. DOEunder contract DE-AC05-76RLO 1830. Transcriptomics work wassupported in part by the University of Colorado Cancer Center’sGenomics and Microarray Shared Resource (NCI grant P30CA046934)with RNA sequencing services performed by K. Diener. Computationalaspects used resources from the University of Colorado BoulderResearch Computing Group, which is supported by the NationalScience Foundation (awards ACI-1532235 and ACI-1532236), theUniversity of Colorado Boulder, and Colorado State University.Proteomics work at Oak Ridge National Laboratory (ORNL) wassupported by the Genomic Science Program, U.S. DOE, Office ofScience, BER as part of the Plant Microbe Interfaces Scientific FocusArea (http://pmiweb.ornl.gov). ORNL is managed by UT-Battelle,LLC, for the DOE under contract number DE-AC05-00OR22725. Thiswork was supported by an OSU Center for Applied Plant SciencesGrant (to F.R.T.) and the Genomic Science Program, U.S. DOE, Office ofScience, BER under award number DE-SC0019338 (to F.R.T., K.C.W.,and W.R.C.). Author contributions: F.R.T., R.L.H., K.C.W., W.R.C., andJ.A.N. designed the experiments. W.X. and R.L.H. performedproteomics. J.A.N. and A.B.N. performed transcriptomics,bioinformatics, and comparative genomics analysis. J.A.N., K.M.B.,S.J.Y., G.Z., J.A.W., and S.M. performed the growth, genetics,and metabolite analysis assays. W.R.C. performed thermodynamicsimulations. J.A.N., W.X., and A.B.N. analyzed the data. J.A.N.and F.R.T wrote the manuscript, with contributions and approvalfrom all other authors. Competing interests: The authors declareno competing interests. Data and materials availability: All dataare available in the main text or the supplementary materials,except as follows: All raw mass spectra and searched data files forthe proteome measurements have been deposited into theProteomeXchange repository: MassIVE accession numberMSV000084455 (FTP link to files: ftp://massive.ucsd.edu/MSV000084455/) and ProteomeXchange accession numberPXD015818. Raw transcriptomics reads are available in the NCBISequence Read Archive under accession numbers SRR10887327through SRR10887338 and BioProject accession numberPRJNA601207.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/369/6507/1094/suppl/DC1Materials and MethodsSupplementary TextFigs. S1 to S15Tables S1 to S6References (29–65)MDAR Reproducibility ChecklistData S1 to S3

View/request a protocol for this paper from Bio-protocol.

7 March 2020; accepted 12 June 202010.1126/science.abb6310

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A nitrogenase-like enzyme system catalyzes methionine, ethylene, and methane biogenesis

Wildenthal, William R. Cannon, Kelly C. Wrighton, Robert L. Hettich and F. Robert TabitaJustin A. North, Adrienne B. Narrowe, Weili Xiong, Kathryn M. Byerly, Guanqi Zhao, Sarah J. Young, Srividya Murali, John A.

DOI: 10.1126/science.abb6310 (6507), 1094-1098.369Science 

, this issue p. 1094Sciencequestions that are now ripe for exploration.highlights the potential of unexplored diversity in this family of enzymes and raises many mechanistic and evolutionary reductase enzymes found in bacteria and archaea. The involvement of such nitrogenase-like genes in sulfur metabolismappropriate substrates were provided. The key genes involved are distantly related to nitrogenase and several other concomitant ethylene production, through this pathway. Methane and ethane were also observed as products whenGrowing cells in sulfur-limiting conditions enabled them to identify the enzymes involved in sulfur salvage, and the

traced the source of ethylene to a small, sulfur-containing organic molecule produced by certain reactions in cells.al.etCuriously, some of these organisms give off ethylene gas when starved for sulfur under anaerobic conditions. North

Soil bacteria have a range of metabolic pathways that contribute to acquiring and recycling nutrients and carbon.Soil sulfur metabolism surprise

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