Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. uts NR r h w eta nye nti aha.Hr epeetapeiul neotdNir unreported previously Nir a within domains present protein we of re- Here analyses oxide Phylogenetic pathway. nitric impact, this Chloroflexi. and Phylum widespread in of (Nir) ma- has enzymes members reductase pathway from central in Nitrite architecture this two nitrogen domain through the removing climate. are species and and (NOR) nitrogen reducing agriculture, ductase of capacity, cycle, flux carrying nitrogen The ecological global affecting ecosystems. the terrestrial in and role rine central a plays Denitrification SUMMARY 497-1747 (415) +1 [email protected], *Correspondence: Fournier P. Gregory Schwartz L. Chloroflexi Sarah Phylum in in Architecture repertoire Denitrification Novel denitrification TITLE: chimeric SHORT a suggests structure domain Chloroflexi reductase nitrite Novel TITLE: previously eNOR. been have subtype, than reductase PAPER exist oxide RESEARCH arrangements nitric enzyme reported domain denitrification rarely chimeric of a this diversity of produced greater event diversity analyses a fusion genomic Phylogenetic reported. and suggest expanded transfer results Chloroflexi. an horizontal Phylum these identify ancestral of Together, an also members pathway. that We this in indicate in architecture enzymes Nir architecture. domain central terrestrial within capacity, two Nir the carrying domains and unreported are ecological protein marine (NOR) previously affecting of from reductase impact, a oxide nitrogen widespread nitric present removing a and we has (Nir) and Here pathway reductase reducing this Nitrite cycle, through climate. nitrogen species and nitrogen agriculture, global of the flux in The role ecosystems. central a plays Denitrification Abstract 2021 11, June 3 2 1 Fournier Gregory and Schwartz Sarah Chloroflexi Phylum chimeric in a repertoire suggests denitrification structure domain reductase nitrite Novel nvriyo otenCalifornia Southern of University Zurich ETH Technology of Institute Massachusetts Dprmn fBooia cecs nvriyo otenCalifornia Southern of University California Sciences, Southern Biological of of University Department Sciences, 6. Earth Zurich of ETH Department Sciences, Technology 5. Earth of of Institute Department Massachusetts CA Sciences, 4. Pasadena, Planetary Inc., and Exponent, Atmospheric, Technology3. Earth, of of Institute Department Massachusetts 2. Program, Graduate Microbiology 1. 1 1,2* iyMomper Lily , 2 iyM Momper M. Lily , 1 1 .Tiei Rangel Thiberio L. , 2,3 .Tiei Rangel Thiberio L. , 1 1 aaMagnabosco Cara , 2 aaMagnabosco Cara , 4 a .Amend P. Jan , 2 a Amend Jan , 5,6 3 and , , Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. hl eirfiainhsbe otwdl tde n bevdi rtoatra h rcs a also has process diverse, the physiologically Proteobacteria, and ecologically in are observed Chloroflexi and Chloroflexi. including studied phyla, widely other in most identified been been has (S´anchez denitrification flavohemoglobins or While 2002), Gardner, and alternative Helmick al. including (Hendriks (Gardner, et flavorubredoxin possible, oxides and 2002), are (Blomberg nitric Gardner, oxidase detoxification environmental and c oxide cytochrome detoxify nitric as to presumably such pathways enzymes can alternative qNOR— NORs, and quinol-dependent Beyond example, non-denitrifiers NOR—for 2000). of in types other found microbes, denitrifying are in found only to are cNORs oxide a it nitrous not before to but oxide oxide contain gene nitric that nitric detoxifying genomes reduce or find immediately cytotoxic. (Hendriks effluxing to enzymes highly to of NOR thought is chemistry means membrane-bound NirK, are a the using injury, and require Denitrifiers by and avoid NirS cells levels. constrained of taxa so lethal be and product to different may periplasmic, the accumulates across variation are (NO), oxide types this vary Nir nitric of to Both example, Some For functions appears 2014). intermediates. non-denitrifying may genes certain Hallin, for of organisms pathway and Such enzymes Jones denitrification genes. these (Graf, of denitrification environments of co-occurrence four one the the use denitrifers, of may subset others partial while (Hendriks a denitrification, or partial one perform only contain bacteria in characterized Many (Stein and Chloroflexi identified and recently alternative Archaea Sievert more Rarer, Firmicutes, 2008; been respectively. Proteobacteria, Gennis, have donors, the eNOR, of electron widely and members quinol gNOR most limited or sNOR, The cytochrome including 2008). enzymes, their Gennis, NOR by oxide oxygen and distinguished nitric to (Hendriks (Hemp and by 2014), qNOR another, and superfamily catalyzed Hallin, cNOR one oxide—is reductase are to enzymes nitrous oxygen related NOR to closely heme-copper studied and oxide the homologous nitric in are of NORs reductases reduction bacterial pathway—the Most the (NORs). reductases in NirK, step reductase next key nitrite The of copper-based diversity enzymes—the the distinct by two (Braker NirS in underscored al. reductase found proteobacterial is cytochrome-type is Nir pathway among the reductase, functionality the diverse and nitrite Nir of is especially enzyme utility oxide. is denitrification nitric the and canonical The 2002); taxa, enzymes. various Philippot, constituent in 1997; reported (Zumft, widely groups Canfield, been 2002; has (Philippot, Denitrification gas to dinitrogen capacity link to genomic 1 nitrate Figure the alternative reducing have an 1), as microorganisms 2010). Falkowski, nitrite diverse Decleyre and or (Fig. Several 1997; Glazer nitrate denitrification (Zumft, using zones. gases complete facultatively greenhouse oxygen-limited its perform often potent for in extensively denitrification, of acceptor studied source of been a electron capable has as are and and bacteria cycling loss Diverse nitrogen nitrogen global fixed in in pathway key role a is denitrification Microbial cytochromes reductase, INTRODUCTION nitric-oxide reductases, nitrite Chloroflexi, phylogeny, have Denitrification, than exist arrangements eNOR. enzyme subtype, reductase denitrification KEYWORDS oxide of nitric diversity reported architecture. greater reported. rarely domain previously a a chimeric been suggest of this diversity results produced genomic event these expanded fusion Together, an and identify transfer also horizontal We ancestral an that indicate 2016). , 2011). , tal. et nir 00 Sanford 2000; , eeaefrmr omn(Hendriks common more far gene—are tal. et 08 Hemp 2008; , nir tal. et u not but 02 rf oe n aln 04 Roco 2014; Hallin, and Jones Graf, 2012; , tal. et nor hl raim hwn h nes—h rsneo a of presence inverse—the the showing organisms while , 2015). , tal. et 2 dloh 08 roioeuts Grnr Helmick (Gardner, oxidoreductase or 2018) Adelroth, ¨ tal. et 00 re´,Bae n ide 02 Decleyre 2002; Tiedje, and Priem´e, Braker 2000; , tal. et 00 epadGni,20;Ga,Jnsand Jones Graf, 2008; Gennis, and Hemp 2000; , tal. et 00 rf oe n aln 04.While 2014). Hallin, and Jones Graf, 2000; , 00.Prasfrti esn ti rare is it reason, this for Perhaps 2000). , , tal. et tal. et hc eue irt to nitrite reduces which 07.I partial In 2017). , 07 epand Hemp 2007; , tal. et 2016). , tal. et nor et , Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. vlto a eplrzn h eaiepaeet fgop ntete.Ti ako outescue by caused robustness of lack This Chloroflexi tree. in to the differences in unrelated the notable groups patterns of are for that placements there relative responsible suggesting Additionally, the likely Proteobacteria, polarizing S4). are the be Fig. may noise within S3, evolution phylogenetic subclades (Fig. phylogenies and among relative these the placement sampling inverts in domains that both differences suggesting containing observed ORFs Chloroflexi, from exclusively of reconstructed placement trees domain NirS and C2 link 3b Figure proteobacterial large grouping. the polyphyletic link Ep- to the 3a Bacteroidetes, to sister Figure sister Aquificae, placing are comprising group Chloroflexi combined group NirS, Gam- the polyphyletic for and group; with the Beta-, Spirochaetia, within trees: Alpha-, and domain fall diverse from both silonproteobacteria, sequences sequences containing between 3b) slightly proteobacterial Chloroflexi group varies this (Fig of sequences C2, polyphyletic cases, Chloroflexi grouping domains For both a of large NirS placement In to a relative the The Spirochaetia. to related maproteobacteria. and and sister closely is Epsilonproteobacteria, 3a) Chloroflexi group Bacteroidetes, (Fig. of polyphyletic Aquificae, C2 clade from for a sequences topology recover NirS domain. overall both domain NirS-specific similar maximum-likelihood trees the ancestry, a These and different C2, indicate have C1, phylogenies for enzyme Domain event. gene Methods) the fusion (See multiple of and reconstructed of acquisition domains independently horizontal fusion different were ancestral trees lineage-specific the an C1 A if for of evidence determine Chloroflexi. domain inclusion provide the To to the among would histories Chloroflexi limited reductases, evolutionary Different within nitrite and arrangement. subunits C1-C2-NirS in rare novel this co-occur extremely explain frequently could appears domains domains ORF functional the NirS in and C2 4.06x10 the = 2006). Though e-value Rosato, 13442, and (pfam Cavallaro (Bertini, III cytochromes subunit two oxidase Conserved c NCBI’s e-value=5.31x10 cytochrome COG2010, by (NCBI type variant identified diheme and was mono- ORF C Anaerolineales-type 6.4x10 the = e-value in (C1) (Marchler-Bauer Database domain Domains cytochrome first The S1). link Datafile cytochro- MAG 2 two Supplementary SURF and Figure 2, from domain (Fig. NirS (ORF) domains cytochrome-type frame one superfamily reading interest: C of open me regions reductase functional nitrite putative three Anaerolineales-type 4indicated the of analysis Domain NirS Chloroflexi Information’s in Biotechnology fusion for chimeric Center as Apparent National well as the genomes, from databases. metagenome-assembled protein metagenomes deni- Chloroflexi (NCBI) and of sequenced analysis genomes recently comprehensive available 100 a previously over performed we in Chloroflexi, homologs and in trification genes bacteria, denitrification of divergent investigate subset origin. To this their and for of metabolisms unusual variant denitrification be microbial divergent Hemp RESULTS may of a 2008; understanding denitrification broader harbor Gennis, of a also biochemistry and for may and/or informative (Hemp evolution and Archaea the 2018), of that Fischer, members and in McGlynn reported Ward, possess Anaerolineales—may 2015; Order , of reduction (Wei members nitrite for NirK Chloroflexi—especially capacity the copper-type have and may the sludge Thermomicrobia and via anaerobic Chloroflexia including Classes environments, of members light-limited Chloroflexi, and (Hug nutrient-, oxygen-, sediments in subsurface players key often and -4 .Tescn yohoedmi C)wspeitdwt ihseict sacytochrome a as specificity high with predicted was (C2) domain cytochrome second The ). tal. et tal. et 03 Ward 2013; , tal. et 05 Decleyre 2015; , 05 Lu 2015; , tal. et tal. et 3 08.Peiu uvy aeidctdta,within that, indicated have surveys Previous 2018). , tal. et 00 sactcrm 5152(CICOG4654, (NCBI c551/552 cytochrome a as 2020) , 06.Rcn tde niaeta certain that indicate studies Recent 2016). , -9 .C nlddargo rdce sacbb3- a as predicted region a included C2 ). tal. et -7 ;sc uuisfeunl contain frequently subunits such ); nirS 05.Teefidnssuggest findings These 2015). , nta of instead nirK nor (Hemp previously tal. et . Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 2Nr eino h uaiegn SplmnayDtfieS) ffrst neednl oe h C1 the model independently to Efforts S3). Datafile (Supplementary gene putative the of (Bienert region modeling homology C2-NirS using structure al. enzyme full et nitrite the containing chimeric visualize Chloroflexi-type ORFs the to NirK of in Attempts or diversity identified missing NirS no were metagenomes. with to existing little homologs co-occurred is in none C1 there reductase query that subunits, putative a suggest or these data as domains These domain of domains. well superfamily C1 identified some as the were enzyme domain using While cytochrome-type hits C1 database hits. several the additional env-nr included overall While the these of construct. few of search gene none returned SURF independent domains, full An from C2-NirS S2). the joint ORF Datafile the of (Supplementary full to examples homology the additional putative with reflected identify that (env-nr) not other database in did observed environmental 42 not is non-redundant MAG and Chloroflexi, NCBI’s to Querying unique Nitrospirae, appears groups. Proteobacteria, architecture in C1-C2-NirS (largely full these reductases The respectively, Nitrospinae). nitrite regions, and cytochrome-type C2-NirS or and cytochrome C1 different constituent reflect the hits for independently exist homologs putative Chloroflexi Though to transfer Unique independent an is Nitrospinae. Architecture for and similar Domain source Nitrospirae architecture—or C1-C2-nirS the into Gammapro- C2-NirS been domain and the have C1 Beta-, lineage, may free Alpha-, donor Chloroflexi the the do- the that of of C1 infer appears members the it reliably among Additionally, of to widespread teobacteria. fusion us domains—is of allow functional result of not the arrangements does strongly is domains, trees Chloroflexi NirS gene in and Proteobacterial these C2 observed horizontally-acquired with a structure compared with domain as main C1-C2-NirS C1, the of that distribution taxonomic suggest copper-containing and other phylogeny or distinct cupredoxins The contained ORF, the also analyzed NirK) of the S1). of copper-type several (Fig. length (not However, domains the NirS artifact. across cytochrome-type identical assembly a NirK are which an and copper-type MAGs, fusion represent the of this with number may As small cytochrome domain. this a cupredoxin/plastocyanin Chlo- C1 N-terminal within the the an apparent included pairing only to also is ORF restricted ORF an NirK is This contain genes instead. MAGs domain reductase Chloroflexi nitrite C1-containing of number predicted these small of within homologs majority domain The data, roflexi. C1 additional Nitrospirae of limited the yielded to occurrence of genes similarity members The ORF these sequence several The of detectable in S1). analysis showed reductase (Table domain sequences free oxide domains representative S1); a nitric isolated (Table but protein as a or genome structural as either Geobacteraceae rare or annotated homolog one In membrane was and domain domains. with homolog C1 superfamily C1 ORFs the cytochrome the in contain or containing co-occur tree cytochrome-type homologs multiple domain C1 of C1 cases, one the as in or represented domain cytochrome, ORFs other of the majority in The seen that from link different 4 is Figure and Chloroflexi placement tree Additional the C1 However, domains. the Proteobacteria. domains C1 related in NirS two distantly trees. Chloroflexi Proteobacteria more or other of of of C2 the clade representation number small for taxonomic large than a a trees history with within evolutionary group domain sequences together different the NirS grouping much and to Nitrospinae C2 a contrast and the shows In that C1 hypothesis 4). null for (Fig. reflective the phylogeny not reject inferred Chloroflexi. and to within artefactual, reason The origins no be evolutionary may be same to topology the seems tree clade have this There in domains between events. differences or reticulation the clade gene that polyphyletic of the suggest within Proteobacteria, values the support and poor with combined sampling, alternative 08 eeuscesu;srcua oeswr nyal opeitacoemthfrteconserved the for match close a predict to able only were models structural unsuccessful; were 2018) , nir ee aetectcrm-yeNr oan oee,a however, domain; NirS cytochrome-type the have genes nir 4 ee huhtplg n xattxnsmln of sampling taxon extant and topology Though gene. , h 1te hw eune rmNitrospirae from sequences shows tree C1 the nir suooa norC Pseudomonas ee htcnandaC homolog C1 a contained that genes tal. et 07 Waterhouse 2017; , genes. Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. R nClrflx ihu h ue 1dmi rsn,tefso rbbyocre eyso fe the after soon very occurred probably fusion ta- the any incorporated present, likely in domain was C1 ORF cytochrome fused C1-C2-NirS the C1 full without the a domains Chloroflexi that other of in suggest the absence ORF data of in apparent these that the domains than Chloroflexi, with different into C2-NirS than strikingly Combined other paired is ORF. group the tree reductase xonomic of domain taxono- nitrite C1 the presence taxo- the the contrast, the the In of in distributions, phylogeny and Proteobacteria. or the and similar, throughout histories distribution very broadly evolutionary mic is appears identical reductases have groups nitrite not these cytochrome-type do of domains representation NirS nomic and C2 an the also Although is and cycling treatment as characteriza- waste nitrogen such and or discovery genes applications. global The agricultural of in variants 2014). with novel Stensel, role of associated and Chandran tion crucial those Lu, as a 2011; such Dannenmann, eco- plays and systems, microbial the (Butterbach-Bahl species applied smaller-scale of nitrogen and of understanding uni- basic fixed component deeper the for essential of a implications to broadly, reduction important adaptations More has The environments. reflect denitrification logy. microbial these may of within which extent cycling enzymes, ecological nutrient denitrification of of challenges diversity que under-described an harbor of analyses phylogenetic The Gennis, and (Hemp DISCUSSION subunits oxidase link heme-copper 5 the Figure of similarity Deltaproteobacteria. structural and Gamma-, the Beta-, of CDD). Alpha-, because the likely of members genes, many putative in these and of Chloroflexi, Many other as putative well Additional as isolated well. one as in least Halobacteria eNOR other at described and have 2008) of studies presence Gennis, the and indicates (Hemp analysis active Hemp Phylogenetic Archaea an 2008; in included Gennis, 42 described (Hemp and MAG previously bacterium SURF (Hemp been Anaerolineales do- in eNOR (Hendriks has electron NOR of Proteobacteria eNOR as predicted characteristic of S4). the quinols However, distribution substitution or 2008). broad glutamine c Gennis, a site cytochrome and as contain Hemp well (which 2005; as enzymes Zumft, novel unusual Chloroflexi, family the in an which qNOR respectively) of in (nitric and nors, presence genome Nir cNOR assembled the of established original reveal product the metagenome—the the observed—did 42 of MAG was toxicity SURF ORF nitric the the (Hendriks a of and counterintuitive contain analysis uncommon, absence to wever, relatively this appear makes is not NO) it do oxide, suggest structure surveys C1-C2-NirS genomic unique previous the ( with gene architecture reductase genomes NirS oxide of novel majority the the Notably, with Chloroflexi diversity (His239) among eNOR His327 of conserved Expansion to (His46, is alignments corresponding it residue NirS S1). though the and interestingly, Datafile conserved, Met125) (Supplementary ORF; universally (His65, (Rinaldo NirS C2 anion not Chloroflexi the nitrite is the within site for conserved active His300) are the His239, residues stabilize to Corresponding believed of 2014). structure are Chloroflexi crystal His369 the the However, and in region. present coding residues In (Benkert cytochrome conserved NirS. the several -4.50 of retain above length does scores short sequence relatively QMEAN the predicted reflect recover may not could structure nirS .aeruginosa P. nagn uineetwti hooei olwn G.A hr sn vdneo h C2-NirS the of evidence no is there As HGT. following Chloroflexi, within event fusion gene a in nor iS i5 n e8 oriaehm ;Hs8 oriae eed;adHis327 and d1; heme coordinates His182 c; heme coordinate Met88 and His51 NirS, eNOR .Tog h bec fthe of absence the Though ). nirS isapa ohv enmsnoae rmsaee sctcrm oxidase c cytochrome as mislabeled or misannotated been have to appear hits tal. et Natronomonas and nirS eNOR 2015). , and Rsi hooeisgetta usraeeoytm may ecosystems subsurface that suggest Chloroflexi in ORFs eNOR eNOR eNOR hs aaidct lse of cluster a indicate data these ; 5 na xadddvriyo eoe Fg ) Previous 5). (Fig. genomes of diversity expanded an in a hrfr aetewyfrftr biotechnological future for way the pave therefore may ee peri utpemmeso Anaerolineales, of members multiple in appear genes nor tal. et eei eoe with genomes in gene nor 00 rf oe n aln 04.Ho- 2014). Hallin, and Jones Graf, 2000; , ooo.Peiu tde aeidentified have studies Previous homolog. tal. et tal et nirS 01 aaadMoura, and Maia 2011; , suooa aeruginosa Pseudomonas eNOR 01.Tepo scores poor The 2011). sntunprecedented, not is tal. et ee throughout genes 05 (Table 2015) , tal. et nirS 2000; , gene nirS Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. htteeitn nesadn fdnticto a neetmt h eei iest n ecological and diversity genetic the underestimate may denitrification of understanding existing strategies the metabolic different that reflect not may or differently may situ above performs described NirS enzymes Chloroflexi-associated denitrification gene divergent novel The this the Experimen- use domain. if C1 instead determine the NirS and to by canonical constrained denitrification, the necessary than or groups, be the perform enabled denitrifying would not and potentially known validation pathway, function, do from tal metabolic denitrification Chloroflexi NirS different active of a and an for group domain product suggests this this between that NirS in homology remains domain of the C1 possibility presence reflects a the tree of domain while inclusion Additionally, NirS Proteobac- the NirS. to that of similarity possible detectable duct therefore with is lacking reductases, It genomes oxide subunits. within nitric reductase as several oxide annotated nitric in be are teria cyto- enzymes can and membrane-anchored reduction reductase homologs a oxide C1 oxide contain contain Nitric nitric reductases fusion. inclusion oxide of C1 the nitric absence the that chrome cNOR conspicuous with possible well-studied ORF the also the NirS to is cytochrome-dependent; a It linked capacity. containing cytochro- be or genomes upstream could requirement metagenome-assembled the surprising, redox construct of not additional addition this is reflect cytochrome- unusual of It is may the itself However, 2006). (C1) NirS 2006). domain Rosato, reductase. Rosato, nitrite me and with and Cavallaro Cavallaro frame (Bertini, in (Bertini, passage subunits dependent c-containing electron cytochrome allow find to to therefore, enzymes redox to sed superfamily Cytochrome analyses. reductase oxygen metagenomic broader or less- from are genomic distinguished sNOR accurately in and be members eNOR not as may such and enzymes oxide documented divergent nitric However, extensively quinol-dependent evidence well-established. and fairly phylogenetic (cNOR) are cytochrome-dependent The (qNOR) of reductases reductases. function oxide and Diversity nitric do, reductases. oxide however, and bacteria surveyed reductases knowledge, diverse of nitrite our number for between To small correlation 2014). a genomic llin, survey—but the recent Ha a and in harbor Jones genomes to of appear 4% interestingly, than fewer This NirS representing 2014). Hallin, of , and product Jones cytotoxic Graf, reducing a 2007; of highly harbor means the efficient to an oxide, genomes provides nitric reductase nitrite oxide detoxifying in nitric itself. and capacity, role domain redox reduction generalized downstream nitrite providing but a Beyond the important of of absence an profile apparent serve genetic The or may history pattern cytochrome evolutionary distribution C1-type co- the This evolutionary the ORFs. of The cupredoxin general denitrification that reduction—regardless S1). a a into possibility suggests Fig. cytochrome contain domains the this 4, ORFs Nir incorporate raises (Fig. copper-dependent to Chloroflexi architecture and Chloroflexi is Several cytochrome- C1-C2-NirS within surprising both trend tree: the with Similarly to domain C1 2006). N-terminal of C1 Rosato, occurrence domain the and copper-containing Cavallaro in similar (Bertini, revealed or enzyme relationship NirK inverse the the of cytochrome of fold cupredoxin-like co-occurrence The the enzyme. copper-type copper-type a in the is genomes exclusive NirK always—mutually not NirS, Chloroflexi often—though containing (Jones are some genomes and origin, in denitrifier evolutionary known shared found among Chloroflexi. a show were in not gene do domains enzymes the two cytochrome of function C1 containing the of ORFs for necessary homologs be putative may Interestingly, and region C2-NirS the of acquisition u h dnicto fbt novel a both of identification the But . c Hm n ens 08.Frhr h 1dmi rercvr Rsi h irsia that Nitrospirae the in ORFs recovers tree domain C1 the Further, 2008). Gennis, and (Hemp eNOR c rtisfnto seeto rnfrpoen naarbcrsiainadaeotnfu- often are and respiration anaerobic in proteins transfer electron as function proteins nirK nirK ooossget ieyudcmne rudrxlrddvriyfrdvretnitric divergent for diversity underexplored or undocumented likely suggests homologs nvivo in nir a enietfidi aeisacsi rtoatra n oe ssrrsn,given surprising, as noted and Proteobacteria, in instances rare in identified been has not , eNOR eewtota without gene nor hswr,teeoe ugssapoiigdrcinfrftr investigation. future for direction promising a suggests therefore, work, this ; nirS eet oegnrlzdNRlk oei eoicto ftecttxcpro- cytotoxic the of detoxification in role NOR-like generalized some reflects ooo ntemjrt fgnmswt h C1- the with genomes of majority the in homolog nirS Fg ,Fg 1.Tog iSadNr r ucinlyeuvln,the equivalent, functionally are NirK and NirS Though S1). Fig. 4, (Fig. ihu loharboring also without tal. et nir-nor nor nirS 08 rf oe n aln 04.Ulk h cytochrome- the Unlike 2014). Hallin, and Jones Graf, 2008; , ee atclryfrognsswith organisms for particularly gene, R n nepne iest feO nye suggests enzymes eNOR of diversity expanded an and ORF imthi uhrrrfrpttv eirfir with denitrifiers putative for rarer much is mismatch 6 eNOR cNOR a o enicue nsc nlssof analyses such in included been not has . or ti o nrcdne o bacterial for unprecedented not is It qNOR (Heylen nirS c oan nOF with ORFs in domains nirK uini unexpected. is fusion tal. et (Heylen 07 Graf, 2007; , nir tal. et genes nirS in , Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 1 hr o-hooeiht eesvrl iie seblw.Dpiaesqecs(rmsriswith strains for (from removed sequences One removed. also Duplicate were was variant). surveyed) alignment below). filter databases gene Sequence (see multiple genus-level novel in limited The the of or severely of exception entries genus. were genome distribution the hits per multiple with taxonomic 1x10 allowed non-Chloroflexi over- level, the was where of genus capture [?] hit sampling C1, the E fully multispecies biased to with (to divergent and subsampled hits Chloroflexi imprecision additional, were quality; the limiting hits for while of diversity Proteobacteria), assessed members capture and (e.g., To combined groups were represented analyses. S4) initial (Table for databases included SURF all June from from of as data Hits (env-nr, sequence database sequence protein metagenomic environmental using non-redundant 2020)(Agarwala NCBI’s evaluated query were to 42 homologs MAG environmental putative Additionally, (Camacho that scaffolds for on step Sequences simply This sampling sequence not Chloroflexi. and were to construction here also genome. database presented were Chloroflexi Genetic putative interest genes a using of flanking into database genes all binned RefSeq transforming for incorrectly NCBI were nitrogen hits the the top on that that searched individually ensured confirming were algorithm, pipeline study BLASTp review) this the expert in presented Genomes gene Microbial a (Integrated denitrification as IMG-ER interpreted Institute was Genome (Markowitz genes Joint accepted marker DOE widely copy the five single through from these Alneberg values of 2013; composite any , the of (Creevey using contamination copies of calculated Duplicate measure was metrics. (Momper gene Completeness essential in S2. core Table used in methods listed the are to corresponding according as well reconstructed (Lau as were previously fluids, described those from been extractions have Magnabosco DNA data genomic geochemical total and and physical samples fluid all of Collection assembly and investigation. sampling further Genome large—requires at dynamics nitrogen PROCEDURES microbial EXPERIMENTAL subsurface of scope ( the reductase as oxide well nitrous Hug as such 2012; genes , pathway denitrification (Hug to dynamics Chloroflexi cycling subsurface carbon in (Hemp roles metabolisms key play they where Momper systems, aquifer (Hug and sediment cycles subsurface deep biogeochemical microbial those global diverse increasing influence as dynamic, al. garnered to of such have potential et existence biomes, systems metabolic the These subsurface the supports isolated. with deep evidence were ecosystems extensive in study years; true this recent in in especially analyzed attention be Chloroflexi may several which This from enzymes. constituent of distribution Tefl CInnrdnatpoendtbs a f2 etme,2019)(Agarwala September, 25 of (as database protein genomes non-redundant bioreactor NCBI sludge full assembled The MAG recent 3. from genomes 86 data for MAG constructed deep-subsurface databases from Genome assembled 2. genomes Chloroflexi 21 for constructed databases Genome 1. (Parks Momper Rapp´e, 2017; and Amend (Jungbluth, 04 09 Momper 2019; 2014, , tal. et tal. et tal. et tal. et tal. et tal. et nirS 07 Kadnikov 2017; , tal. et 09 he eoi repositories: genomic three 2009) , tal. et 06 Momper 2016; , 06 Heard 2016; , 08 Huntemann 2008; , 07 TbeS3) (Table 2017) , and tal. et 08 SplmnayDtfieS). S2) Datafile (Supplementary 2018) , 04.Idvda eoe eete umte o eecligadannotations and calling gene for submitted then were genomes Individual 2014). , eNOR 05 Denef 2015; , tal. et tal. et ee rmSR A 2(al 1 eeue sqeist BLAST to queries as used were S1) (Table 42 MAG SURF from genes tal. et tal. et 07.Clrflx r rqetyctda elrpeetdmmesof members well-represented as cited frequently are Chloroflexi 2017). , tal. et 07 Momper 2017; , tal. et 00.BtClrflx r nw oas abrdvrenitrogen diverse harbor also to known are Chloroflexi But 2020). , tal. et 07.Terl fClrflx nsbufc irgncycling—as nitrogen subsurface in Chloroflexi of role The 2017). , 01 Dupont 2011; , 05.Frqaiycnrlproe,tegnsflnigevery flanking genes the purposes, control quality For 2015). , 06 Spieck 2016; , tal. et tal. et 7 tal. et 07.AlMG rmNrhAeiaadAfrica and America North from MAGs All 2017). , 07 TbeS1). (Table 2017) , tal. et 02 uadSot 02 Campbell 2012; Scott, and Wu 2012; , tal. et 00,adpeiu tde aelinked have studies previous and 2020), , tal. et 07.MGietfir n sources and identifiers MAG 2017). , 04 06 Osburn 2016; 2014, , tal. et nos (Sanford ) tal. et 03 Osburn 2013; , tal. et tal. et 2018) , -10 2014; , 2013; , tal. et tal. et were Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ottasadapoiaelklho ai et eepromduigI-resutaatbosrpand bootstrap ultrafast IQ-Tree’s using performed were tests (Kalyaanamoorthy ratio likelihood command approximate (-MFP) and ModelFinder bootstraps the (Nguyen by IQ-Tree using defined constructed 2017). were Dagan, trees and Maximum-likelihood Landan (Tria, rooting (MAD) deviation construction Tree ancestor outgroup minimal identify using to rooted were expanded Sequences was S2) increase NirS to and 2017). phyla (Fig. C2 Dagan, all tree and for 10 Landan removed preliminary (Tria, was [?] A rooting filter E genus-level (MAD) with the hits phylogeny. genera), including by domain total sequences (17 the hits of initial of resolution paucity a to Due branch the on rooted C1 manually and construction, outgroup. tree the before to re-aligned a leading were in sequences observed Remaining WP subgroup domains. CCQ74688.1, major sequences per outgroup representative Retained divergent taxon WP other S4). single or Datafile a (Supplementary superfamily to the tree reductase subsampled crosslinking. of preliminary (oxygen cofactor were members sequences in from reductases) Outgroup role also oxide a S5). plays but nitric (Table that reductases, 2008) site oxide this Gennis, in nitric and Tyr other conserved (Hemp from a have only which superfamily, not reductase eNOR oxygen distinguishes site This 333. Ingroup eNOR identification outgroup and Rooting identified the beyond To tree the to Because S4). prior Table realigned (see sequences region remaining N-terminal NirS the conserved and poorly removed, a manually showed was domain construction. region tree. C1 this the the accuracy, in improve for artifacts attraction alignment branch initial taxa. long An other of than evidence sites clear alignment no fewer show and upon C1 residues based site therefore active is key sequences retain (Actinobacteria these All of sequences placement Two the alignment; OQY7467.1; UTPRO2, construction. removed, bacterium tree Rhodocyclaceae manually and RME47896.1; was to bacterium, region (Chloroflexi prior sequences this three and realigned misalignment, avoid were and RBG sequences accuracy bacterium improve remaining to the S4); and Table (see ORF conserved the the for NCBI’s alignment by preliminary identified A domains re-aligned. individual then was of domain Protein eNOR Each length S3). S1). the (Table were Datafile selection to protein (Supplementary model the database trimmed evolutionary of domains were and regions conserved analyses alignments in phylogenetic sequence insertions confounding anomalous avoid or to regions removed missing substantial (Nakamura with auto-parameterization sequences using MAFFT, with al. aligned et were sequences protein homologous Putative hdtemsprofundi Rhodothermus 9206.,WP 097280063.1, 08,advsaie nJliw(Waterhouse Jalview in visualized and 2018), , eNOR nirS eune eeietfidb h rsneo osre l eiu nainetposition alignment in residue Gln conserved a of presence the by identified were sequences 16 oanhdaCtria lcmn nteOFars is -emnlstsextending sites C-terminal hits, across ORF the in placement C-terminal a had domain 68 nirS 2 F7691 and OFW73639.1, 12, 8782.,RC99.,adWP and RLC59399.1, 089728124.1, oanwr nlddi h rme alignment. trimmed the in included were domain WP , eNOR 7751.)wt isn -emnlrgoswr nlddi h final the in included were regions C-terminal missing with 072715415.1) eesoe orycnevdrgo erteCtria n of end C-terminal the near region conserved poorly a showed gene -10 h eutn rewsroe sn iia netrdeviation ancestor minimal using rooted was tree resulting The . Thermus tal. et 8 WP 09.Ainet eemnal uae;partial curated; manually were Alignments 2009). , 8740. r noae suncharacterized as annotated are 083704903.1 1774.)wt isn -emnlregions N-terminal missing with 015717644.1) tal. et tal. et 05,udrteotmlmodel optimal the under 2015), , 07 TbeS) Ultrafast S6). (Table 2017) , 100277903.1, Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. Research S. functions’, Bienert, and 10.1021/cr050241v. Occurrence doi: c: ‘Cytochrome 90–115. (2006) pp. A. Rosato, , and G. Cavallaro, I., Bertini, M. H. Berman, 10.1038/nmeth.3103. doi: J. Alneberg, Research Acids Nucleic R. 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Bio- Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ea,adGmartoatra upr ausfrslce iattosaelbld(LTb) Support (aLRT/bb). labeled Alpha-, are of bipartitions radiation selected broad Epsilonpro- for a including values to clade Support sister diverse is a Gammaproteobacteria. clade in and this Chloroflexi Beta-, Planctomycetes; the and places Bacteroidetes, homologs Aquificae, domain teobacteria, C2 domain. domain of reductase analysis C2 nitrite Phylogenetic for C-terminal tree a Phylogenetic and domains 3a. indicates superfamily RJP53747.1) Figure cytochrome (GenBank reductase distinct nitrite two Chloroflexi of 42 presence MAG the SURF of map analysis oxide domain domain nitrous Conserved frame at and reading (NOR) Open reductase figshare 2. oxide ( of Figure nitric enzyme gene by in first followed reductase canonical 2014), the nitrate (Nos). Hallin, considered available The reductase and is Jones (Nir) reductase gas. (Graf, openly nitrite denitrification dinitrogen organisms; to non-denitrifying are in nitrate distributed reduces denitrification study Complete this Denitrification 1. of Fig. https://github.com/slschwartz/fournierlab-scripts. findings at LEGENDS available FIGURE are the analyses automate to used support Scripts that http://doi.org/10.6084/m9.figshare.14515554. data The heme-copper respiratory, the on STATEMENT AVAILABILITY emphasis DATA with prokaryotes of reductases oxide type’, AM- ‘Nitric oxidase (2005) with G. sequences W. Zumft, archaeal denitrification.’, and of basis MMBR bacterial molecular and : of biology reviews ‘Cell analysis (1997) G. ‘Phylogenomic W. Zumft, (2012) J. A. Scott, PHORA2’, and M. Wu, mej.2015.9. previously’, considered W. Wei, workbench’, M. A. Waterhouse, Research anaerobic Acids divergent Nucleic a of sequence A. spring’, genome Waterhouse, hot ‘Draft 10.1128/genomeA.00571-18. sulfidic (2018) a doi: W. from 1–2. W. Ardenticatenia Fischer, class pp. Chloroflexi and the E. of S. member McGlynn, M., L. Ward, deviation’, ancestor minimal using transfer’, rooting M. ‘Phylogenetic L. (2017) Ward, T. Dagan, and evolution G. & ecology Landan, Nature K., LLC. D. Schrodinger, F. date). Tria, (no System’ Graphics Molecular PyMOL ‘The adaptation’, niche for 10.1111/j.1462-2920.2007.01409.x. Implications C91: tropha Y. L. Stein, tal. et rnir nMicrobiology in Frontiers Bioinformatics Bioinformatics tal. et tal. et ora fIogncBiochemistry Inorganic of Journal 21)‘ihrdvriyadaudneo eirfigmcoraim nevrnet than environments in microorganisms denitrifying of abundance and diversity ‘Higher (2015) tal. et 14,p.5366 o:http://dx.doi.org/10.1016/j.sbspro.2014.08.122. doi: 533–616. pp. 61(4), , 20)‘hl-eoeaayi fteamnaoiiigbceim irsmnseu- Nitrosomonas bacterium, ammonia-oxidizing the of analysis ‘Whole-genome (2007) 21)‘vlto fpoorpyi h hooeipyu rvnb oiotlgene horizontal by driven phylum Chloroflexi the in phototrophy of ‘Evolution (2018) tal. et SEJournal ISME 21)‘WS-OE:Hmlg oeln fpoensrcue n complexes’, and structures protein of modelling Homology ‘SWISS-MODEL: (2018) o:10.1093/nar/gky427. doi: . o:10.1093/bioinformatics/bts079. doi: . o:10.1093/bioinformatics/btp033. doi: . o:10.1038/s41559-017-0193. doi: . 20)‘ave eso - utpesqec lgmn dtradanalysis and editor alignment sequence multiple 2-A Version ‘Jalview (2009) o:10.3389/fmicb.2018.00260. doi: . auePbihn ru,99,p.15–95 o:10.1038/is- doi: 1954–1965. pp. 9(9), Group, Publishing Nature . niomna Microbiology Environmental 91,p.1425 o:10.1016/j.jinorgbio.2004.09.024. doi: 194–215. pp. 99(1), , 13 irbooyadmlclrbiology molecular and Microbiology eoeAnnouncements Genome (2,p.29–07 doi: 2993–3007. pp. 9(12), , nar swidely is ) 6(25), , Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. i.1 value one Fig. with (white); support, 50 following conflicting (grey). [?] (grey); the 90 values 90 with both FIGURES [?] and with indicated other 50 support the between is weak values and (black); nodes 50 both 90 other or [?] values [?] one for values Support with both Support support substitutions. with intermediate support other (aLRT/bb). expanded Strong or labeled an scheme: superfamily) are color reveals reductase bipartitions 42 (oxygen selected MAG Tyr-323 for have SURF tips) from (blue reductase sequences oxide nitric Putative phyla. the diverse to homologs putative of of diversity tree support, phylogenetic conflicting A (grey); (grey). values 90 90 both and [?] with 50 other support between 5. the weak values with Figure and (black); both indicated 50 90 or is [?] one [?] nodes value with values one other support both with for intermediate is with (white); Support support Chloroflexi 50 Strong (aLRT/bb). diamonds in [?] labeled scheme: magenta genes color are with reductase following bipartitions indicated the nitrite selected are for into reductase values nitrite fused Support with have ORFs in (NirK). to in diamonds sequences inferred yellow co-occur of or (See is this that (NirS) group hits Within C1 homologs largest distant Deltaproteobacteria. which C1 and more The along Nitrospinae, labeled. of branch Nitrospirae, domain. inclusion in the and found the domains clade, filter of to genus-level sister distribution no places taxonomic Chloroflexi domain—with limited C1 a the (grey). Methods)—indicates for 90 tree [?] and phylogenetic 50 other A between tree the values and domain (black); both 50 C1 90 or [?] one [?] 4. value values with Figure one both support intermediate with with (white); support support, 50 Strong conflicting Support (grey); scheme: [?] (aLRT/bb). values color 90 labeled following both are the with bipartitions with support indicated selected weak scheme is for color nodes values simplified other Support a Aquificae, for using values. Epsilonproteobacteria, displayed bootstrap were the values aLRT/rapid of support for by members Statistical dominated including Planctomycetes. clade a and also within Bacteroidetes, but Chloroflexi the Gammaproteobacteria, places homologs Beta-, domain (grey). Alpha-, type) 90 (cd-1 NirS domain [?] and of NirS analysis 50 other Phylogenetic between for the values and tree (black); both 50 Phylogenetic 90 or [?] one [?] 3b. value values with Figure one both support intermediate with with (white); support support, 50 Strong conflicting (grey); scheme: [?] values color 90 following both the with with support indicated weak is nodes other for eNOR oantree domain eNOR eNOR ooosi o nyAcaaadClrflx,btas rtoatraadother and Proteobacteria also but Chloroflexi, and Archaea only not in homologs eune rdtp)hv h hrceitcGu33i h lgmn;outgroup alignment; the in Glu-323 characteristic the have tips) (red sequences 14 Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. otdfile Hosted chloroflexi reductase-domain-structure-suggests-a-chimeric-denitrification-repertoire-in-phylum- image6.emf file Hosted 4 Fig. chloroflexi reductase-domain-structure-suggests-a-chimeric-denitrification-repertoire-in-phylum- image5.emf file Hosted 3b Fig. chloroflexi reductase-domain-structure-suggests-a-chimeric-denitrification-repertoire-in-phylum- image4.emf file Hosted 3a Fig. chloroflexi reductase-domain-structure-suggests-a-chimeric-denitrification-repertoire-in-phylum- image3.emf file Hosted 2 Fig. vial at available at available at available at available https://authorea.com/users/419181/articles/525743-novel-nitrite- https://authorea.com/users/419181/articles/525743-novel-nitrite- https://authorea.com/users/419181/articles/525743-novel-nitrite- https://authorea.com/users/419181/articles/525743-novel-nitrite- 15 Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. i.5 Fig. chloroflexi reductase-domain-structure-suggests-a-chimeric-denitrification-repertoire-in-phylum- image7.emf vial at available nitrate NO Ala-7 3 NB O 4654) COG (NCBI superfamily Cytochrome - C1 nar https://authorea.com/users/419181/articles/525743-novel-nitrite- Leu-119 NO nitrite 2 - Ala-142 nir NB fm13442) pfam (NCBI III subunit cbb3-type, oxidase, C Cytochrome 2010) COG variants diheme mono/ C Cytochrome ircoxide nitric C2 NO 16 NB (NCBI Glu-256 nor Arg-266 oan irt euts reductase nitrite domain, iru oxide nitrous yohoec1hm idn binding heme cd1 Cytochrome N 2 O NirS 02239) nos NB fm pfam (NCBI dinitrogen Thr-657 N 2 Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 86/97 Proteobacteria Nitrospirae Chloroflexi te phyla Other iatto support Bipartition 97.9/100 Intermediate Strong Weak hur WP_004255893.1 Thauera 77.8/91 yrgnvracaldilitoris Hydrogenivirga yrgnbce thermophilus Hydrogenobacter niai thermothiophila Inmirania odtlapetrii Bordetella Persephonella_hydrogeniphila_WP_097000987.1 odtlaWP_028352686.1 Bordetella lhpoebcei bacterium Alphaproteobacteria qiia bacterium Aquificae ieoyaslithotrophicus Sideroxydans suooaae atru 32-61-5 bacterium Pseudomonadales atooaae bacterium Xanthomonadales aioatrsegnicrescens Marinobacter egao p 4572_84 sp. Beggiatoa aioatrWP_082905200.1 Marinobacter ufrfsi variabilis Sulfurifustis eartoatrabceimSG8_39 bacterium Betaproteobacteria lntmctsbacterium Planctomycetes eoatrsp. Ketobacter hoaoatrthiocyanaticus Thiohalobacter ufrcui limicola Sulfuricaulis holc ingrica Thioploca addtsTimraianelsonii Thiomargarita Candidatus zvbi restrictus Azovibrio aooa p PBN3 sp. Halomonas yrgnpiae atru 28-61-11 bacterium Hydrogenophilales rshcmcoimhirschii Prosthecomicrobium ellnacaldifistulae Bellilinea hdccae atru CG17_big_fil_post_rev_8_21_14_2_50_68_7 bacterium Rhodocyclales irsia atru RBG_16_64_22 bacterium Nitrospirae trioatrdenitrificans Steroidobacter ogoas.LCSB751 sp. Zoogloea hoaooa denitrificans Thiohalomonas addt iiinN1 bacterium NC10 division candidate SA13_2710641719 znxshydrophilus Azonexus eiihlsthermophilus Tepidiphilus atoi p C 69-89 SCN sp. Lautropia cdtibclae atru SG8_45 bacterium Acidithiobacillales Candidatus_Magnetobacterium_casensis_WP_040333821.1 ezoxaglaWP_116899358.1 Wenzhouxiangella yhmcoims.32-62-53 sp. Hyphomicrobium ael chejuensis Hahella ufrsm sediminicola Sulfurisoma addtsMtyoiaii oxyfera Methylomirabilis Candidatus yrgnpiu thermoluteolus Hydrogenophilus eietcl thiotaurini Sedimenticola caoprlae bacterium Oceanospirillales 0.3 addtsTisminoneisti Thiosymbion Candidatus laioaaeebceimGenoA1_TS13_700 bacterium Alcanivoracaceae addtsMpoebcei bacterium_RBG_16_62_13 Muproteobacteria Candidatus aigou lutimaris Halioglobus hoas roseopersicina Thiocapsa Sulfurihydrogenibium_subterraneum_WP_051524441.1 atru HR39 bacterium addtsDdbcei bacterium Dadabacteria Candidatus hoolx atru HGW-Chloroflexi-10 bacterium Chloroflexi aaruoocss.DB1506 sp. Paracraurococcus hdccaeebceimUTPRO2 bacterium Rhodocyclaceae Azospirillum krael aerolata Skermanella antsiilms.ME-1 sp. Magnetospirillum anarxalpica Balneatrix addtsRkbcei atru RIFCSPHIGHO2_02_FULL_73_26 bacterium Rokubacteria Candidatus hoailsdenitrificans Thiobacillus am rtoatru HdN1 proteobacterium gamma aoiros.YL5-2 sp. Halovibrio hru thermophilus Thermus holv marina Thioclava hooatraee(4) Chromobacteriaceae anrooatrs.MME-103 sp. Paenirhodobacter LPB0142 sp. Rhodobacter hohi caldifontis Thiothrix hdfrxfermentans Rhodoferax rnmnsmetalli Arenimonas neoie thermophila Anaerolinea trlbceimdenitrificans Sterolibacterium SA12_2710640794 rmtlu aromaticum Aromatoleum zacss.PA01 sp. Azoarcus osi oceani Woeseia suoubninas.MAI-1 sp. Pseudogulbenkiania hoolx atru HGW-Chloroflexi-8 bacterium Chloroflexi aiasalexigens Haliea hdtemsmarinus Rhodothermus Nitratiruptor_sp._SB155-2_WP_012083162.1 atooaaeebceimSCN_69-123 bacterium Xanthomonadaceae hraartrxdaxensis Thermanaerothrix qioa voraii Aquimonas aiaeebceimLZ-16-2 bacterium Halieaceae ezoxaglamarina Wenzhouxiangella doaiaWP_110075452.1 Idiomarina ufrtlahydrogenivorans Sulfuritalea ehltnr p RIFCSPLOWO2_02_FULL_45_14 sp. Methylotenera ampoebcei bacterium Gammaproteobacteria addtsTidaorpaendolucinida Thiodiazotropha Candidatus doaiaatlantica Idiomarina aaocslutimaris Paracoccus suooa euioaBWHPSA037 aeruginosa Pseudomonas aiiel litoralis Marinicella hosuooa denitrificans Thiopseudomonas antvbi blakemorei Magnetovibrio suooa WP_003129144.1 Pseudomonas aaocsWP_028713157.1 Paracoccus Arcobacter_sp._LA11_WP_072679979.1 addtsCmeiatrdenitrificans Competibacter Candidatus SA14_2710648716 addtsPoinvbi aalborgensis Propionivibrio Candidatus oasbce gelatinilyticus Cohaesibacter neoieebceimUTCFX3 bacterium Anaerolineae UBA7227 holaiironitratireducens Thioalkalivibrio ercmcoi bacterium Verrucomicrobia UFMG68 MAG SURF ei oryzae Leeia dR61 JdFR hoolx atru RMG62435.1 bacterium bacterium 3 Chloroflexi Clade Thermofonsia Candidatus UFMG71 MAG SURF agel spongicola Kangiella hilaaffigens Ahniella rdrioica bacterium Bradyrhizobiaceae Campylobacteraceae_bacterium_4484_4_OQX73487.1 aooa WP_108132129.1 Halomonas elvioa denitrificans Defluviimonas hdsiillsbceimRIFCSPLOWO2_12_FULL_67_15 bacterium Rhodospirillales rdrioimoiorpiu S58 oligotrophicum Bradyrhizobium odnlaimmobilis Dokdonella ehlbclu p MM3 sp. Methylobacillus ufrcladenitrificans Sulfuricella irvruaaerodenitrificans Microvirgula oiiioauu sp. Robiginitomaculum hoolx atru HGW-Chloroflexi-6 bacterium Chloroflexi hdbceaee(7) Rhodobacteraceae eeiatrneptunius Celeribacter addtsAcmlbce p 66-26 sp. Accumulibacter Candidatus hoolx atru 44-23 bacterium Chloroflexi hoolx atru RIK46552.1 bacterium Chloroflexi hlsopr WP_063095021.1 Thalassospira otccaeebacterium Porticoccaceae noyboto aelbahaluymesi Lamellibrachia of endosymbiont zsiaoryzae Azospira UFMG42 MAG SURF yooclsbacterium Myxococcales aloellsbceimRIFCSPLOWO2_02_FULL_59_110 bacterium Gallionellales hlsopr alkalitolerans Thalassospira hur phenylacetica Thauera ehoooa denitrificans Dechloromonas aloelca atru CG_4_10_14_3_um_filter_60_1069 bacterium Gallionellaceae oeel p LIG4 sp. Vogesella suoiros.Tun.PSC04-5.I4 sp. Pseudovibrio oglnaarvoryzae Longilinea rtoatrabacterium Proteobacteria ukodrae (25) Burkholderiales embce p LW-1 sp. Gemmobacter UBA5797 hoolx atru HGW-Chloroflexi-2 bacterium Chloroflexi UBA5796 ioilalaminariae Kiloniella sulfur-oxidizing_symbionts emtmndtsbacterium Gemmatimonadetes suohdbce aquimaris Pseudorhodobacter holu denitrificans Thioalbus Deltaproteobacteria_bacterium_GWC2_42_11_OGP32269.1 hoolx atru HGW-Chloroflexi-3 bacterium Chloroflexi uraiu pauculus Cupriavidus hdsiilca atru R c57 BRH bacterium Rhodospirillaceae UBA6663 hdbceaeebceimHLUCCO07 bacterium Rhodobacteraceae hoolx bacterium_RBG_13_50_21 Chloroflexi Candidatus_Kuenenia_stuttgartiensis_CAJ74898.1 asoi p 25mfcol4.1 sp. Ralstonia qaprlu sp._LM1 Aquaspirillum yrgnbclms.Y04AAS1 sp. Hydrogenobaculum irblie donghaiensis Microbulbifer caihru profundus Oceanithermus iioapingtungensis Rivicola oyopu gilvum Polymorphum eapoebceimL13 proteobacterium beta horcae atru HS_08 bacterium Thiotrichales 17 oiprlu itersonii Novispirillum Candidatus_Scalindua_sp._husup-a2_WP_096896200.1 horcaeebacterium Thiotrichaceae Aliiarcobacter_faecis_WP_026806151.1 oefeu castenholzii Roseiflexus yrgnpiaeebacterium_CG1_02_62_390 Hydrogenophilaceae rcyoa eirfcn DSM_15123 denitrificans Brachymonas hdbu orientis Rhodobium uraiu WP_111516662.1 Cupriavidus ansiilmsalinarum Caenispirillum oiel viscosa Moritella suoleooa eirfcn S 6059 DSM denitrificans Pseudoalteromonas cioatrabacterium Actinobacteria cdbcei atru RIFCSPLOWO2_02_FULL_68_18 bacterium Acidobacteria oiirtu p MSK22-1 sp. Motiliproteus hdbceae (18) Rhodobacterales hroerbce p TC1 sp. Thermopetrobacter toi thiooxydans Ottowia Oleiphilus SAR324_cluster_bacterium_PCI30783.1 hzbae bacterium Rhizobiales lvbceilsbceimCG18_big_fil_WC_8_21_14_2_50_32_9 bacterium Flavobacteriales oelaee(3) Cowelliaceae atriee atru HGW-Bacteroidetes-12 bacterium Bacteroidetes uiatrprofundi Lutibacter lipr antarctica Oleispira prcatebceimHGW-Spirochaetae-10 bacterium Spirochaetae etnm illini Leptonema qiaias.MAR_2010_214 sp. Aquimarina ufrvms.UBA12169 sp. Sulfurovum Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. Proteobacteria Nitrospirae Chloroflexi Archaea te phyla Other 100/100 iatto support Bipartition Intermediate Strong Weak 95.3/100 73.7/12 37.6/10 antvbi blakemorei Magnetovibrio oiirtu p MSK22-1 sp. Motiliproteus lihlss.HI0132 sp. Oleiphilus hdbceaee(5) Rhodobacteraceae holu denitrificans Thioalbus hlsooa viridans Thalassomonas addtsTidaorpaendoloripes Thiodiazotropha Candidatus suoleooa eirfcn S 6059 DSM denitrificans Pseudoalteromonas owli mytili Colwellia oiel viscosa Moritella hlsoae p ND16A sp. Thalassotalea lipr antarctica Oleispira oasbce gelatinilyticus Cohaesibacter ellnacaldifistulae Bellilinea SA_13_2710641719_1-397 dR6 2730422191 61 JdFR hraartrxdaxensis Thermanaerothrix SA_12_2710640794_1-397 neoie thermophila Anaerolinea UBA6663 UFMG71 MAG SURF UFMG42 MAG SURF hroerbce p TC1 sp. Thermopetrobacter oefeu castenholzii Roseiflexus oglnaarvoryzae Longilinea eiie saccharolytica Levilinea UBA5797 aoiom nitratireducens Halobiforma UFMG68 MAG SURF aourms.WN019 sp. Halorubrum addtsSaidarubra Scalindua Candidatus aoergn jeotgali Haloterrigena arnm ejinorense Natrinema UBA7227 SA_14_2710648716_1-397 UBA5796 0.4 addtsMtyoiaii oxyfera Methylomirabilis Candidatus roooa aquaeolei Arhodomonas SA_20_2710662197_1-388 hoailsbrandeum Thiolapillus 18 ufrmnss. sp. Sulfurimonas espoel hydrogeniphila Persephonella addtsKeei stuttgartiensis Kuenenia Candidatus ufrcui limicola Sulfuricaulis addtsMgeoatru bavaricum Magnetobacterium Candidatus ufrhdoeiimsubterraneum Sulfurihydrogenibium ufrfsi variabilis Sulfurifustis yrgnbce thermophilus Hydrogenobacter ehl clavipes Nephila euftgu phosphitoxidans Desulfotignum zvbi restrictus Azovibrio atoi p C 69-89 SCN sp. Lautropia zacscommunis Azoarcus aplbceae (3) Campylobacterales ehltnr p RIFCSPLOWO2_02_FULL_45_14 sp. Methylotenera antsiilms.64-120 sp. Magnetospirillum ehlbclu p MM3 sp. Methylobacillus zsiilmWP_059399790.1 Azospirillum hlsoiuss.CG17_big_fil_post_rev_8_21_14_2_50_53_8 sp. Thalassolituus addtsCmeiatrdenitrificans Competibacter Candidatus oiebsiilmautotrophicum Noviherbaspirillum ukodracepacia Burkholderia eiihlsthermophilus Tepidiphilus ieoyaslithotrophicus Sideroxydans yrgnpiu thermoluteolus Hydrogenophilus rmtlu rmtcmEbN1 aromaticum Aromatoleum tetccu pneumoniae Streptococcus emnioa p CN sp. Herminiimonas hdtemsprofundi Rhodothermus irtrpo p SB155-2 sp. Nitratiruptor trlbceimdenitrificans Sterolibacterium rcyoa eirfcn S 15123 DSM denitrificans Brachymonas WP_107889100.1 Microvirgula hru thermophilus Thermus ufrcladenitrificans Sulfuricella asoi p 25mfcol4.1 sp. Ralstonia uraiu pauculus Cupriavidus ehoooa denitrificans Dechloromonas iioapingtungensis Rivicola zsias.I13 sp. Azospira qaprlu p LM1 sp. Aquaspirillum ogoas.LCSB751 sp. Zoogloea addtsPoinvbi aalborgensis Propionivibrio Candidatus caihru profundus Oceanithermus oeel p EB sp. Vogesella eietcl thiotaurini Sedimenticola krael aerolata Skermanella yrgnvras.128-5-R1-1 sp. Hydrogenivirga hoailsdenitrificans Thiobacillus holc ingrica Thioploca znxsfungiphilus Azonexus hur humireducens Thauera hoaoatrthiocyanaticus Thiohalobacter addtsAcmlbce phosphatis Accumulibacter Candidatus niai thermothiophila Inmirania ufrsm sediminicola Sulfurisoma ufrtlahydrogenivorans Sulfuritalea egao p 4572_84 sp. Beggiatoa hohi caldifontis Thiothrix ehlccaee(7) Methylococcaceae suoiros.Tun.PSC04-5.I4 sp. Pseudovibrio aioatrguineae Marinobacter hdfrxs.OTU1 sp. Rhodoferax ioilalaminariae Kiloniella ael p CCB-MM4 sp. Hahella hoas roseopersicina Thiocapsa anarxalpica Balneatrix toi thiooxydans Ottowia 67-35 SCN sp. Comamonas odtlaWP_028352686.1 Bordetella ipiipr psychrophila Simplicispira hoaooa denitrificans Thiohalomonas eoatrsp. Ketobacter hooatraee(6) Chromobacteriaceae oiiioauu sp. Robiginitomaculum itersonii Novispirillum aoiros.YL5-2 sp. Halovibrio addtsTisminoneisti Thiosymbion Candidatus aooa aestuarii Halomonas ansiilmbisanense Caenispirillum liroatrfaecis Aliiarcobacter hlsopr profundimaris Thalassospira addtsTimraianelsonii Thiomargarita Candidatus suooa aeruginosa Pseudomonas crmbce p DBTN3 sp. Achromobacter yrgnbclms.Y04AAS1 sp. Hydrogenobaculum roatrbivalviorum Arcobacter hosuooa denitrificans Thiopseudomonas trioatrdenitrificans Steroidobacter rshcmcoimhirschii Prosthecomicrobium TSA1 sp. Bradyrhizobium qiaias.MAR_2010_214 sp. Aquimarina ukodrae (16) Burkholderiales uiatrprofundi Lutibacter aiiel litoralis Marinicella liouu xiamenense Albidovulum hdbu orientis Rhodobium oyopu gilvum Polymorphum atooaae (4) Xanthomonadales ufrvms.UBA12169 sp. Sulfurovum etnm illini Leptonema hdbceaee(4) Rhodobacteraceae osi oceani Woeseia suohdbce ferrugineus Pseudorhodobacter eliroae (3) Cellvibrionales hdbceaee(4) Rhodobacteraceae ezoxaglamarina Wenzhouxiangella aiiiatrs.55A14 sp. Maritimibacter hdbce p SW2 sp. Rhodobacter holv indica Thioclava holaiironitratireducens Thioalkalivibrio aaocshalophilus Paracoccus agel profundi Kangiella doaiasp. Idiomarina yhmcoims.32-62-53 sp. Hyphomicrobium hdbceaee(10) Rhodobacteraceae ehlccmsoryzae Methylocucumis ehlmgu ishizawai Methylomagnum Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. Nitrospina Nitrospirae Chloroflexi Proteobacteria te phyla Other iatto support Bipartition i oani ORF in domain Nir Intermediate Strong NirS Weak 1Nr fusion C1-NirS NirK Apparent retctnabceimRME11532.1 bacterium Ardenticatenia retctn aiiaWP_054492141.1 maritima Ardenticatena 0.3 1Nr fusion C1-NirK Apparent retctn aiiaWP_054493624.1 maritima Ardenticatena ukodras.OK806 sp. Burkholderia aalrnaptereochthonis Caballeronia etpoebcei atru GWC2_42_11 bacterium Deltaproteobacteria addtsTemfni ld atru PJF48684.1 bacterium 3 Clade Thermofonsia Candidatus hoolx atru RME48917.1 bacterium Chloroflexi hoolx atru RME74748.1 bacterium Chloroflexi irsia atru ICPOO 23_6OGW07928.1 12_39_16 RIFCSPLOWO2 bacterium Nitrospinae adlna atru RME59657.1 bacterium Caldilineae addtsTemfni ld atru PJF48408.1 bacterium 3 Clade Thermofonsia Candidatus etpoebcei atru GWA2_43_19 bacterium Deltaproteobacteria hoolx atru RMF78192.1 bacterium Chloroflexi irsia atru ICPIH20_91 OGV95443.1 02_39_11 RIFCSPHIGHO2 bacterium Nitrospinae cdbcei bacterium Acidobacteria 19 etpoebcei atru GWF2_42_12 bacterium Deltaproteobacteria irsia atru 314C__21 OLD42076.1 13_1_40CM_2_62_10 bacterium Nitrospirae hoolx atru RME46417.1 bacterium Chloroflexi ehlhlbu crimeensis Methylohalobius atru addtsBaklbcei CG18_big_fil_WC_8_21_14_2_50_49_26 Blackallbacteria Candidatus bacterium addtsMliaatrabceimHGW-Melainabacteria-1 bacterium Melainabacteria Candidatus irsia atru 13_2_20CM_2_63_8 bacterium Nitrospirae OLD38719.1 13_1_40CM_2_62_10 bacterium Nitrospirae irsia atru 314C__21 OLD37429.1 13_1_40CM_2_62_10 bacterium Nitrospirae hoolx atru GWC2_70_10 bacterium Chloroflexi irsia atru RMH38294.1 bacterium Nitrospirae oiebsiilmautotrophicum Noviherbaspirillum UBA5796 adlna atru RME50437.1 bacterium Caldilineae neoieebceimS81 KPK06759.1 19 SG8 bacterium Anaerolineae addtsPoieflmbreve Promineofilum Candidatus dR6 2730423366 JdFR-61 neoieebceimPWH12756.1 bacterium Anaerolineae neoieebceimRME88945.1 bacterium Anaerolineae neoieebceimPWH17682.1 bacterium Anaerolineae hoolx atru RPI81993.1 bacterium Chloroflexi hoolx atru HGW-Chloroflexi-10 bacterium Chloroflexi neoieebceimRIK21833.1 bacterium Anaerolineae addtsRkbcei atru PYM78808.1 bacterium Rokubacteria Candidatus uetlapratensis Luteitalea irsia atru 13_2_20CM_62_7 bacterium Nitrospirae atru R2GBD09014.1 HR22 bacterium irsia atru 13_1_40CM_2_62_10 bacterium Nitrospirae addtsRkbcei atru PYO50356.1 bacterium Rokubacteria Candidatus hrolxshgnoti WP_088571740.1 hugenholtzii Thermoflexus hoolx atru RME76307.1 bacterium Chloroflexi UBA7227 UTCFX3 bacterium Anaerolineae SA14_2710648716_17-122 rtoatrabceimRIK95915.1 bacterium Proteobacteria irsia atru PHX90395.1 bacterium Nitrospirae neoieebceimRCK74925.1 bacterium Anaerolineae addtsMpoebcei atru RIFCSPHIGHO2_01_60_12 bacterium Muproteobacteria Candidatus A22cutrbceimI1 Chloro-G9 Io17 bacterium cluster SAR202 irsia atru RIFCSPLOWO2_01_FULL_62_17 bacterium Nitrospirae RIFCSPLOWO2_12_FULL_63_8 bacterium Nitrospirae irsia atru 314C__21 OLD41619.1 13_1_40CM_2_62_10 bacterium Nitrospirae neoie hroioa thermolimosa Anaerolinea irsia atru G491__mfle_15PJA78332.1 CG_4_9_14_3_um_filter_51_5 bacterium Nitrospirae irsia atru ICPOO_2FL_21 OGW64727.1 RIFCSPLOWO2_02_FULL_62_14 bacterium Nitrospirae addtsMpoebcei atru RIFCSPHIGHO2_02_FULL_60_13 bacterium Muproteobacteria Candidatus irsia atru RMH34907.1 bacterium Nitrospirae hoolx atru RMF33815.1 bacterium Chloroflexi irsia atru RMH04440.1 bacterium Nitrospirae irsia atru ICPOO_2FL_21 OGW64275.1 RIFCSPLOWO2_02_FULL_62_14 bacterium Nitrospirae addtsRkbcei atru PYM25573.1 bacterium Rokubacteria Candidatus neoie thermophila Anaerolinea hoolx atru PIE82360.1 bacterium Chloroflexi addtsTemfni ld atru PJF48624.1 bacterium 3 Clade Thermofonsia Candidatus RMG62435.1 bacterium Chloroflexi hoolx atru B_35_1OGN92417.1 RBG_13_50_21 bacterium Chloroflexi irsias.N1WP_080880425.1 ND1 sp. Nitrospira WP_013249804.1 defluvii Nitrospira hoolx atru PID87170.1 bacterium Chloroflexi irsiagracilis Nitrospina irsiajpnc SLM46303.1 japonica Nitrospira silclrstihie WP_044200009.1 trichoides Oscillochloris nutrdClrfeibceimBAL57500.1 bacterium Chloroflexi uncultured irsias.OB KXJ99429.1 OLB3 sp. Nitrospira hoolx atru G-hoolx- PKN91058.1 HGW-Chloroflexi-6 bacterium Chloroflexi irsmndls() (5) Nitrosomonadales hoailsdenitrificans Thiobacillus UFMG71 MAG SURF addtsRkbcei atru PYM13089.1 bacterium Rokubacteria Candidatus hraartrxdxni WP_054521542.1 daxensis Thermanaerothrix albce p FWC2 sp. Caulobacter irsiaeebceimRPH80748.1 bacterium Nitrospiraceae irsia atru G491__mfle_15PJA78181.1 CG_4_9_14_3_um_filter_51_5 bacterium Nitrospirae addmnsbauzanensis Candidimonas irsias.SCGC_AG-212-E16 sp. Nitrospira irsiamsoini WP_053380952.1 moscoviensis Nitrospira neoieebceimRIK22595.1 bacterium Anaerolineae hoolx atru UTCFX4 bacterium Chloroflexi UFMG68 MAG SURF SA12_2710640794_15-120 UBA2414 UBA2323 UBA2395 UBA2356 dR6 2730422191 JdFR-61 neoieebceimS81 KPK02806.1 SG8_19 bacterium Anaerolineae ioiie eohl OUC09162.1 aerophila Litorilinea ellnacaldifistulae Bellilinea irsia atru CG11_big_fil_rev_8_21_14_0_20_56_8 bacterium Nitrospinae oefeu castenholzii Roseiflexus uilmnss.J169-3-13 JR1 sp. Pusillimonas A22cutrbceimIo17-Chloro-G2 bacterium cluster SAR202 atru R1GBD45706.1 HR41 bacterium irsias.SG-bin2 sp. WP_087473935.1 Nitrospira SBR1015 moscoviensis cf. Nitrospira SA13_2710641719_17-122 irsia atru CG22_combo_CG10-13_8_21_14_all_47_10 bacterium Nitrospinae irsiadfui WP_041186672.1 defluvii Nitrospira hoolx atru PIE81805.1 bacterium Chloroflexi irsialnaWP_121989999.1 lenta Nitrospira ebceaeebceimRPI69070.1 bacterium Geobacteraceae irsias.N1WP_080880290.1 ND1 sp. Nitrospira irsias.S-i5OQW67753.1 ST-bin5 sp. Nitrospira irsia atru PHX90100.1 bacterium Nitrospirae UFMG42 MAG SURF uilmnss.J169-2-13 JR1 sp. Pusillimonas irsialnaSPP65905.1 lenta Nitrospira hoolx atru RIK28123.1 bacterium Chloroflexi irsia atru GBL39781.1 bacterium Nitrospirae irsia atru RBG_19FT_COMBO_58_9 bacterium Nitrospirae UBA5797 oglnaavrzeWP_075072401.1 arvoryzae Longilinea irsias.OB KXK06844.1 OLB3 sp. Nitrospira irsias.S-i5OQW63335.1 ST-bin5 sp. Nitrospira irsia atru CG11_big_fil_rev_8_21_14_0_20_45_15 bacterium Nitrospinae irsiaeebceimRPH76514.1 bacterium Nitrospiraceae UBA6663 Nitrospira irsia atru RIFCSPLOWO2_12_FULL_47_7 bacterium Nitrospinae hoolx atru 44-23 bacterium Chloroflexi rtoatrabceimPZN26450.1 bacterium Proteobacteria irsiamsoini ALA60174.1 moscoviensis Nitrospira irsias.S-i1OQW37650.1 SG-bin1 sp. Nitrospira irsias.UW-LDO-01 sp. Nitrospira irsiajpnc WP_080885133.1 japonica Nitrospira suooaae (7) Pseudomonadales uilmnscaeni Pusillimonas irsiac.msoini B11 WP_087475037.1 SBR1015 moscoviensis cf. Nitrospira eohzbu Mezorhizobium hdnbce fulvus Rhodanobacter irsiac.msoini B11 WP_086425991.1 SBR1015 moscoviensis cf. Nitrospira (4) irsias.S-i4OQW59715.1 ST-bin4 sp. Nitrospira rnlsccu antarcticus Granulosicoccus hoolx atru RIK46552.1 bacterium Chloroflexi addtsNtopr npnt WP_062486230.1 inopinata Nitrospira Candidatus hoolx atru RLT35450.1 bacterium Chloroflexi hdnbce p Root627 sp. Rhodanobacter hoolx atru HGW-Chloroflexi-8 bacterium Chloroflexi (9) hoolx atru RMF03298.1 bacterium Chloroflexi hoolx atru HGW-Chloroflexi-2 bacterium Chloroflexi hoolx atru HGW-Chloroflexi-3 bacterium Chloroflexi Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 100/100 100/100 24.5/51 iatto support Bipartition Intermediate Strong Weak Proteobacteria Nitrospirae Chloroflexi te phyla Other Nitrospinae Archaea hoolx atru RME47896.1 bacterium Chloroflexi hrarbce p PB12_4term sp. Thermaerobacter hoolx atru RME48457.1 bacterium Chloroflexi hoolx atru RMH02092.1 bacterium Chloroflexi atru RIL08691.1 bacterium irsia atru RIFCSPLOWO2_01_FULL_39_10 bacterium Nitrospinae UBA4733 retctn maritima Ardenticatena irsia atru RBG_16_64_22 bacterium Nitrospirae atru HR22 bacterium hoolx atru RBG_16_57_8 bacterium Chloroflexi hrolxshgnoti JAD2 hugenholtzii Thermoflexus unrel parva Turneriella hroirbu roseum Thermomicrobium eartoatrabceimRIFCSPLOWO2_02_64_14 bacterium Betaproteobacteria A34cutrbacterium cluster SAR324 etpoebcei atru GWA2_45_12 bacterium Deltaproteobacteria neoieebceimRIK31904.1 bacterium Anaerolineae adtihet bacterium Calditrichaeota GWF2_70_14 bacterium Rokubacteria Candidatus UBA2395 UBA2323 UBA2414 UBA2356 parbce thermophilus Sphaerobacter UFMG4 Ga0104751_100161138 42 MAG SURF 100/100 addtsMpoebcei atru RBG_16_60_9 bacterium Muproteobacteria Candidatus RIFCSPLOWO2_01_FULL_47_190 bacterium Gammaproteobacteria emtmndtsbacterium Gemmatimonadetes horcae atru SG8_50 bacterium Thiotrichales hoolx atru RIK54890.1 bacterium Chloroflexi rtoatrabceimRIK92331.1 bacterium Proteobacteria addtsKytnu thompsoni Kryptonium Candidatus ufrfsi variabilis Sulfurifustis hdsiillsbceimRIFCSPLOWO2_12_FULL_67_15 bacterium Rhodospirillales cdbcei atru SCN_69-37 bacterium Acidobacteria yrgnbce thermophilus Hydrogenobacter ufrcui limicola Sulfuricaulis qiia bacterium Aquificae lhpoebcei atru RMF00900.1 bacterium Alphaproteobacteria pigbceilsbceim44-15 bacterium Sphingobacteriales yrgnvracaldilitoris Hydrogenivirga ukodrae bacterium Burkholderiales antsiilmmarisnigri Magnetospirillum ercmcoi bacterium Verrucomicrobia yhmcoims.CS1BSMeth3 sp. Hyphomicrobium atriee atru OLB10 bacterium Bacteroidetes antvbi blakemorei Magnetovibrio rdrioica bacterium Bradyrhizobiaceae auipar limnophila Lacunisphaera SW132 sp. Rhodohalobacter atoi p C 70-15 SCN sp. Lautropia aigou p U0301 sp. Halioglobus holaiirodenitrificans Thioalkalivibrio icnbce caeni Piscinibacter uvbce p PAE-UM sp. Curvibacter otccaeebacterium Porticoccaceae addtsHidlacaoaacao LC_2 archaeon Heimdallarchaeota Candidatus 98.2/98 hdsiilca bacterium Rhodospirillaceae eohzbu p Root552 sp. Mesorhizobium hoolx atru RLC59405.1 bacterium Chloroflexi Wenzhouxiangella hzbae atru 68-8 bacterium Rhizobiales addtsDdbcei bacterium Dadabacteria Candidatus hdccaeebceimUTPRO2 bacterium Rhodocyclaceae trioatrdenitrificans Steroidobacter atooaae atru 63-13 bacterium Xanthomonadales eufrccaeeacao AG1 archaeon Desulfurococcaceae aloellsbacterium Gallionellales lntmctsbacterium Planctomycetes liouu xiamenense Albidovulum oevru p GCL-8 sp. Roseovarius ufrtlahydrogenivorans Sulfuritalea ioebce arenae Litoreibacter urvvxs.SN71-131 SCN sp. Rubrivivax hru WP_015717644.1 Thermus hdccae atru GWA2_65_20 bacterium Rhodocyclales addtsPoinvbi aalborgensis Propionivibrio Candidatus SK-01 sp. Accumulibacter Candidatus hru thermophilus Thermus cioatrabceimRBG_16_68_12 bacterium Actinobacteria addtsMgeoatru bavaricum Magnetobacterium Candidatus ukodrae (8) Burkholderiales bauzanensis Candidimonas hdtemsprofundi Rhodothermus aousss.HD8-51 sp. Halorussus aolnss.CBA1112 sp. Haloplanus aiiatrs.10B sp. Salinibacter 0.4 aoatru jilantaiense Halobacterium aoaiasediminis Halolamina aiirnmrubrum Salinigranum aorhebu iranensis Haloarchaeobius aoavmsedimenti Haloparvum pharaonis Natronomonas aourmWP_094527696.1 Halorubrum 20 aorhen3A1_DGR haloarchaeon aourmpersicum Halorubrum malekzadehii Halopenitus aoeu aranensis Halovenus arabca (6) Natrialbaceae atru RIL08403.1 bacterium UFMG4 Ga0104751_10079453 42 MAG SURF dloiroae atru RIFOXYB2_FULL_36_6 bacterium Bdellovibrionales hoolx atru RBG_16_63_12 bacterium Chloroflexi ailsmethanolicus Bacillus aoirbu mukohataei Halomicrobium yoauu ferrireducens Pyrobaculum uyrhet rhenPXF18200.1 archaeon Euryarchaeota aaocsaestuarii Paracoccus addtsTisminoneisti Thiosymbion Candidatus hdbceae atru RIFCSPHIGHO2_02_FULL_62_130 bacterium Rhodobacterales hdfrxs.OTU1 sp. Rhodoferax oiirtu p MSK22-1 sp. Motiliproteus hoolx atru RLC59399.1 bacterium Chloroflexi antsias.QH-2 sp. Magnetospira ansiilmbisanense Caenispirillum embce megaterium Gemmobacter ehlfrl stellata Methyloferula hdbc barguzinensis Rhodobaca Mariprofundus_aestuarium cdau hospitalis Acidianus Posted on Authorea 11 Jun 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.162337328.84022836/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. time xatChloroflexi Extant Chloroflexi Ancestral yohoe1 Cytochrome C1 O 4654 COG C1 oanfusion Domain C2 C2 nirS nirS 21 hmrcntierdcaei nqet to unique is reductase nitrite Chimeric ebr fPyu hoolx Chloroflexi Phylum of members yohoe2 Cytochrome oiotlgn rnfr transfer gene Horizontal O 2010 COG Proteobacteria irt reductase nitrite nirS