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Gene 349 (2005) 15 – 24 www.elsevier.com/locate/gene Alternative oxidase and plastoquinol terminal oxidase in marine prokaryotes of the Sargasso Sea Allison E. McDonald, Greg C. VanlerbergheT Department of Life Sciences and Department of Botany, University of Toronto at Scarborough, 1265 Military Trail, Scarborough, ON Canada M1C 1A4 Received 13 September 2004; received in revised form 6 December 2004; accepted 22 December 2004 Available online 10 March 2005 Received by G. Theissen Abstract Alternative oxidase (AOX) represents a non-energy conserving branch in mitochondrial electron transport while plastoquinol terminal oxidase (PTOX) represents a potential branch in photosynthetic electron transport. Using a metagenomics dataset, we have uncovered numerous and diverse AOX and PTOX genes from the Sargasso Sea. Sequence similarity, synteny and phylogenetic analyses indicate that the large majority of these genes are from prokaryotes. AOX appears to be widely distributed among marine Eubacteria while PTOX is widespread among strains of cyanobacteria closely related to the high-light adapted Prochlorococcus marinus MED4, as well as Synechococcus. The wide distribution of AOX and PTOX in marine prokaryotes may have important implications for productivity in the world’s oceans. D 2005 Elsevier B.V. All rights reserved. Keywords: Endosymbiosis; Metagenomics; Photosynthesis; Respiration 1. Introduction Alternative oxidase (AOX) is a member of the membrane-bound diiron carboxylate group of proteins (Berthold and Stenmark, 2003). In eukaryotes, AOX is an additional terminal oxidase in mitochondrial electron transport (in addition to cytochrome oxidase) that catalyzes the oxidation of ubiquinol and reduction of O2 to H2O. It is a non-energy conserving branch of electron transport, bypassing the last two sites of proton translocation associated with the cytochrome pathway (Siedow and Umbach, 2000; Vanlerberghe and Ordog, 2002). Besides its well-established presence in the kingdoms Plantae, Fungi and Protista, AOX has also been very recently described in the aproteobacterium Novosphigobium aromaticivorans (Stenmark and Nordlund, 2003) and in three different animal Abbreviations: AOX, alternative oxidase; PTOX, plastoquinol terminal oxidase. T Corresponding author. Tel.: +1 416 287 7431; fax: +1 416 287 7642. E-mail address: [email protected] (G.C. Vanlerberghe). 0378-1119/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.12.049 phyla (McDonald and Vanlerberghe, 2004). However, while AOX respiration appears ubiquitous in the Plantae, it is clearly more sporadically distributed among the species of other kingdoms. Hence, the full extent of its distribution, particularly among bacteria, protists and animals, is unknown. Plant chloroplasts contain a protein that shares some sequence similarity with AOX and which is also a member of the membrane-bound diiron carboxylate group of proteins (Wu et al., 1999; Berthold and Stenmark, 2003). This oxidase (called the IMMUTANS terminal oxidase or plastid terminal oxidase) is associated with the photosynthetic electron transport chain and catalyzes the oxidation of plastoquinol with reduction of O2 to H2O. IMMUTANS terminal oxidase is involved in carotenoid biosynthesis and may also have a general role in maintaining the reduction state of electron transport chain component(s) (Aluru and Rodermel, 2004; Kuntz, 2004). Thus far, IMMUTANS proteins have only been found in photosynthetic organisms, being described in higher plants (Wu et al., 1999); algae (Acc. No. AF494290), and recently in diatoms (McDonald and Vanlerberghe, 16 A.E. McDonald, G.C. Vanlerberghe / Gene 349 (2005) 15–24 2004) and cyanobacteria (Finnegan et al., 2003). It is, however, clearly not present in all photosynthetic organisms (e.g., absent from the genome of Synechocystis sp. PCC 6803) and the extent of its distribution, particularly among cyanobacteria (and potentially other photosynthetic bacteria) is unknown. We recently suggested that the cyanobacterial protein be termed plastoquinol terminal oxidase (PTOX) (McDonald et al., 2003). Recently, Venter et al. (2004) reported the results of a massive environmental genome sequencing analysis of the Sargasso Sea. They sequenced 1.6 Gb of DNA (collected from surface water that was filtered to retain prokaryotes but largely eliminate eukaryotes) that generated over one million translated proteins, thus almost doubling the number of protein sequences in public databases. Their analyses of the sequence data estimated that at least 1800 genomic species (and likely far more) were present in the samples. The Sargasso Sea dataset provides a wealth of new information considering the usual short-fall of genome sequence data from marine microbes (Hess, 2004). Here, we used the Sargasso Sea metagenomic data (Venter et al., 2004) to assess the presence and diversity of AOX and PTOX genes in a marine microbial community. We find that, in the Sargasso Sea, AOX is widely distributed among Eubacteria (and possibly Archaea) while PTOX is widespread among different strains of the cyanobacteria Prochlorococcus and Synechococcus. 2. Materials and methods AOX and PTOX homologues were uncovered by BLASTp searches of the Sargasso Sea protein set (under t h e e n v i r o n m e n t a l s am p l e s h e a d i n g at N C B I ; www.ncbi.nlm.nih.gov) using a subset of known protein sequences (Table 1). Using the scheme outlined previously Table 1 AOX and PTOX sequences used in this work Kingdom Phylum or Division Species Protein, Acc. No. Animalia Fungi Chordata Mollusca Ascomycota Plantae Anthophyta Ciona intestinalis Crassostrea gigas Aspergillus niger Candida albicans Gelasinospora sp. Neurospora crassa Penicillium chrysogenum Arabidopsis thaliana Acrasiomycota Apicomplexa Bacillariophyta Capsicum annuum Glycine max Hordeum vulgare Lycopersicon esculentum Mesembryanthemum crystallinum Nicotiana tabacum Oryza sativa Populus tremula Sauromatum guttatum Sorghum bicolor Triticum aestivum Zea mays Dictyostelium discoideum Cryptosporidium parvum Thalassiosira pseudonana AOX, TC17302a AOX,BQ426710 AOX, AB016540 AOX1, AF031229 AOX, AY140655 AOX, L46869 AOX, AY425133 AOX1a, AF370166; AOX2, NM_125817; PTOX, AF098072 PTOX, AF177981 AOX2a, U87906 PTOX, TC121698a PTOX, AF302932 PTOX, TC3799a AOX1, S71335 AOX1b, AB004813; PTOX, AAC35554 AOX, AJ251511 AOX, Z15117 PTOX, TC41730a AOX1c, AB078883 PTOX, TC16323a AOX, BAB82989 AOX, AY312954 AOX1, scaffold 278, 4692-5382b; PTOX, scaffold 70, 79626-80180b AOX, CF258325 PTOX, AY267664 AOX1, AF047832; PTOX, AAM12876 AOX, AB070617 AOX, CAE11918 AOX, AP006491; PTOX, AP006491 PTOX, NP_486136 PTOX, gll0601c PTOX, NP_892455 PTOX, NP_896980 AOX, ZP_00095227 Protista Chlorophyta Eubacteria Euglenozoa Oomycota Rhodophyta Cyanobacteria Proteobacteria Acetabularia acetabulum Bigelowiella natans Chlamydomonas reinhardtii Trypanosoma brucei brucei Pythium amphanidermatum Cyanidioschyzon merolae Anabaena variabilis PCC7120 Gloeobacter violaceus Prochlorococcus marinus MED4 Synechococcus sp. WH8102 Novosphigobium aromaticivorans All sequences are available at Genbank unless otherwise indicated. a Available at TIGR. b Available at Department of Energy Joint Genome Institute Database. c Available at CyanoBase. A.E. McDonald, G.C. Vanlerberghe / Gene 349 (2005) 15–24 (McDonald et al., 2003; and see Results) it was possible to readily distinguish between AOX and PTOX proteins. Multiple sequence alignments were then used to compare the recovered sequences to AOX or PTOX proteins from a wide range of taxa (Table 1). Sequence bounded by the first and fourth iron-binding motifs (McDonald et al., 2003) was used in this analysis and deduced amino acid sequences were aligned using Clustal X Version 1.81 with default gap penalties (Thompson et al., 1997). This approach allowed us to at least provisionally assign the proteins as either prokaryotic or eukaryotic in origin. Several approaches were then taken to further narrow the taxonomic origin of the likely prokaryotic proteins. First, sequence alignments of the uncovered proteins with the four prokaryotic (cyanobacterial) PTOX sequences of known origin (Table 1) or the lone prokaryotic (a-proteobacterial) AOX sequence of known origin (Table 1) was used to establish %similarity with the proteins of these species. Second, the Sargasso Sea dataset was used to establish AOX- or PTOX-containing synteny groups (i.e., groups of genes present in a continuous region of DNA sequence in two or more species/strains). In some cases, we were able to use the completed genomes of Prochlorococcus marinus MED4 and Synechococcus sp. WH8102 or the incomplete genome of N. aromaticivorans as scaffolds for this analysis. Only a subset of the uncovered proteins could be analyzed in this manner, as others exist in the dataset as singletons. Third, proteins other than AOX or PTOX within the synteny groups were used in BLASTp searches to establish their similarity to proteins of known taxonomic origin. BLASTp searches of the non-redundant protein database at NCBI and the deduced protein database at CyanoBase (www. kazusa.or.jp/cyanobase) were used. Protein phylogenies were generated using MEGA version 2.1 with the neighbor-joining method and the p-distance model (Kumar et al., 2001). Phylogenies generated using the number of distances, gamma, or Poisson models yielded identical topologies. 3. Results 3.1. Identification and analysis of AOX and PTOX proteins from marine prokaryotes The massive dataset generated by bwhole-genome shotgun sequencingQ (Venter et al., 2004) was used to uncover numerous AOX and PTOX proteins present in Sargasso Sea microbial populations. In all, 96 different putative proteins (69 AOX, 27 PTOX), including many partials, were retrieved using our search methods. Based on further analyses (see below), 89 of these proteins (67 AOX, 22 PTOX) are likely from prokaryotic organisms. The finding of some eukaryotic proteins (despite the steps taken to filter out eukaryotes in the Sargasso Sea samples) has been noted before (Venter et al., 2004). 17 It was previously shown that AOX proteins from diverse taxonomic groups nonetheless all share key conserved amino acid residues in the central region of the protein (Berthold and Stenmark, 2003; McDonald et al., 2003; McDonald and Vanlerberghe, 2004). These include the six iron-binding residues distinctive of these diiron carboxylate proteins, other residues within the four iron-binding motifs, and several other amino acids. All of these residues are also completely conserved in the Sargasso Sea AOX and PTOX proteins (Fig. 1). Previously, an analysis of the four iron-binding motifs of all known AOX and PTOX proteins (a total of 67 proteins including just 1 prokaryotic AOX and four prokaryotic PTOX proteins) showed that eleven amino acid residues in these regions were consistently different between AOX and PTOX (McDonald et al., 2003). Collectively, these residues could thus be readily used to distinguish between AOX and PTOX proteins. When the large new Sargasso Sea dataset is taken into account, the predictive power of three of these eleven residues (residue 3 in iron-binding motif two, residue 4 in iron-binding motif three and residue 11 in iron-binding motif four) is lost (Fig. 1). Nonetheless, each iron-binding motif still has at least two residues with predictive power and so, collectively, the model can still readily and reliably distinguish AOX from PTOX. As noted for some eukaryotic AOX sequences (McDonald et al., 2003), most of the prokaryotic AOX proteins are likely not to be recognized by a widely used monoclonal antibody to AOX (Elthon et al., 1989) since a critical residue for recognition (an A at the third residue in ironbinding motif 4; Finnegan et al., 1999) differs in most of these proteins (Fig. 1). Most PTOX proteins possess an insert between the third and fourth iron-binding motifs that AOX proteins lack. The inserts of higher plants, green and red algae, diatoms and hcyanobacteria range from 15 to 20 amino acids in length (Fig. 1). With one exception (the red alga C. merolae), the insert is similar (9 amino acids completely conserved) among these groups. However, in the a-cyanobacteria Synechococcus the insert is only about half this length while in the a-cyanobacteria P. marinus the insert is missing entirely and this protein is in fact missing an additional 23 amino acids in this region. Compared to other PTOXs, this represents a 30–40 amino acid deletion in this region. 3.2. Synteny and BLASTp analyses Initial multiple sequence alignments suggested that many of the Sargasso Sea AOX and PTOX proteins were likely prokaryotic. To confirm this and to further narrow the taxonomic origin of these proteins, we used the Sargasso Sea dataset for synteny analyses. Proteins within the synteny groups were then used in BLASTp searches to establish their similarity to proteins of known taxonomic origin. The majority of the Sargasso Sea AOX sequences that were not 18 A.E. McDonald, G.C. Vanlerberghe / Gene 349 (2005) 15–24 * * * O.sativa AOX1a A.thaliana AOX2 C.merolae AOX1 EAK46738 EAI84676 EAK49986 EAH88150 N.aromaticivorans AOX D.discoideum AOX C.parvum AOX T.brucei brucei AOX A.acetabulum AOX C.reinhardtii AOX1 A.niger AOX N.crassaAOX P.aphanidermatum AOX C.gigas AOX C.intestinalis AOX T.pseudonana AOX1 O.sativa PTOX A.thaliana PTOX A.variabilis PTOX T.pseudonana PTOX B.natans PTOX C.reinhardtii PTOX EAK14890 EAD66202 C.merolae PTOX P.marinus MED4 PTOX Synechococcus PTOX LETVAAVPGMVGGMLLHLRSLRRFEQSGG- - - - WI RTLLEEAENERMHLMTFMEVA- NPKWYERALVI TVQGVFFNAYFLGYLLSPKF LETVAAVPGMVGGMLLHLKSI RKFEHSGG- - - - WI KALLEEAENERMHLMTMMELV- KPKWYERLLVMLVQGI FFNSFFVCYVI SPRL LETVAAVPGMVGGMMLHLQCLRRFRQSGG- - - - WI RVLLEEAENERMHLMVYMSI A- QPRALERALVI LAQAGFFSFYTLLYTI SPKT LETVAAVPGMVAGMLNHLKSLRNMEQDRG- - - - WI KTLLDEAENERMHLMTFI NI A- KPSVFERFLVI I VQGI FFNLYFVMYLVSPRT LETVAAVPGMVAGMLLHLRSLRKI EDDKG- - - - WI KTLLDEAENERMHLMTFI HVA- KPTTLERFI I MVAQFI FI VTYAI I YLVSQRT LETVAGVPGMVAGVWMHFKSLRVMKAGYGE- - - QI REMLAEAENERMHLMFFI EI A- KPNYFERFI VLFAQVI FGLFYLFMYI FFTRT LETVAAVPGMVAGMLHHFKSLRSMTDDDG- - - - I I KELLDEAENERMHLMTFI EI S- KPTLFERLLVLGAQI VFATFYFFLYVFFRGT LETVAAVPGMVGATI NHLACLRRMCDDKG- - - - WI KTLMDEAENERMHLMTFI EI S- KPTLFERAVI MGVQWVFYLFFFGLYLVSPKT LETVAAVPGLVAGMFLHLKTLRNMQ- SNN- - - - WI KI LMDEMENERMHLLSFMELT- KPTLLERGMVAVTQAI YWNLFLVFYVLSPKT LETVAGVPGMVGAMLRHFSSLRKMKRDNG- - - - WI HTLLEEAENERMHLLI SLQLI NKPSI LTRVSVI GTQFAFLI FYTVFYI I SPKY LETVAGVPGMVGGMLRHLSSLRYMTRDKG- - - - WI NTLLVEAENERMHLMTFI ELR- QPGLPLRVSI I I TQAI MYLFLLVAYVI SPRF LETVAGVPGMVGGMLRHLRSLRTMRRDHG- - - - WI HTLLEEAENERMHLLTFLKLR- EPGPLFRGFVI LTQGI FFNTFFLAYLVSPTL LETVAGCPGMVAGMLRHLKSLRSMSRDRG- - - - WI HTLLEEAENERMHLI TFLQLR- QPGPAFRAMVI LAQGVFFNAYFI AYLLSPRT LESVAGVPGMVGGMLRHLRSLRRMKRDNG- - - - WI ETLLEEAYNERMHLLTFLKLA- EPGWFMRLMVLGAQGVFFNGFFLSYLMSPRI LESI AGVPGMVAGMLRHLHSLRRLKRDNG- - - - WI ETLLEESYNERMHLLTFMKMC- EPGLLMKTLI LGAQGVFFNAMFLSYLI SPKI LETVAGVPGMVGGMARHLRSLRSMRRDYG- - - - WI HTLLEEAENERMHLLI FMNMK- QPGPLFRLLVLGAQGVFFNMFFVSYLVSPRT LETVAGVPGMVAAMTRHLHSLRRLKRDHG- - - - WI HTLLEEAENERMHLMTALQLR- QPSWLFRSGVI VSQGAFVTMFSI AYMLSPRF LETI AGVPGMVGAMVRHLVSLRRLKRDHG- - - - WI HTLLEEAENERMHLMTAMRI A- NPGI I MRTSI VVAQGI FVSGFSLAYLI SPRF LETVAAI PGMVAAI I RHFRSLRNMARDGG- - - - MLNMFLEEANNERMHLLTFI RMK- DPGYLFRATVI GGQFAFGSAFLTMYMI SPAF LETI ARVPYFAFI SVLHMYETFGWWRR- - - - ADYI KVHFAESWNEFHHLLI MEELGGNSLWVDRFLARFAAFFYYFMTVAMYMVSPRM LETI ARVPYFAFMSVLHMYETFGWWRR- - - - ADYLKVHFAESWNEMHHLLI MEELGGNSWWFDRFLAQHI ATFYYFMTVFLYI LSPRM LETVARVPYFSYLSVLHLYETLGWWRK- - - - ADWLKVHFAESWNELHHLLI MESLGGAGFWGDRFLAKTAALI YYWI I I AVYFVSPHS LETI ARVPYFSYLSVLHLYETLGKWRR- - - - VKYLKLHFAESWNEMHHLLI MEELGGSERFFDRFLAQHCAFGYFLI VI TLYLI NPVQ LETVARMPYLSYVTMLHLYESFGWWRRA- - - AAVKRVHFAEEWNEFHHLLTFEALGGDRSWATRFLAQHAAI VYYWVLVLMWLLSPTL LETVARMPYFSYI SMLHLYESLGWWRAG- - - AELRKI HFAEEWNELHHLQI MESLGGDQLWFDRFAAQHAAI LYYWI LLGLYVFSPRL LETVARVPYFSFVSVLHLYETI GLWRK- - - - VDYMETHFGQTMNEYHHLLI MEDLGGDKRFI DKFFAQHTAFFYYWI TCLI YMASPCM LETVARVPYFSFVSVLHLYETLGLWRK- - - - VDYMETHFAQTMNEYHHLLI MENLGGDERFI DRFFAQHTAFFYYWLTCLI YMVSPCM LEMVARVPYFSFLSVLHLYESLDLAHL- - - - TELRRAHFI EEWNEMHHLLI MQALGGDGRWLDRFLAYHVSLVYYWALVLLYMI APAV LEVI ARSPYFAFLSVLHFKESLGLKNDI - - TMFLMKEHFYQAI NETEHLKEMEKRGGDKFWI DRFLARHLVLVYYWI MVFYYFCSPRN LEI I ARTAYTAEESACHYLETI GLDKEGTI RDTLELARYQDT- NEQTHEDI FARDLD GLKNWGDRFLARHI AVVI YWVFAI TTLI DHEM O.sativa AOX1a A.thaliana AOX2 C.merolae AOX1 EAK46738 EAI84676 EAK49986 EAH88150 N.aromaticivorans AOX D.discoideum AOX C.parvum AOX T.brucei brucei AOX A.acetabulum AOX C.reinhardtii AOX1 A.niger AOX N.crassaAOX P.aphanidermatum AOX C.gigas AOX C.intestinalis AOX T.pseudonana AOX1 O.sativa PTOX A.thaliana PTOX A.variabilis PTOX T.pseudonana PTOX B.natans PTOX C.reinhardtii PTOX EAK14890 EAD66202 C.merolae PTOX P.marinus MED4 PTOX Synechococcus PTOX AHRVVGYLEEEAI HSYTEFLKDLEAGKI DNV- - - - - PAPAI AI DYWRLPA- - - - - - - - - - - - - - - - - - - - NATLKDVVTVVRADEAHHRDVNH AHRVVGYLEEEAI HSYTEFLKDI DNGKI ENV- - - - - AAPAI AI DYWRLPK- - - - - - - - - - - - - - - - - - - - DATLKDVVTVI RADEAHHRDVNH AHRLVGYLEEEAI VSYTEYLKDI DDGRI PNI - - - - - PAPPI AI DYWQLDP- - - - - - - - - - - - - - - - - - - - NARLRDVVLATRADEAHHRDVNH CHRI VGYFEEQAI I SYTEYLDEI ENGNI ENV- - - - - KAPQI AI DYWGLSQ- - - - - - - - - - - - - - - - - - - - FAKLKDVI I AVRNDEMGHRDVNH AHRI VGYFEEEAVI SYTEYLNELEAGTI PDQ- - - - - PAPLI AI NYWNLPL- - - - - - - - - - - - - - - - - - - - HATLKDVVRVI RDDEAGHRDVNH AHRMI GYFEDEAVKSYTEYLELVESGKVENI - - - - - QAPKLAI NYYKLGT- - - - - - - - - - - - - - - - - - - - DAKLSDLI RCVRADEEHHSETNH AHRMI GYFEEEAVTSYTEFLDEI DKGTI ENV- - - - - AAPKI AVDYWNLGN- - - - - - - - - - - - - - - - - - - - KATLRDVVVAVRNDEAGHRDKNH AHRVVGYFEEEAVI SYTHYLAEI DQGRSANV- - - - - PAPAI AKRYWGLPD- - - - - - - - - - - - - - - - - - - - NAMLRDVVLVVRADEAHHRDVNH AHRFTGYLEEQAVVTYTHMLEDI DSGKVPNY- - - - - KAPQI AI EYWGLPE- - - - - - - - - - - - - - - - - - - - DATLRDLI LVI RQDESDHRLVNH SHRFVGYLEEEAVSTYTHLI EEI DKGLLPGF- - - ERKAPKFASVYYGLPE- - - - - - - - - - - - - - - - - - - - DATI RDLFLAMRRDESHHRDVNH VHRFVGYLEEEAVI TYTGVMRAI DEGRLRPT- - - KNDVPEVARVYWNLSK- - - - - - - - - - - - - - - - - - - - NATFRDLI NVI RADEAEHRVVNH CHRMVGYLEEEAI KTYSHCLHDI ETG- - LGW- - AERPAPPI AI EYWKLPA- - - - - - - - - - - - - - - - - - - - DASMRDVVLAVRADEACHSHVNH CHAFVGFLEEEAVKTYTHALVEI DAG- - RLW- - KDTPAPPVAVQYWGLKP- - - - - - - - - - - - - - - - - - - - GANMRDLI LAVRADEACHAHVNH CHRFVGYLEEEAVI TYTRAI KEI EAGSLPAW- - EKTEAPEI AVQYWKMPEG- - - - - - - - - - - - - - - - - - - QRSMKDLLLYVRADEAKHREVNH THRFVGYLEEEAVHTYTRCI REI EEGHLPKWSDEKFEI PEMAVRYWRMPEG- - - - - - - - - - - - - - - - - - - KRTMKDLI HYI RADEAVHRGVNH CHRFVGYLEEEAVKTYTGLLKDI EDGHLKEW- - EKMTAPAI ARSYYKLPD- - - - - - - - - - - - - - - - - - - - EASVYDMI KCI RADEANHRDVNH CHRFVGYLEEEAVFTYSKCLKDI ESGSLKHW- - QTKAAPDVAI RYWKLPE- - - - - - - - - - - - - - - - - - - - TASMKDVVLAI RADEAHHRVVNH CHRFVGYLEEEAVKTYTHCLEELDSGNLKMW- - CRMKAPEI AVEYWKLPD- - - - - - - - - - - - - - - - - - - - DAMMRDVI LAI RADEAHHRSVNH CHRFVGYI EEEACATYTKI I KAI EEDLGNWR- - - TEEAPKI AKGYWHLGE- - - - - - - - - - - - - - - - - - - - HGSVLDLMLAVRADEAEHRDVNH AYHFSECVERHAYSTYDKFI KLHEDELKKLP- - - - - - APEAALNYYLNEDLYLFDEFQTARV P- CSRRPKI DNLYDVFVNI RDDEAEHCKTMK AYHFSECVESHAYETYDKFLKASGEELKNMP- - - - - - APDI AVKYYTGGDLYLFDEFQTSRT P- NTRRPVI ENLYDVFVNI RDDEAEHCKTMR AYNFMEQVEQHAYSSYDKFLTTHEAELKTQP- - - - - - APEVAKTYYRDGDLYMFDEFQTAHS P- SERRPNI DNLYDVFVAI RDDEMEHVKTMV AYNLNQDVEEHAFATYDTFLKENAEMLKTKP- - - - - - APKVAI EYYRHGDMYMFDEFQTEL- - - - - RRPEI NNLYDVFVAI RDDEMAHVKTME AYNFSELI EAHAVDTYGEFADANEELMKELP- - - - - - APGI AI QYWMGGDMYLYDEFQTERR LGDERRPNI TNLYDVI CAI RDDEAEHVATMA AYNFSELI EYHAVDTYGEFWDANEELLKSLP- - - - - - PPLVAAVYYRSQDLYMFDSFQTSQP MQNPRRPSCKTLYDVFKNI CDDEMEHVKTMK AYNLSEQI EEHAYHTYDEFLKNHKASLSLEK- - - - - - APVVASEYYDD- - - - - - - - - - - - - - - - - - - - - - VENLYDVFTRVRDDEAEHVKTMQ AYNLSEQI EEHAYHTYDEFLKNHGVSLSLEK- - - - - - PPPVAVNYYDN- - - - - - - - - - - - - - - - - - - - - - VESLYDVFVNVRDDEGKHVKTMQ AYNFSELLEKHAYDTYAVFI EQNETLLRTLP- - - - - - APSVARAYYESGERFRFRADTI NAE THACEGPPVATLFDAFVNI RDDEGEHI KMME AYDVNI KI EEHAFNTYTKYLKDHPEDQK- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I KEI AQDELNHVEELN AALLGEAVEVEAVKTYRRMLKEQPEEWLDQP- - - - - - AAPTATHYWEKPNSMWRVRGDHMP- - - - - - - - - - GSMRDVVEAI VRDEADHVKANS * PTOX Insert * * Fig. 1. A multiple sequence alignment of AOXs and PTOXs from diverse taxa and including representatives from the Sargasso Sea dataset (identified by Acc. Nos.). Black bars above the alignment indicate the four iron-binding motifs (McDonald et al., 2003). Asterisks indicate the conserved iron-binding residues. Black boxes indicate other completely conserved residues. The black arrows indicate amino acids that can be used to distinguish between AOX and PTOX proteins (McDonald et al., 2003). The insert present in most PTOX proteins is indicated. singletons could be divided by synteny analysis into seven groups (Groups 1–7 in Fig. 2). Significantly, the closest BLASTp hits for proteins on all these scaffolds were from prokaryotes. One AOX sequence in Group 1 (Acc. No. EAG77159) was 88% similar to that of the only previously known prokaryotic AOX, that being from the a-proteobacterium N. aromaticivorans (Stenmark and Nordlund, 2003). Further, the adjacent protein on the scaffold (Acc. No. EAG77158) displayed the best BLASTp hit with a protein in N. aromaticivorans that also resides beside AOX in the DOE JGI N. aromaticivorans genome project (AOX, 416543– 417019; hypothetical protein, 417077–417358) (Fig. 2). Besides sharing a common synteny, AOX proteins in Group 2 all shared a high degree of sequence similarity (Fig. 2). The closest BLASTp hits to other proteins on the scaffold were to proteins from various Eubacteria. While displaying a different synteny, AOX proteins in Group 3 shared a high degree of sequence similarity with those in Group 2 (Fig. 2). However, the closest BLASTp hits for the A.E. McDonald, G.C. Vanlerberghe / Gene 349 (2005) 15–24 1 hypothetical N. aromaticivorans EAG77158 2 homoserine dehydrogenase EAK46735 3 4 AOX AOX EAG77159 fructose 1,6bisphosphatase EAK46736 hypothetical conserved EAI84676 EAJ22698 EAH44440 EAJ02071 EAF25139 EAE14290 EAI84677 EAJ22699 EAH44439 EAJ02072 EAF25140 EAE14291 monoxygenase hypothetical protein EAI62228 EAI66230 EAK49985 EAI62227 EAC01006 5 beta-lactamase EAI79088 6 7 copper-binding family EAI79089 EAH00432 ssDNA exonuclease AOX EAK46737 EAI41827 EAB62271 EAG28808 EAK46738 EAI41828 EAB62270 EAG28807 transformylase EAK46739 EAK46741 EAJ02073 AOX aminopeptidase EAK49986 EAI62226 EAI66229 EAC01007 EAK49987 EAI62225 EAI66226 hypothetical protein EAI62224 quinone oxidoreductase EAI62223 AOX EAI79090 EAH00433 transcriptional regulator EAH88146 EAH88148 EAH88149 EAF54105 EAI06377 EAF93774 EAK46740 EAI41829 glutathione peroxidase EAI84678 EAJ22700 beta-lactamase EAH88147 photolyase histidinol dehydrogenase sodium-dependent proteorhodopsin drug pump AOX 19 AOX desuccinylase EAH88150 EAH88151 hypothetical conserved archaeal? EAF54106 EAI06376 EAF93775 Fig. 2. Different AOX-containing synteny groups (numbered 1–7) seen in the Sargasso Sea dataset. Boxes indicate the synteny and putative proteins while corresponding accession numbers from the Sargasso Sea dataset are indicated below each box. In one case (Group 1), the incomplete genome of N. aromaticivorans acted as the scaffold. hypothetical protein were from the cyanobacteria Synechococcus sp. WH8102 and P. marinus MIT9313, while the best hit for histidinol dehydrogenase was from the hproteobacterium Ralstonia eutropha. In Group 4, the closest BLASTp hits for proteins on the scaffold were Eubacterial. In Group 5, only two partial AOX sequences were available so it was not possible to examine sequence similarity with the other groups. The closest BLASTp hit for other proteins in this group was for proteins from the a-proteobacterium Mesorhizobium and the cyanobacterium Trichodesmium erythaeum. The AOX protein in Group 6 was similar to those in Group 4. The scaffold here includes a gene encoding proteorhodopsin (Fig. 2). The best BLASTp hit against this protein was from isolate Hot75m4, a marine gproteobacteria (Béjà et al., 2001). Each of the AOXs in Group 7 is partial sequences. The only other protein on this scaffold has the best BLASTp match to a hypothetical protein from the marine Archaea Cenarchaeum symbiosum. 20 A.E. McDonald, G.C. Vanlerberghe / Gene 349 (2005) 15–24 A Rhomboid family N-acetyl transferase PMM0332 PMM0333 Conserved hypothetical PMM0334 NAD(P)H quinone oxidoreductase PMM0335 PTOX PMM0336 Conserved hypothetical PMM0337 EAC56372 (77%) EAC56371 (94%) EAC58003 (72%) EAC58002 (93%) EAI02077 (98%) EAI02078 (98%) EAI02079 (99%) EAI02081 (100%) EAJ24023 (90%) EAJ24021 (93%) B Conserved hypothetical PTOX SYNW0886 SYNW0887 EAH17817 (99%) EAH17818 (99%) Fig. 3. Different PTOX-containing synteny groups in Sargasso Sea samples in comparison to the synteny displayed by the genome of P. marinus MED4 (A) or Synechococcus WH8120 (B). Boxes indicate the synteny and putative proteins. Just below the boxes are the coding region identifiers from the MED4 or WH8120 genome. Just below these are the accession numbers of the corresponding Sargasso Sea proteins. Numbers in brackets indicate the percent similarity between the Sargasso Sea proteins and those in the MED4 or WH8120 genome. searches, all of the closest hits were to putative NAD(P)H quinone oxidoreductase genes from different strains of P. marinus (data not shown). Two PTOX proteins were found that shared a high degree of sequence similarity with the PTOX of the acyanobacterium Synechococcus WH8102 (Acc. No. SYNW0887). In one case (Acc. No. EAH17818), the protein shared 99% sequence similarity and a synteny in common with that of the WH8102 genome (Fig. 3). In the second case (Acc. No. EAC79126), the similarity was reduced somewhat (to 82%) suggesting that this PTOX was from a Synechococcus strain less related to WH8102. However, this gene was a singleton so its synteny could not be confirmed. The proteins share 47% sequence similarity. No potential orthologs were identified in Eubacteria or eukaryotes. Many PTOX proteins were uncovered that displayed a high level of sequence similarity with the PTOX of the acyanobacterium P. marinus MED4 (Acc. No. PMM0336). This included the 30–40 amino acid deletion distinctive of this PTOX (Fig. 1). Further, several of the scaffolds containing these PTOXs displayed a synteny common with that of the P. marinus MED4 genome (Fig. 3). Interestingly, a putative NAD(P)H quinone oxidoreductase gene is adjacent to PTOX in this genome (Fig. 3) and in DNA from the Sargasso Sea (Fig. 3). When the putative Sargasso Sea NAD(P)H quinone oxidoreductases (Acc. No. EAC56372, EAC58003; Fig. 3) were used in BLASTp A B 69 EAJ02071 EAJ22698 100 EAI84676 92 EAK46738 EAI41828 EAH88150 71 62 75 98 98 98 67 78 53 Group 2 EAI66229 100 EAK49986 98 EAI62226 N. aromaticivorans C. merolae AOX1 69 89 Group 3 Group 6 Group 4 G. max Aox2 O. sativa Aox1a A. thaliana Aox1a D. discoideum Aox T. brucei brucei Aox T. pseudonana AOX1 C. intestinalis AOX C. gigas AOX C. reinhardtii Aox1 A. niger Aox P. aphanidermatum Aox 59 L. esculentum C. annuum 84 M. crystallinum 97 A. thaliana 100 H. vulgare 96 O. sativa 58 S. bicolor 67 Z. mays T. pseudonana A. variabilis 81 72 G. violaceus EAK14890 100 EAC98174 98 100 EAD66202 C. merolae 89 B. natans 96 C. reinhardtii 62 EAD86912 P.marinus MED4 100 100 EAI02081 EAC56371 96 EAC58002 91 EAF06364 Synechococcus 0.05 0.05 Fig. 4. A protein phylogeny of AOXs (A) or PTOXs (B) from a wide variety of taxa and including representatives from the Sargasso Sea dataset (identified by Acc. Nos.). The identified groups (2, 3, 4, 6) in panel (A) correspond with the synteny groups in Fig. 2. A.E. McDonald, G.C. Vanlerberghe / Gene 349 (2005) 15–24 3.3. Phylogenetic analyses A subset of AOX or PTOX sequences from the Sargasso Sea dataset and other known organisms (Table 1) was used to generate an unrooted protein phylogeny. This analysis used that portion of the proteins bordered by the first and fourth iron-binding motifs (as in Fig. 1). Proteins not complete in this region were excluded. The AOX phylogeny generated two groups, one containing the fungal, green algal, diatom, animal and parasitic protist AOXs, the second containing the AOXs of D. discoideum, higher plants, red algae, N. aromaticivorans and all of the other presumed prokaryotic AOXs (based upon the synteny and BLASTp analysis) (Fig. 4). These presumed prokaryotic AOXs grouped most closely to one another and then to the AOX of N. aromaticivorans. Among the prokaryotic AOXs from the Sargasso Sea, the phylogeny generated the same groupings of proteins as suggested by the synteny analysis. However, no Group 1, 5 or 7 proteins 21 could be included as none were complete enough for this analysis. The PTOX phylogeny confirmed the synteny analysis, indicating that several of the Sargasso Sea PTOX proteins derive from organisms highly related to P. marinus MED4. As seen before (McDonald et al., 2003), the a- and hcyanobacteria grouped separate from one another, with the green (and red) algae between them. Two other uncovered PTOX singletons (including Acc. No. EAD86912, Fig. 4) grouped closely with the green algae C. reinhardtii and B. natans and hence are likely green algal in origin. Three other singletons (Acc. No.’s EAK14890, EAC98174, EAD66202) grouped closely together but separate from all other PTOX-containing groups (Fig. 4). 3.4. Analysis of the AOX N-terminus Many of the presumed prokaryotic AOX sequences (based upon similarity, synteny and phylogenetic analyses, O.sativa T.aestivum N.tabacum P.tremula S.guttatum A.thaliana AOX2 G.max AOX2a EAI62226 EAK49986 EAI66229 EAI79090 EAH00433 EAJ02071 EAI41828 N.aromaticivorans EAK46738 EAH88150 D.discoideum Gelasinospora N.crassa A.niger P.chrysogenum T.bruceibrucei C.parvum P.aphanidermatum C.albicans C.reinhardtii - - MSSRMAGSAI LRHVG- - - - - - - - - - - - GVRLFTASATSPAAAAAAAARPFL- - - AGGEAVP- - - GVWGLRLMSTSSVASTEAAAKAEAKKADAEKE- - - - - - - - - MSSRVAGSVLLRHLGPRVF- - GPTTPAAQRPLLAGGEGGAVAVAMWARPLS- - - TSAAEAAREEATASKDNVASTAAATAEAMQAAKAGAVQAAKEGK- - - - - S - - MMTRG- ATRMTRTVLGHMGP- RYFSTAI FRNDAGTGVMSGAAVFMHGVPANPSEKAVVTWVRHFPVMGSRSAMSMALNDKQHDKKAENGSAAATGGGD- - GGDE MMMASRGEGVKLASSMMLFS- - - RSFSTAI SRGI I AKEAVTAKAVECHGDVVR- - KNI GEFWVRG- SVFGVRHGSTMSFGEKDQQKVEMKQTQSVAEGGD- - KEEK - MI SSRLAGTALCRQLSHVPVP- QYLPALRPTADTASSLLHRCSAAAPAQRAG- - - LWPPSWFSP- - - - - PRHASTLSARAQDGGKEKAAGTAGKVPPGEDGGAEK - - MSQLI TKAALRVLLVCGRGNCNMFVSSVSSTSVMKSPYEI TAPMRI HDWCGGFGDFKI GSKHVQGNFNLRWMGMSSASAMEKKDENLTVKKGQNGGGS- - - - - - - MKLTALNSTVRRALLNGR- - - NQNGNRLGSAALMP- - - - - YAAAETRLLCAG- G- - - - - - - - ANGWF- FYWKRTMVSPAEAKVPEKEKEKEKAKAEKS- - - - - ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- - MFKYVTVLNKNNNLN- - - - - - KLLLNSI SKGNTLNSNNGLASTSI S- - - - - - - - - - - - - - - - - - - - I NSSI RSFSSFSSSKVMLDKRSEK- - - - - - - - - - - - - - - - MNTPKVNI LYSPGQAAQLSRTLI STCHTRPFLLGGLRVATSLHPTQ- - - - - - - - - - - - - - - - - - - TNLSSSPPRGFTTTSVVRLKDFFPA- - - - - - - - - - - - - - - MNTPKVNI LHAPGQAAQLSRALI STCHTRPLLLAGSRVATSLHPTQ- - - - - - - - - - - - - - - - - - - TNLSSPSPRNFSTTSVTRLKDFFPA- - - - - - - - - - - - - - - MNSLTATAPI RAAI PKSYMHI ATRNYSGVI AMSG- LRCSGSLVANR- - - - - - - - - - - - - - - - - - - HQTAGK- - RFI STTPKSQI KEFFPP- - - - - - - - - - - - - - - MNTLSVRAPLRAAAKPQYLHLAVRTYSGVAATTLNPACGANKRTSI - - - - - - - - - - - - - - - - - - - FSLTSK- - RPI SSTPQNQI TDYFPP- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MFRNHASRI TAAAAPWVLRTACRQK- - - - - - - - - - - - - - - - - - - SDAKTP- - - VWGHTQLNRLSFLETV- - - - - - - - - - - - - - - - - - MYYVRNLSNTNKLRYFYGRHLWLLSSKVNLNNLCSI VHSNKGQKI TSKLYI TLEKDRSSNNQGDFSKKRTLECKSDQI NKFDAENEEKVGS- - - - - - - - - - - - - - - - - - MLALSPSTRLLKRSALRMTSVTPLGKALSQTI TNNHVLS- - - - - - - - - - - - - - - - - - - - - FSTSPDAKDVTEEKPLI AHFSQSS- - - - - - - - - - - - - - - - - MI GLSTYRNLPTLLTTTTVI STALRSKQLLRFTTTTSTKSRSSTSTAATTVGNSNPKSPI DEDNLEKPGTI PTKHKPFNI QTEVYN- - - - - - - - - - - - - - MLQTAPMLPGLGPHLVPQLGALASASRLLGSI ASVPPQHGGAGFQAVRGFATGAVSTPAASSPGHKPAATHAPPTRLDLKPGAGSFAAGAVAPHPGI N- - - - - - - O.sativa T.aestivum N.tabacum P.tremula S.guttatum A.thaliana AOX2 G.max AOX2a EAI62226 EAK49986 EAI66229 EAI79090 EAH00433 EAJ02071 EAI41828 N.aromaticivorans EAK46738 EAH88150 D.discoideum Gelasinospora N.crassa A.niger P.chrysogenum T.bruceibrucei C.parvum P.aphanidermatum C.albicans C.reinhardtii VVVNSYWGI E- QSKKLVREDGTEWKWSCFRPWETY- TADTSI DL TKHHVPKTLLDKI AYWTVKSLRFPTDI - - - - - - - - - - - - - - - - - - - - - - - - FFQRRYGCRAMML PAASSYWGI V- PAK- LVNKDGAEWKWSCFRPWEAY- TSDTTI DL SKHHKPKVLLDKI AYWTVKSLRVPTDI - - - - - - - - - - - - - - - - - - - - - - - - FFQRRYGCRAMML KSVVSYWGVQ- PSK- VTKEDGTEWKWNCFRPWETY- KADLSI DL TKHHAPTTFLDKFAYWTVKSLRYPTDI - - - - - - - - - - - - - - - - - - - - - - - - FFQRRYGCRAMML KEI ASYWGVP- PSR- VTKEDGAEWKWNCFRPWETY- SADLSI DL KKHHVPATFLDKMAYWMVKALRFPTDL- - - - - - - - - - - - - - - - - - - - - - - - FFQRRYGCRAMML EAVVSYWAVP- PSK- VSKEDGSEWRWTCFRPWETY- QADLSI DL HKHHVPTTI LDKLALRTVKALRWPTDI - - - - - - - - - - - - - - - - - - - - - - - - FFQRRYACRAMML VAVPSYWGI ETAKMKI TRKDGSDWPWNCFMPWETY- QANLSI DL KKHHVPKNI ADKVAYRI VKLLRI PTDI - - - - - - - - - - - - - - - - - - - - - - - - FFQRRYGCRAMML VVESSYWGI S- - RPKVVREDGTEWPWNCFMPWESY- RSNVSI DL TKHHVPKNVLDKVAYRTVKLLRI PTDL- - - - - - - - - - - - - - - - - - - - - - - - FFKRRYGCRAMML - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MQI L DKLNK- QNI SDAFALSMTKFFRI I ADT- - - - - - - - - - - - - - - - - - - - - - - - FFAKKYGHRAVVL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MQI L DKI NK- QNI SDAFALSMTKFFRI I ADT- - - - - - - - - - - - - - - - - - - - - - - - FFAKRYGHRAVVL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MQI L DKI SR- ENI SDAFALSMTKFFRFI ADT- - - - - - - - - - - - - - - - - - - - - - - - FFAKRYGHRAVVL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MNKP- - - KNFSDFFALSMTKFFRFI ADT- - - - - - - - - - - - - - - - - - - - - - - - FFAKRYGHRAVVL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MNKP- - - KNFSDYFALSMTKFFRFVADT- - - - - - - - - - - - - - - - - - - - - - - - FFAKRYGHRAVVL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MSDL NKHFQPKNFSDKVALSFTKFLRLLADT- - - - - - - - - - - - - - - - - - - - - - - - FFKKRYGHRAVVL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MNR- - - NL NTHYKPENLSDKI AFAFTKLLRFTADF- - - - - - - - - - - - - - - - - - - - - - - - FFAKRYGHRAVVL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MI PPFI DL SVHHKPGGLSDRI AFGFTKALRWCADT- - - - - - - - - - - - - - - - - - - - - - - - FFAERYGHRAVVL - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MFKHYKPVNNSDRI ALLFTKALRLFADL- - - - - - - - - - - - - - - - - - - - - - - - FFKKRYGHRAVI L - - - - - - - - - - - - MKN- - - - - - - - - - - - - - APFEK- - TI GFTDQD STHKYPHGLSDRSAYLI TRALRI AADL- - - - - - - - - - - - - - - - - - - - - - - - FFRKRYGHRAVVL - - - - - - - - - - - PQNPLYHRNSTLYS- - - FTPRDVLI KI EKDFVMPPSYEAKSLSDNFAKFSVLFLRKFSNL- - - - - - - - - - - - - - - - - - - - - - - - FFKEKFLHYAI VL - - - - - - - - - - - KETAYI RQTPPAW- - - - - - PHHGWTEEEMI SVV PEHRKPETVGDWLAWKLVRI CRWGTDI ATGI RPEQQVDKNHPTTATSADKPLTEAQWLVRFI FL - - - - - - - - - - - KETAYI RQTPPAW- - - - - - PHHGWTEEEMTSVV PEHRKPETVGDWLAWKLVRI CRWATDI ATGI RPEQQVDKHHPTTATSADKPLTEAQWLVRFI FL - - - - - - - - - - - PTAPHVKEVETAW- - - - - - VHPVYTEEQMKQVA I AHRDAKNWADWVALGTVRMLRWGMDLVTGYRHPP- - - - - - PGREHEARFKMTEQKWLTRFI FL - - - - - - - - - - - PKAPNVKEVQTAW- - - - - - VHPVYTESQMQNI R I AHRQAANWSDWVALGTVRI FRWGMDTATGYRHPK- - - - - - PGQELPDMFKMTEHKWMNRFI FL - - - - - - - - - - - PVVPLRVSDESSE- - - - - - DRPTWSLPDI ENVA I THKKPNGLVDTLAYRSVRTCRWLFDTFSLYRFGS- - - - - - - - - - - - - - - - I TESKVI SRCLFL - - - - - - - - - - HFMKKSNHAASI LEGKEYGFNSPI WDLEEVNNVQKTHLCPNGFKDKMSYYLVI ALRKSFDLLTRYKKG- - - - - - - - - - - - - - - - - HNEYQWCRRI I FL - - - - - - - - - - - TRHPLDKAQEPVWEN- - PVPHAVYDLQKI EDI P QTHHDPKKI HERAAYVAVKLVRKGFDI ASGYRGPG- - - - - - - - - - - - - - GAMTEKDWLHRCLFL - - - - - - - - - - - - KAGI EANDDDKFLTKPTYRHEDFTEAGVYRVH VTHRPPRTI GDKI SCYGTLFFRKCFDLVTGYAVPDP- - - DKPDQYKGTRWEMTEEKWMTRCI FL - - - - PARMAADSASAAAGASGDAALAESYMAHPAYSDEYVESVR PTHVTPQKLHQHVGLRTI QVFRYLFDKATGYTPTG- - - - - - - - - - - - - - - SMTEAQWLRRMI FL Insert Fig. 5. A multiple sequence alignment of the N-termini of AOX proteins from a wide range of taxa. Black ovals denote Cys residues involved in biochemical regulation of the plant proteins (Siedow and Umbach, 2000). Also indicated is an insert present in several eukaryotic groups. 22 A.E. McDonald, G.C. Vanlerberghe / Gene 349 (2005) 15–24 see above) appeared complete at the N-terminus. Their Ntermini were compared with those of a wide taxonomic range of AOXs. Arbitrarily, the first residue (L) in the Fig. 1 alignment served as the last residue in the alignment of Ntermini (Fig. 5). As observed before (Stenmark and Nordlund, 2003), the N. aromaticivorans AOX had a truncated N-terminus compared with those of eukaryotes. Significantly, the presumed prokaryotic AOX proteins from the Sargasso Sea all showed the same truncated N-terminus (Fig. 5), which ranged in length from 37–47 residues. This is compared to ranges of 151–179 (higher plants), 152–178 (fungi) and 121–186 (protists). AOXs of the fungi, oomycota, green algae and the parasitic protists contain an insert (ranging from 7 amino acids in C. parvum to 24 amino acids in N. crassa) near the C-terminal end of the alignment that is absent in the AOXs of higher plants, prokaryotes and D. discoideum (Fig. 5). The absence of this indel indicates that these groups arose from the same ancestral AOX protein. It is likely that the indel was introduced into the common ancestor of the fungi, oomycota, green algae and parasitic protists. Such signature sequences have recently been used to explore the evolutionary relationships among bacteria (Gupta, 2001). Conservation in the last 13 amino acids of the alignment also suggests the same two robust groupings of organisms suggested by the indel analysis. 4. Discussion and Dasgupta, 1993; Robaina et al., 1995; Eriksen and Lewitus, 1999). Also, isotope discrimination experiments (which use the difference in isotopic discrimination against 18 O by cyt oxidase and AOX to estimate the partitioning of electrons in respiration) in Lake Kinneret suggest a largescale uptake of O2 by AOX in this environment (Luz et al., 2002). Similar experiments examining microbial respiration in soils also suggest a significant contribution by AOX (Angert et al., 2003; Lee et al., 2003). The Sargasso Sea is a very nutrient-depleted environment, which prokaryotes are known to dominate (Whitman et al., 1998; Venter et al., 2004). Interestingly, in both phytoplankton (Weger and Dasgupta, 1993; Eriksen and Lewitus, 1999) and higher plants (Vanlerberghe and Ordog, 2002), AOX can be strongly induced by nutrient limitation and may thus play an important role in such environments. In some bacteria, AOX shares synteny with a gene encoding proteorhodopsin (Fig. 2). Proteorhodopsin is a light-driven proton pump and the resulting electrochemical gradient can drive ATP synthesis. Once thought to be exclusively found in the Archeae, this protein is now known to be widely distributed among divergent marine bacterial taxa, including both a- and g-proteobacteria (de la Torre et al., 2003; Sabehi et al., 2004; Venter et al., 2004). Proteorhodopsin phototrophy could have a significant impact on the overall carbon and energy metabolism of the ocean, by reducing the energy needing to be supplied by respiration (Béjà et al., 2001). It is interesting, then, that at least some of these bacteria also harbour the non-energy conserving AOX respiration pathway. 4.1. AOX respiration in marine bacteria Analysis of a bwhole-genome shotgun sequencingQ metagenomic dataset derived from marine microbes in the Sargasso Sea (Venter et al., 2004) allowed us to uncover 69 different AOX genes, approximately doubling the number of AOX genes in public databases. The AOX proteins encoded by these genes contain the characteristic conserved features of these diiron carboxylate proteins (Fig. 1). Further, the synteny/BLASTp analyses (Fig. 2) and analyses of the N-termini (Fig. 5) indicate that the majority of these genes (67 of the 69) are from marine bacteria. The above findings suggest that AOX could contribute significantly to the respiratory O2 consumption by bacteria in the Sargasso Sea. No studies have specifically addressed this question. In fact, few studies have attempted to establish the presence or activity of AOX respiration in any freshwater or marine species, although AOX genes are beginning to be found in such organisms (Table 1). Studies which have been done suggest that AOX may indeed be widespread in aquatic environments. For example, a significant capacity for AOX respiration (i.e., O2 uptake resistant to CN but sensitive to AOX inhibitors) has been found in members of the Euglenophyceae, Chlorophyceae, Rhodophyceae, Bacillariophyceae, Cryptophyceae, Chrysophyceae and Dinophyceae (Benichou et al., 1988; Weger 4.2. Plastoquinol terminal oxidase in Sargasso Sea cyanobacteria Prochlorococcus and Synechococcus are dominant photosynthetic microbes of the ocean. Previously, it was concluded that ~90% of the cyanobacterial-like scaffolds in the Sargasso Sea dataset formed a conglomerate of closely related Prochlorococcus strains, while much of the remaining cyanobacterial sequence could be attributed to Synechococcus (Venter et al., 2004). Accordingly, most of the prokaryotic PTOX proteins that we uncovered (19 or 20) were closely related to that of P. marinus MED4, while two other sequences were attributed to Synechococcus (see Results). In higher plants, PTOX could function to oxidize plastoquinol generated by a chloroplast NAD(P)H quinone oxidoreductase, a process called chlororespiration (Aluru and Rodermel, 2004; Kuntz, 2004). In this regard, the PTOX synteny displayed by P. marinus MED4 and several Sargasso Sea strains is intriguing, suggesting that NAD(P)H quinone oxidoreductase and PTOX represent a functional and possibly transcriptional unit in these cyanobacteria (Fig. 3). Indeed, a chlororespiration-like pathway is hypothesized to exist in cyanobacteria (Berry et al., 2002; Schreiber et al., 2002). A.E. McDonald, G.C. Vanlerberghe / Gene 349 (2005) 15–24 The complete genomes of three strains of P. marinus (MED4, MIT9313, SS120) are available but we find that PTOX is only present in MED4, a strain that is adapted to the higher light environment of surface waters (Ferris and Palenik, 1998; Rocap et al., 2003). We see changes in cyanobacterial PTOX gene expression in response to changes in light intensity (McDonald AE and Vanlerberghe GC, unpublished), suggesting a role for PTOX in adaptation of the photosynthetic apparatus to changing light environments. 4.3. Origin and distribution of AOX and PTOX proteins Prior to this study, the only example of a prokaryotic AOX was from the a-proteobacterium N. aromaticivorans (Stenmark and Nordlund, 2003). Since an a-proteobacteriallike organism is thought to have given rise to mitochondria, the origin of AOX in eukaryotes may have been the endosymbiotic event that gave rise to mitochondria (Finnegan et al., 2003; McDonald et al., 2003; Atteia et al., 2004). Alternatively, phylogenies showed that the bacterial AOX groups closely with plant AOXs (Stenmark and Nordlund, 2003; McDonald et al., 2003), suggesting the possibility of a horizontal gene transfer of AOX from plants to this bacterium. An analysis of both the N-termini of AOX (Fig. 5) and a phylogeny based on the core of the protein (Fig. 4) indicates a robust grouping of not just higher plants and bacteria, but the slime mold D. discoideum as well. These findings, along with the finding of AOX in numerous groups of bacteria (Fig. 2), support the hypothesis of a vertical inheritance of AOX from bacteria to eukaryotes via endosymbiosis (see McDonald et al., 2003 for further discussion). Interestingly, one of the AOX genes that we found (Acc. No. EAF76395) was incomplete but the partial sequence (180 amino acids) shared 100% amino acid identity with AOX1a of tomato, Lycopersicon esculentum (Acc. No. AAK5882). We can only speculate that this represents degrading plant material brought into the area from the gulf stream, or from bird or animal waste. Another AOX gene (Acc. No. EAK50703) shares 96% similarity with an AOX of the small marine green alga Ostreococcus sp. CCE9901 (Acc. No. AC152104). While the Sargasso Sea dataset is dominated by sequence from Eubacteria (particularly proteobacteria; Venter et al., 2004), no evidence was found to suggest the presence of PTOX in prokaryotes other than cyanobacteria. Hence, the known distribution of PTOX continues to be limited to photosynthetic organisms (see Introduction). This is consistent with the hypothesis that modern-day eukaryotic PTOXs arose from the cyanobacterial symbiont that gave rise to chloroplasts (Finnegan et al., 2003; McDonald et al., 2003; Atteia et al., 2004). Further discussion of the endosymbiotic events that may have given rise to eukaryotic AOX and PTOX proteins as well as the relationship between these two proteins can be found elsewhere 23 (Finnegan et al., 2003; McDonald et al., 2003; Atteia et al., 2004; McDonald and Vanlerberghe, 2004). We found two PTOX singletons (one of which was complete enough for the phylogenetic analysis) that were similar to those known from freshwater green algae (Fig. 4), suggesting that marine algae (which would have been largely excluded from the Sargasso Sea samples) also have PTOX. We also uncovered three PTOX singletons that were closely related to one another but which grouped only distantly with the PTOXs of higher plants, a diatom and hcyanobacteria (Fig. 4). At present, we have assumed that these are eukaryotic and representative of some other photosynthetic group such charophytes, chrysophytes, dinoflagellates or brown algae. However, these PTOXs are quite unique in that, similar to the a-cyanobacteria, they have an ~22 amino acid deletion compared to all other known PTOXs (Fig. 1). 4.4. Conclusions Prokaryotes are ubiquitous members of aquatic environments and are known to dominate the biomass of oligotrophic open ocean waters (Whitman et al., 1998). 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