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Alternative oxidase and plastoquinol terminal oxidase in marine

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Alternative oxidase and plastoquinol terminal oxidase in marine
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). As
such, bacteria are key contributors to Earth’s biogeochemical cycles. This study finds that AOX and PTOX are
prevalent in marine bacteria. It now remains to be
established how AOX respiration and PTOX activity may
be impacting productivity of the world’s oceans.
Acknowledgements
Work was supported by a research grant from the Natural
Sciences and Engineering Research Council of Canada (to
G.C.V.) and by an Ontario Graduate Scholarship (to
A.E.M.).
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