Genomic Makeup of the Marine Flavobacterium Nonlabens

13 Pages·2013·0.75 MB·English

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Genomic Makeup of the Marine Flavobacterium Nonlabens
(Donghaeana)dokdonensisandIdentificationofaNovelClass
of Rhodopsins
Soon-Kyeong Kwon1,2, Byung Kwon Kim3, Ju Yeon Song3, Min-Jung Kwak1,2, Choong Hoon Lee3,
Jung-Hoon Yoon4, Tae Kwang Oh1,5, and Jihyun F. Kim1,3,*
1SystemsandSyntheticBiologyResearchCenter,DivisionofBiosystemsResearch,KoreaResearchInstituteofBioscienceandBiotechnology,
Yuseong-gu,Daejeon,Korea D
o
2BiosystemsandBioengineeringProgram,SchoolofScience,UniversityofScienceandTechnology,Yuseong-gu,Daejeon,Korea w
n
3DepartmentofSystemsBiology,CollegeofLifeScienceandBiotechnology,YonseiUniversity,Seodaemun-gu,Seoul,Korea loa
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4DepartmentofFoodScienceandBiotechnology,CollegeofBiotechnologyandBioengineering,SungkyunkwanUniversity,Jangan-gu,Suwon, ed
Gyeonggi-do,Korea fro
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521CFrontierMicrobialGenomicsandApplicationsCenter,Yuseong,Daejeon,Korea h
ttp
*Correspondingauthor:E-mail:[email protected]. s
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Accepted:December 21, 2012 c
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Datadeposition:CP001397 em
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Abstract o
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Rhodopsin-containingmarinemicrobessuchasthoseintheclassFlavobacteriiaplayapivotalroleinthebiogeochemicalcycleofthe be
/a
euphoticzone(FuhrmanJA,SchwalbachMS,StinglU.2008.Proteorhodopsins:anarrayofphysiologicalroles?NatRevMicrobiol. rtic
6:488–494).Decipheringthegenomeinformationofflavobacteriaandaccessingthediversityandecologicalimpactofmicrobial le
-a
rhodopsinsareimportantinunderstandingandpreservingtheglobalecosystems.Thegenomesequenceoftheorange-pigmented b
s
marineflavobacteriumNonlabensdokdonensis(basonym:Donghaeanadokdonensis)DSW-6wasdetermined.Asamarinephoto- tra
c
heterotroph, DSW-6 has written in its genome physiological features that allow survival in the oligotrophic environments. The t/5
sequenceanalysisalsouncoveredageneencodinganunexpectedtypeofmicrobialrhodopsincontainingauniquemotifinaddition /1/1
toaproteorhodopsingeneandanumberofphotolyaseorcryptochromegenes.Homologsofthenovelrhodopsingenewerefound 87
/7
inotherflavobacteria,alphaproteobacteria,aspeciesofCytophagia,adeinococcus,andevenaeukaryotediatom.Theyallcontainthe 3
1
characteristicNQmotifandformaphylogeneticallydistinctgroup.ExpressionanalysisofthisrhodopsingeneinDSW-6indicatedthat 78
3
itisinducedathighNaClconcentrations,aswellasinthepresenceoflightandtheabsenceofnutrients.Genomicandmetagenomic b
y
surveysdemonstratethediversityoftheNQrhodopsinsinnatureandtheprevalentoccurrenceoftheencodinggenesamong g
u
e
microbialcommunitiesinhabitinghypersalineniches,suggestingitsinvolvementinsodiummetabolismandthesodium-adapted s
lifestyle. t on
1
Key words: heterotrophic picoplankton, Bacteroidetes, bacteriorhodopsin, xanthorhodopsin, sodium pump, metagenome. 0 A
p
ril 2
0
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Introduction 9
animportantpositioninthephotometabolicsysteminmarine
Marineecosystems,inwhichhalfofglobalprimaryproduction environments.Acomprehensivecommunityanalysisindicates
occurs, are home tooligotrophs that are responsible for the that the phyla Proteobacteria (63%), Bacteroidetes (13%),
biogeochemical cycle and add an important axis to the Cyanobacteria(7.9%),Firmicutes(7.5%),andActinobacteria
Earth’s energy balance (Falkowski et al. 1998; Whitman (4.6%) are among the dominant members of the oceanic
et al. 1998; Azam and Malfatti 2007). Flavobacteria, which picoplankton (Venter et al. 2004). At the class level, Alpha-
belong to the phylum Bacteroidetes, previously called the and Gammaproteobacteria and Flavobacteriia are believed
Cytophaga–Flavobacterium–Bacteroides (CFB) group, occupy to be major carriers of microbial rhodopsins called
(cid:2)TheAuthor(s)2013.PublishedbyOxfordUniversityPressonbehalfoftheSocietyforMolecularBiologyandEvolution.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/by-nc/3.0/),which
permitsunrestrictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013 187
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Kwonetal.
proteorhodopsins(PRs) (Giovannoni etal. 2005; Ruschet al. et al. 2006; Yi and Chun 2012). The genome information
2007). Among metagenome fragments recruited from the provides a glimpse to the survival strategy of DSW-6 as a
GlobalOceanSampling(GOS)expedition,twoassembledfla- photoheterotroph in the oligotrophic ocean. Importantly, in
vobacterial genomes harboring the PR gene turned out to addition to a typical PR, we found a new type of rhodopsin
represent the dominant taxa in the Northwest Atlantic whose retinal-binding sequences are distinct from those of
(Ruschetal.2007;Woykeetal.2009). well-studiedrhodopsins.Tobetterunderstandthecharacter-
Solar energy is captured and converted into chemical isticsofthisnewtypeofrhodopsin,itsgeneexpressionlevelin
energy by phototrophs that depend either on the chloro- DSW-6wasmonitoredundervariouslightintensities,nutrient
phyll-harboring photosynthetic reaction center or on the concentrations, and NaCl concentrations. Similarity searches
photoactive retinal-binding rhodopsin. Contrary to the against completely sequenced genomes and expressed
multicomponent photosynthetic reaction centers, which are sequencetagswereperformed,revealinganumberofhomo- D
o
restrictedtosixbacterialphyla,single-moleculemicrobialrhod- logspresentintheclassesFlavobacteriia,Alphaproteobacteria, wn
opsins show wide taxonomic distribution, possibly through Cytophagia, and Deinococci, and even a eukaryotic diatom. loa
d
horizontalgenetransferbetweendomainsandphyla(Bryant Finally,thefrequencyofthisrhodopsinfamilyindiverseaqua- ed
and Frigaard 2006; Sharma et al. 2006; Bryant et al. 2007). tic ecosystems was investigated by searching through public fro
m
Despitethegreatdiversityofmicrobialrhodopsins,thesepro- databasesofenvironmentalsequencedata. h
teins share structural features such as seven-membrane- ttp
s
spanning helices (Fuhrman et al. 2008). The structure and ://a
functionofarchaealbacteriorhodopsins(BRs)withtheretinal Materials and Methods cad
chromophorehavebeenstudiedmostintensivelytodate.BRs em
moveprotonsacrossthemembraneoutofthecellusinglight StrainandCultureConditions ic
.o
energytogenerateanelectrochemicalprotongradient,which Donghaeana dokdonensis DSW-6, recently reclassified as up
in turn can be used for ATP production (Lanyi 2004). Nonlabens dokdonensis comb. nov. (Yi and Chun 2012), .com
Metagenomic approaches enabled the discovery of PRs, the was isolated from the surface seawater collected between /g
b
firstrhodopsinofbacterialoriginfromtheunculturedmarine the two main islands of Dokdo, Republic of Korea (Yoon e/a
gammaproteobacterialSAR86group(Bejaetal.2000,2001). etal.2006).Thisnonmotilestraingrowsunderstrictlyaerobic rtic
PRssharehighsequencesimilaritywithBRs,andlight-driven conditionsandexhibitsoptimalgrowthinthepresenceof2% le-a
chemiosmoticprotontranslocationwasobservedafterheter- NaClat25(cid:2)C(Yoonetal.2006).CellsweregrownonMarine bs
ologousexpressioninEscherichiacoli(Bejaetal.2000,2001). tra
Agar2216(Difco,USA)orArtificialSeaWater(ASW)prepared c
Recently,inaPR-containingmarineflavobacterialsuspension, from sea salts (Sigma-Aldrich, USA) enhanced by 2.5% w/v t/5/1
light-drivenprotontransportactivitysufficientforATPgener- peptone and 0.5% w/v yeast extract. The strain produces /1
8
ationwasdemonstrated(Yoshizawaetal.2012). 7
orange-coloredcarotenoidpigments. /7
Among other well-known rhodopsins that generate 31
7
proton-motive force, xanthorhodopsins (XRs), which have 8
been discovered first in Salinibacter ruber, are unusual in GenomeSequencingandAnnotation 3 b
y
thattheyrequiretwochromophores,thecarotenoidssalinix- AhybridapproachofRoche/454pyrosequencingandSanger gu
e
anthin and retinal, to broaden the spectral range for light sequencing followed by manual gap filling was applied to s
t o
harvesting (Balashov et al. 2005). Actinorhodopsins (ActRs), deciphertheN.dokdonensisDSW-6genome.Shotgunpyro- n
1
whichwererecentlyfoundinactinobacteriafromahypersa- sequence reads of approximately 30-fold genome coverage 0
A
linelagoon,anestuary,andafreshwaterlake,areabundantin were generated from GS FLX (NICEM, Korea) and were p
microbial communities in freshwater ecosystems (Sharma assembled into 98 contigs using gsAssembler. A total of ril 2
0
et al. 2009). Halorhodopsins (HRs), which are light-driven 2,035 paired-end Sanger sequence reads (GenoTech Co., 19
inwardchloridepumps,existinhalophiles.Theserhodopsins Korea) from a 35-kb genomic library were incorporated to
may participate in the regulation of ionic content and the yieldtwoscaffolds.Genomicregionscontainingnonribosomal
osmotic state (Mongodin et al. 2005). Although most rhod- peptidesynthetasegenesorISelementscouldnotbeproperly
opsins function as ion transporters, sensory rhodopsins (SRs) assembled because of their highly repeated sequence pat-
mediatephototaxisorsignaltransduction,likephotoreceptors terns. To disentangle these overcollapsed contigs, additional
do (Fuhrman et al. 2008). The amino acid sequences of the Sangersequenceswereprovidedbyrandomshotgunsequen-
proton-pumpingrhodopsinsnecessaryforretinalbindingare cing of 35-kb fosmid clones spanning each gap. All the re-
highlyconservedanddifferfromthoseofHRs,andSRseven maining small gaps were closed by sequencing polymerase
lackthefunctionalresiduesintheretinal-bindingpocket. chain reaction (PCR)-amplified genomic fragments. The
In this study, we determined and analyzed the complete PHRED/PHRAP package (Ewing and Green 1998) was used
genomesequenceofanorange-pigmentedmarineflavobac- for Sanger read base calling and partial mini-assembly, and
terium,Nonlabens(Donghaeana)dokdonensisDSW-6(Yoon all sequence editing procedures were conducted using
188 GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013
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GenomicMakeupoftheMarineFlavobacterium
CONSED (Gordon et al. 1998). The final assembly led to a and MrBayes v3.1.2 (Zhang et al. 2012) for Bayesian infer-
singlechromosomewithoutplasmids.Thesequencewasvali- ence.PhylogenetictreeswereconstructedbytheNJmethod
datedanderrorswererectifiedbycomparingthefinalassem- basedontheJukes&Cantor distancemodel, followedby a
bly with independent sequence data to further increase the 1,000-replicate bootstrap analysis for statistical support. For
accuracyoftheassemblyandtoavoidsequenceerrorsinthe theapplicationofMLandBayesianmethods,weranProtTest
homopolymeric nucleotides. Trimmed high-quality 102-bp v2.4(Abascaletal.2005)todeterminetheappropriatemodel
shotgunreadsat806-foldgenomecoveragewereproduced ofaminoacidreplacement.Eachcomputationwasaccompa-
using the Illumina/Solexa GA II system and were mapped to nied with 500,000-generation runs of four chains. Finally,
the assembled sequence with CLC Genomics Workbench Bayesianposteriorprobabilityfrom thesetofnonparametric
(CLCbio,Inc.,Denmark). bootstrapreplicatesamplestreeswassupportedbySumTrees
GenepredictionofthefinishedDSW-6genomesequence programinDendroPypackage(SukumaranandHolder2010). D
o
was conducted using Glimmer 3.0 (Delcher et al. 2007). The 0.25 fraction of initial number of trees in each file was wn
Functional assignment of the predicted genes was achieved excluded from the analysis. The image of trees obtained by loa
d
bysearchingforhomologsinpublicproteindatabases,suchas MrBayeswasillustratedbyusingtheDendroscope3program ed
UniRef100, Swiss-Prot, the GenBank nonredundant protein (HusonandScornavacca2012). fro
m
database, KEGG, and SMART, using the Basic Local h
Alignment Search Tool (BLAST) program (Altschul et al. NaCl-DependentExpressionoftheNQRhodopsinGene ttps
1997). The outputs were automatically parsed using ://a
ForthestudyofNQrhodopsingeneexpression,DSW-6was c
AutoFACT (Koski et al. 2005), and then all the annotations a
d
grown in 30ml of ASW enriched with an additional carbon e
weremanuallycurated.tRNAscan(LoweandEddy1997)was m
applied to search for tRNA genes in the genome, and rRNA source.ASWpreparedwithseasalt(Sigma-Aldrich,USA)was ic.o
passedthrougha0.2-mm-pore-sizefilter,enrichedbyadding u
geneswereidentifiedusingBLAST.Metabolicpathwayswere p
0.15g of peptone (Bacto Peptone, Difco) and 0.03g .c
examined using the KEGG database (Aoki-Kinoshita and o
m
Kanehisa 2007) and BioCyc, which was generated by the yeastextract(BactoYeastExtract,Difco)andthenautoclaved. /g
PathoLogic program of the Pathway Tools software (Karp The final dissolved organic carbon concentration was be/a
et al. 2010). The sequence and annotation have been de- approximately 2,200ppm, as measured using a TOC-V total rtic
organiccarbonanalyzer(Shimadzu,Japan).Triplicatecultures le
positedinGenBankundertheaccessionnumberCP001397. were incubated at 25(cid:2)C under continuous light -ab
The genome information is also available from the Genome s
Encyclopedia of Microbes (GEM; http://www.gem.re.kr, last (135.05mmolm(cid:3)2s(cid:3)1 photons). When the culture’s optical trac
accessedJanuary9,2013). dtuerneswityasatce6n0t0rinfumgerdea,cahneddthaepppreollxeitmwaateslsyto0r.4ed,3immmloefdtiahteelcyuinl- t/5/1/1
8
500mlofRNAlater(Ambion,USA)toyieldtheuntreatedcon- 7
PhylogeneticAnalysisofMicrobialRhodopsins /7
trol(UC)samples.Fortheremainingcultures,4MNaClorthe 3
1
To infer the phylogenetic groups of microbial rhodopsins, carbon-enrichedASWwasaddedintoeachcellculturetoyield 78
3
exemplary representatives of each class of ActRs, BRs, HRs, NaClconcentrationsof2.5%(0.43M),5.0%(0.86M),7.5% b
y
SRs, PRs, and XRs were retrieved from the UniProt or (1.28M), 10% (1.71M), and 12.5% (2.14M). The culture g
u
e
GenBank databases. The two rhodopsins of N. dokdonensis volumeofeachsamplewasadjustedto50ml,causingadi- s
t o
DSW-6 and nine NQ-type rhodopsins identified from com- lutionofapproximately2-fold.Tomakethe4MNaClstock, n
1
plete genomes and expressed sequence tags were included NaCl was dissolved in the ASW enriched with peptone and 0
A
in the analysis. They are from Gillisia limnaea R-8282 (Van yeast extract to maintain the nutrient concentration of the p
Trappen et al. 2004), Krokinobacter (reclassified into culturemedium.WhentheOD600nmofthe2.5%NaClcul- ril 20
Dokdonia; Yoon et al. 2012) sp. 4H-3-7-5 (Klippel et al. turereachedapproximately0.4again,induction(ID)samples 19
2011), Hymenobacter roseosalivarius DSM 11622 (Hirsch were collected in 500ml of RNAlater by the same method.
et al. 1998), Citromicrobium bathyomarinum JL354 (Jiao Samplesof3mlwerecollectedforthe2.5%NaClIDcultures,
et al. 2010), Citromicrobium sp. JLT1363 (Zheng et al. 5ml samples were collected for the 5.0% NaCl ID cultures,
2011), Fulvimarina pelagi HTCC2506 (Kang et al. 2010), and 6ml samples were collected for the 7.5%, 10%, and
Truepera radiovictrix DSM 17093 (Albuquerque et al. 2005), 12.5%NaClIDcultures.AlloftheRNAlater-treatedsamples
andChaetocerosneogracileKOPRIAnM0002.Amultiplese- were stored at 4(cid:2)C until RNA extraction. Total RNA was ex-
quencealignmentwasobtainedusingtheMUSCLEalgorithm tracted using the RNeasy kit (Qiagen, USA). The remaining
(Edgar2004),andambiguouslyalignedregionswereadjusted chromosomal DNA was eliminated using a Turbo DNA-free
withtheGblocksprogram(TalaveraandCastresana2007).To Kit (Ambion, USA). No amplification was observed after 40
construct phylogenetic trees, we used the MEGA5 package cyclesofPCRwhenusingtheRNAsamplesastemplateswith-
(Tamura et al. 2011) for maximum parsimony (MP) and outthereversetranscriptionstep,confirmingcompleteDNA
neighbor-joining(NJ),andmaximumlikelihood(ML)methods, removal. cDNAs were obtained from reverse transcription
GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013 189
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Kwonetal.
polymerase chain reaction (M-MLV cDNA Synthesis Kit, localizedwithintheNQ-rhodopsingrouporshowslowphylo-
Enzynomics,Korea). geneticcorrelations(bootstrapvalue(cid:5)70),thecorresponding
Real-time PCRs were carried out on a CFX Connect readwasdiscarded.
Real-Time PCR Detection System (Bio-rad, USA) with the Relationships among the 10 NQ-type rhodopsins and the
iQ SYBR Green Supermix (Bio-rad). Each cDNA sample was 55 metagenomic reads were further looked into through a
amplifiedwithspecificprimers(qNR_F5’-GAGAATTATGTA clusteringanalysis,becausemanyoftherhodopsinsequences
GGTGCTACAGACG,qNR_R5’GTGCCAAATTACCAA frommetagenomedatasetsareinsmallfragmentsandthey
GTAATACACCA,q16S_F5’-AGGACTTAACCTGACACC often do not overlap to each other. We made a simple as-
TCAC,q16S_R5’-GGGTTGTAAACTACTTTTGTACAG, sumption that the identity values of a test sequence against
qPR_F5’-CTAAAATGGCCACAGATGATTATGTAG,and the10referencesequenceswillbesimilartothoseofitsclose
qPR_R 5’-TTG CAT CAC CAA TGT TGT AAA CTA CG) and relatives. Gap regions among the 10 references were deter- D
o
quantifiedintriplicate.ThePCRconditionswereaninitialde- minedbymultiplesequencealignmentbasedontheMUSCLE wn
naturation step at 95(cid:2)C for 5min, followed by 40 cycles of algorithm(Edgar2004),andaminoacidpositionscorrespond- loa
d
amplification at 95(cid:2)C for 30s, 55(cid:2)C for 30s, and 72(cid:2)C for ingtothegapswereexcludedfromfurtheranalysis.Identity ed
1min.Finally,anadditionalsteptoestablishthemeltingcurve, values between the test sequences and the references were fro
m
in which the temperature was decreased from 95 to 65(cid:2)C calculatedusingtheMEGA5programwiththepairwisedele- h
(0.05(cid:2)Cs(cid:3)1), was performed. Threshold cycle values (C) for tion option. To estimate the similarities between the test ttp
t s
eachmeasurementweredetermined.Therelativequantifica- sequences,theidentityvalueswereusedasinputcharacters ://a
c
tionofgeneexpressionwascalculatedbyusingthecompara- for the UPGMA algorithm (Murtagh 1984) with root mean a
d
tivecriticalthreshold2(cid:3)(cid:2)(cid:2)CTmethod,inwhichtheamountof squaredeviation(Huangetal.2005). em
theRNA of interestis adjustedtoaninternalreferenceRNA The prevalence of prokaryotic cells that possess the ic.o
u
(LivakandSchmittgen2001).The16SrRNAgenewasusedas NQ-type rhodopsins in the metagenomic data sets was pos- p
.c
theinternalcontrolinthisstudy.Thefollowingequationswere tulatedbytheratiooftheNQ-typerhodopsinreadstoRecA, o
m
used: RpoB, and EF-Tu reads. These three proteins are commonly /g
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(cid:2)CT ¼Ct of NQ rhodopsinðgene of interestÞ eBnLAcoSdTeXdsbeyarachsiensgolef-ctohpeymgeetnaegiennmomosictpreroakdasrfyrootmicgCeAnMomEReAs. e/artic
(cid:3)Ct of 16S rRNA geneðinternal controlÞ andIMG/MagainstRecA,RpoB,andEF-Turetrievedfromthe le-a
b
(cid:2)(cid:2)C ¼(cid:2)C of ID samples(cid:3)(cid:2)C of UC samples GenBank and UniProt databases followed by BLASTX of the s
RelatiTveexpreTssionlevel¼2(cid:3)(cid:2)(cid:2)CT T r2e0s0u3lt)inidgecnatnifideiddattheesaregaadinsshtothmeoCloOgGoudsattoabthaseet(hTraeteusporovteetinasl.. tract/5
/1
Whenestimatingthenumberofeachprotein,thelengthof /1
8
theproteinshouldbetakenintoaccount;thelongeraprotein 7/7
3
MetagenomicDataAnalysisoftheNQRhodopsins is, the higher the chance of that protein being sequenced 17
from the metagenome pool. Thus, the number of reads as- 83
Toidentifythenewtypeofrhodopsingenesfromthemeta- b
signedtoeachproteinwasdividedbytheaveragelengthof y
genomic data sets and to assess their abundance in diverse g
the protein. The normalized proportion (r ) of prokaryotes u
a e
aquatic environments, approximately 58Gb of unassembled s
reads from metagenome sequencing projects were down- cdoantatasientinwgatshcealncuelwatetdypaesofofllorhwosd:opsins in a metagenomic t on 1
loaded from the CAMERA (Sun et al. 2011) and IMG/M 0
(sMeaarrckhoewsoitfzinetdiavli.d2u0al1r2e)addsat(aidbeansteitsyi(cid:4)nO30c%tob,eErv2a0lu1e1(cid:5).B1LeA(cid:3)ST5X) ra ¼ Njd=(cid:3)ld April 2
were performed against an in-house database consisting of 1nPNi=(cid:3)li 019
i
prokaryotic rhodopsins selected for phylogenetic reconstruc-
whereN isthenumberofreadsdesignatedasNQrhodopsins
tion retrieved from the GenBank and UniProt databases. d
andN isthenumberofthei-threferenceprotein.l andl are
Identity of the NQ-type rhodopsins among the recruited i d i
the lengths of the nucleotide sequences of each protein. n
reads was assured by carrying out BLASTP searches of the
(¼3)indicatesthenumberofreferenceproteins.
translationproductsagainsttheGenBanknonredundantpro-
tein database and selecting ones that matches the NQ-type
rhodopsinsinthedatabaseasoneofthetopthreebesthits. Results
The metagenomic reads confirmed as NQ-type rhodopsins
GenomePropertiesforMarineHeterotrophy
werealignedwith10full-lengthNQ-typerhodopsins(seeear-
lier),andtheirphylogeneticpositionswereassignedbyrecon- IsolatedfromtheseawatersampletakenatDokdointheEast
structing trees using the NJ method in the MEGA5 package SeaofKorea,N.dokdonensis(basonym:Donghaeanadokdo-
(Tamura et al. 2011) with 1,000 iterations. If a read is not nensis)DSW-6isamemberoftheclassFlavobacteriia(Yoon
190 GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013
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GenomicMakeupoftheMarineFlavobacterium
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FIG.1.—OverviewofmetabolismandtransportinNonlabens(Donghaeana)dokdonensisDSW-6.Transportersaregroupedbytheirtransportmech- tra
anisms:secondarytransporters(yellow),ATP-driventransporters,ionchannels(orange),P-typeATPasesuperfamily(purple),andunclassifiedtransporters. ct/5
Directionsofsubstratetranslocationareindicatedbyarrows. /1
/1
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7
etal.2006).Wedeterminedthecompletegenomesequence of proteins by secreted peptidases and then transported in /73
1
ofthismarineflavobacterium,whichharborsasingle3.9-Mb across the membrane, where they serve as the main source 7
8
3
chromosome (supplementary fig. S1 and table S1, ofnitrogen.Theureacycledoesnotexist,butapathwayfor b
y
SupplementaryMaterialonline).Phylogeneticanalysisof16S the conversion of nitrite to ammonia to glutamine is con- g
u
rRNA genes suggested Gillisia, Gramella, Psychroflexus, and served. Enzymes involved in the synthetic pathway for the es
ZunongwangiaasthesistergeneraofNonlabens,whereasa three branched-chain amino acids leucine, isoleucine, and t o
n
treebasedonbroadlyconservedproteinsoftheflavobacteria, valinearemissing,butgenesencodingtheenzymesrespon- 10
whosegenomeshavebeencompletelysequenced,indicated sible for the degradation of these amino acids exist in the Ap
that Croceibacter, Gramella, Krokinobacter, Lacinutrix, and genome. ril 2
0
ZunongwangiaarecloselyrelatedtoNonlabens(supplemen- 1
9
taryfig.S2,SupplementaryMaterialonline). PhysiologyforSurvivalintheMarineEnvironment
The genome sequence allowed us to reconstruct in silico Asamarinebacteriumthatinhabitssurfaceseawater,DSW-6
themetabolic networkof thebacterium. The primarymeta- hasmanyattributesthatmayendowthebacteriumwithmeta-
bolicnetwork,includingtheaminoacidbiosyntheticpathways bolicadvantagesthatallowsurvivalunderoligotrophiccondi-
andtransportsystems,issummarizedinfigure1andsupple- tions.Enzymescompensatingforcarbonlimitationincentral
mentarytableS2,SupplementaryMaterialonline.Thegenes metabolism are well conserved in DSW-6. Anaplerotic
encoding the enzymes for glycolysis and the pentose phos- enzymes replenish the pools of metabolic intermediates in
phatepathwaywereallpresent.Thesepathwaysprovidethe the TCA cycle, which are used as precursors for the biosyn-
precursorsofmetabolitesfornucleotideandfattyacidbiosyn- thetic pathways, and maintain the oxidative carbon flux.
thesis.Alltheenzymesinthetricarboxylicacid(TCA)cycleare Phosphoenolpyruvate (PEP) carboxylase (DDD_3148) and
also well conserved in this genome. Most amino acids are pyruvate carboxylase (DDD_1224) each create oxaloacetate
synthesizedbyDSW-6itselforarederivedfromthedigestion from PEP or pyruvate by adding bicarbonate (Owen et al.
GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013 191
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Kwonetal.
2002). Bicarbonate is imported into the cell by transporters online).Carotenoidsandthesephotoproteinsarealsorelated
encoded by a SulP-type Na+-dependent bicarbonate trans- totherhodopsin-basedphotosysteminthemarineeuphotic
porter (DDD_2127) or the Na+-dependent bicarbonate sec- zone. b-Carotene, one of the carotenoid pigments, is a
ondary transporter SbtA (DDD_0780). Carbonic anhydrase precursor of retinal that binds to rhodopsin, and phylogen-
interconvertsCO andbicarbonatetosustainasufficientsub- etic analysis illustrates the coexistence of photolyase/crypto-
2
stratelevel. chromeandPRsamongmarineflavobacteria(Gonzalezetal.
The CFB group bacteria are known to be adapted 2008).
tousehigh-molecular-weightorganicmatter, primarily poly-
saccharides and proteins (Kirchman 2002). In particular, IdentificationofaUniqueTypeofMicrobialRhodopsin
during a mesocosm study of phytoplanktonic blooms, dom-
When mining through the DSW-6 genome, two rhodopsin
inatingflavobacteriawereshowntoplayleadingrolesinthe D
genes,aswellastheblhgene,whoseproductisresponsible o
degradation of organic matter in micronutrient-rich environ- w
ments (Riemann et al. 2000). Genome analysis of several fortheoxidativecleavageofb-caroteneintotworetinalmol- nloa
ecules,wereuncovered.Retinalbindingisessentialforrhod- d
marine flavobacteria revealed that these organisms are e
adaptedtousepolymeric organicmatterthat isoccasionally orhposdinopfusinncstioann.dE.thceolipBuLr2ifi1e(dDEp3r)oetexpinreswsienrgeepitihnekrisho-fretdhesine d from
available (Bauer et al. 2006; Gonzalez et al. 2008, 2011). colorduetothebindingofrhodopsintotrans-retinal,which h
Similartothegenomesofthesestrains,theDSW-6genome ttp
wasprovidedseparately(datanotshown). s
encodesnumerousdegradingenzymes,including14glycosyl Primarysequenceanalysisofthetranslationproductssug- ://a
transferases,20glycosylhydrolases,and25predictedpeptid- ca
gestedthatoneisatypicalPR.Itssequenceishighlysimilarto d
ases.Thisgenomealsoencodes24proteinswithcell-surface- em
thoseofflavobacterialPRs.Thefunctionalresiduesintheion
adhesion domains that may function in binding to organic ic
transferpathwayareallconserved,asinthenumerousother .o
particles for efficient breakdown, even though the bacter- up
ium is not motile. These proteins benefit the survival of proton-pumpingrhodopsins.Retinalispredictedtocovalently .co
bindtothe"-aminogroupoftheLys-233residueinDSW-6PR m
DSW-6 in nutrient-poor oceans where blooms occur only /g
(Lys-216forBR),yieldingaprotonatedretinylideneSchiffbase. b
occasionally. e
Whenstimulatedbylight,Asp-87(Asp-85forBR)becomesa /a
DSW-6 shares features with other ocean-inhabiting bac- protonacceptorresidueoftheprotonfromthedeprotonated rtic
teria that establish a Na+ gradient instead of a H+ gradient le
retinalmolecule.TheprotonreleasegroupofArg-84(Arg-82 -a
forgeneratingmotiveforceforthefirststepoftheirrespira- b
tory chain (Unemoto and Hayashi 1993). DSW-6 possesses forBR)pumpsouttheprotontotheextracellularsideofthe stra
Na+-translocating NADH:quinine reductase (NQR) subunits membrane.Glu-97(Asp-96forBR)restorestheoriginalpro- ct/5
tonatedformoftheretinalmolecule.Then,aprotonfromthe /1
as parts of NADH dehydrogenase. In addition to the NADH /1
cytosol reprotonates the donor residue, and the protonated 8
dehydrogenase subunits, genes encoding the cytochrome c 7
acceptorresiduegivesaprotontothereleasegroup,andthe /7
oxidase, cytochrome c, and ATP synthase are present, but 31
proton-pumpingcyclerepeats(Hayashietal.2003). 7
genesforthebc complexdonotexist(supplementarytable 8
S3, Supplementa1ry Material online). Instead, the alternative Interestingly,theotherrhodopsingeneappearstoencodea 3 b
complex III (Refojo et al. 2010) is present to feed electrons newtypeofmicrobialrhodopsinaccordingtotheaminoacid y gu
sequence analysis. Although this rhodopsin has seven trans- e
to cytochrome c. DSW-6 has numerous genes to cope with s
membrane domains, as do other rhodopsins, its sequence t o
internalorenvironmentalstresses,suchasoxidativestressor n
differs considerably. Notably, although carotenoid-binding 1
osmotic shock (supplementary table S4, Supplementary 0
Lys-255 (Lys-216 for BR) and proton-releasing Arg-109 A
Materialonline). p
Many nonphotosynthetic marine bacteria use carotenoids (Arg-82forBR)areconserved,thekeyactivesiteresiduesin ril 2
todealwiththedamagecausedbysolarradiation(Haderand typical proton-pumping rhodopsins (Asp-85 and Asp-96 for 019
BR)arereplacedbyAsn-112andGln-123(fig.2A),suggesting
Sinha2005)ortoadapttothecoldenvironmentbymodulat-
that this protein is functionally unique. We will refer to this
ingmembranefluidity(Jagannadhametal.2000).DSW-6is
Asn-(Xaa )-GlnsequenceastheNQmotifhenceforth.
orangecoloredbecauseofcarotenoidpigments(Yoonetal. 10
2006),whicharesynthesizedbyclusteredcrtgenes.Therep-
TheNQMotif-ContainingRhodopsinsFormaDistinct
ertoireandorganizationofthecarotenoid-biosyntheticgenes
PhylogeneticClass
are conserved between DSW-6, G. limnaea R-8282,
and Krokinobacter sp. 4H-3-7-5 (supplementary fig. S3, Homologousproteinsweredetectedinbroadlydifferenttaxa
SupplementaryMaterialonline).DSW-6mayexpressnumer- through similarity searches of public databases of microbial
ous photolyases/cryptochromes related to repairing UV- genomes.Genesencodingrhodopsinsofthenewtypewere
induced DNA damage and PAS/BLUF/PAC light-sensing present in the recently sequenced genomes of three CFB
domainslocatedinmembrane-boundsensormolecules(sup- strains and three marine alphaproteobacterial strains
plementary fig. S4 and table S5, Supplementary Material (fig. 2B). Among these strains, two flavobacterial strains,
192 GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013
GBE
GenomicMakeupoftheMarineFlavobacterium
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FIG.2.—CharacteristicfeaturesofthenewfamilyofrhodopsinsthatcontaintheNQmotif.(A)Aminoacidsequencealignmentofthethirdtrans- s
membranehelixoftheNQrhodopsinofNonlabens(Donghaeana)dokdonensisDSW-6andrepresentativemicrobialrhodopsins.Asterisks,invariantresidues; tra
c
shaded,activesiteresiduesofmicrobialrhodopsins.(B)Phylogeneticrelationshipsamongmicrobialrhodopsins.Atreeinferredfrom199conservedamino t/5
/1
acidpositionswasconstructedusingMEGA5.Bootstrapvaluesfor1,000replicatesareshownnexttothebranches.NQ,NQrhodopsin;BR,bacteriorho- /1
8
dopsin; PR, proteorhodopsin; XR, xanthorhodopsin; ActR, actinorhodopsin; HR, halorhodopsin. Accession numbers: Citromicrobium sp. JLT1363, 7
ZP_08702831; Citromicrobium bathyomarinum JL354, ZP_06860850; Fulvimarina pelagi HTCC2506, ZP_01440547; Chaetoceros neogracile KOPRI /73
1
AnM0002, EL620625; Truepera radiovictrix DSM 170931, YP_003705905; T. radiovictrix DSM 170932, YP_003706581; Gillisia limnaea R-8282, 7
8
ZP_09669334;Krokinobacter(Dokdonia)sp.4H-3-7-5,YP_004429763;HymenobacterroseosalivariusDSM11622,geneID2502504185orlocustag 3 b
HrosDRAFT_3745intheIMGdatabase. y g
u
e
s
G. limnaea R-8282 and Krokinobacter sp. 4H-3-7-5, and a (fig. 2B and supplementary fig. S5, Supplementary Material t o
n
cytophagal species, H. roseosalivarius, have both the PR online). 1
0
gene and the new type of rhodopsin. The deinococcus A
p
T. radiovictrix, a radiation-resistant species recovered from TheExpressionoftheNQRhodopsinGeneofDSW-6Is ril 2
sodium-richhotspringrunoff,carriedtwoof thesegenesin InducedatHighConcentrationsofNaCl 01
9
addition to an HR gene and a rhodopsin gene of an un-
Tracking gene expression patterns can be a reliable clue for
assigned family. All these rhodopsins contain the NQ motif
deducingthefunctionofagivengene,ascellschangetheir
instead of the DD motif, and the existence of genome se- geneexpressioninresponsetoenvironmentalperturbations.
quences containing only the new type suggests that these ToinfertheroleoftheDSW-6rhodopsins,thegeneexpres-
organismsapparentlydonotrequireblhandidiforfunction- sionlevelsundervariousnutritionalandenvironmentalcondi-
ing(table1).Surprisingly,ahomologoussequencewasfound tions were measured (see supplementary materials and
amongtheexpressedsequencetagsoftheAntarcticmarine methods, Supplementary Material online). Quantitative
planktonicdiatomC.neogracile,suggestingthatthisclassof real-timePCR(qRT-PCR)analysisindicatedthatbothrhodop-
rhodopsins is not restricted to prokaryotes. Comparative singenesarehighlyinducedwhencellsareincubatedinthe
analyses based on NJ, ML, and Bayesian methods confirm, presenceoflightorintheabsenceofsufficientnutrients(sup-
supported by (cid:4)99% bootstrap values, that the NQ motif- plementaryfig.S6,SupplementaryMaterialonline).However,
containing rhodopsins form a distinct phylogenetic group the absolute levels of the gene encoding the NQ
GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013 193
GBE
Kwonetal.
Table1
Rhodopsin GenesandRetinal Biosynthetic Genesin Nonlabens(Donghaeana) dokdonensis DSW-6and Other Strainswith theNQ Motifin
Rhodopsin Sequences
StrainName NQ PR blh crtE crtB crtI crtY ispA idi GenBankAccession
Nonlabens(Donghaeana)dokdonensisDSW-6 + + + + + + + + + CP001397
Krokinobacter(Dokdonia)sp.4H-3-7-5 + + + + + + + + + CP002528
GillisialimnaeaR-8282 + + + + + + + + + AHKR00000000
HymenobacterroseosalivariusDSM11622 + + (cid:3) + + + + + (cid:3) TaxonID:2502422321(IMG)
TrueperaradiovictrixDSM17093 ++ (cid:3) (cid:3) + + + (cid:3) + (cid:3) CP002049
FulvimarinapelagiHTCC2506 + (cid:3) (cid:3) + + + + + (cid:3) AATP00000000
CitromicrobiumbathyomarinumJL354 + (cid:3) (cid:3) + + + + + (cid:3) ADAE00000000
D
Citromicrobiumsp.JLT1363 + (cid:3) (cid:3) + + + + + (cid:3) AEUE00000000 o
w
n
NOTE.—++indicatestwocopiesofthesamegene.NQdenotestheNQrhodopsingene;PRdenotestheproteorhodopsingene.CrtEBIY,Blh,Idi,andIspAaredirectly lo
involvedinbeta-caroteneandretinalbiosynthesis. ad
e
d
fro
m
motif-containing rhodopsin were much lower than those of even Antarctica (supplementary table S6, Supplementary h
thePRgeneundertheseconditions. Material online). Sixteen NQ motif-containing reads are ttp
s
ThepresenceoftheuniqueNQmotifinthenewlyfound shown in supplementary figure S7, Supplementary Material ://a
c
classofrhodopsinsandthepresenceoftworhodopsinscon- online. a
d
e
tainingthismotifinabacteriumthatthrivesinasodium-rich Phylogeneticrelationshipsofthegenome-derivedNQrhod- m
ic
hotspringpromptedustoanalyzetheexpressionoftheNQ opsinsinferredthroughMP,NJ,ML,andBayesianapproaches .o
u
rhodopsingeneatvariousconcentrationsofNaCltoinferthe indicatedthatoverallbranchingpatternsarealmostidentical p
.c
functionoftheNQrhodopsins.Theartificialseawater(Huang among the trees (fig. 4A and supplementary fig. S8, om
etal.2005)usedfortheculturemediumcontains2.5%NaCl Supplementary Material online). However, grouping of the /g
b
e
andrepresentstheisosalinecondition.DSW-6cellswerefirst branches of an NQ rhodopsin in T. radiovictrix DSM 17093 /a
growninASWenrichedwithadditionalcarbonandnitrogen andthatinH.roseosalivariusDSM11622intheMP,NJ,and rtic
le
sourcesuntiltheearlylogphase,andthentheconcentration BayesiantreescouldnotbesupportedintheMLtree(supple- -a
b
oMfaNtearCiallswaansdseMtteoth2o.5d%s f,o5r.0d%eta,i7ls.)5.%Ce,ll1s0e%xp,oosred12t.o5%2.5(s%ee, mofetnhteamryefitga.gSe8nAom,SicuprepaledmsoennttahreyNMJattreereiaolfotnhleinfeu).ll-PleonsigtitohnNinQg strac
5.0%,or7.5%NaClkeptgrowing,whereasthoseexposedto rhodopsins indicated that 40 of them (72.7%) can be an- t/5/1
10%NaCldidnotgrowfurtherafter6h(fig.3A).At12.5% chored to the branches that comprised the ones present in /18
7
NaCl,theOD600nmvaluesdecreased.After3hofexposureto N. dokdonensis, G. limnaea, and Krokinobacter sp., and /7
3
each NaCl concentration, total RNA from each sample was 1
YP_003706581 of T. radiovictrix (fig. 4A). Among the reads 7
8
extracted,andtherelativeexpressionleveloftheNQrhodop- included in this subfamily are four out of four reads from a 3 b
singenewasdetermined.Nodifferencebetweenthebefore y
hypersalinemicrobialmatand12of13readsfromtheGreat g
and after treatment time points was observed for the 2.5% u
Salt Lake. YP_003706581 has the largest number (15) of e
s
NaCl culture, as expected. As the NaCl concentration reads that are closest. Results obtained from clustering of t o
n
increased,geneexpressionwasinduced.Therelativeexpres- the metagenome reads and the full-length NQ rhodopsins 10
sionlevelwashighestat10%NaCl(of375±31.4).In12.5% usingUPGMAshowedthatNQrhodopsinsfromthemetage- Ap
NaCl,thelevelofgeneinductionwaslowerthanthatin5.0% nomic data sets can be divided into at least four groups ril 2
NaCl(fig.3B).Incontrast,theexpressionlevelsofthePRgene 0
(fig. 4B). Similar to the results from phylogenetic anchoring, 1
were not affected by the NaCl concentration (data not 9
39 reads were clustered with those of the four species
shown).
(YP_003706581 for T. radiovictrix). Out of the 13 reads
fromtheGreatSaltLake,10readsseemtobecloselyrelated
NQRhodopsinsAreFrequentlyFoundinHypersaline
to YP_003706581. Among the reads from Antarctica, one
Environments
clusters with the NQ rhodopsin of C. neogracile, whereas
TogaininformationonthediversityandtheprevalenceofNQ six others form another group and appear more distantly
rhodopsinsinnature,wesearchedforthisrhodopsintypein related.
metagenomicsequences.Atotalof55readsorthologousto The occurrence of NQ rhodopsin-carrying prokaryotes in
NQrhodopsingenesinthemicrobialgenomesdescribedear- themetagenomicdatasetswasevaluatedusingtheratioof
lier were recruited from metagenomic data sets originating rhodopsingenestoconservedsingle-copygenes(supplemen-
from various aquatic environments, including a hypersaline tary table S7, Supplementary Material online). A hypersaline
microbial mat, saltern and freshwater lakes, oceans, and microbial mat sample from Guerrero Negro, Mexico,
194 GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013
GBE
GenomicMakeupoftheMarineFlavobacterium
Discussion
ThegenomeanalysisofDSW-6demonstratesthatthisstrainis
highly adapted for living in the oligotrophic surface of the
ocean. Similar to other ocean-inhabiting bacteria, DSW-6 is
thoughttousetheNa+gradientinsteadoftheH+gradientto
generatethemotiveforcefortherespiratorychain(Unemoto
andHayashi1993)andhasmanyNa+-dependenttransporter
genes. Several stress-response gene products and pigments
mayprotectDSW-6fromtheharshsunlight.Inlightoflight
utilization,genesforaPRandretinalbiosynthesisinaddition
D
tothoseforlight-sensingproteinsexistintheDSW-6genome. ow
n
In the case of the PR-carrying marine flavobacterium lo
a
Polaribactersp.MED152,uptakeofbicarbonateincreasedin d
e
d
thepresence of light, suggestingthata PR-mediatedproton fro
gradient drives anaplerotic inorganic carbon fixation toward m
more efficient anabolism (Gonzalez et al. 2008). Similar to http
MED152, enzymes involved in the anaplerotic pathway are s://a
wellconservedinDSW-6. c
a
BecauseBejaetal.(2000)discoveredPRinanuncultured de
m
marinebacterialclade,extensiveresearch,includingmetage- ic
nomic and photochemical approaches, has been conducted .ou
p
to discover the reasons for the great success of this wide- .c
o
spreadrhodopsinfamily.Currently,theselight-drivenproton m
/g
pumps are considered to power cell growth or extend the be
survival of marine oligotrophs. Light stimulates growth of /artic
the PR-containing marine flavobacterium Dokdonia sp. le
MED134 (Gomez-Consarnau et al. 2007). Vibrio sp. AND4, -ab
s
anotherPR-containingstrain,exhibitedlongersurvivalduring tra
c
NonlaFbIGe.n3s.(—DoTnhgehareelaantiave)deoxkpdroesnseionnsisleDvSeWls-o6faNtQdiffrehroednotpNsainClgceonnecsenin- s(Gtaorvmaetizo-nConthsaarnnauitsetcaol.rr2es0p1o0n).diWnghenPRexpdreelsesteiodninmE.utcaonlit, t/5/1/1
trations. (A) Cell growth was determined by optical density values at a PR from the SAR-86 clade of the Gammaproteobacteria 87
/7
600nmforculturesincubatedin2.5%NaCl(circles),5.0%NaCl(down- promotes proton-motive force that turns the flagellar motor 3
1
wardtriangles),7.5%NaCl(square),10%(upwardtriangles),and12.5% duringlightillumination(Walteretal.2007).Severallinesof 78
3
NaCl(diamonds).After3h,cultureswerecollectedforRNAextractionto evidencesuggestthatthePRinDSW-6shouldbeafunctional b
y
analyzethegeneexpression.Errorbarsindicatethestandarddeviationsfor equivalentofotherPRsandmayplaysimilarrolestocontrib- g
u
triplicatesamples.(B)TheNQrhodopsinexpressionlevelwasquantified e
utetothegrowthorsurvivalofthebacteriuminoligotrophic s
usingqRT-PCRandthecomparativecriticalthreshold(2(cid:3)(cid:2)(cid:2)CT)method environments.First,itexhibitsthekeyfeaturesofatypicalPR, t on
(LivakandSchmittgen2001).The16SrRNAgenewasusedasaninternal 1
andallthefunctionalresiduesintheprotontransferpathway 0
control,andtheNQrhodopsintranscriptat3hwasquantifiedrelativeto A
areconserved.Second,theencodinggeneishighlyexpressed p
rtahnegNeQs frrhoomdo6p0sintotra3n3s0c.ripEtrrionrabnaUrsCin(2d.i5ca%teNtahCel)s.tTahnedabrrdeadkeivniatthioenysafxoisr inthepresenceoflight orintheabsenceofsufficient nutri- ril 20
triplicatereactions. ents. Most significantly, purified PR protein binds to trans- 19
retinal and pumps out proton (Han S-I, Kwon S-K, Kim JF,
andJungK-H,unpublisheddata).
Microbial rhodopsins are functionally versatile and may
containing approximately 90 practical salinity units had an transit nonproton ions across the plasma membrane. HRs
abundance of the NQ rhodopsin genes as high as 17.36%. existinginhalophilespumpinchlorideinsteadofprotonina
Inaddition,theywerefoundfrequentinthesamplesfromthe light-dependentmanner(Kolbeetal.2000).Thechannelrho-
GreatSaltLake(1.48%)andLabonteLake(2.75%).Basedon dopsinsofgreenalgaearelight-gatedcationchannels(Kato
theabundanceanalysisofGOSdata,however,0.09%ofthe etal.2012).Inthisarticle,weproposetheexistenceofanew
prokaryotesinhabitingtheseasurfacemaypossessthisrhod- type of rhodopsin with a function that is unique among
opsintype.Noprokaryotespossessingthisgenewerefoundin proton pumps and is related to salinity; this proposal was
high salinity ponds from Chula Vista solar salterns, where based on the following: These proteins, which we dubbed
haloarchaeadominate(Pasicetal.2009). the NQ rhodopsins, have seven transmembrane domains
GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013 195
GBE
Kwonetal.
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FIG.4.—NQrhodopsin-likesequencesinthemetagenomicdatasets.(A)PhylogeneticpositionsoftheNQrhodopsin-likemetagenomicreads.The
cylindersymbolrepresentsametagenomicreadoriginatingfromhypersalinemicrobialmat(brown),GreateSaltLake(red),YellowstoneLake(orange),urban
lake(yellow),Antarctica(white),oroceans(blue).Aphylogenetictreeoffull-lengthNQ-rhodopsinswasconstructedbasedontheNJdistanceanalysisof243
positions.(B)UPGMAclusteringoftheNQrhodopsin-likemetagenomicreads.ThedendrogramwasderivedfromUPGMAclusteranalysisusingidentity
valuesagainstcompletelysequencedgenes.MetagenomeprojectsfromwhichNQrhodopsin-likesequenceswererecruited:hypersalinemat,Guerrero
Negrohypersalinemicrobialmat(CAM_PROJ_HypersalineMat);GreatSaltLake,GreatSaltLake(Gm00191ofGOLDIDinIMGdatabase);YellowstoneLake,
Yellowstone Lake (CAM_PROJ_YLake); urban lake, LaBonte Lake (Gm00212 of GOLD ID in IMG database); Antarctica, Antarctica aquatic microbial
metagenome (CAM_PROJ_AntarcticaAquatic); ocean, Global Ocean Sampling expedition (CAM_PROJ_GOS). Truepera radiovictrix DSM 170931,
YP_003705905;T.radiovictrixDSM170932,YP_003706581.
196 GenomeBiol.Evol.5(1):187–199. doi:10.1093/gbe/evs134 AdvanceAccesspublicationJanuary3,2013