Environmental Switching During Biofilm Development in a Cold Seep System and Functional Determinants of Species Sorting

Environmental switching during biofilm development in a cold seep system and functional determinants of species sorting

Item Type Article

Authors Zhang, Weipeng; Tian, Renmao; Yang, Bo; Cao, Huiluo; Cai, Lin; Chen, Lianguo; Zhou, Guowei; Sun, Jingya; Zhang, Xixiang; Al- Suwailem, Abdulaziz M.; Qian, Pei-Yuan

Citation Environmental switching during biofilm development in a cold seep system and functional determinants of species sorting 2015:n/a Molecular Ecology

Eprint version Post-print

DOI 10.1111/mec.13501

Publisher Wiley

Journal Molecular Ecology

Rights This is the peer reviewed version of the following article: Zhang, W., Tian, R., Yang, B., Cao, H., Cai, L., Chen, L., Zhou, G., Sun, J., Zhang, X., Al-Suwailem, A. and Qian, P.-Y. (2015), Environmental switching during biofilm development in a cold seep system and functional determinants of species sorting. Mol Ecol. Accepted Author Manuscript., which has been published in final form at http://doi.wiley.com/10.1111/mec.13501. This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.

Download date 02/10/2021 12:20:10 Link to Item http://hdl.handle.net/10754/583482 Zhou Keywords: species sorting; biofilm;brine pool; Running ba title:Functional Kong Hong Bay,Water Clear and Technology, Email: [email protected] Qian Pei-Yuan This article is protected Allrights bycopyright. reserved. 10.1111/mec.13501 of Record.versiondifferences and article Please between the cite this Version this as doi: through the copyediting, typesetting, pagination for publication accepted This articlehasbeen # 2 Zhang Weipeng Authors and functional determinants of species sorting Environmental switchingduring biofilmcolddevelopment seepsystemin a Article type : Original Article Date:24-Nov-2015 Accepted Revised Date : 22-Nov-2015 :26-Apr-2015 Date Received 1 King Abdullah University of Science and Tec and King ofScience AbdullahUniversity Science of University Kong Hong Life Science, Program, Divisionof GlobalPartnership KAUST Accepted Corresponding author: Article 1 , Jin Sun , Jin 1 , Xixiang Zhang , Xixiang 1 , Renmao Tian , Renmao ses for species sorting sorting ses forspecies 2 , Abdulaziz Al-Suwailem , Abdulaziz 1 , Yang Bo , Yang 1 , Huiluo Cao , Huiluo and undergone full peer review buthasnot been review undergone fullpeer and hnology, Thuwal, The Kingdom of Saudi Arabia ofSaudiArabia TheKingdom Thuwal, hnology, Marinobacter and proofreading process, which may lead to 2 , Pei-Yuan Qian , Pei-Yuan 1 , Lin Cai ; Oleispira 1 , Lianguo Chen ;

metagenome metagenome 1# 1 , Guowei , Guowei This article is protected Allrights bycopyright. reserved. Accepted (Jones form a communityas a result ofselection by abiotic local and biotic environmental conditions sorting theory,the frameworkofspecies particular community microbial of understanding A mechanistic Introduction theory. light onmicrobial community assembly special habitatsgap in a and inspecies bridges sorting habitat generalists inboth thebrinepooland NB metal ion resistance and amino acid metabolism as functional traits conferring the survival of ( species twobacterial reconstruction of pathway and binning Genome communities. sortingthe succession determinants for biofilm of identities; taxonomic me biofilm communityassembly that was based pr incubation conditions. Moreover, a comparis biofilms establishedin new envi of16S Analyses rRNA genes of 71 biofilmsample environmentalcould bacterial filtering reflect immigration environments. effects tonew during Articleof environmentalswitching be toanothe patch organisms fromone normal bottomwater toformametacommunity (NBW) via the potentialimmigration of II coldseeps Thuwal the cold seepsystem, successiondu taxonomic revealed work our recent environments deep sea in assembly community microbial for especially elusive, remains theory sorting species for basis The functional Abstract (852)23581559 Fax: Telephone: (852)23587331 et al. et 2009; Langenheder Langenheder 2009; tal ion resistance and amino acidme amino and tal ionresistance tween the brine pool and the NBW onbiofilm thebrinepoolandNBW tween assembly, which ronments wasdeterminedby bot et al. et r. Here, we designed an experiment to investigate the effects effects the investigate to experiment an designed we Here, r. 2011;Stegen Marinobacter Marinobacter , which comprises a brine pool and the adjacent pool andtheadjacent brine comprisesa which , on of 18 metagenomes provided evidence for on of18metagenomes providedevidencefor . Usingartificial. surface-based biofilmmodels, W. TheresultsofW. thepresent study new sheds species are selected from apoolofbacteriato from speciesareselected ring biofilm development in ring development biofilm defineda newly s suggested thatthe microbial composition of imarily imarily on than rather features functional et al. al. et sp. and assembly in nature is of great interest. In interest. great isof in nature assembly 2012; Székely Oleispira tabolism were the major species major species tabolism werethe h the source community and the h thesourcecommunityand the sp.) further demonstrated sp.) further demonstrated et al. et 2012; Zhang 2012;Zhang et al. et

brine poolisthecoldest temperature (21.7°C)a seeps II (Batang normal 850 mandadjacent and 840 depthof a water al. In (Zhang topic. studies recent anew seepsis cold of ecology The microbial ecological lead tointeresting would theories findings a from functional perspective. Moreov remain mostly undiscovered. rise (Öztürk environments have undergone deep the sequencing, diversity ofmicrobial activities continues to of number ofanincreasing microbiomes the Because surveys suchasmetagenomics. culture, a greater awareness oftheir activities could be achieved through cultivation-independent Although systems. of subsurface majority the been achieved over the past few years in reveal years in few past overthe been achieved deep sea environments. Significant progress has especially microbial for communities in elusive, remain effects largely functiona andthe unknown; Stegen is available evidence confined proposed microbial communitiesand the rolesof Although studies demonstrated have g are generalists habitat processes, whereas commu microbial innatural the habitatspecialists re plasticity lowfunctional have a generalists, a plasticity. whichfunctional high possess In of level contrast, habitat specialists who conditions. the Furthermore, filteringeffect by communities hasbeenproposedtobeinfluenced adaptenvironments by tonew adjusting their This article is protected Allrights bycopyright. reserved. 2012; Orsini bacterial immigration into 2014). The species sorting has inthemetacommunity framework concept been applied to explain

Accepteddevel were 2015),biofilms Article et al. al. et 2012; Székely et al. et et al. et et al. et 2013; Li 2013; 2013),anditassumes that microbialco 2012). 2012). The thininterface. a by separated are NBW and pool The brine newenvironments(Peay et al. et et al. al. et oped at two locations intheRedS oped attwolocations to the taxonomic level (Jones (Jones level taxonomic tothe 2012;Zhang spond primarily to environmental primarily spond 2014). However, microbial lifestyles in deep sea environments environments sea deep in microbiallifestyles However, 2014). er, the application ofmetagenom application er, the general principles general as me vre anyb ipra rcse. mainly dispersalprocesses. by overned et al. et species compositions. The establishment ofnew compositions. The species nd leastamountofsaline (7.4%)among thebrine environmental isrelatively parameters for weak these organisms could not be obtained in pure couldnotbeobtainedinpure organisms these by both the source community and the incubation communitythe source and theincubation both by ing the functions of mi functionsof the ing nities are modulated according to environmental to according aremodulated nities 2014).Thefunctionalbase species sorting in the assembly of natural natural of sortingintheassembly species et al. et bottom water (NBW) next to the Thuwal cold totheThuwalcold next bottom water(NBW) l features of specialists and generalists are generalists are and ofspecialists l features 2012; Sullam 2012; mmunities havethe potentialtorapidly et al. et ea, namely, a brine pool located at a brinepoollocated ea, namely, 2009; Langenheder Langenheder 2009; ntioned above, most of the above,mostofthe ntioned factors. Thus, the dynamics of of Thus, thedynamics factors. ics to explain the effects of theeffects explain ics to et al. et croorganisms innatural croorganisms et al. et 2012; Székely 2012; s for species sorting sorting s forspecies 2014; Zhang Zhang 2014; et al. et 2011; et al. et et et

immigrated and communities identify bases functional the processes. forthese by sorting influence ofspecies that pool inthebrine continuously were developed This article is protected Allrights bycopyright. reserved. (Zhang study a previous in introduced The samplinglocationswere Experimental design Methods and Materials ofthesw and metagenomes genes biofilmsswitchedfrom for brine and“S-NBW” biofilm toasswitched referred (hereafter days moved toNBW and brine, respectively, and foran then additional incubated three before being allowedtodevel were environment. Thebiofilms environmental oncommunity effect esta filtering framework the in II system seeps cold Thuwal study, In present the we designed an to experiment assembly community study microbial in the shapingthe communities the of structures inthiscold seep system. andbacter sorting species another, andthus,the toanothe organisms fromonepatch distinct communitiesare connect thebr between different community structures Based on16SrRNAge brine pool,respectively). car organic dissolved of concentrations higher (0.20 ppm),andiron(0.10ppm)(Batang including relativetotheNBW, concentrations is water brine The Sea. Red pools reportedinthe by the remotely operated vehicle (ROV); and iii)the and experimental(ROV); set-up could be easily by the remotely vehicle operated the substrates completely were ii)the immersed inwater; biofilmswere stable during movement particular a Briefly, 1. Figure design isshownin Accepted study Basedonaprevious recovery. that showing variations inthemi manipulated toreduce Article itched biofilms were sequenced a biofilms were sequenced itched particular environmental parameters inthe assembly ofthe ed to form a metacommunity ed toforma r. However, the local conditions of the water differ from one fromone thelocalconditionsofwaterdiffer r. However, et al. et crobial community produced during launch and and launch community during produced crobial bons (48.23 bons mg/L NBW inthe andand 0.48 mg/L aluminum (0.24ppm),ar s: “S-brine” for biofilms switched from NBW to to NBW switchedfrom for biofilms s: “S-brine” ine biofilmsand NBW biofilms. These spatially experimental toguarantee set-up built was i) that of the metacommunity concept, particularly the the particularly concept, metacommunity the of 2012). The NBW contains morethan100-fold contains 2012). TheNBW op separately in brine and NBW for three days days three for NBW and brine in op separately blishment after bacterialimmigration toanew ial functional plasticitymight be important in brine toNBW). Subsequently, the16SrRNA the structureofbiofilms ne and metagenomics analyses, we ne andmetagenomics we analyses, observed or NBW. Thus, we were abletoevaluate were the Thus,we NBW. or enriched for metals at ten-fold or higher higher atten-foldor for metals enriched nd compared with the biofilms biofilms the with nd compared via the potentialdispersalof via the et al. et senic (0.34ppm),copper 2014). The experimental experimental The 2014). developed in deep-sea developed indeep-sea This article is protected Allrights bycopyright. reserved. environments couldbeinfluenced proteinase K and 10% SDS weread proteinase Kand10%SDS cotton tips in Tris-HCl buffer we (Zhang described aspreviously was performed extraction and then weobtained Collectively, recovered. additional days) days forthree total (a ofsix environments incubated inthewere further two frames four remaining The pool. brine tothe NBW from the transferred was one and pool totheNBW, another from Concomitant the NBW recovered. were (PTFE), stainlesssteel(SS),and aluminum (Al),polyether ether included substrates different Thesix analysis. DNA the usedfor were that samples same density measurements arenotshownin which extraction, 10forthe tworeplicat DNA metagenomic for 10slides type: substrate same ofthe 30slides contained frame Thus, each slots. the in embedded were which 10slides, contained carousel same the in substrate of each type carousels, three each contained carousel Each frame the brinepool.In threesteelframeswe parallel, First,three surfaces. steel frames, substr we appliedsixdifferent (5’-GTGYCAGCMGCCGCGGTAA-3’) and and (5’-GTGYCAGCMGCCGCGGTAA-3’) samplesas templa amplified usingthebiofilm ofthe region The V4-V8 at260nm. Kong) Hong (ND- device a Nanodrop with and electrophoresis gel agarose by checked were DNA ofthe andquantity Thequality instructions. manufacturer’s usingaccording Germany) Hilden, tothe MiniKit(Qiagen, the AllPrep DNA/RNA included S-brine and S-NBW biofilm included S-brineandS-NBW Accepted-80 at stored were samples The buffer. inTris-HCl resuspended and tips cotton sterile using wereharvested biofilms sea,the the from recovery after Immediately PCR of amplification DNA extraction, biofilmsincludedand theNBW Article ates to demonstrate common mechanisms of microbial life on life ofmicrobial mechanisms common demonstrate to ates ketone (PEEK), polyvinyl chlori 3d-NBW and 6d-NBW biofilms. and6d-NBW 3d-NBW titanium (Ti). After threedays, one titanium (Ti). re pelleted by centrifugation at4,000×gcentrifugation re pelletedby for 10min.Lysozyme, es of 16S rRNA amplicons, and10fo es of16SrRNAamplicons, each containing six differenttype containing six each by the type of substrate (Meier (Meier ofsubstrate type the by ded to the suspended cells before cells tothesuspended ded s; thebrinebiofilmincluded 16S rRNA genesa three groups ofbiofilms: three re placed in NBW and incubated for three days. days. andincubated placedinNBW forthree re the present work; RNA was extracted using the the using was extracted RNA work; present the 1000 spectrophotometer, DiaMed China Limited, DiaMed China 1000 spectrophotometer, 16S rRNA genes in Bacteria and Archaea were were Archaea and Bacteria genesin rRNA 16S te and the universal forward primer U515F primer forward universal te andthe contained six different containedsix ly, one frame was transferred from the brine brine the from wastransferred frame ly, one the reverse primer U1390R primerU1390R the reverse et al. et nd 454pyrosequencing 2014). Briefly, bacterial cells from cells Briefly, bacterial 2014). de (PVC), polytetrafluoroethene polytetrafluoroethene de (PVC), et al. et o C until DNA extraction. DNA DNA DNA extraction. C until 3d-brine and 6d-brinebiofilms; 3d-brine frame from thebrinepooland frame from s of substrate, were placed in in placed were substrate, s of lysis purification and further 2013),inthepresentstudy, r other analysis such as cell r cell such as analysis other theswitchedbiofilms types of substrate, and types ofsubstrate, This article is protected Allrights bycopyright. reserved. homopolymers reads containing reads toremove performed control was Quality Sequence quality filtering and study. present 23brinebiofilm including biofilms, 24switched amplicons, 16SrRNA (Shanghai,In total,Titanium ofthe 71samples platform China). inCHGB the3d-brin and tworeplicates(excluding prev inour conditions asdescribed using thesame reacti PCR The (5’-GACGGGCGGTGTGTRCAA-3’). each OTU for subsequent analysis. analysis. subsequent for each OTU Representatives of 97%. identity withan (OTUs) an clustered, barcodes, totheir samples according (Lee study inourprevious described (Caporaso Acceptedwas pe distance Bray-Curtis on based analysis (Hammer (PAST) package statistics software usingand visualized principalc rela with a genera using Bray-Curtis distances 16S rRNAdatasets of analysis Statistical replicat two oneofthe least at genus that or theclass thatis, wasused; replicate aredisplayed,the classandgenus andacu levels rRNA gene displayedamplicons are S1 inFigure S1. Rarefaction detailing curves the numberofgeneragiven the number ofsequenced 16S Table of50%.Theread info bootstrap confidencelevel cla (RDP) Project Database Finally, were thereads removed fromthedataset. for representative Chimeras OTUs. inthe aligned reads were identified by ChimeraSlayer and then alignmen reference-based and QIIME available in Article et al. et , 2010) was used to analyze the 16S rR the was usedtoanalyze 2010) , ssifier version 2.2 (Wang (Wang version2.2 ssifier swsdfnda mnr. “minor”. as es wasdefined

taxonomic classificati oordinate analysis (PCoA)impl analysis oordinate ≥ 6 bp. Subsequently, the QIIME 1.7.043 software package softwarepackage 1.7.043 theQIIME 6 bp.Subsequently, et al et De novo De ., 2012). The reads were assigned to their corresponding to theircorresponding assigned were The reads 2012). .,

e-SS) wasconductedus e-SS) alignment using (Edgar MUSCLE et al. et tive abundance of >0.1% as input were generated >0.1%asinput generated tive of were abundance did not achieve an abundance greater than 1% in than1% greater abundance an notachieve did with ambiguous nucleotides: reads <150 bpand <150 reads with ambiguous nucleotides: ious study. Pyrosequencing of thePCRproducts of ious study. Pyrosequencing rformed. Furthermore, one-way permutational permutational one-way Furthermore, rformed. t against the Silva108 database were performed performed were t against theSilva108database s and 24 NBW biofilms were analyzed in the inthe analyzed biofilmswere NBW s and24 of the most abundant reads were selected from from selected were reads abundant of themost d then assigned to operational taxonomic units taxonomic tooperational d thenassigned t-off of 1% relative abundance for at least one foratleastone t-off of1%relativeabundance rmation fortheswitchedbiofilms is shown in assigned to different taxa using the Ribosomal Ribosomal using the taxa different assigned to . The microbial composition of the samples at samples ofthe composition The microbial . 2001). To confirm the confirm the 2001). To no h 6 RA theon of 16SrRNA on of 26 thermal cycles was performed performed cycles was thermal on of26 NA pyrosequencing data following steps steps following NA pyrosequencing data et al. et 2007) against Silva108witha against 2007) emented inthePaleontological ing theROCHE454FLX PCoA results, cluster cluster PCoA results, et al. et 2004) 2004) for conducted database were (Kanehisa Genomes(KEGG) and the Kyoto ofGenes Encyclopedia and 2000) al.

This article is protected Allrights bycopyright. reserved. S-brine and NBW biofilmcommunities, compar and 6d-brine biofilms separately.In addition, toidentify bacterial genera differentiating the 6d-NBW biofilmsseparately. Similarly, theS-NB and 3d-NBW the with compared were biofilms S-brine the and replicates, as together grouped community ofswitchingeffect composition. onthe varian multivariate analysis of using Programming theProkaryotic Dynamic Ge (Zhang study ourprevious described in wa control 2012). Briefly, quality (Mason studies previous to referring by performed were analyses Metagenomic S2. Table Thedetailedinformati reads. paired-end 100-bp Illuminaa meta HiSeq2000platformtogenerate an using performed was Thesequencing analysis. metagenomic the for selected were materials three onthese biofilms the Therefore, metal). to contrast (in material a plastic represented biomass, theTibiofilm greatest sample hadthe total; switched and the six biofilmswere inthe study). present documented The Al biofilm switched biofilms,brinebiofilms themeta for were used that The DNAsamples Sequence quality filtering, assembly andannotation ofmetagenomic sequences 1993) (reads of <100 bp were removed) (reads of<100bpwere removed. Each steps ofthese was followed by thefilteringspecific reads witha length of cut-off toolkit (Patel

Acceptedagainst theclus 2010),andBLAST searches Article

analysis based on the Bray-Curtis distanceus Bray-Curtis onthe analysis based et al. et 2012), and reads containing homopol 2012), andreads the functional annotation. annotation. the functional ce (PERMANOVA) implementedin (PERMANOVA) ce and NBW biofilmsdevelopedonAl and NBW s conducted using thenext-gen s conductedusing . Subsequently, qualif et al. et ters of orthologous groups (COGs) (Tatusov oforthologousters groups(COGs) 2015). For the annotation, ORFs were predicted predicted ORFs were annotation, the For 2015). isons wereSIMPER by performed (Clarke sample lowest had the nefinding (Prodigal, Algorithm v2.50) (Hyatt genomic datasetof~70 on for the switched biofilms issummarizedbiofilms in on fortheswitched genomic sequencing were extracted from the from the extracted were genomic sequencing W biofilms were compared biofilmswereW withthe3d-brine ing relative abundance of genera as input. asinput. genera of abundance ing relative The biofilms on all the six substrates Thebiofilmsonallthe were six ied Illumina reads ymers and reads were low-quality eration sequencing (NGS) QC (NGS) sequencing eration PAST wasusedtoassessthe , Ti, and PVC(18samplesin, Ti,and biomass, whereas PVC PVC whereas biomass, Gb, whichconsistedof Gb, were assembled as were assembledas

et al et . 2000) et al. et et al. et et al. et et et

using SAMtools(Li assembled contigsBowtie2 using (Langmead the to mapped were reads pyrosequencing Illumina The frequency. tetranucleotide and content, 2013; Tian to according performed was genome binning Draft Genome binningandvalidation and pathway analysis biofilms andthesourcebiofilms. switched the between differences the to COG ofeach contribution the determine to performed This article is protected Allrights bycopyright. reserved. SIMPER distance. Bray-Curtis onthe based performed was analysis cluster results, PCoA the in at least one us sample were of composition andrelativeabundance d toaccountforth standardized Li coverage sequencing 2012), andthe aligned to were control quality obtained after ca ofCOG abundance therelative To calculate the Statistical analysisof metagenomic datasets representation of two bacterial species bacterial oftwo representation datasetsAll ofthe switched biofilm assemb were proteins. tothephylum-lcontigs labeled according were with BLASTP <1e-05)andassigned (e-value the HMMdatasets. The identified essential were proteins searched againstthe NCBI NRdatabase of esse Markov models(HMM) the following section were obtained using the S-brine-Ti/S-NBW-Ti metagenome pairs. the For greater metagenomic coverage than the S-brine-Ti was selected, which contained ~52% GC ~52%GC contained which was selected, S-brine-Ti the than coverage metagenomic greater genome binning of Accepted Articleistances/similarities among different microbial communities were based onthe determined et al. et 2009). The normalized counts of COG functi countsofCOG normalized The 2009). et al. et 2014). Briefly, the genomes were sepa genomes were Briefly, the 2014). Marinobacter Marinobacter et al. et 2009); whereas thecontigs2009); whereas weresear ed) and visualized usingand visualized PCoAim ed) e unequal sequence coverage betw coverage e unequalsequence ntial proteins (Albertsen (Albertsen ntial proteins sp of the contigs was calculated with SAMtools (version 0.0.19; with SAMtools(version0.0.19; wascalculated contigs the of ., a group of contigs 10.0× thatexhibited intheT-NBW-Ti agroupof . COGs (only COGs with an abundance of more than 0.05% ofmorethan0.05% COGs withanabundance COGs(only

Marinobacter ORFs using Bowtie2 (version 2.0.0; Langmead 2.0.0; Bowtie2 using (version ORFs et al. et taxonomically using 5.046. Finally, MEGAN the evel taxonomic affiliati taxonomic evel led together. Genome binning resulted in an over inan resulted binning Genome together. led previously described methods (Albertsen methods previously described tegories in the two metagenomes, raw reads reads raw tegories inthetwometagenomes, 2012); the genome coverage was calculated was calculated 2012); thegenome coverage sp onal annotations per bi onal annotationsper et al. et rated based on the genome coverage,genome onthe based rated GC . and 2013) using default cut-off values in in values default cut-off 2013) using Oleispira ched against asetof107 hidden against ched plemented inPAST.Toconfirm een the samples.Bray-Curtis een the on based ontheessential sp . genomes described in genomes in described ofilm sample were ofilm samplewere et al. et et al. et

was

rpsC pyrG This article is protected Allrights bycopyright. reserved. (Castresana clustermethod UPGMB gap -50, parameters: open molecular evolutionary genetics BLASTP using NCBI-Nr database sequenceswere protein aligned concatenated based aligned genes were marker dow genomes reference andthe genomes extracted genomes extracted of the phylogeny To identify the Phylogenetictree construction based onessential genes non-redundant (Nr) database. Similar genome. to revealadraft assi via ataxonomical confirmed findings were to themethoddescribedabove.Fi assigned in eachcontig taxonomically were contaminatinggenomes, resulting ina core set ofcontigs. The ORFs were and obtained, the ORFs the genome from target the separate further to was used frequencies tetranucleotide ofthe (CA) ofthelabeled most content andincluded Acceptedprotein was constructedusing from hydrolase 4-dienedioate 2-hydroxy-6-oxonona-2, of tree The phylogenetic replicates. tree. ML phylogenetic the 2013) toconstruct 31 essentialgenes. essential 31 genes( The marker Articlekey wasva enzymes of annotation (http://www.genome.jonline toolKEGGMAPPER databases witha maximum e-value 1e of cut-off a were genomes The ORFs intheextracted , , rpsB pgk , , nusA rpoB et al. et , , infC , 2000) was applied to eliminate theless wasappliedto , 2000) rpmA , frr, frr, , rplT and , rplS dnaG analysis (MEGA 6.0) (Tamura 6.0) (Tamura (MEGA analysis on the protein sequences separately proteinsequences on the methods were used toobtainthe methods wereused nally, a draft genome ofthe genome nally, adraft sequences. The reference seque reference The sequences. , lidated based ontheresults of lidated based rplP . Aalignment was c . Aalignment ) were predicted by AMPHORA (Wu (Wu AMPHORA by predicted were ) , Gammaproteobacterial rplN using the BLASTP and MEGAN programs according programsaccording andMEGAN BLASTP using the nnotated by BLAST against the KEGG and COG BLASTnnotated by against theKEGG , then importedintoMega 6.05)(Hall then (version rplM gnment using the BLASTP and MEGAN programs programs BLASTPand MEGAN gnment using the The bootstrap values were calculated with 1000 calculated were bootstrapvalues The -05. To construct-05. To themetabolism pathways, the nloaded fromtheNCBI da , phylogenetic trees were , and mindiag 24.TheGblocksoftware length tsf rplL , p/kegg/mapper.html) was applied. The p/kegg/mapper.html) wasapplied.The smpB onducted with Muscle in the software with Muscleinthesoftware onducted , rplK informative sites in the alignments. inthealignments. sites informative , Marinobacter rpsS , contigs. Correspondence analysis analysis Correspondence contigs. rplF et al et Oleispira the BLAST searchagainst the , nces were obtained from the obtained were nces and then linked together. The linkedtogether. The andthen rpsM , ., 2013) using the following following usingthe ., 2013) rplE , , rpsK rplD sp.wasobtained.The constructed based on constructedbasedon et al. et sp. tabase. The essential

, , rf eoe draft genome. rpsJ rplC 2008) from the from 2008) Oleispira , , rpsI rplB , , rpsE et al. et rplA sp. , ,

were toperforming the determined prior qPCR. and0.5 primers, andreverse forward Palo Alto,CA).The 20- MA)forbothDNAWoburn, andcDNAwithaMx The PCRreactionswereconducte and the specificities ofthe sequences by wereBLAST verified analysis the against NCBI database. Primers we andDNA. gene(cDNA) target inRNA ALLPREP RNA/DNAkit,andthis allowedus to This article is protected Allrights bycopyright. reserved. AcceptedHalm the genes intheDNAsamplesandreverse-transc functional ofselected expression the To examine q-RT-PCR detection ofkey functional genes for thereplicate) quan extracted were replicates oftheRNAsamples(10slides the IIISuperScript reverse transcript coli Escherichia to measure the RNAconcentration.was subjected The RNA toa tailing PolyA reaction using ArticleDiaM spectrophotometer, (ND-1000 device a Nanodrop was gel electrophoresis provided inthekit.Agarose SD and10% K with lysozyme, proteinase lysed RNAlaterbu in resuspended were Briefly, bacterial cotton tips(forthe cells biofilm from samples) and filters (forthe water samples) fromthe same RNA sample.which(Qiagen, and allowed the extraction Germany), ofDNA Hilden, inst manufacturer’s tothe according performed harvested onaboardusing sterile biofilmsondifferentsubstr For RNAextraction, transcription reverse and RNA extraction Nearest-Neighbor-Inter model,the Tamura-Nei The constructionofmaximum et al. et (2011). fr extracted DNA andRNAwere Both PolyA Polymerase I andthen (Austin,TX) PolyA Polymerase μ L 10 contained qPCRreaction likelihood trees ofwasconducte ase (Invitrogen, Carlsbad,CA)a ase (Invitrogen, cotton tips and stored in RNAlater buffer. RNA extraction was extraction RNA buffer. and RNAlater in tips stored cotton ffer, pelleted by centrifugation at 4,000 × g for 10 min and then then 10minand gfor × at4,000 centrifugation by pelleted ffer, d using theKapaSYBR Fast qPCR Kit (KapaBiosystems, titative PCR(q-RT-PCR)assay. real-time μ L of template. The optimal dilutions of DNA and cDNA and cDNA L TheoptimaldilutionsofDNA oftemplate. S. The DNA was removed using the DNA column DNA the using removed was The DNA S. 1000bootstrapvalue. and method (NNI) change ructions using the AllPrep DNA/RNA Mini Kit DNA/RNAMini AllPrep ructions using the samepooled together substratetype asone were genes, fluorogenic PCR The cycling parameters were 5 min at 95 °C, were5minat95 °C, The cycling parameters useq-RT-PCR toqualify ofa abundance the ates in the brine pool and in the NBW were were ates inthebrinepoolandNBW ribed the cDNA as described inthestudy ribed thecDNAasdescribed by re designed (Table S3) usingre (Table designed S3) Primer5 software, 3000P qPCR machine(Agilent Technologies, used to estimate the integrity of the RNA, and RNA,and of the estimate theintegrity used to ed China Limited, Hong was Kong) used μ om one sample at thesample one sameom time using reverse-transcribed intocDNAusing reverse-transcribed L of 2 × Master Mix, 1pmol/ L Mix, 2×Master of nd oligo(dT) primers. The three nd oligo(dT) primers.Thethree d usingMEGA6.06withthe was applied to quantify was appliedtoquantify μ L of L of developed continuously inthebrine pool developed continuously community structures at thegenuslevelbetween biof ~50%oftheS-NBW ~10%and accounted for the with compared biofilms S-NBW in enriched groups taxa core dissimilarity between (Fig. S3). the 3d-NBW biofilms,ascommunity illustrated ofthe bythe 6d-NBW SIMPER analysis and For example, withthesourcebiof the genus levelcompared inFigure 2.The summarized >1.0% are abundance arelative with genera and genus level, the at classified further were biofilms the reads from To further illustrate the effects ofenvironmen diminished intheS-brine-PV a 15%-20% inthe6d-brine-PVC brine poolorNBW. For example, switchedbiof the between differed Candidatedivisi Deferribacteria, Hyd24-12, (namely bacteria several the proportionsof ofthe abundance were assigned mostly were assigned tothe are shownaswell). phyla the corresponding First, profilesbetweenth distincttaxonomic revealed communities, which lacked ahigh level ofdiversity.class Assignment thereads the of at level Lines S1. reaching the characterize asymptotes to sufficient were reads the that suggested This article is protected Allrights bycopyright. reserved. Accepted Article curves Rarefaction Taxonomic of structure Results ratios(Halm cDNA/DNA in triplicate, and the relative levels expression ofthe genes based were normalized ontheir assessed samplewas °C.Each at60 and15s at 50°C, °C,15s 15 sat95 of cycles 40 followed by Marinobacter Marinobacter Gammaproteobacteria of 16SrRNAgene amplicons the switched biofilmsthe switched et al. Gammaproteobacteria and C and S-brine-Ti biofilms. C andS-brine-Tibiofilms. 2011). Oleispira nd 6d-brine-Ti biofilms (Zhang nd 6d-brine-Tibiofilms ilm and the biofilms that were developed continuously in the the in continuously developed thatwere biofilms ilm andthe Firmicutes ofBac (consisted was lower inthe lower S-brinebi was were enriched in the S-brine biofilms compared with with compared biofilms S-brine the in wereenriched on WS3, OP9, OP11, OD1 and Acidobacteria) OP9,OP11,OD1andAcidobacteria) WS3, on ofthe microbial communities are inFigure shown ilms, biofilms. and6d-NBW namely the3d-NBW tal switchingtal onbiofilmcommunity composition, Verrucomicrobia, TM6, Verrucomicrobia, e S-brine and S-NBW biofilmsamples (Fig. S2; the the switched biofilmsand the biofilmsthat were 3d-brine and 6d-brine biofilms (Fig. S4);they biofilms 6d-brine 3d-brine and (85% to 95% within each sample), whereas the the whereas eachsample), 95%within (85%to or NBW were confirmed using one-way usingconfirmed one-way were NBW or S-brine biofilmsdisplayed patternsat different the bacterial reads from the S-NBW biofilms theS-NBW the bacterialreadsfrom ilms, respectively. Significant differences in ilms, respectively. in Significant differences Interestingly, these two genera were also Interestingly, were twogenera these illi and Clostridia) accounted accounted illi andClostridia) for et al. et ofilms (25%-62%). Moreover, Moreover, ofilms (25%-62%). 2014), whereas they were they were whereas 2014), TA06, Planctomycetes, Planctomycetes, TA06, biofilm S-brine andS-NBW dendr S5B) the because (Fig. cluster analysis Fig. ofthevariance; by 35.67% PC2(explaining distinguished thesw functional contentclearly based onthe16SrRNAgenes andthefunctiona Based onthePCoA,weobservedsubstan S2. Table switched biofilmsdevel the switchedbiofilms, characterize To further composition (Fig. S5A). s analysis cluster the supportby could be biofilms andthe thatwe PCoA tothetaxonomic plot (Fig. according 3A), biofilms, suggesting increasedcolonization pressure inNBW than inbrine water. However, the 6d-brine biofilms,while S- biofilms. Furthermore, the biofilmswere S-NBW more similar tothe 6d-NBW biofilmsthan to andNBW brine the from different weresignificantly biofilms switched the that observation whic biofilmswereobserved, and theS-NBW (Fig Asaresult 3d-brine-SS). excluding replicates (a 71samples all ofthe for was performed abundance inthebrinepool continuously were developed structuralsimilarityTo study be thetaxonomic Similarities basedontaxonom 6d-brine: Pseudo-F versus =0.0001; S-NBW 6d-NBW: Pseudo-F 6d-NBW: This article is protected Allrights bycopyright. reserved. Accepted Article Pseudo-F versus 3d-NBW: PERMANOVA (S-brine 194,4485 oped on Al, PVC and Ti. The metagenome information is summarizedin is information metagenome oped onAl,PVCandTi.The =41.4, P =0.0001; S-NBW versus 3d-brine: Pseudo-F versus3d-brine: =41.4,P=0.0001;S-NBW ntesm rnh branch. same s onthe brine biofilms were located between the 6d-NBW and 6d-brine and6d-brine the6d-NBW brine biofilmswerelocatedbetween ic and functional contents ic andfunctional contents re developedcontinuously in 165,3818 ograms based on the COG abundance positioned basedontheCOGabundance ograms the howing two major groups major howing two itched biofilms from the brine and NBW biofilms NBW and biofilmsfrom thebrineitched tween the switched biofilms and the biofilms that theswitchedbiofilmsand tween metagenomic analyses were conducted for the the for conducted were metagenomic analyses or NBW, PCoA based on the relative genera genera on therelative PCoAbased NBW, or there was no clear border between the border clear wasno there switched 3B). These results could be supported by the the couldbesupportedby results 3B). These h wasconsistentwith =75.91, P =0.0001). =75.91,P=0.0001). . 3A), similarity the S-brine between biofilms l profile based onthe l profile tial differences between between tial differences ll of the biofilms consisted of two biological oftwobiological ll ofthebiofilmsconsisted 212,4899 =6.59,P=0.0009; S-brineversus the brine pool or NBW, whichthe brinepoolorNBW, the above-mentioned theabove-mentioned COG annotation.The the taxonomic profilethe taxonomic based on taxanomic taxanomic based on 196,4531 =73.83,P NBW. We reconstructed the ge reconstructed We NBW. bacteriapossessafu speculated thatthesetwo in those than higher weresignificantly S-NBW This article is protected Allrights bycopyright. reserved. Accepted two the for information The 6). (Fig. illustrate the functional switched organization biofilms ofthe of the abundances above, mentioned singlegenome binning bacterial toobtain ofenriched organization the elucidate To further bacteria dominant and representative two of reconstruction Pathway biofilms. S-NBW the in enriched functions,suchas signal transduction-related (COG aminopeptidases/acylaminoacyl-peptidases acidpermea amino branched-chain an COG0569 pumps(COG0841, cation efflux acidmetabolism. amino functions involvedin Fo biofilms were enriched largely for metal ion resistance-related as wellfunctions, as some bi two the between contributing tothedifferences functions COG wasperformed. biofilms brine and S-NBW the between comparison a Similarly, Article (P<0 COG0471 (P<0.005),COG0553 biofilm significantgroups. For example, among different functions COG ofselected changes significant the check to was performed biofilms,re NBW in abundance a very low (Bakker transport system (COG1135) ion metal ABC-type the example, For biosynthesis. protein and metabolism acid amino resistance, stress ion The majority ofthe significantly functions changed was individual related to metal contributedCOG functions to that strongly of biofilm 6d-NBW) and (3d-NBW NBW S-brine and the metagenomesswitched ofthe biofilmwithth To identifyfunctional switching of theeffects on biofilms switched the for functions enriched and comparison Metagenomic nomes of the two bacteria ( bacteria two ofthe nomes Oleispira et al. et .001), andCOG2206 (P <0.05). differences were demonstratedfor were differences COG gardless of the substrate type (Fig. ofthesubstratetype gardless inS-brinebiofilmsbutdisplayedprevalent was 1987) transcriptional regulators (COG5001 and COG2909) andCOG2909) regulators (COG5001 transcriptional thesecommunities twodifferent types (Fig. of intheS-brinebiofilmsandof genomes fromthe metage genomes functions inthefunctions switchi the brine and NBW biofilms. Thus, it can be can it Thus, the biofilms. brineandNBW nctional adaptationtoboththebrine poolandnctional d COG3696), aminopeptidase N (COG0308), N(COG0308), COG3696), aminopeptidase d ofilm groups are listed in Figure 5. The S-NBW S-NBW listedinFigure 5.The groupsare ofilm 1506). Moreover, the comparison also revealed alsorevealed comparison the 1506). Moreover, eir sourcebiofilms.A direct comparison of the ses (COG1114), and dipeptidyl anddipeptidyl (COG1114), ses profile changes in the biofilms, wechanges comparedprofile inthe r example, theenrich r example, metagenomes SIMPER using revealed a set Marinobacter sp. and sp. ng functions,weapplied ed functionsincluded nomic datasets. As Marinobacter

4). Student’s t-test 4). Student’st-test Oleispira 1135 (P <0.005), sp.) to inthe

4).

This article is protected Allrights bycopyright. reserved. ofthisbacterium. on thegenomicannotation (Kube bacterium an oil-degrading Secondary MetaboliteAnal ferrochelatase, ferric-chelate reductase, and cytochrome genes. The AntiSMASH(Antibiotics & dismutase, superoxide catalase, including wereidentified, stresses oxidative to resistance suggesting present, glycolysis were pathways 6A). (Fig. andironuptake metal transport,zinc, genome, the (Singer environmental conditions marine bacteria sp. was closely related (clustered intothe same branch inthe phylogenetic tree) to the reported Based onthe phylogenetic trees using the31 essential concentrated genes (Fig. S6), (TableS4). and ~65%,respectively was >97% of genome out 70 contamination. However, du its that indicated genomes compared of the oftw thepresence strATCC49840. Furthermore, pr were genes two copiesofthese thegenomebinof in recovered havebeen not (TIGR01032)L20 may (Albertsen genomes isdisplayed inTableS4.Following th AcceptedThe regulation network. identified, aswellmany two-component regu biosynthetic cluster of ectoine. aminoMoreover, acid metabolism and utilization pathways were Articleribosomal protein L35 (TIGR00001) by comparison withtheir close Basedone onthe relativesgenes copy results, ofthe S5). (Table essen ofexpected Thenumber sequenced bacteria. investigated using asuiteofHMMencompassing Oleispira et al. et Oleispira Marinobacter sp. was closely related to M. aquaeolei 2013;Tian sp. Thus, we assumed that the co the that Thus, weassumed sp. sp. identified in the present study possesses numerous genes for heavy heavy genes for numerous possesses study present identified inthe sp. ysis Shell)(Medema VT8, which is an “opportunitroph” that can survive under various various surviveunder can that “opportunitroph” an whichis VT8, et al et of 107essential single et al. et esent inthe complete genome of et al. et , ribosomal protein S5(TIG , ribosomal . 2014), the completeness of the two genome bins was genomewas bins two ofthe the completeness 2014), . 2013).Themetabolicpathways O. antarctica 2011). complete Based ofthenearly ontheanalysis a heterotrophic lifestyle. Many genes involved in involvedin genes Many lifestyle. a heterotrophic o GTP-binding Era proteins (TIGR00436) in all (TIGR00436) in proteins o GTP-bindingEra lators, suggestinglators, thepr plication inourgenome binswasnot dueto Complete tricarboxylic acid (TCA) cycle and and cycle (TCA) acid tricarboxylic Complete e methods described in previous publications inpreviouspublications e methodsdescribed tial single-copy genes wa et al et 107 proteinsthatarec The results suggested thisbacteriumisa The resultssuggested strain RB-8, which has been reported tobe has beenreported which RB-8, strain . 2011) search also revealedacomplete . 2011) search also -copy genes were identified inthedraft identified genes were -copy mpleteness ofthe R01021), andribosomalprotein R01021), M. hydrocarbonoclasticus Marinobacter were reconstructed based based reconstructed were esence of a complicated a complicated of esence onserved in95%ofall s further investigated s furtherinvestigated two draft genomes two draft Marinobacter sp. because

nitrogen metabolism and envir nitrogen and metabolism amin and coenzymes acids, amino of biosynthesis in the Glutamine source amidotrans biofilms). (the numberoftranscript bacteria glutamine amidotransferase and Intriguingly, the revealed results an up-regulation ofamino acid metabolism-relatedgenes, such as metabolism and metal ionstress resistance in acid amino to related enzyme encoding genes ofkey transcription active the examine to To evaluate thefunctional activities expressed in Expression of key Oleispira astepin thatcatalyzes (K05714, anenzyme of thephylogeny example, For events. transfer me acid inarachidonic sp., suchasgenesinvolved of bins genome the in identified also were characteristics special diff acids have amino different bacteria appeared to prefer different amino that notion the with wasconsistent which acids, Marinobacter aquaeolei furtherWe compared the potential ability toutilize different in amino acids both thebrinepoolandNBW. ABC-type Fe ABC-type This article is protected Allrights bycopyright. reserved. Acceptedi In metal 2003). contrast, in participates O-acetyltransferase metabolism cysteine and (Takagi metabolism sulfur Articleversatility, the functional ofthe because acceptor. trans export metal heavy Furthermore, asanelectron use to nitrate potential the suggesting werepresent, reduction nitrate dissimilatory L-arginineL-glutamine to and L-glutamate were the of example, complete transformation For6B).for pathways (Fig. lipid metabolism speciesthatutilizes ala heterotrophic bacterial sp. isshownin Figure S7. 3+ -siderophore transporter, were up-regulated up-regulated were -siderophore transporter, functional genes functional VT8, on stress resistance-related genes, such astheMn genes,such on stressresistance-related Oleispira onmental adaptation (McDonagh B (McDonagh onmental adaptation erent metabolic costs (Behrends (Behrends costs metabolic erent s per genes per was significantly inthe higher switched biofilmsthan serine O-acetyltransferase, in the S-NBW biofilms for both forboth biofilms S-NBW the in O-acetyltransferase, serine Oleispira sp., and sp., and rge proportion ofgenes toperformaminoacidand 2-hydroxy-6-oxonona-2, 4-dienedioate hydrolase hydrolase 4-dienedioate 2-hydroxy-6-oxonona-2, phenylpropanoid compound degradation) from degradation)from compound phenylpropanoid the switched biofilms, q-RT-PCR was performed was performed q-RT-PCR biofilms, switched the ferases utilizeglutamine the amide of inthe Marinobacter sp. would also be a heterotroph that can adapt to adaptto thatcan a heterotroph sp. wouldalsobe o sugars, and they are important for microbial for important are and they o sugars, Oleispira antarctica identified.Moreover, tabolism, suggesting the occurrence of gene of tabolism, suggesting occurrence the porters were also identified. Collectively, in S-brine biofilms. in S-brine sp.and Oleispira et al. et et al. et RB-8 (Table S6). These These S6). (Table RB-8 other 2013). Moreover, Oleispira 2+ pathways involvedin sp. /Zn 2012). The serine 2012).Theserine

and Marinobacter 2+ Catalase is an Catalaseis sp.(Fig. S8). transporter and transporter and Marinobacter Marinobacter et al. al. et sp., This article is protected Allrights bycopyright. reserved. 2012; Székely Deterministic processes occur inbacterial assemblages across ecosystems various (Stegen sorting species function-based and environments new in assembly community Microbial and genomicanalyses. biofilm communities switched succession ofthe identified asmajor species sortingdeterminants inthe metabolism-related were functions character primarily onfunctional results conditions. Moreover,the provided eviden environmental communitiesenvironments new determined and source was by boththe biofilm developmentrevealedth environmental Environment switchi conditions. The majoraimofthe present study wastotest Discussion up-regulated inthe biofilmsforbothbacteria. S-brine by metalions(Scharf which canbeinduced (ROS), oxygen species reactive by damage oxidative against cells protects that enzyme important the switchedbiofilmsand thesourcecommunitie environments. Furthermore, PCoA pl the taxonomic coloni results suggestedmore Additionally, the sw structureofthe taxonomic theswitchedbiofilm 3A) separated parameters, and competition withlocal communities. The PC1 ofthe taxonomic PCoA plot (Fig. changeto copeforces: selective caused the changes by twomain withthe inenvironmental communities. the After immigration thebiofilm of effect ofspecieschanging sorting by the conditions incubation forthe immigrated biofilm environments has rarely the revealed been study present the in obtained results The performed. in deep sea residing regions,investigation surface in the species of sorting hypothesis Accepted functional plasticity. which couldbe assembly, Article et al. et 2012). Because most of the related related mostofthe Because 2012). attributed to the observation that many taxa possess a high degree of of degree possessahigh thatmany taxa attributed totheobservation at the composition ofthe biofilmcommunity established in new itched biofilms wasinfluenced istics. Furthermore, metal istics. Furthermore, fromthebrine biofilms,which andNBW suggested that the et al based on evidence from integrated from based onevidence metagenomic ng between the brine pool and NBW during and NBW ng pool the brine between sation pressure with the NBW than the brine brine thanthe NBW withthe pressure sation ce that biofilm community assembly community biofilm ce that was based to the new environment, the bacterialto the new taxa had ot alsorevealedacl thespeciessorting inresponseto effect s, suggesting source-dependent community community s, suggesting source-dependent . 2014). Consistently, catalases were also . 2014).Consistently, also were catalases studies were performed incommunities performed studies were ion resistance and amino acid acid andamino resistance ion by the newenvironments. by the ose relationshipbetweenose et al. et

3d-NBW biofilms; after immigration ofthe bi biofilms; after 3d-NBW on particularionsaselectron donorsinthebr environmental parameters, cold-seep system for a long andthe analyses metagenomic the Bytime. coordinating sp. and ability metal toresist ions, particular, totheir due In 6). (Fig. biosynthesis protein complete pathways for metal ion transport and resistance as well acid as amino metabolism and the bacteria, and dominant representative two survivalinbothth bacterial importantfor were acid metabolism-related genes fo amino and resistance stress ion formetal enrichment an revealed comparison The metagenomic Metal ionresistance amino acid and meta functions” that confer theability ofthe bacteriaadapt to tobothenvironments. Therefore, inhabiting the bacteria S-brine a pools, whichwouldbethemain different species selectedfrom two The species further were communities. source different biofilms contain communities ofa particular habitat. However, This article is protected Allrights bycopyright. reserved. Accepted Articlefunctions species withequivalent spac first; arrives species is wonby whichever 2011). Thatis,withinagroup ofspecieswithsim community could structure be explai communities associatedin with algaewerefrom the selected species same and the pool, variations the previousandpresentstudy, the underlying (Burke algae with associated rather than species composition, as demonstrated in a previous study investigating communities observation that microbi also consistentwiththe adaptive functionstoboth thebrinepooland NBW biofilms.This andNBW fromthebrine separated revealed a tight between relationship the S-br Interestingly, the PCoAplotandclusteranal Oleispira sp. mightgeneralists be versatile that have adapted environments tothe ofthe we propose that both habitat specialis thatboth we propose et al. et r the switched biofilms, which biofilms, switched r the 2011). Although asimilarphe 2011).Although ; there should be a ; thereshould core functions ned by“lottery” model(Sale the bolism asspecies sorting determinants nd S-NBW biofilms should share “core ecological ecological “core share biofilmsshould nd S-NBW e colonization israndomfromwithinagroup e colonization of ofilms from the NBW to the brine pool, only the the tothebrinepool,only fromtheNBW ofilms e brine pool and NBW (Fig. (Fig. NBW brine pooland & Consistently, 5). 4 e ysis derived fromtheCOGfunctions(Fig. 3B) ysis ine and S-NBW biofilms, andS-NBW ine clearly which were for cold seep biofilms, the S-brine and S-NBW cold seepfor the biofilms, ine pool) and generalists were present inthe werepresent ine pool)andgeneralists ilar ecological functions, the “lottery” for space the“lottery” forspace functions, ilar ecological Marinobacter al community assembly assembly community al causes of their different taxonomic structures. causes oftheirdifferent mechanisms would be different. Bacteria in Bacteria wouldbedifferent. mechanisms result ledusto proposethatonly with taxa can surviveandproliferate. Thisnotionwas sp. and sp.and nomenon was observed between wasobservedbetween nomenon ts (such as autotrophs that rely rely as autotrophsthat ts (such suggests that these functions thatthese suggests consistently present inall present consistently et al. al. et Oleispira is based on functional functional basedon is 1976; Burke Marinobacter sp.,showed et al. et

catalyses a step in the pathway ofphe astepinthepathway catalyses 2-hydroxy-6-oxonona-2,4-dienedi (Zhang al Marinobacter a tooxidative and resistance ala they adopted that suggest present study Genome analysisgenomic ofthe information. of and geneticfeatures degradation, lipiddegradati (Kube bacterium marine (Singer resistance metal ofenvi involved intheperception anddefense electron donor, iron asan mechanisms VT8 revealed itsmetabolic potential toutilize oxygenandas nitrate terminal electronacceptors, poolandthe can surviveinboththebrine Results ofthe present that study suggest environmental condition(Feehily facilitates intracellu useth genera bacterial species inseveral This article is protected Allrights bycopyright. reserved. environments (Feehily response bacterial the roles in acids play concentration. ion metal high the by caused stress oxidative of occurrence the suggested brine the sp. in metabolism-related (Fig.genes S8). Notably, theup-regulation catalase ofthe of levels expression the by further supported competitive and proliferate after the being (Fig. NBW moved to results 7).These could be utilization and amino acid metabolism forthecommunity were members important tobe biofilms.Similarly, intheS-brine proliferate generalistsbiofilm withmetal resistance-related inthe ionstress and could survive functions

Accepted. sy of thetype VI2005). Thepresence secretion Article et al et . 2013a; Lin . 2013a;

sp., is a compound that is used by many taxa to cope with osmotic stress (Teixidó (Teixidó stress tocopewithosmotic thatisusedby taxa many is acompound sp., The importance of amino acids could be amino acidscould The importanceof lar pHhomoeostasisbylar consumin Marinobacter Marinobacter et al. et al et et al et et al. et on, and siderophore pr on, andsiderophore . 2011). 2013;Horcajo . 2013) and possesses an array of array an and possesses . 2013) nd metal ion stresses. The ectoine, Theectoine, nd metalionstresses. may by produced which be , 2015).Moreover,uniquefeatures ronmental fluctuations andantibio fluctuations ronmental et et al oate hydrolase (K05714) in (K05714)in hydrolase oate O. antarctica and 03. . 2013). nylpropanoid compounds degradation (Lam compounds degradation nylpropanoid Oleispira Marinobacter NBW. Genome analyses of the bacterium bacterium ofthe analyses Genome NBW. e glutamate decarboxylase (GAD) system, which system, (GAD) decarboxylase glutamate e rge proportion of genes amino for acid metabolism et al. et for the S-NBW biofilms, heterotrophic carbon carbon biofilms,heterotrophic theS-NBW for Marinobacter Marinobacter of the ion transporter genes and amino acid acid genes andamino theiontransporter of to numerous stresses and adaptation to new adaptationto and numerousstresses to stem might alsocontributetostressresistance oduction pathways. Howe oduction pathways. has been reported to be ahydrocarbonoclastic tobe has beenreported 2012;Behrends remain largely uncharacterized due tolimited acquired via horizontal gene transfer that are that genetransfer acquired viahorizontal and explained byconclusion thataminoexplained the Oleispira g protonsto copewithacidic sp. metabolism,alkane amino acid

and Oleispira tics, phages, toxins, and heavy andheavy tics, phages,toxins, were also observed, such as observed,suchas also were Oleispira et al. et are habitat generalists that 2013). For example, sp. This enzyme sp.Thisenzyme ver, thefunctional sp. binnedin the Marinobacter Marinobacter M. et al. et

aquaeolei aquaeolei 1997). 1997). et University of Science and Technology and Science University of HKSAR from the (661611) grant a GRC 2012CB417304), This article is protected Allrights bycopyright. reserved. National CAS) (SIDSSE-201206), SanyaInstitute ofDeep Sea Science and Engineer for samp Science andTechnology of University Kong Hong the from Zhang Gen and Wong YueHim to grateful are The authors Acknowledgements Thus, the assembly inthisspecial habitat. w inotherbacteria pathways Future effortsinthe ofmetabolic reconstruction strategies. adaptation bacterial regarding insight herein as examplesofthe generalists inhabiting the Thuwal which cold providedII, seeps some so thespecies governing principles the general additionaldatasets fromlarger-scalemetagenomes, used forbiofilmdevelo were types ofsubstrates different community structures in provided also We process. sorting species the the present study the bridge gap inthe species sorting theory by presenting functional bases for Through the integrationof experimental strategies withmetagenomic comparisons, the results of Concluding remarks bacteria inthe switched biofilms. the revealing by process sorting species for the survive inboth the brinepooland Thes NBW.

Accepted Article Oleispira sp.

and Marinobacter ill facilitateabetter observed between the brine pool and the NBW. Although six Although NBW. thebrinepooland between observed le collection.Thisstudy fromthe by supported awards was

Basic (SA-C0040/UK-C0016) toP.Y.Qian. (SA-C0040/UK-C0016) sp. have developed specialized adaptation strategies specialized to developed have sp.

Research rting process. Two genome bins were analyzed analyzed genome binswere Two rting process. genomic andmetabolicf genomic pment and core mechanisms were revealed in the the in wererevealed andcoremechanisms pment ing, the Chinese Academy ofSciences (SIDSSE, dications that species explain sorting the may e resultsfurtherelucid cold seep environment are required to confirm toconfirm required are seep environment cold understanding ofthemicrobialcommunityunderstanding

Program government, and the King Abdullah Abdullah theKing government, and

of China(973Program, No: eatures oftwodominant eatures ate thefunctionalbases ate phylogenetic analysis. phylogenetic (2000)Sel Castresana J Austral Ecology community sequencing data. species. than rather R Doug S, Peter Burke C, 398-408. systems in Hyatt D, Chen GL, Locascio PF GL, Hyatt D, Chen translation initiation site identification. This article is protected Allrights bycopyright. reserved. BakkerIR, EP,Booth DinnbierU,EpsteinW, Biotechnology bacteriadifferential cove by obtained P,SkarshewskiA Hugenholtz Albertsen M, References in the South Pacific Gyre. Gyre. Pacific South the in TG Ferdelman Lam P, Halm H, and Ecology acid andotherstresses. glutam Roleof (2013) KAG Karatzas, Feehily C, Research Acids Nucleic throughput. high and accuracy with high alignment MUSCLE:Edgar RC(2004) multiplesequence Clarke KR(1993)Non-pa Stombaugh J J, Kuczynski Caporaso JG, aeruginosa V, Ryall Zlosnik B, JEA Behrends continental margin ofthecentral Red Sea. BatangZB, E,Al-Suwailem Papathanassiou A education and data analysis. analysis. education anddata PAST:Pa Ryan PD(2001) DAT, Hammer Ø,Harper tr phylogenetic Building Hall BG(2013) Accepted acids. peptidoglycannoncanonical D-amino editing by Horcajo P,dePedroMA,Cava Article Escherichia coli Escherichia during cystic fibrosis chronic lung infections. lung duringcystic fibrosischronic , 30 ,

31 , , 1229-1235. , 1229-1235. 18 , 533-538. , 533-538. Proceedings oftheNationalAcademySciences Proceedings , 117-143. Molecular BiologyandEvolution Molecular Journal ofAppliedMicrobiology Journal ,

ection of conserved blocks from multiple alignments for their use in fortheiruse frommultiplealignments blocks conserved ection of 32 The ISMEJournal et al. et . , 1792-1797. rametric multivariate analyses of analyses multivariate rametric Palaeontol Electron Journal ofBacteriology Nature Methods (2011) Bacterial community (2011)Bacterialasse et al et F (2012)Peptidoglycan plasticit et al et . (2011) Heterotrophic organisms dominate nitrogen fixation nitrogen organisms dominate Heterotrophic . (2011) BMC Bioinformatics et al. et ees frommolecular ees with data MEGA. et al. et . (2010) Prodigal: prokaryo . Journal ofMarineSystems Journal rage binningrage multiple metagenomes. of , et al. al. et , Gajewska A (1987) Evidence formultipleGajewska A(1987)Evidence K (2013) Metabolic (2013) 6 7 (2010) QIIME allows analysis of high-throughput ofhigh-throughput analysis allows QIIME (2010) , 1238-1249. , 1238-1249. et al. et , 335-336. , 335-336. , 4 ate metabolism in bacterial responses towards towards responses ate metabolisminbacterial (2013) Genome sequences sequences Genome (2013) , 1-9. , (2012) First discovery of a cold seep onthe ofacoldseep (2012)First discovery leontological statistics leontological statistics 169 Microbial DrugResistance , , 7334. , 3743-3749. 17 11 , 540-552. , 12. , 11-24. 8 changes in community structure in changes Environmental Microbiology Environmental 1. , 119. mbly based onfunctionalgenes mbly based adaptations of adaptations y stress-induced inbacteria: , ,

108 94 tic gene recognition and gene recognition tic 4-5. , 247-253. , 14288-14293. software package for for software package of rare, uncultured uncultured rare, of Molecular Biology Molecular , Pseudomonas 18 0-1. , 306-313. + Nature export , 15 . , sequences. metabo ofsecondary and analysis Langmead B,Langmead Salzberg (2012)FastSL the initial assembly ofbacterial communities. (2011)Speciessorti AJ S,Székely Langenheder 12242–12251. bacterium oil-degrading the of analysis Al-RamahiY Kube M,ChernikovaTN, Research by colonization. differentiation isolation by inthe wild: dispersal limitation, isolation byadaptation and isolation I, Swillen J, L, Mergeay J, Orsini Vanoverbeke deepsea. the in formed surfaces engineering Mowlem MC,Keevil NM, Meier A,Tsaloglou, P K,Cimermancic Blin MH, Medema Microbiology Reports This article is protected Allrights bycopyright. reserved. environments. LN Huang ZS, Hua SJ, Li Bioinformatics 2-hydroxy-6-keto-nona-2,4-d hydrolase: C-C a of analysis andstereochemical characterization, Purification, (1997) TD Bugg WW, Lam kyotKEGG: Kanehisa M,SusumuG(2000) 905-913. dynamics. community bacterial onfreshwater immigration SE,McMahonKD(2009)Specie Jones Li H, Handsaker B, Wysoker A B,Li Wysoker H,Handsaker Microbiology in bacterial communities associated withcorals the from Red Sea. S Bougouffa J, Yang Lee OO, 357-359. extensive changes in the redox proteome of proteome redox inthe changes extensive Lin J, Cheng J, Chen K AcceptedMcDonagh B, Domínguez 1715-1727. respons microbial reveal sequencing Borglin S TC, Mason OU,Hazen Infection Microbiology virulence-related characteristics in Article , 28 Nucleic Acids Research Acids Nucleic , 27-30. , 27-30. , AEM-01111. , AEM-01111. Scientific Reports , 25 Molecular Ecology Molecular , 2078-2079. , , 4 5 5-6. , 257-267. et al. et doi: 10.3389/fcimb.2015.00070. - Martín MA,Gómez et al et et al et (2015) The icmF3locusisinvol The (2015) et al et , 4 lite biosynthesis gene clusters in bacterial and fungal genome genome andfungal gene clustersinbacterial lite biosynthesis , 6205. , et al et . (2012) Pyrosequencing reveals sp reveals Pyrosequencing . (2012) , . (2014) Microbial communities evolve faster in extreme inextreme faster communitiesevolve Microbial (2014) . Pseudomonas aeruginosa Pseudomonas 39 . (2009) The sequence alignment/map formatandSAMtools.alignment/map sequence The . (2009) 22 e to Deepwater Horizon oilspill. Horizon Deepwater e to , 339-346. , 339-346. gapped-read alignment with Bowtie 2. with Bowtie alignment gapped-read et al et , 5983-5999. , 5983-5999. Oleispira antarctica . (2012) Metagenome, . (2012) et al et s-sorting may explain an apparent minimal effect of of minimaleffect anapparent explain mays-sorting iene-1,9-dioic acid 5,6-hydrolase. acid5,6-hydrolase. iene-1,9-dioic . (2011) antiSMASH:rapid . (2011) o encyclopedia of genes and genomes. genes and of o encyclopedia Biofouling . (2013) Genomesequence . (2013) The ISMEJournal Prochlorococcus - ng and neutral processes are both important during bothimportantduring are processes ng andneutral Baena G CW, Connelly DP (2013) Hyperbaric Connelly biofilms on DP (2013) CW, Meester L (2013) Drivers of population genetic ofpopulation genetic L(2013) Drivers Meester , 29 et al. et 0914. , 1029-1042. . Nature Communications (2012)Nitrogen induces starvation PAO1. PAO1. metatranscriptome andsingle-cell metatranscriptome Environmental Microbiology Environmental sp. strain SS120. sp. strain , 5 ved inmultipleadaptation-and , 1086-1094. , 1086-1094. Applied andEnvironmentalApplied atial and species variations and speciesvariations atial Frontiers inCellularand Frontiers identification, annotation identification, and functional genomic functionalgenomic and The ISME Journal The ISME Nature Methods Biochemistry Environmental Environmental Nucleic Acids Nucleic , 4 , 2156. , , , , 36 11 9 6 , , , , This article is protected Allrights bycopyright. reserved. subsurface microbial communities. WC Singer EA,Nelson E,Webb Reports by encoded identifying novelhalogenases Öztürk B, de Jaeger H L, Smidt Sullam KE, Essinger SD, Lozupone CA SD, Sullam KE,Essinger LinStegen JC, X,KonopkaAE “opportunitroph”. biogeochemical data. sequencing Patel RK, Jain M (2012) NGSQCToolkit: a t Biology M,Eisen (2008) Asimple,fast,and Wu JA 5261-5267. Microbiology capacity ofa potentially symbiotic sulfur oxidizing bacterium in sponge. bacterialcommunities innew environments. Mech S(2012) Langenheder M, Berga AJ, Székely shapegut the bacterial communities offish:a meta-analysis. dispersal: macroecological patterns dr Nguyen NH,BrunsTD Peay SchubertMG, KG, 4122-4136. taxonomy. newbacterial intothe rRNA sequences (2007)Na Cole JR JM, GM,Tiedje Wang Q,Garrity Tian RM, Wang Y, Bougouffa S agglomerans CPA-2. biocontrol agentPantoea glycine–betaineand ectoine, inosmotic stress ad 6.0. genetics version analysis G,PetersonD,FilipskiTamura K,Stecher A, Sale PF (1976)Re Teixidó N, Cañamás TP, Usall J N,Cañamás UsallTeixidó J TP, andevolution. proteinfunctions analysis of biosynthesis. cysteine in O-acetyltransferase AwanoN, Yoshioka K, H, Takagi ScharfB, Clement CC,Zolla V, Accepted Natale DA Tatusov Galperin RL, MY, Articletoxicity. , , Scientific Reports 3 9 , 2780. , R151. , 16 PloS one ,

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et al et ISME Journal et al. et Crambe crambe Crambe . (2005) Accumulation of the compatible solutes, solutes, thecompatible of . (2005)Accumulation Nucleic Acids Research Acids Nucleic accurate method of phylogenomic inference. inference. methodofphylogenomic accurate . (2000) The COG database: a tool for genome-scale genome-scale tool for a COGdatabase: The . (2000) (2003) Roleof (2003) The FEMS MicrobiologyLetters FEMS Kumar S. (2013) MEGA6: molecular evolutionary Kumar S.(2013)MEGA6:molecularevolutionary (2012) Environmental and ecological factors that that factors ecological and Environmental (2012) Letters inAppllied Microbiology

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aptation and heat shock cross-protection inthe cross-protection shock aptation andheat 85 oolkit forquality contro Applied and Environmental Microbiolog Environmental Applied and ïve bayesian classifier for rapid assignment of for rapidassignment classifier ïve bayesian (2012) Measuring ectomycorrhizal fungal fungal ectomycorrhizal (2012)Measuring , 60-65. , anisms determining the fate of dispersed dispersed fate of the anisms determining 6 6316. , 1653-1664. nd deterministicassembly in processes (marine sponge) microbiota. sponge) (marine ndent and independent approaches for for approaches ndent andindependent Molecular Ecology Molecular Saccharomyces cerevisiae Saccharomyces

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veals versatile heterotrophic versatile heterotrophic veals 28 Marinobacter aquaeolei , 33-36. , 33-36. Molecular Ecology Molecular , 218 l ofnextgeneration , 77 9-9. , 291-297. , , , 2763-2771. , 2763-2771. 41 21 Environmental Environmental , 248-252. , 248-252. , 3363-3378. , 3363-3378. Scientific Genome Genome serine serine , , 21 73 , a , , substrates deployed in a cold seep system. seepsystem. cold a in deployed substrates by metal ionand stresses. oxidative 10.1111/1462-2920.12978. biofilm functions during S Bougouffa Y, Wang W, Zhang Zhang W, Wang Y, Tian RM OO Lee Y, Wang Zhang W, Microbiology This article is protected Allrights bycopyright. reserved. Figure 1. Figure legends q-RT-PCR data,aligne metagenome data,16S comparative annotation, metagenome assembly, metagenome sequences, Illumina The raw Data accessibility pseudotuberculosis Yersinia Y, Song WangZhang Y W, Microbial Ecology (QIIME) w pipeline. Genera Microbial (QIIME) Ecology classifier(RDP) inthe QuantitativeInsights level into using the ribosomal database project samples. Operationaltax Figure 2. (PTFE), and stainless titanium steel (Ti). (SS) (Al), polyether ether aluminum biofilms The to NBW). for biofilmsfrombrine ( incubated foranadditional three days being before days forthree the brineandNBW and Acceptedthan 1%inatleas greater of an abundance achieve not did that genera The shown. are 2replicates from calculated values andmean replicate Article Oleispira Schematic model of the experi Schematic model ofthe Taxonomic classificationof Taxonomic , p r ouetdi ra o:056/ra.n8. inDryad doi:10.5061/dryad.9ng87. sp. aredocumented 15 , 557-569. , 557-569. d sequences for tree constr for tree sequences d development in a cold seep brine pool. brine coldseep development ina onomic units (OTUs) with an identity of 97% were classified at the genus classifiedatthe (OTUs) withanidentity of97%were onomic units 454 sequences, aligned 16Ssequen aligned 454 sequences, functions tomaintain intracellular pHhomeostasis. et al et et al et et al et ketone (PEEK), polyvinyl chloride (PVC), polytetrafluoroethene polytetrafluoroethene (PVC), polyvinyl chloride (PEEK), ketone . (2013b) Adaptation ofintertidal . (2013b)Adaptation et al et . (2013a) A type VI secretion system regulated by OmpRin by regulated VI system secretion Atype (2013a) . . (2014)Speciessorting during bi Scientific Reports . (2015) Synchronized dynamics of bacterial niche bacterial of dynamics (2015) Synchronized . “ qualified bacterialreadsretr mental design. The biofilms we S-brine Scientific Reports Scientific t one of two replicates were defined as defined t oneoftworeplicateswere ” moved to the NBW and moved totheNBW brine, respectively, and for biofilms from NBW to brine and brine to NBW biofilmsfrom for ith a relative abundance >1% in at least one inatleast >1% abundance ith arelative uction, and genome of uction, and bins were developed on six different substrates: substrates: different onsix developed were , 3 10 , 3180. , 4 , 6647. Environmental Microbiology Environmental ces, OTU table, SIMPER data, ces, OTU biofilmcommunitiesisdriven ieved from switchedbiofilm ieved ofilm assembly re developed separately in in separately re developed Marinobacter Environmental Environmental “ by artificial minor “ S-NBW ‐ specific specific , doi: ” . A, . A, sp. ”

proliferated. proliferated. for amino acid and metabolism metal ionstressresistancebecame competitive and more immigratedcommunities. withthegeneralists environments, capacity biofilms After the tonew Figure 7. also identified. were and ectoinebiosynthesis secretion system are system assemblyIII, pathways fortype andVI secretion Complete shown. relatedamino acid metal to metabolism, stress oxidative resistance ionand and the bacterial Figure 6. indicatedyellow in and respectively. red, Inorganic ion metabolism(P), transport and a theircontributiontoth of presented intheorder were 6d-brine). Comparisons Figure 5. indicatedyellow in and respectively. red, Inorganic ion metabolism(P), transport and a theircontributiontoth of presented intheorder 6d-NBW). Comparisons were performed based based were performed Comparisons 6d-NBW). This article is protected Allrights bycopyright. reserved. Accepted ArticleFigure 4. andredforTi. SS for orange forPTFE, green dark PVC, for green ondifferent biofilms represent colors The figure. of varian eigenvalues (percentage (B). The (COG) annotation groups orthologous of clusters and (A) composition taxanomic the by Figure 3. biofilm communities are showninFigureS4. S3and S-brine biofilms;B, biofilms.Bacteria S-NBW Schematic model ofspecies Schematic Similarity of the microbial communities in the switched biofilm samples, as samples, illustrated switched biofilm the microbial communities the Similarityof in Genomic construction of construction Genomic SIMPER analysis oftheCOGs from the SIMPER analysis oftheCOGs from the performed performed onthemetage based ce for the first two principalco thefirsttwo ce for Marinobacter sorting during the establishment ofswitched biofilm nd amino acid metabolism transport and (E) are nd amino acid metabolism transport and (E) are l genera differentiating the switched and source switchedandsource the l genera differentiating e difference (highest to lowest, top to bottom). to e difference (highest tolowest, top e difference (highest to lowest, top to bottom). to e difference (highest tolowest, top materials: purple for Al, blue for PEEK, bright PEEK,bright blue for Al, for materials: purple sp. (A) and and sp.(A) on themetagenomica S-NBW and and S-NBW (3d-brineand biofilms source S-brine and S-brine and (3d-NBW biofilms source Oleispira mponents) are indicated in the inthe areindicated mponents) nomic annotation. COGsare nomic sp. (B). sp. nnotation. COGs are nnotation. COGsare

The pathways The This article is protected Allrights bycopyright. reserved. Accepted Article

This article is protected Allrights bycopyright. reserved. Accepted Article