Metaproteogenomic insights beyond bacterial response to naphthalene exposure and bio-stimulation

ORIGINAL ARTICLE Me ap o eogenomic insigh s beyond bac e ial esponse o naph halene exposu e and bio-s imula ion Ma ı ´a-Eugenia Guazza oni 1,9,11 , Flo ian-Alexande He bs 2,9 ,I a ´nLo es 3,9 , Ja ie Tamames 4,9 , Ana Isabel Pela ´ez 3 , Nie es Lo ´pez-Co e ´s 1 , Ma ı ´a Alcaide 1 , Me cedes V Del Pozo 1 , Jose ´Ma ı ´a Viei es 1 , Ma in on Be gen 2,5 , Jose ´Luis R Gallego 6 , Ra ael Ba giela 1 , A an xa Lo ´pez-Lo ´pez 7 , Die ma H Piepe 8 , Ramo ´n Rossello ´-Mo ´ a 7 , Jesu ´sSa ´nchez 3,10 , Jana Sei e 2,10 and Manuel Fe e 1,10 1 Depa men o Bioca alysis, Ins i u e o Ca alysis, CSIC, Mad id, Spain; 2 Depa men o P o eomics, UFZ-Helmhol z-Zen um u¨ Umwel o schung GmbH, Leipzig, Ge many; 3 A ´ ea de Mic obiologı ´a, IUBA, Facul ad de Medicina, Uni e sidad de O iedo, O iedo, Spain; 4 CSIC, Cen o Nacional de Bio ecnologı ´a, Mad id, Spain; 5 Depa men o Me abolomics, UFZ-Helmhol z-Zen um u¨ Umwel o schung GmbH, Leipzig, Ge many; 6 IUBA, A ´ ea de P ospeccio ´n e In es igacio ´n Mine a, Uni e sidad de O iedo, Mie es, Spain; 7 IMEDEA (CSIC-UIB), Espo les, Spain and 8 Helmhol z Zen um u¨ In ek ions o schung–HZI, B aunschweig, Ge many Mic obial me abolism in a oma ic-con amina ed en i onmen s has impo an ecological implica ions, and ob aining a comple e unde s anding o his p ocess emains a ele an goal. To unde s and he oles o biodi e si y and a oma ic-media ed gene ic and me abolic ea angemen s, we conduc ed ‘OMIC’ in es iga ions in an an h opogenically in luenced and polya oma ic hyd oca bon (PAH)-con amina ed soil wi h (Nbs) o wi hou (N) bio-s imula ion wi h calcium ammonia ni a e, NH 4 NO 3 and KH 2 PO 4 and he comme cial su ac an I eysol, plus wo naph halene-en iched communi ies de i ed om bo h soils (CN2 and CN1, espec i ely). Using a me agenomic app oach, a o al o 52, 53, 14 and 12 dis inc species (acco ding o ope a ional phylogene ic uni s (OPU) in ou wo k equi alen o axonomic species) we e iden i ied in he N, Nbs, CN1 and CN2 communi ies, espec i ely. App oxima ely 10 ou o 95 dis inc species and 238 ou o 3293 clus e s o o hologous g oups (COGs) p o ein amilies iden i ied we e clea ly s imula ed unde he assayed condi ions, whe eas only wo species and 1465 COGs con o med o he common se in all o he mesocosms. Resul s indica ed dis inc biodeg ada ion capabili ies o he u ilisa ion o po en ial g ow h-suppo ing a oma ics, which esul s in bio-s imula ed communi ies being ex emely i o naph halene u ilisa ion and non-s imula ed communi ies exhibi ing a g ea e me abolic window han p e iously p edic ed. On he basis o compa ing p o ein exp ession p o iles and me agenome da a se s, in e -alia in e ac ions among membe s we e hypo hesised. The u ilisa ion o cu a ed da abases is discussed and used o i s ime o econs uc ‘p esump i e’ deg ada ion ne wo ks o complex mic obial communi ies. The ISME Jou nal (2013) 7, 122–136; doi:10.1038/ismej.2012.82; published online 26 July 2012 Subjec Ca ego y: in eg a ed genomics and pos –genomics app oaches in mic obial ecology Keywo ds: biodi e si y; bio-s imula ion; label- ee p o ein quan i ica ion; me agenomics; me ap o eomics; polya oma ic hyd oca bon In oduc ion Polya oma ic hyd oca bons (PAHs) a e widely dis- ibu ed in he en i onmen owing o hei abundance in c ude oil and hei widesp ead use in chemical manu ac u ing (Ka ¨s ne , 2000). PAHs a e pollu an s o g ea conce n owing o hei oxici y, mu agenici y and ca cinogenici y. A numbe o mic oo ganisms, ia he ac ion o Rieske non-haem i on oxygenases, ha e he abili y o g ow on PAHs as a sole ca bon and ene gy sou ce (Beil e al., 1998; Roling e al., 2003; Wi zig e al.,2006;Penge al., 2008). The majo i y o e o s aimed a unde s anding mic obial esponses o a oma ic compounds ha e Co espondence: J Sa ´nchez, A ´ ea de Mic obiologı ´a, IUBA, Facul ad de Medicina, Uni e sidad de O iedo, O iedo, Spain. E-mail: [email p o ec ed] o J Sei e , Depa men o P o eomics, UFZ-Helmhol z-Zen um u ¨ Umwel o schung GmbH, Leipzig, Ge many. E-mail: [email p o ec ed] o M Fe e , Depa men o Bioca alysis, Ins i u e o Ca alysis, CSIC, Ma ie Cu ie 2, Mad id 28049, Spain. E-mail: [email p o ec ed]s 9 These au ho s con ibu ed equally o his wo k. 10 These au ho s con ibu ed equally o his wo k. 11 Cu en add ess: Labo a o y o Molecula Ecology, Cen o de As obiologı ´a (CSIC-INTA), Ca e e a de Ajal i km 4, To ejo ´nde A doz 28850, Mad id, Spain. Recei ed 9 Janua y 2012; e ised 11 June 2012; accep ed 11 June 2012; published online 26 July 2012 The ISME Jou nal (2013) 7, 122–136 & 2013 In e na ional Socie y o Mic obial Ecology All igh s ese ed 1751-7362/13 www.na u e.com/ismej been ocused on genomic (Jime ´nez e al., 2002; Nogales e al., 2008; Pucha"ka e al., 2008), an- sc ip omic (Domı ´nguez-Cue as e al., 2006; Yus e e al., 2006) and p o eomic (San os e al., 2004; Segu a e al., 2005; Kim e al., 2006; Ku ba o e al., 2006; Toma ´s-Galla do e al., 2006) analyses con- duc ed in pu e cul u es. Among PAH-(including naph halene, luo ene, phenan h ene, py ene and dibenzo u an, o ci e some) mine alising s ains, a numbe o bac e ia ha e ecei ed special a en ion. Those include membe s o he common gene a o Pseudomonas,Sphingomonas,Cycloclas icus, Bu kholde ia,Rhodococcus,Pola omonas, some no el gene a o Nep unomonas and Janibac e ,some he mophilic bac e ia o Noca dia and Bacillus, some anoxic bac e ia o Del ap o eobac e ia and Alcaligenes, and high molecula weigh PAH- deg ading bac e ia o Mycobac e ium,S eno opho- monas and Pas eu ella; a lis wi h known pu e bac e ial cul u es capable o deg ading PAHs can be seen in a ecen e iew by Lu e al. (2011). Howe e , a la ge numbe o cul u e-independen echniques ha e shown ha pollu an -deg ading o ganismsen ichedin helabo a o yo endono ha e an impo an ole in he in si u biodeg ada ion o pollu an s and ha he di e si y o pollu an -deg ad- ing o ganisms in pollu ed en i onmen al samples is much g ea e han he di e si y de e mined om cul i a ion (Abulencia e al., 2006; Liu e al., 2009; Bo onin and Koshele a, 2010; Yagi e al., 2010; Liang e al., 2011). Unde his scena io, i is ele an o use molecula mic obial ools o de ine key ca abolic playe s a con amina ed si es o p edic pollu an deg ada ion ne wo ks in he en i onmen and o sugges me hods o a ional in e en ion associa ed wi h he implemen a ion o bio emedia ion (Vilchez- Va gas e al., 2010). Howe e , he numbe o in e- g a i e ‘omic’ in es iga ions ha ha e been ca ied ou in PAH-associa ed mic obial communi ies is limi ed (Kweon e al., 2007; Powell e al., 2008; Selesi e al., 2010), because o he incomple e genomic in o ma ion and cu a ed da abases a ailable (Pe ´ ez-Pan oja e al., 2009, 2012). Taking all o he abo e in o ma ion in o consid- e a ion, we pe o med a ho ough and holis ic (o ecosys ems biology app oach) phylogene ic, unc- ional and p o eomic analysis o he key playe s in a sample o s ongly an h opogenically in luenced, PAH-con amina ed soil (N) and a naph halene- en iched communi y de i ed om his soil (CN1). This s udy was ca ied ou using also samples o he same soil bio-s imula ed wi h calcium ammonia ni a e, NH 4 NO 3 and KH 2 PO 4 , and he comme cial su ac an I eysol (I ey In e na ional Inc., Campbell Ri e , BC, Canada) (he ein e e ed as Nbs), and he naph halene-en iched communi y de i ed om i (CN2). In all cases, he biodeg ada ion ne wo ks o he espec i e whole communi ies we e econ- s uc ed. This s udy cla i ied he genomic and p o eomic basis o he pu pose o unde s anding mic obial biodi e si y, ecology and unc ion in esponse o bo h PAHs ( ep esen ed by naph halene) and bio-s imula ion. I should be no ed ha ou me ap o eomic in es iga ions we e es ic ed o naph halene-en iched communi ies because o hei lowe complexi y as compa ed wi h he soil samples as well as he la ge assembled sequences ob ained h ough di ec py o-sequencing o bo h samples. Ma e ials and me hods Gene al me hods and ‘OMIC’ da a analysis and p ocessing Full desc ip ions o he ma e ials and me hods used o soil cha ac e isa ion; hyd oca bon analysis; DNA ex ac ion; cons uc ion o 16 S RNA gene clone lib a ies, sequencing and phylogene ic analysis; dena u ing g adien gel elec opho esis analysis; me agenomic se up and sequencing, assembly and gene p edic ion; me ap o eomic se up and p o ein ex ac ion, sepa a ion and iden i ica ion and da a p ocessing a e a ailable in he Supplemen a y Ma e ials and me hods. Soil sample collec ion and p epa a ion o bio- s imula ed soil and en ichmen cul u es Soil samples (he ein e e ed o as N) we e ob ained om a pa cel on he no he n Ibe ian Peninsula con iguous o a chemical plan (Lugones, O iedo, Spain; 4011803300N, 313903100W, a an al i ude o 300 m). The chemical plan (Supplemen a y Figu e S1) was used o se e al decades o he p oduc ion o naph halene, phenols and o he compounds om coal a p ocessing as well as o manu ac u ing esins. A conside able amoun o o he chemical p oduc s (pes icides, sol en s, e c.) was s o ed, al hough p obably no manu ac u ed, in he plan . In 1989 he plan was closed and hen used o yea s o s o e chemical was e, pa icula ly polychlo i- na ed biphenyls, coolan s and o he unspeci ied p oduc s. In 2006 mos o he buildings we e demolished and he cha ac e isa ion o soil con am- ina ion s a ed. Thus, a signi ican amoun o he PAH de ec ed we e concei ably o med by he en iched bac e ial popula ions p esen in he soil h ough a na u al a enua ion e ec . The op soil was sampled a a dep h o 0–30 cm on Feb ua y 2007 (soil empe a u e 18 1C). Th ee eplica es (1 kg each) we e collec ed wi hin a 1 m dis ance, and he samples we e kep in open plas ic bags in he da k a 4 1C. Vege a ion and o he non-soil ma e ials, including cobbles, we e emo ed p io o homo- genising he samples. Immedia ely a e acqui ing he soil samples, hey we e sie ed (2-mm mesh size), ollowed by mixing o ep esen a i e subsam- ples o he iplica es samples, and 100 g aliquo s o sample N we e used o chemical analysis. In addi ion, 10 g aliquo s we e s o ed a 20 1Cin s e ile lasks o DNA- and p o eome-based analyses. Bio emedia ion was pe o med o e app oxi- ma ely 900 m 3 o he con amina ed soil (N), whe e Mic obial esponse o polya oma ics M-E Guazza oni e al 123 The ISME Jou nal 9 ons o dehyd a ed calcium ammonia ni a e and 3 ons o a mix u e o dehyd a ed NH 4 NO 3 and KH 2 PO 4 combined wi h 4500 l o he comme cial su ac an I eysol we e applied o he homogenised soil. The C:N:P a io ha was applied was 100:10: 1, as ecommended o bio emedia ion pu poses (Gallego e al., 2007a). The ea men was pe o med o e 231 days, and he biopile was ou inely wa e ed and illed o main ain humidi y (15–20%) and adequa e oxygena ion. Samples o he op bio-s imula ed soil, he ein named Nbs, we e aken and p ocessed using he same p o ocols as desc ibed abo e o he o iginal soil. En ichmen cul u es we e pe o med in Bushnell Haas (Sigma Chemical Co., S Louis, MO, USA) mine al medium ha con ained naph halene a a concen a ion o 0.1% (w/ ) as he sole ca bon sou ce as desc ibed p e iously (Gallego e al., 2007b). The composi ion o he medium was as ollows: MgSO 4 H 2 O (0.20 g l 1 ), CaCl 2 2H 2 O (0.02 g l 1 ), KH 2 PO 4 (1.0 g l 1 ), K 2 HPO 4 (1.0 g l 1 ), NH 4 NO 3 (1.0 g l 1 ) and FeCl 3 (0.05 g l 1 ). Two di e en inocula we e used. The CN1 en ichmen cul u e was ob ained by inocula ing 1 g o he pollu ed soil (N) in o a lask con aining 100 ml o he cul u e medium; he CN2 cul u e was inocula ed wi h 1 g o he same soil ha had been subjec ed o a massi e bio emedia ion (bio-s imula ion) p ocess (Nbs). The en ichmen cul u es we e incuba ed a 30 1C and 250 .p.m., in which 0.1% ( / ) o he cul u e was ans e ed o esh medium each week. Resul s and discussion Sample cha ac e is ics An ag onomic analysis o he soil N e ealed a loamy clay soil wi h a pH o 8.2 and a conduc i i y o 0.13 dS m 1 clea ly con aining low amoun s o he ypically p edominan ions (calcium, magne- sium, po assium and sodium); he de ec ed na u al o ganic ma e , ni ogen and phospho us le els in he soil (Supplemen a y Table S1) a e cha ac- e is ic o in e ile soils. Gas ch oma og aphy–mass spec ome y (GC–MS) was used o quan i y he le els o he 16 EPA (En i onmen al P o ec ion Agency) p io i y PAHs p esen in he soil (Supplemen a y Figu e S2). To ally, he soil exhib- i ed an a e age concen a ion o 805 mg PAH pe kg (Table 1), which is in he ange o sligh ly lowe o he le el ound in p e iously epo ed PAH con amina ed soils ( ha is, 1667 mg kg 1 in Nı ´ Chadhain e al. (2006); 589 mg kg 1 in Richa dson e al. (2012); 335–8645 mg kg 1 in Tha amani e al. (2012); 3000 mg kg 1 in Lo s e al. (2012)). The bio- s imula ed soil exhibi ed a o al concen a ion o 221.6 mg kg 1 (a e age) o he 16 EPA p io i y PAHs and he quan i ied compounds a e lis ed in Table 1. The deg ada ion a e o naph halene in N was oo low and was di icul o be es ablished owing o he long ime anscu ed in he p esence o he con aminan s; howe e , once submi ed o bio- s imula ion, we calcula ed he deg ada ion a e o naph halene in soil om ime 0 o 231 days: 1.818 mg kg 1 soil pe day. Dena u ing g adien gel elec opho esis was used o su ey he de elopmen o he mic obial commu- ni ies in he en ichmen cul u es and o deduce he ime poin a which a s able communi y was ob ained. We obse ed ha he dena u ing g adien gel elec opho esis p o iles o CN1 en ichmen cul u es changed be ween he 45 and 60 ans e s (Supplemen a y Figu e S3); changes in he p o iles we e also obse ed o CN2 a e 35 and 60 ans e s. These changes we e subsequen ly main ained and samples subjec ed o a leas 60 ans e s we e he ein used o u he in es iga ions. The majo bands we e excised and sequenced, and hei a ilia ion e ealed he p esence o mic oo ganisms iden i ied as membe s o he gene a Ach omobac e , Fla obac e ium and Acido o ax in bo h en ich- men s, and Pseudomonas,Mic obac e ium,Lysobac- e and o an endosymbion o Acan homaeba in CN2. Resul s indica ed ha conso ia CN1 and CN2 we e able o deg ade 97% naph halene a an ini ial concen a ion o 1000 p.p.m. wi hin 72 and 80 h, espec i ely. Bac e ial di e si y and composi ion bluep in s DNA isola ed om each in es iga ed mic obial communi y was employed o a PCR-based 16S ecombinan DNA ( DNA) gene di e si y su ey o he communi y s uc u es in he samples. Fo his pu pose, clone lib a ies we e cons uc ed as Table 1 Le el o 16 EPA p io i y PAHs p esen in he o iginal (N) and bio-s imula ed (Nbs) soils PAH Concen a ion (mg kg 1 ) Soil N Soil Nbs Naph halene 607 174 Acenaph hylene 1.60 0.40 Acenaph hene 16.60 4.40 Fluo ene 20.80 5.96 An h acene 21.20 4.99 Phenan h ene 46.40 24.23 Fluo an hene 30.40 4.73 Py ene 19.40 4.02 Benzo(a)an h acene 12.00 2.83 C ysene 12.50 2.64 Benzo(b) luo an hene 4.40 1.34 Benzo(k) luo an hene 3.10 0.90 Benzo(a)py ene 4.90 1.27 Indene(1,2,3-cd)py ene 3.40 1.58 Benzo(g,h,i)pe ylene 1.50 0.82 Dibenzo(a,h)an h acene 0.79 0.18 Abb e ia ions: N, non-bio-s imula ed polya oma ic hyd oca bon- con amina ed soil; Nbs, bio-s imula ed polya oma ic hyd oca bon- con amina ed soil; PAH, polya oma ic hyd oca bon. Gas ch oma og aphy–mass spec ome y (GC–MS) was used o quan i ica ion. Mic obial esponse o polya oma ics M-E Guazza oni e al 124 The ISME Jou nal desc ibed in he Supplemen a y Ma e ials and me hods, and he clones we e ully sequenced o ob ain he almos comple e cloned 16S DNA gene sequence. Addi ionally, and because o he low yields in he clone lib a ies o Nbs, we used he 16S DNA gene pa ial sequences ob ained in he me agenome su ey. Fo his pu pose, we used only hose sequences ha had a leng h 4600 nucleo ides. All sho e sequences we e disca ded. A o al o 670 sequences ( ha is, N: 212; Nbs: 261 (86 clones þ175 454 pa ial sequences); CN1: 90; CN2: 107) we e ob ained, and analysed. The o e all phylogene ic composi ion in he lib a ies is shown in Figu es 1–3, in which all ope a ional phylogene ic uni s (OPUs) a ilia ed wi h wel e phyla o he domain Bac e ia, namely, P o eobac e ia ollowed o much lowe ex end by Bac e oide es,Ve ucomic obia,Fi mi- cu es,Ac inobac e ia,Chlo o lexi,Cyanobac e ia, Planc omyce es,Spi ochae es and he candida us phyla OP8, TM7 and WCHB1 (Figu e 4). The clones we e classi ied in o 124 bac e ial ope a ional phylogenic uni s (OPUs) (Lo ´pez-Lo ´pez e al., 2010) (Table 2 and Supplemen a y Figu e S2). Highe alues o Shannon–Wiene ’s and Good’s co e age indexes indica ed ha communi ies N and Nbs we e much mo e complex (51 OPUs and 53 OPUs we e de ec ed, espec i ely) han he CN1 and CN2 (13 OPUs and 12 OPUs, espec i ely) commu- ni ies, which we e domina ed by ew mic obial species o s ains and exhibi ed a a he simple s uc u e (Figu es 1–3). I should be ecalled ha each o he dis inc OPUs de ec ed was iden i ied as a pu a i e single species owing o he high sequence iden i y (Ya za e al., 2008). Howe e , i should also be no ed ha he sequencing su ey co e ed o e 75% o he expec ed OPU di e si y in all cases as i can be deduc ed om he high Good’s co e age in all samples (Table 2), and he a e ac ion cu es indica ing closeness o sa u a ion (Supplemen a y Figu e S4). A la ge p opo ion o he ob ained sequences a ilia ed wi h known amilies and clus e ed wi h b anches ep esen ed by cul u ed mic oo ganisms ha ha e p e iously been ound in con amina ed en i onmen s (Figu es 1–3 and Supplemen a y Figu es S5 and S6), and hus, hey do no appea o be speci ic o he soil and he en ichmen samples ha we e in es iga ed he e. Wha e e he case, despi e soil cha ac e is ics and speci ic pollu an s composi ion may di e , he biodi e si y ound in he o iginal soils (N and Figu e 1 A neighbou -joining ee o he p o eobac e ial 16S DNA gene sequences om he gene sequences o igina ed om N, Nbs, CN1 and CN2 communi ies. Fo he econs uc ion, only he almos comple e sequences o he clones om he ou samples had been used. Besides, pa ial 454 sequences o 4600 nucleo- ides we e inse ed in he ee using he Pa simony ool implemen ed in ARB. The numbe o sequences comp ised in each iden i y clus e (OPU) is speci ied o each sample. The colou code o he 16S DNA sequences is as ollows: N (g een), Nbs (pu ple), CN1 (blue), CN2 ( ed). Whe he an OPU con ains sequences o mo e han one sample, he OPU denomina ion is w i en in black ollowed by he code colou and sequence numbe o each s udied sample. Mic obial esponse o polya oma ics M-E Guazza oni e al 125 The ISME Jou nal Nbs) (Shannon indexes o 2.81 and 3.32, espec- i ely) a e in he ange o ha ound in o he PAH- con amina ed soils. Fo example, Shannon indexes o 1.74–2.78 (Vi as e al., 2008), 2.38–2.96 (Tha amani e al., 2012) and 4.87–5.01 (Ma in e al., 2012) we e obse ed in soils ha had been his o ically con amina ed wi h PAHs. The dominan g oup among he Gammap o eo- bac e ia consis ed o membe s o he genus Pseudo- monas (Figu es 2 and 4). The N sequences we e dis ibu ed a he e enly wi hin his genus, wi h app oxima ely 40% o he o al gammap o eo- bac e ial sequences b anching in he P. luo escens, P. ede iksbe gensis and P. amygdale lineages, ollowed by hose a ilia ed wi h he Pseudoxan ho- monas spp. (20%) and P. pu ida (14%) lineages (Supplemen a y Table S2). A simila e en dis ibu- ion was obse ed in he sample Nbs, being as well he membe s o Pseudomonadaceae he mos abun- dan . Howe e , in his case, he Pseudomonas species composi ion shi ed owa ds he majo ep esen a i es P. uomou ensis,P. pe ucinogena, P. s u ze i genomo a 1, and ye undesc ibed Pseudomonas species. These esul s clea ly con as wi h he dis ibu ion o he CN2 sequences, whe e jus ep esen a i es o P. s u ze i (46%) lineages (Supplemen a y Table S2) we e obse ed in acco - dance wi h his being one o he majo ep esen ed lineages in Nbs. Finally, in CN1, no ep esen a ion o he Pseudomonas genus was ound, bu six sequences ep esen ing Pseudoxan homonas we e iden i ied (6% o he o al) (Figu es 1 and 4 and Supplemen a y Table S2); his is in ag eemen wi h p e ious obse a ions made in PAH mic ocosm expe imen s ha sugges ha , al hough, Pseudomonas spp. can be easily en iched and use a a ie y o o ganic subs a es, hey may be ep essed by o he P o eobac e ia (Rhee e al., 2004). The alphap o eobac e ial sequences de ec ed in N we e widely dis ibu ed among Rhizobium, De osia,No osphingobium,Sphingomonas and B e undimonas, which we e he mos ep esen ed ;;)1VG1UPO (Pseudomonas s u ze i 1N 12Nsb 48CN2 sbN3)(2UPO Pseudomonas alcaligenes Pseudomonas o i idis, AY953147 ;)3VG(3UPO Pseudomonas s u ze i 6N 1Nsb ,).ps(4UPO Pseudomonas s u ze i 8Nbs 1CN2 ;)C41M(5UPO Pseudomonas s u ze i 5N 10Nbs Nbs132 (OPU 96) bsN12)(6UPO Pseudomonas uomue ensis Pseudomonas mendocina, Z76674 N-179 (OPU 7) Pseudomonas a gen inensis, AY691188 Pseudomonas la escens, U01916 Nbs117 (OPU 97) sbN8)(8UPO Pseudomonas pe ucinogena OPU 9 ( sp.) 20NbsPseudomonas )(01UPO Pseudomonas guineae 1Nbs4N, Pseudomonas anguillisep ica, X99540 Pseudomonas bo bo i, AM114527 N56)(11UPO Pseudomonas amygdali N-193 (OPU 12) Pseudomonas sp. VET-8, EU781734 Pseudomonas b assicacea um, AF100321 Pseudomonas luo escens, AJ308308 Pseudomonas o ien alis, AF064457 N22)(31UPO Pseudomonas pu ida Pseudomonas japonica, AB126621 OPU 14 sp.) 3Nbs(Pseudomonas Azo obac e inelandii, AB175657 N-05 (OPU 15) Pseudomonas balea ica, U26418 uncul u ed bac e ium, GQ979955 OPU 16 ( sp.) 3NbsPseudomonas 10% N clones CN1(N) clones CN2 (Nbs) clones Nbs clones and 454 sequences Figu e 2 A subse o he ee shown in Figu e 1, whe e he genealogical composi ion o he membe s o he amily Pseudomonadaceae is shown. Layou , econs uc ion and colou codes used a e he same as in Figu e 1. Mic obial esponse o polya oma ics M-E Guazza oni e al 126 The ISME Jou nal gene a (Figu e 1 and Supplemen a y Table S2). On he o he hand, membe s o his lineage we e ba ely ep esen ed in Nbs wi h jus se en sequences, being B e undimonas he mos ep esen ed. None o hese gene a we e de ec ed in any o he en ichmen cul u es (Figu e 1). In con as , mos o he alpha- p o eobac e ial clones in CN1 (73%) we e a ilia ed wi h Azospi illum species, ha is, Azospi illum o yzae, whe eas only h ee alphap o eobac e ial sequences (ou o a o al o 108) we e ound in CN2, and only one was a ilia ed wi h Azospi illum (Figu e 5 and Supplemen a y Table S2). Finally, he wo N clones (ou o 214) ep esen ing Be ap o eobac e ia a ilia ed wi h Acido o ax and Ach omobac e spp. (Supplemen a y Table S2). On he o he hand, Nbs sequences we e impo an ly ep esen ed in his lineage wi h 27 sequences (10%), a ilia ed wi h Te a hiobac e and Acido o ax.In his ega d, he pe cen age o Be ap o eobac e ia ound in he en ichmen cul u es was much highe (Figu es 1 and 4); we obse ed an o e ep esen- a ion o sequences a ilia ed wi h he gene a Comamonas and Ach omobac e in CN1 and Ach omobac e in CN2 (Figu es 1 and 5). In addi- ion o Del a-andEpsilon-p o eobac e ia, membe s o Tene icu es, Ve ucomic obia, Fi micu es and Cyanobac e ia we e only de ec ed in samples N and Nbs (Figu es 3 and 4). The abo e da a demons a ed ha he CN1 and CN2 communi ies displayed conside ably di e en phylogene ic s uc u es (Figu es 4 and 5), sha ing only wo OPUs: Be a- and Gamma-p o eobac e ia ep esen a i es o he deni i ying Ach omobac e ( ha is, Ach omobac e spanius) and Azospi illum ( ha is, Azospi illum doebe eine ae) gene a, espec- i ely. Whe eas he i s one was mos abundan in CN2 (29 s 7 sequences), he second was pa icu- la ly en iched in CN1 (35 s 1 sequences) (Supplemen a y Table S2). In addi ion o supplying biosu ac an s, he addi ion o di e en ni a e compounds may ha e s imula ed he g ow h o deni i ying Ach omobac e spp. and P. s u ze i (Song and Wa d, 2003) in he bio-s imula ed com- muni y, whe eas deple ion o ni a e may ha e a ou ed he p esence o he ni ogen- ixing Azos- pi illum species in he non-s imula ed communi y. In addi ion o deni i ica ion, P. s u ze i is well known as naph halene deg ade , which has been isola ed in such hyd oca bon en ichmen s om a wide ange o en i onmen al samples (Rossello ´- Mo a e al., 1994), and was al eady an impo an componen o he o iginal Nbs soil. Howe e , he possibili y ha he selec i e deso p ion and libe a- ion o ecalci an compounds om soils du ing he bio-s imula ion p ocess may ha e key unc ions in shaping he genomes and communi y s uc u es o he associa ed mic oo ganisms should no be uled ou . The indings o p e ious s udies using BTEX biodeg ada ion co-cul u es (Shim e al., 2005) ag ee wi h his hypo hesis. In any case, al hough bac e ia ela ed o Pseudomonas, Ach omobac e Figu e 3 A neighbou -joining ee o non-p o eobac e ial 16S DNA gene sequences om he clone lib a ies es ablished om N, Nbs, CN1 and CN2 communi y DNA. Layou , econs uc ion and colou codes used a e he same as in Figu e 1. Mic obial esponse o polya oma ics M-E Guazza oni e al 127 The ISME Jou nal and Comamonas spp. ha e been widely associa ed wi h PAH deg ada ion in a numbe o con amina ed ecosys ems and mic obial conso ia (Goyal and Zyls a, 1997), he p esence o he Azospi illum species in con amina ed soils and PAH-deg ading bac e ial conso ia has only p e iously been epo ed in a hea ily c eoso e-con amina ed soil (Vin ˜as e al., 2005). This is o special in e es because i is known ha he ecen ly sequenced Azospi illum sp. B510 ha bou s gene se s o deg ada ion pa hways, hough hei unc ions ha e no ye been expe imen ally elucida ed (Kaneko e al., 2010). Due o he clea shi s in communi y composi ion de ec ed among he in es iga ed condi ions, i is impo an o es ablish he oles and deg ada ion capabili ies o he indi idual mic obial membe s o he communi ies o unde s and he o e all unc ion o each communi y and i s membe s. Genomic signa u es associa ed wi h he ae obic deg ada ion o a oma ics DNA isola ed om each mic obial communi y was sequenced using a Roche GS FLX DNA sequence , a Li esequencing SL (Valencia, Spain) which p o- duced 1 471 821 eads (N: 165 791; Nbs: 438 821; CN1: 448 713; CN2: 418 293) wi h an a e age leng h pe ead o 395 bp o CN1 and CN2, 336 bp o N and 531 o Nbs. Acco dingly, a o al o 55.7, 233.3, 177.2 and 165.2 Mbp o aw DNA sequences o N, Nbs, CN1 and CN2 we e ob ained, which we e assembled in o 2.6 Mbp (4335 con igs), 17.9 Mbp (16 032 con igs), 20.0 Mbp (20 809 con igs) and 13.0 Mbp (9915 con igs), espec i ely. CN1 (266) and CN2 (256) con ained a highe numbe o con igs wi h leng hs g ea e han 10 kbp compa ed wi h Nbs (68) and o majo ex en N (only 1); his may be due Figu e 4 Compa ison o bac e ial phylo ypes (cu -o o 497% sequence iden i y) based on he 16 S DNA sequences ex ac ed om N, Nbs, CN1 and CN2 communi y DNA. The pe cen ages o bac e ial phylogene ic lineages de ec ed we e based on OPUs. (a) Pe cen age o 16S DNA sequences dis ibu ed by phyla. (b) Con ibu ion o dominan g oups among Gammap o eobac e ia.(c) Rela i e dis ibu ion o majo bac e ial phylo ypes (based on 16S DNA gene sequences (OPUs)) o soil communi ies CN1 and CN2 a he genus le el. Table 2 S a is ical indexes o he ou samples N Nbs CN1 CN2 Taxa (OPUs) 51 53 13 11 Indi iduals (sequences) 212 261 90 107 Dominance_D 0.13 0.06 0.20 0.30 Shannon_H 2.81 3.32 2.02 1.50 Good’s_G 0.76 0.80 0.86 0.90 Abb e ia ions: CN1, naph halene-en iched communi y de i ed om N soil; CN2, naph halene-en iched communi y de i ed om Nbs soil; OPU, ope a ional phylogene ic uni ; N, non-bio-s imula ed polya oma ic hyd oca bon-con amina ed soil; Nbs, bio-s imula ed polya oma ic hyd oca bon-con amina ed soil. PAST so wa e 1.82b (Hamme e al., 2001) was used o calcula e he s a is ical indices o he bac e ial sequences. The ollowing o mulas we e used. Shannon–Weine index: H ¼S(ni/n ) Ln (ni/n ), whe e ni is he numbe o sequences o a pa icula OTU and n is he o al numbe o sequences. Dominance_D: D ¼S(ni/n )2. Good’s co e age: C¼1(ni/n ), whe e ni is he numbe o OTUs obse ed exac ly once and n is he o al numbe o sequences. Mic obial esponse o polya oma ics M-E Guazza oni e al 128 The ISME Jou nal o he high di e si y o he N sample, which esul ed in a la ge pool o eads ha could no be assembled (Supplemen a y Figu e S7). Fu he in o ma ion ega ding he ob ained me agenome cha ac e is ics is gi en in Supplemen a y Table S3. Wi h a h esh- old o highe han 95% iden i y and an aligned leng h o mo e han 150 bp, 91.7% ( o N), 95.2% ( o Nbs), 92.3% ( o CN1) and 89.9% ( o CN2) o he p edic ed genes we e assigned o pa icula gene a, he analysis o which showed esul s compa able o hose ound in he 16S DNA assign- men s (see Figu e 5 o CN1 and CN2 compa ison). App oxima ely 89% o he p edic ed genes (63 974 in o al) we e assigned o COG p o ein amilies, and 65% we e assigned o KEGG pa hways (Supplemen a y Table S3). Among he 1538 ( o N), 2751 ( o Nbs), 2775 ( o CN1) and 2507 ( o CN2) KEGG o hologs de ec ed, 891 we e sha ed be ween he wo en ichmen cul u es, and 66% o hese we e pu a i ely a ibu ed o Ach omobac e species. We i s calcula ed he o e ep esen a ion o unc ions be ween me agenomes; o ha , we applied a z- es o independen p opo ions p o- posed by Li (2009) o s a is ically analyse he changes o he unc ional ca ego ies be ween sam- ples. I should be no iced ha he compa ison was es ic ed o samples Nbs, CN1 and CN2 because he lowe numbe o py o sequences and COGs in sample N may ha e impac on he compa a i e esul . We ound a a he s able dis ibu ion o he unc ional ca ego ies composi ion be ween he sam- ples wi h signi ican di e en con ibu ion o only 238 ou o 3293 COGs (Supplemen a y Table S4). Among hem, 56/23 did show a signi ican inc ease in he bio-s imula ed soil as compa ed wi h he CN1/CN2 en ichmen s, 58/26 inc eased in CN1 as compa ed wi h Nbs/CN2 and 41/55 we e o e - ep esen ed in CN2 as compa ed wi h Nbs/CN1. O e all, we obse ed ha bo h bio-s imula ed com- muni ies we e pa icula ly en iched in COGs ela ed o ‘Replica ion and epai ’ and ‘T ansla ion’ (29 s 4 dis inc COGs in o al in CN1). O e ep esen a ion o such unc ions is ypical o mic obial commu- ni ies de eloped unde e y dynamic en i onmen al condi ions, such as bio-s imula ion; he addi ion o di e en ni a e compounds may ha e s imula ed a compe i ion be ween as -g owing o ganisms and o ganisms capable o me abolising poly-a oma ics. By con as , i is no ewo hy ha COGs ela ed o ‘T ansc ip ion’ we e mos likely cha ac e is ics o en ichmen cul u es (13 and 7 COG en iched in CN1 and CN2, espec i ely), whe eas only wo (COG0789 and COG1974) we e en iched in Nbs (Supplemen a y Table S4). This sugges s a plausible scena io in which he s ess caused by a oma ic compounds du ing he naph halene cul i a ion led o an en ichmen o genomes con aining ansc ip- ional elemen s ha could be equi ed o s ess endu ance (Domı ´nguez-Cue as e al., 2006) and/o ac i a ion o unc ions equi ed o subs a e up ake. Ten dis inc COGs wi hin he ‘Cell wall, memb ane, en elop biogenesis’ ca ego y we e ound o e ep e- sen ed in CN1, a numbe much highe ha hose ound in Nbs ( ou COGs) and in CN2 ( wo COGs) (Supplemen a y Table S4). In addi ion, CN1 was also pa icula ly en iched in ‘ABC T anspo Sys- ems’ by meaning o genes dis ibu ed in 11 dis inc o e ep esen ed COGs, o which only h ee and wo we e ound in Nbs and CN2, espec i ely. COG0318 ace yl-CoA syn he hase was also associa ed o CN1 communi y; enzymes o he COG0318 ca alyse he o ma ion o ace yl-CoA om ace a e, sugges ing ha ace a e may be a majo end p oduc used o p oduce ace yl-CoA eeding he K ebs pa hway in membe s o CN1 (in ag eemen wi h p o eomic da a ha will be discussed below). By con as , CN2 was pa icula ly en iched in genes coding p o eins Figu e 5 Rela i e abundances and dis ibu ions o gene coding sequences in he CN1 and CN2 communi ies based on axonomic bins o bac e ial 16S DNA gene sequences (OPUs) (blue), axonomic bins o me agenome-de i ed genes encoding p o eins ha could be assigned a axonomic anno a ion ( ed) and axonomic ca ego ies o p o eins ha we e iden i ied in he me ap o eomes (g een). As shown, he me agenome and he 16S DNA analyses yielded cong uen esul s. Fu he , only a mino numbe o bac e ial membe s seemed o be me abolically ac i e in bo h communi ies, by meaning o exp essed p o eins binned o hem. Mic obial esponse o polya oma ics M-E Guazza oni e al 129 The ISME Jou nal con aining Fe–S clus e s, namely COG3210 (la ge exo-p o eins in ol ed in haem u ilisa ion o adhesion), COG1049 (aconi ase B), COG0348 (poly e edoxin) as well as COG4773 (ou e memb ane ecep o o e ic cop ogen and e ic- hodo o ulic acid) (Supplemen a y Table S4), indica ing i on being pa icula ly ele an o CN2 communi y membe s. Taken oge he , he mos no able obse a ions d awn om all hese da a se s a e ha bio-s imula ed communi ies appea o be ex ensi e ese oi s o genes in ol ed in eplica- ion, epai and ansla ion and i on adhesion and u ilisa ion whe eas bac e ial componen s o non-s imula ed communi ies seems o be mos ac i e in cell wall, memb ane and en elop biogen- esis; in addi ion, en ichmen mos likely a ou ed ansc ip ional e en s. The ob ained co e age was su icien o p oduce a u he pa ial heo e ical me abolic econs uc ion (including bo h exclusi e and common capabili ies) o he ae obic a oma ic ca abolic ou es in he in es iga ed communi ies; al hough hese econs uc ions we e incomple e and likely ep esen composi e cell ne wo ks, he in o ma ion ob ained may be su i- cien o achie e a be e unde s anding o how he ou communi ies beha e ega ding hei biodeg a- da ion capabili ies on a genomic scale. To do ha , a p ope unc ional assignmen o he p edic ed genes was pe o med using an in-house da abase con ain- ing p o ein sequences wi h biochemical unc ions shown o be in ol ed in biodeg ada ion ( o de ails see Supplemen a y Ma e ials and me hods). Acco d- ing o his p o ocol, 428 (N: 38; Nbs: 115; CN1: 132; CN2: 143) open- eading- ame agmen s we e iden- i ied as showing close sequence simila i y o genes ha encode enzymes known o be in ol ed in he ae obic me abolism o a oma ics ia di- and i- hyd oxyla ed in e media es and, acco dingly, p e- sump i e unc ions we e assigned. The o e all gene ea u es and p esump i e ca abolic ou es mos likely associa ed o hem, a e shown in Figu e 6 Po en ial ae obic deg ada ion ne wo ks o a oma ics ia di- and i-hyd oxyla ed in e media es in he ou in es iga ed communi ies. The colou code used o he espec i e pa hways is as ollows: black, all samples; g een, N and Nbs (speci ic o soil communi ies); blue, CN1 (N) (speci ic o non bio-s imula ed communi ies]; ed, CN2 (Nbs) (speci ic o bio-s imula ed communi ies). Illus a ions we e c ea ed by ChemD aw g aphic p og amme Chem D aw Ul a 8.0 (Camb idgeSo ; h p://www.camb idgeso .com/) based on subs a e speci ici y o enzymes lis ed in Supplemen a y Table S5, and he co esponding me abolic pa hways we e es ablished based on bibliog aphic eco ds. I should be no iced ha he iden i ica ion o a pa icula ac i i y in CN1 and CN2 implies i s p esence in N and Nbs, espec i ely, al hough hei signa u es we e no iden i ied in he la e mos likely owing o hei highe biodi e si y and low co e age o he me agenome. Acco dingly, hose ac i i ies we e indica ed as CN1 (N) and CN2 (Nbs). Mic obial esponse o polya oma ics M-E Guazza oni e al 130 The ISME Jou nal