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Characterzaton of novel antibotc resistance genesdentifed by functonaI metagenomcs on sol samplesGloria Torres-Corts,r Vcanta Milldn,lHu go C. Ramfrez-Saad,2 Raf ael N lsa-Martinez,lNcolis Torol and Francsco Martinez-Abarca1*1Genetic Ecology Group, Estacin Expermental delZaidn, consejo Supeior de lnvestgaciones Centficas,Cale Profesor Albareda 1, 18008 Granada' Spain.2Laboratorb de Ecologia Molecular, Deparamento deSisfemas Biolgicos, Universidad AutnomaMetrapoltana _ Xachimlco, 0496a M6xco.SummaryThe soil microbal communi is highly complex andcontans a high densty of antibiotc-producing bac-teria, making it a likely source of diverse antiboticresstance determnants. We used unctional metage-nomis to seareh ,or antbiotc resstance genes inlibrares generated from three different soil sample,contaning s.5 Gb of DHA in total. we identfied 11ne$, antbiotc rgsstance genes: 3 conferring resis-tanc to ampcillin, 2 to gentamicin, 2 to choram-phenicol and 4 to trimethoprim. ona of the clondentfed UYas a ngw trimethoprim resistance geneencoding a 26.8 kDa protin closely resembling un-assigned reductases o he dihydroolate reductasegroup. Ths protein, Tn&3, conerred trmethoprimresistance in Eschericha col and Snorhzobiummelilot {a and a-proteobacteria respectvely). Wedemonsrated that this gene encoded an enzyme withdihydroolate reductase actvity' wth knetc con-stants similar to other type and ll dihydrofolatereductases {K, of 8.9 M for NADPH and 3.7 pM fordihydrofolate and lCsg of 20 pM for trimethoprm). Thisis the first description o a new type of reductaseconerring ress_ance to trimethoprm. our reultsindicate tat ol bactera display a high level ofgenetc diversi and are a resgrvoir o antibiotcresstance genes' supporting the use ol ths approachfor the dscovery of novel enzymes wilh unex-pected activities unpredictable from their amino acidseguences.Received 27 october,20]o; accepted t5 Decembe 20'l0."For conespondence. E-mail fmabarca@eez.csic.es; Tet. (+34)9581816O0; Fax (+34) 958129600.

Environmental Microbiology {201 1 ) doi: 1 0. 1 1 1 t /j. 1 462-2920.201 O -824?2x

lntroductionThe diversity of the microbes hidden in soil (up to 10'gmicroorganisms/g; Trevors, 2010) has been exploredwith a view o developing new clinical and rnedical appli-cations {Daniel, 2004; Da Costa et aL, 2007; Molinari,2o09). The most signiicant application to date hasundoubtedly been the imp|ementaion of naural anti-biotic products, uhich has revolutionized our approachto treating inectious diseases. More than 80% of theantibiotics in clinical use originate from soil bacteria,either directly, as natural products, or as heir semi_synthetic derivatives (Martin and Liras, 1989; Kieseretal.,1AOO\. Thus, soil may serve as a hidden reservoirfor antibiotic resistance that has already emerged or hasthe potential to emerge in clinically important bacteria(Riesenfeld etal., 2ao4; Da Aosta eaL, 2o06; Allenetal., 2oo9; Donato eal, 2010}. Consequently, anunderstanding of resislance deerminants present in soilwil provide information not only abo antibiotic resis-tance requencies, but also about novel mechanismsthat may emerge as clinical problems and about the roleof antibiotic resitance genes in natural environrnents(Wright, 2007).The success of antibiotics for treating inections and,conversely, the risk to human health posed by antibioticresitance have focused research principally on the clini-cal setting (Aminov, 2009; Martinez, 2009). By contrast,the unction of antibiotics in naural (non-clinical) environ-ments has received relalivey little atention. Do antibioticsand antibiotic resistance genes play the same role innon-clinical environments? This may be the case in someinstances, but lhere is compelling edene o indicaevery different roles in mediating the interactions o micro-bial communilies in natural environrnents (Fajardo eaL,2009; Martinez et al,,2OO9\.ln recent years, metagenomic tools have identified anti-biotic resistance genes in DNA libraries from environ-rnental samples {Riesenfed etal.' 2oa4i Allen ea.'2o09; Mori et a.,2ao8; Kazimierczak etaL' 20o9; Donatoe ar., 2o10). The analysis of metagenomic clones is oftenbased on random sequencing (Venter e aI', 2o04; Tringee ar., 2005) or the PCR ampliication o target genes (Dela Torre et al., 2Oo3:' Henriques e aL, 2006; Demaneche

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2 G' Torres-cort6s el a'et aL' 2oo8|. Aftematively, unctional metagenornics'which involves the heterologous expression of metage-nomic DNA in a surrogate host and activity-based screen-ing, provides a means of discovering genes, the functionof whih may not be obvious from their sequence. Theterm 'antibiotic resisome'was proposed for the collectionof all antibiotic resistance genes in microorganisms,including those from pathogenic and non-pathogenic bac-tera. This erm explots the concept o a unigue reservoirof antibiotic resistance genes in environmental samples(Wright,2007).ln this study, we aimed to discover new antibiotic resis-tance genes, using rnetagenomic libraries constructedfrom DNA extracted rom three different soils, followed byheterologous expression and the screening of resistanceactivity against different antibiotics. We hypothesized hatgenes conferring resistance to antibiotics are presenin the environmenl, even in the absence o strong selec_ion pressure that would be associated wih high anti_biotic conentrations. The idenlfication of new resistancegenes, patcularly hose potentially present in non_cutfurable microbes, may faciliate prediction of the emer-gence of resistance.We conslructed libraries rom fragments of clonedsoil DNA and selected clones expressing resistance tovarious antibiotics targeting different bacterial functions.We identified genes encoding proteins that inactivatedthe antibotic (p-lacamases for ampicilin, Ap, and acetyl-transerases for gentamicin, Gm), genes encoding multidrug pumps conferring resistant to chloramphenicol (Cm)and genes conferring resistance to the folate antagonisttrirnethoprim {Tmp).Trimethoprim eompelively inhibits the enzyme dihy-drofolate reduclae {DHFR)' which is reonsible forreducing dihydrofolate to tetrahydroolate (HuovineneaL, '1995; skd' 2001). Cells that cannot regeneraetetrahydroolae suffer defective DNA synthesis, eventu_ally leading to death. Acquired resistance o Tmp includesmutations in the promoter region or in he DHFR structuragene (dff; Huovinen etaL, 1995; Skld etal.,2oo1), butthe most common mechanism of Tmp resistanc is thecomplementation of a Tmp_sensitive DHFR wth a Tmpresistance_mediating DHFR. ln lhis sudy, we isolateda new Tmp-resstanl gene encoding a 26.8 kDa proteinclosey resembling reductases not assigned la dhfrgenes. This protein, Tm8-3, conlers Tmp resistance toEscherchia coli and inorhzabium melioti- We demon_strate that thiE gene encodes a bona fde DHFR enzyme.This sudy thus prodes an additional exarnple of the useof functional metagenomics for the identificaon of resis_tance ges encoding protns vh activities that cannotbe predic{ed from their amino acid sequences. This mayfacilitate prediction of the emergence of new resistancedeterminants.

ResultsConstrucion of metagsnomc libraries and isolation ofcones expressn g antbioc resstanceThree metagenomic libraries were constructed with DNAextrac{ed fmm differen ols: one from an agricuttural soilclose to our research centre in Granada, Spain {the EEZsample) and two from Mexican soils in the vicini ofMammilaia camea plants from the Tehuacan-Cuicatlanbiosphere collected at the end o the rainy and dryseasons and named R-Mex and D-Mex respectively-Using he lambdaZAP_expressing phagemid sysem, wobtained about 550 000 recombinant clones only about'll1o o which conesponded o the EEZ library. Restrictionanalysis of 1O randomly selected clones from each libraryshowed the mean inset size to be 6.5-7 kb. About 3.5 Gbo enronmental DNA was cloned in these libraries.Clones were selected from the libraries on the basis otheir ability to grow in the presence o each o six antibi-otics: Ap, Gm, streptornycin, Cm, erythromycin and Tmp-We obtained no streptomycin-resistant clone and a largenumber o erythromycin-resistant c{ones, most of whiehrvere found to be alse positives in a secondary screeningtes't. We idenied 11 clones containing metagenomicDNA conferring resistance lo four of the six antibioticsused in E coli (Fig. 1; Table 1).We rneasured the minimum inhibitory concentrations(MlCs; Table 1} o the antibiotics used for selection of the1] resislance dones- An analysis othe MlC values forthehre cones resistant to Amp showed tha pBKAp6-8 fromthe D-Mex library had an MlC twce that of pBKApE1 andpBKApE't4 from the EEZ library. A similar situationwas observed for the two clones resistant to the aminogly-coside Gm, with the MlC value o pBKGm8_S from theR_Mex library being 2-5 times higher han that ofpBKGmEl from the EEZ library. This pattem was evenmore marked for clones conerring Tmp resistance, withthe MIC value of pBKTmS-3 (R-Mex library) being 3.0times higher than the three clones obtained from the EEZlibrary (pBKTmEl, pBKTmES and pBKTmEZ5; Table 1).ln general, the Glones obtaned rom the Mexican soillibraries conferred resistance at hgher MlC values in Ecof han did thqe obtained from the EEZ soil. Atthoughthis feature can be due to position and orientation of theanbiotic-resistant open reading frame (oRF) respect tothe Lac promoter (Fig. 1)dentification of antbiotic resistance genes andphylogenetic analysesThe size, gene organization and genetic similaries o einserts are summarized in Fig, 1 and Table 1. The insertsof resistant clones were between 4 and 10 kb in size. Fordentification of the oRFs responsible for the resstance

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Nove antibiotic rsance genes from soil melagenome 3

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kbFig. 1, Schemac diagan of the organizaion of the oRFs rom the 11 inserts conferring anbiotic resistance in the metagenomic soilibaies. The size o the inserts is indicated, to scale, as a dashed line, and the sequence deposited in the daabase is indicated as a soidline. oRF are epresened by arrows, which are shaded in grey for oRFs involved in anbioiic resisane. Asterisks indicate incompleeoRFs. The percenlage similar to database entries (where appropria) is shown in brackets below the oRFs.

TmE2dh{479} {0rentationof lapomoter

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Novel antiboic ressance genes fmm soi metagename 5phenotype' clones were deleted with restricton enzymsand tested for maintenance o{ the reistance phenotype inE eol. The sequences obtained from 11 asmids showed31 complete and 10 incomplee hoteroogous oRFs. TheG+C ontent of the sequenced insers vaiedrom 52.7a/oto 66.6%' consisent with their diverse origins. The onlineprogram PhyloPythia (McHardy e al.' 2007| was used toidenly the possible oigin o these metagomic DNAfragments. lt showed a predomnance of the phy|um Po-teobacteia in he clones analysed (10 of the 'll clone;Table 1). The subphylum u-Proteobace,1b was the mostfrequently represented among the isoaled clones four ofhe 1't).The putative genes responsible or the resistance phe-notypes were identified by sequencng of the fuIl_lengthinsert for the pBKGmS-3 and pBKGmE-E clones, whichconfer resistance to Gm and Cm, respectively, in E. nli.We sequenced only derivaye inserts that wer smallerthan the oigind plasmid for which the resigtance pheno-type \/as maintained, for the othe nine clones {Fig. 1}.Thus, ORFs encoding B-lactamases were found in thelhree cones conferring resi*ance toAp on the E colihost(Fg- 1 ' Table 1). These esults sggest that the cnferredresistance is due to inactivation o the Ap by disrupticn ofthe p-actarn ring. Clustering analysis of the amino ackisequence grouped the ApSB and ApEl proteins withclass A p-lactamase$. By contrast, the ApE14 proein wasfound to be reated to class C ftlactamases. The con-served residues constituting e our elements surround-ing the active ste were present in all three clones (Ambler,198o; Fig 51 and S2). The sequences o the three ORFsdiffered both from each other and from their best match{57oh,45o/" and 63% amino aeid dtty or Ap6-s, ApEIand ApE14 respec{ively; Tabe 1}.Phylogenec analysis revealed that the clones rom theEEZ library ApEl and ApE14, were c{osely related o thechmmosomal p-lactamases o Phenylobadorium andRhizobum respectively. However, the clone found in theD-Mex (Ap6-8) library belonged to a ganus from a verydifferent ader Solibaer (Table 1 }.Two clones conferred resistance o Gm in E. nh. onefrom the EEZ library (pBKGmEI) and the other from theR-Mex ibrary (pBKGms3}. Both lones contained oRFslikely o encode enzymes confening resisance to ami-nogycosides by 3N-acetylaion (Fig. 'l and Table 1). ThepBKGm8.3 clone conferring Gm restance also conainsputative genes related to elurite and bleomycin resis_tance (Fig. 1 and Table 1). Bo aminoglycoside 3,_N_acetyl tran$ferase contain the'GNAT' motil charateristicof GCN5-related nzymes (Dyda etaL, 2000; Azucenaand Mobashery 2001). Howeve amino acids involved inthe transfer o the acetyl group lrom actyl_conzymo Atothe primary amine of the aminoglycoside vree idertifiedonly in Gm8-3 (Fig. S ). Gm8-3 is related to an AAC{3)

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6 G. Torres-Cortds e al.cluster consisting o lhe six AAC (3)- proeins (Fig. Sa;Riccio ef a/., 2003; Ahrned et al.,2OA4r. GmEl was foundto be the most similar {69% amino acid ideni) to a31N-acetyltransferase conerring resistance to the ami-noglycoside kanamycin and identified by a rnetagenomicapproach to the analysis of soil samples flable 1; Cour-tois eal., 2003). GmE1 was found to be related to threeAAC(3)-| enzymes' alt o{ which have idential resistanceprofiles and the genes of which have been cloned fromPseudomanas spp. (Shaw ef a/., 1993). lt was also oundto be more distantly related to a ciuster o four AAC{3)enzymes encoded by genes from actinomycetes. Phylo-genetic analysis suggesed that GmE1 may be a newAAC(3) lll cluster gene {Fig. S4).on|y he EEZ library conained clones conferring resis-tante o Cm (pBKCmE1 and pBKCmE6}. The 0RF2 otboth clones was simlar to proteins of the Bcr/CflA subfam-ily of drug transporters (Fig. 1 and Table 1). Phylogeneticanalysis showed that these proteins, mE1 and CmE6,were very similar to each other (65% amino acid identity)and to other drug transporter present in described soilproteobaceria (57% and 630/" amino acid identity forCME6 and CMEI respectively; Fig. S5)^ CmEl and CmEGare located upsream from the gene encoding the pre-dicted multidrug efflux pump and have a transcriptionalregulator resembling the genome o Mesorhizobium loti{MAFF3O3O99 strain), which belongs to the subphylumg-Proteobacerla. The proteins o the Bcr/CflA family arepredicted lo have .12 membrane-spanning alpha-helixregions, and lhese regions are present in he CME6and cME1 proteins (F;g. S6i. Furthermore, one ohe bestmatches o both CmE1 and CmE6 was found to be an oRFencoding a multidrug ransporter (n access: AAs90611)ound in clone CR4 rom an unculturable soil bacterium andcon{erring resistance to tetracycline (Fig. 55; Riesenfeldet al.' 2o4l* Boh the pBKCME1 and pBKCMEG cloneswere teted and found to confer resistane o tetracyclinein E coli Dh5o cells (data not shown).By Conrast t0 previous reports {Donato e a/., 2o1o), weidentified no genes related to horizontal ransfer or mobil-ity, such as repeated elements, insertion sequences ortransposons, in any of the 41 ORFs analysed fi-able i).Resbance to rimethoprimpBKTmEl, pBKTmES and pBKTmE2S (found in EEZ soillibrary) and pBKTmS-S (found in R-Mex soil library) con-ferred resistance to Tmp in E. coli. They have inserts of4.5, 6.5, 6.8 and 7.5 kb respectively, The three clonesfound in EEZ soil contained oRFs resembling a ype l dhfr(Table 1 and Fig. 2). A gene encoding a subunit of the HflKprotease was ound downstream lrom the dfr genes.This gene is normally related to chromosome-encodeddlfr genes' Phylogenetic analysis showed TmE] to be

closely relaed to other DHFR proteins ound principally inthe bacteria of the order Rhizobiales and more distantlyrelated to DHFRs from p- and yproteobacteria. TmES andTmE25 are closely related proeins orming a separatecluser (Fig. 2). All three proteins conain conservedamino acid residues that interact with the folate andNADP groups of the active centre and variations of thesepositions involved in Trnp binding in the Tmp_resistanDHFRs o S- haemolyticus (Fig. 3A, Sekiguchi et al.,205}" TmE1, 5 and 25 were identified as chromosomaltype I dhfrgenes distantly related to the plasmid-encodedype ll DHFRs (Fig' 2}.The pBKTm8-3 clone conferring resstance to Tmp wasfound in the R-Mex library. By using restriction enzymes tomake derivatives of the original 7,5 kb clone, we wereab|e o restrict he DNA fragmen conferring he pheno-pe of resistance to Tmp to as little as 1.B kb of metage-nomic DNA {Fig. aA; pBKTm8-3X)" None of the predicedORFs within this '1.8 kb DNA fragment resembled a puta-tive DHFR-encoding gene. ORFI encodes a protein dis-playing 427" amina acid identity to some two-componentresponse regulators (Accession No.: YP_002545888.1)and ORF2 encodes a protein displaying 41% amino acididentity to an 3-oxoacyl_(acyl_carrier-protein) reducase{Accession No.l ZP_01470074^1). We generated afrarne-shift mutaion in each RF to deermine which of theseproteins was responsible for resistance (Fig. a). ORF2disruption resuted in sensitivity o Tmp. Furthermore, aplasrnid carrying only the coding sequence of ORF2under the control of a constitutive promoter conferredresistance to Tmp in E. coli and S. meloti hosl cells(Fig. aB). The gene was named Tm8-3.ldentficatian and bochemcal characteization o a newTm gene, Tm8-3, n a so metagenomic ibraryIrnS-3 corresponds to a749 nl ORF encoding a putative249-amino-acid protein. This protein confers resistance toTmp, but its sequence very diferent rom those of theDHFR proteins described to date {Fig. 2). lt displays simi-larity to other reductases involved in lipid rnetabolism{3-oxoacyl-(acyl-carrier-protein) reductase). A Rossmann-{old NAD(P)H/NAD(P)(+) binding domain found in numer-ous dehydrogenases and rnany other redox enzymes canbe identified in Tm8-3. Fuhermore, some specific con-served residues o the OAR proteins are also conservedi this protein (Fig. 3B}' Two o he three active_site resi-dues conserved in all bacterial and plant OARs are con-served in Tm8-3 (Y160 and K14), and the group of twoarginine residues playing a key role in ACP binding {Wick-ramasinghe eaL' 2006) is partially conserved in Tm8_3(R135 and H181; Fig.3B).For functiona analysis of the oRF2 o the pBKTm8_3gene, we overproduced and purified recombinant. After

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Nove antibotic resisance genes from so metagenome 7alexadr (zP oo952,+oo}Paruibacuum ammtioros tYP oo 41 371 5}

Burlcofur|3|Tbrednbater bmea@ rP oo3o72o2aMarire gmra proteobacterum EP 05096826}Burknlderin thandes (yP 4s5655}sp (zP 0166690)MarifE g orn proteobac'aim (zP o 6 58 1 }Mar]omorids |' Maribact |l|' oembaer {1|agg regata |zP 1 54a258|Sinorhmbm |2|' Rhzabm |1| DHFR type IhraeIa|2|alw]a 2|, Odzrobader |1|Me6hZnbium(2|

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Fig. 2. Neighbou-joining tree of aligned amino acid sequnces of th Tm esistancs genes 'ound in is study. The alignmer was peromedwih pe l and ll df genes. Tm8-3 and i closest relative, the oxo_acy carfer protein reductase (EAU70455), wele used for tree roong.Genera, species and strains ale followed by eir GenBank accession numbers' Some of the branches were grouped to improve the sibiityo the tree {the number of sequences grouped is indicated in parentheses). Boostap values fere cacuated as a percetage of 1oo0replicates and those above 7oolo are shown at the branching points. The sequences obtaioed in this stu are depiced in bold. see Table s4for arnino acid seguene accsion numbers. Scale bar = 0.2 changes/site.

1,,,.",.,,

loading the soluble crude extrac on a Mono Q column,we recovered 75"/" o the initial DHFR activity in a singlefracon in which the protein was 95% pure (Fig.58 lane3). SDS-PAGE of the crude extracl and this fractionsho'ed to be enriched in a 26'8 kDa protein. For identi-licaion of the principal activeform of Tm8_3, the g5% purefraction was subjected to gel filtration coumn chromatog_

raphy. Tvyo fractions with DHFR actvity were obtained {Dand M; Fig. 5), containing proteins with estimated molecu-lar weights o 6o and 28 kDa, respecVey, consistent withthe calculated expected molecuar weights of dimeric andmonomeric lorms repe0tivey. As most of the proten waseluted in fraction D, we can conclude that TrnS-3 is foundmostly as a dimer in its nave orm.@ 2011 Socie for Applied Microbiology and Blackwell Publishing Lld' Environmental Miclobioogy

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I G. Iorres-Corfds et al.A

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Fig. 3- A|ignrnents o' the trimehopm-resistant proteins anaysed in is study' Muliple-sequence aignrnents were Carried out with heClustalW online ioo (hp://aign.bmr.ushu_u.acjp/maffysoftware4 for protein sequences. The degree of conservaon o{ residues is indiatedby shading, running rom dark grey for the most strogly conseved residues o no shading {or non_conserved residues (clamp ear.' 2004}.A. Partia alignrnen and identification of conserved moifs lor type l dihydrofolate 'edudases and the TmE1, TmES and Tm25 proteins. Thefigure shows he amino acid residues interacling with the NADP cofacitor {closed triangles), with the folat substrate (closed circles) and wihtimethoprim {sta.s), based on studies of the E coriChromosoma DHFR (Howell, 2005; sekiguchi ef a/., 2o05).B' Parial alignment of the Tm8_3 Clone with othe 3'-oxoacyl-acyl canier protein reductases (oARs). Active-site residues conserved in albacterial and pant oARs ae indicated by open trianges. open cicles indicate he two arginine residues paying a key role in ACP binding{Wic*ramasinghe e a/.' 20o6). See Table S4 for amino acid sequence accession numbers'

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Some o e kinetic parameters of the purified Tm8-3are presented in Table 2. The Michaelis consiant (Km)Values o{ Tm8-3 for DHF and NADPH substraes were 3.7and 8.9 pM respectively. These values are similarto thosereported {or other type I and ll DHFRS. The 5O% inhibitoryconcenration of Tmp or Tm8-3 was 150 times higherthan that for chromosoma E coliDHFR {Sekiguchi ef a/.,2005), but only one-fifth that of for the S2DHFR describedin Staphylococcus haemaficus Oable 2).ln summary, our resus identify Tm8_3 as a previousyundescribed, new type o DHFR.Table 2. Enzyme kineics, inhibiion and Strucural properties o1purified recombinant DHFRS.

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DiscussionMetagenomic technology is a powerful too tha has beenappied to the discovery of novel nalura| produts andenzymes o biotechnological interest. ln this work, weidentified 11 c|ones conlerring resstance o various typesof anlibioics in E. coli, through the screening 0 lunctonalmetagenomic libraries. Eight of those clones were foundin the library othe distuted soil sample (EEZ), indicatinga major incidence of anthropogenic activity as the mainorigin of these resistant cones as has been describedin other soi samples (Nwosu' 2001; Aminov and Macke,2007; Yang efaL, 2o1o). The geetic determinansof antibiotic resistance within these clones displayed4143o/" amino acid dentty to known functionalproeins (Table 1)' These resistance genes encodedF-lactamases, aminoglycoside acetyltranslerases, multi-drug efflux pumps and DHFRs. one of he genesidentified, Tm8-3' belongs to a new group o antibioticresistance genes never betore described.As in a previous study (Donato ear., 2010}, wefound no streptomycin'resistanl clones in our functionascreens. The most likely explanation for this is thatsreptomycin-resistan genes are regulated by non-functional genetic eemens in the E coli host. onlythree of the 11 clones found ,ere closely relaed to resis_tance proteins identified in analogous metagenomic

S. pneumoniae chrDHFR {t)S. aureus S1DHFR()S. haemolyticusS2DHFR (r)E colichr DHFR (l)867 DHFR {il)Tm8-3

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a. Lee and co|eaglues {20'to}.b. Sekiguchi and colleagues (2005).c. Howl (2oo5).

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frffipBBSyn:Tmt-a-PBsyag. . Characteristics of the pBKTm8-3 timelrop'im_resistantclone.A. Schematic diagram of he organization of the oRFs in plasmidpBKTmS-3. Black bars indicate the extent of the pBKTmS-3 clone,pBKTm&3A and pBKTm8_3X derivative. Significan resrictionsies and their locations are indicated. Arrows indicate the locationsand e diections of transcription o{ the oRFs. The oRF involvedin tarnetroprim resistance s indicated by a grey anow.B. Drop assay of pBKTm&Sfi.S kb) and derivative plasmids on LBplates containing 20 mg Fr timetropim, carried out with seialdilutions (1 o 't0{) ol overnight cultures. pBKTm&SA (3 kb) andpBKTmS-3X (1.8 kb) are deletion-derived plasmids generated bydigestion with Asp718 and Xhol respectively. pBKTmS-SXE andpBKTm8-3XB are derivatves of pBKTm&3X in which oFtF'l orORF2 have been mutated by frameshift, with EcoRl or BamHlsites, respectively, filled in with the Klenow fragment of DNApolynerase |. We assessed the resistanc phenotype basedon Tm&3 {oRF2) in o-proteobacsria, such as S. meitot, byexpressing this ge under the conlrol of a cnstitutive promoter npBBCMs Vectos (Kovach et aI'' 1gg4; pBBSyn:Tm8_3). Emptyvector (pBBSyn) was used as a control. Each assay was performedat least three times, vrith independent cultures.

approaches. CmEl and CmE6, both o{ which confer Cmresislanc display 55% and 65% amino acid idenity,respectively, to tcaB of the CR4 clone identified asa tetracydine resitance gene in another soil library(Accession No.: AY566822; Rieserfeld ef ar., 2004). TheGm-resistant gene GrnEt encodes a protein with anamino acid seguence 69% identical to that of anotherkanamycin-resistant Glone aso isolated from a metage-nomic soil cosmid library (Coutois eaL,20o3).The Ap resistance genes identified in this study encodeB-lactamase enzymes. By contrast to previous studes, inwhich class B p_Iactamases were ound to be abundan inAlaska soils (Allen eal., 2009), the clones present in our

Nove anbioc resisance genes from sa metagenome 9sampes belonged to c|ass A (ApE1 and Ap6-8) and classC {ApE1a).Both clones with Gm resistance phenopes encodedN-acetyltransferase enzymes. Gm8-3 is related to heproleins of he y-proeobacteria AAC (3]-| family {Riccioe al.,2oo3; Ahmed et al.' 2ao4|' and GmE1 is related tothe AAC (3'}-lll amily, a}though it could also be assignedto a new subclass (Figs 53 and S4). The absence o otheraminoglycoside-modifying enzymes (such as phospho-transferases or adenyl lransferases) is consisent with thefindings o preous clinica studies that the ue o gen-amicin is correlated with the occurrence of AAc3 genes(Vakulenko and Mobashery, 2003). A the isolated clonesconfening Cm resistance were found to encode putativeextrusion pumps. We identified no Cm resistance genes

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Fig.5. Puricaon and characteizao of the Tm&3 protein.A. Results obtained by gel filtraon cdumn chomatography as afinal step in the purification of Tm8-3 exracs. The loading mateiawas the peak of DHFR ac,tivity obtained on Mono Q columnchromatography. Absoance at 28B nm (A28o) was moniloredcorinuously ding protein elion. The molecular masses of theprotein species were obtained by comparison with standards(indicated in kDa above the figure), using the same column andelution conditions' Anows indicate the lraons (D and M) in whichDHFR activi ,Vas detec{ed.B. SDS gel electrophoresis of sample obtained during Tm8_3purification. The gel contained 12% (w/v) polyacrylamide and warun under educing conditions, Lane l, mo|ecula mass standardsin kDa; lane 2, crude extract ane 3, concentrated fraction sampeafter MonoQ column chromatography; lane 4, concenrated fractionsample D conesponding to the Tm8-3 dime; lane 5, concentlatedfraction sampe M conesponding to he Tmsg monomer.

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1o G. Tones-Cots eal.encoding enzymes, such as Cm acetyltranserases. Thismay reflect poor represenlation of these types of resis-ance determinans in the study population or the limia_tions of the E colibased screening. The two isoatedclones confening Cm resistance were very similar toeach other and to tetracycline resisance genes. Bothclones conferred tetracycline resistance in E- coi (datanot shown).Trimethoprim resistance in bacteria has often beenassociated with changes in the sensitivity of DHFR actityin resislant isolates (Dale e al' 1 997). only two types odhfr genes have been described to dale (Howell, 2005):dhtr type l, generally presen on the bacterial chromo-some, and dhtr ype ll genes, he location of which isunknown (Howell' 2005). Three o e four identified Tmp-resislant clones (pBKTmE1, pBKTmES and pBKTmE2S)encoded a gene meeting the consensus criteria for a dhfrtype I gene. Houvever, the gene in clone pBKTmS-3 wasfound to encode a reductase very similar to a group ofenzymes involved in lipid metabolism [3-oxoacyl-(acyl-carrier-protein) reductase]. bu with low levels o aminoacid identi {41%). We idenfied the conserved residuesinvolved in NADPH binding, but not the binding site fordihydroolate. The differences with respec to the DHFRsdescribed bekw (Figs 1 and 2) led us to purify and char_acterize this enzyme biochemically. Tm8-3 has beenshown to reduce dihydroolae, but not acetoacetyl CoA(data not shown), the substrate of 3-oxoacyl-(acyl-canier-proten) redutase enzymes. The specificities o Tm8_3 forthe NADPH and dihydrofolae substrates were Yerysirnilar to those of other DHFR enzynes (Fierke ef ar.,1987; Lee eta.' 2o1o|. The lCso of Trn showed Tm&3to be a DHFR conferring Tm resistance, and hat theresistance nnlerred lo E. cali was not purely due tooverproduction o the enzyme by e recombinant system'The nave form o Tm8-3 appears to be a dimer (Fig. 5).This ontrast with other known DHFRs, as the chromo-somal DHFR enzyrnes (Type ) are usualy monomers,whereas the plasmd enzy{nes (type ) are acve in lhetetrameric lorm (Howell, 2005).Our findings highlight the benefit o{ functional metage-nomics, as we were able to identify novel enzymeswithout limitation to genes recognizable as resistancedetenninants on he bais of seqence. We idantiiedvarious antibioic resistance mechanisms and a novelresistance determinant. This finding could exain non-canonical resistance mechanisms found in clinical set-tings. The low level of sequence similarity between theproteins conferring antibioc resistance characterized inthis study and those deposited in database supports thehypotheses developed over th last few years, accordingto which lhese proeins may play a very differert roe innafural onditions. t seems plausible that moleculesselected by pharmaceutica| companies because o their

antibiotic properles at therapeic concenrationswould have also dis,tinct unctions al naturally occurringlower concentrations (Martinez, 2OO8). Thus, it is widelyaccepted that the enzymes deactivating aminoglycosideanibiotics may play a metabolic role in the cell (Millerear.' 1967) or be involved in the acelation of pepti-doglycan (Macinga ear'' '1999}. Similarities in structuebetween the antibiotic and he ubstrae of e enzymemay result in the development of resistance, even if themolecules concemed have very diferent res' The Char_acterized enzyme conferring Tmp resistance, Tm8-3, mayconsttute a new example supporting this hypothesis.Experimental proceduresSoi descriptonThree soils were used to consrut the metagenomic librariesdescribed in this work. Soils were collected, sieved {2 mmpores) and stored at 4'C. DNA vas extracted within 't week ofcolection. The EEZ soil (May' 20o5) was a oamy soilrom anagricultural field with a long history o cropping (> 20 years}ocated at the Zaidin Expeimenta| Station (Granada Spain37"9.957'N, 3"35.493"U\t). This soil had a pH of 8.1 (soil todeionized water, 1:'l ratio); and 1.81% organic matter; itscharacteristics are described esewhere (Ruiz-Lozano et aL,2oo1}. Mexican soil sampes were taken in the vicini oMammillaria camea plants collected lrom he Tehuacan-Cuicaan nature reserye in oaxaca, Mexico, from a sitelocated at 17'44.675'N, 96'58.5ffi11' with little human ilu_ence. The Mexican samples were mlleted a the end of therainy season (R-Mex, late August 2@5}' and during e dryseason (D-Mex, March 2006), These soils were ooarse loamyarid soils with a pH ot 7.3 (soil to deionized water, 1l1 rao),poor in nitrogen and organic maer (1.8%), with waterconent differing by a actor of five to six between the rainyand dry samples (J.F. Aguirre_Ganido, D. MontieFlugo, C.H.Hemeindez-Rodriguez, G- Torre-Cos, V. Millin, N. Toroet al., in preparation).Bacteial strains and cuture condionsAll chemicals and antibio{ics were purchased from Sigma-Aldrich. Escheicha cof strain XL1-B|ue MRF was used forlibrary construction and srain XLoLR was used for expres-sion of the antibiotic resistance determinants from phagemids(Stratagene' La Jd|a' AA, UsA)' Edeicha coli cells weerouinsy cultured in LB medium at 37'c, and plang mediacontained '1.5% Bacto Agar. When appropriate, media rvereamended with 10 mg Fl tetracycline and 50 mg l-r kanamycinor strain and plasmid mainlenance reectively.We used pBK-cMV and he ZAP Express vec{o digestedwith BamHl (Stratagene) to construct the soil metagenomiclibrarie.DNA isoa{on and metagenomic libnry cnstruconToal DNA was extracted rom 5 g soil samples, ith aCETAB-based extraction pfotoco {Porteous et al., 1997).

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Expression libraries were construcled rom the extracted oiDNA with lhe ZAP Express BamHl Predigested Vecor Kit(Stratagene). The extracted metagenomic DNA was patiallydigested with Sa and subjected to eectrophoresis. DNAragments of 9-10 kb in size were isolated from the gel andconcentraled on Microcon ilters (Millipore, Billerica, MA,USA}. We ligated 5G-150ng of DNA to lambda vecoaccording to the recommended protocols. Library titre wasdetermined by mixing various dilutions of the packaged liga-tion product with E coli XL1-Blue cells, according to themanufacturer's instructions, and counted the plaques formed.The ZAP Express vector is designed to allow simple, efficientin viva excision and recircularization of any cloned inserlcontained wihin the lambda phage vector, to orm aphagernid (pBCK-CMV plasmid). Thus, it is possible toconvert a lambda phage DNA library into a phagemid libraryby using the mass excision protoeol described by the manu-facturer (Sratagene). Library size was estimated by multiply_ing the number of clones by mean insert size for l0 cloneso each library.Isolation of clones expressing antibiotc reStancePools of phagemids (1C6 phagemids per plate) from eachlibrary were plated on LB plates supplemened with inhibi_tory concenlIations of Ap {50 mg 1), Gm (20 mg l), Cm(50 mg 1), erythromycin (60 mg l_1), streptomycin (5o mg l_1)and Tmp {5o mg _J). Plates were incubated for 24 or 48 h al37"C. Plasmids were isolated rom antibiotic-resis{ant coo_nies and analysed by DNA restriction. We lransJerred E collDHSa with the plasmids, and the resistance phenotype waschecked by plating the tansfomed cells on appropriateselecive media.Minimum inhibitory concentration were deernined byserial dilution assays of the conesponding antibiotic in LBbroth, with approximately I x1O5 cellsmFl- Experimentswere done in triplicate.ldentfication af antbiatic resistan ce genesThe {ull-|ength sequences of the metageno'nic DNA insetswere oblained by primer walkin with universal primers, lnsome cases, the size o the inserts o, plasmids containing theresis{ance genes was reduced with a different set o resric-tion enzymes. This strategy made i possible lo decrease theamour of sequencing requred to identify the gene respon-sible o he resisance phenotype. The putative oRFs wereannolated with o_asr {Basic Local Alignment Searc-h Tool,Atschul e ar., 1990). Prediced oRFs within the DNA insertsof the various clones are listed in Table l.Phylogenetic anaysisThe 't0 top hits or each oRF encoding the antibiotic resis-tance protein were identiied and collected with Bastp (Ats-chul eal., 't990). Where appropriate, relerence sequencesfrom the corresponding protein families were alo includedflables S1 and S4). The amino acid sequences of all non-redundant enzymes yere downloaded and aligned, withMAFT versin 6 (Kaoh and Toh' 2008), whch offers multiple

Nove anibatic resisance genes from soil metagenome 11aignment 1rategies; we used ClustalW, a simpe progressivemethod. Dendrograms were constructed by he neighbour_joining method, and boottrap analyses o he respective datasets were perfomed with he MEGA 4.1 software package(Kumar et al.' 28).Expression and puifcaon af the Tm8-3 protenThe Tm8-3 gene was amplified rom phagemid pBKTm8by PCR with the Pfu polymerase (Promega, Madison,MA, UsA)' in 28 cycles, rth primers 678Tm8-3f{5'-GCGGATCGTcaTCATCTGAGGTGCCGGCCAC-3J and1427Tm8-3r is'-GCGCATATGATGACGACCACCTTGGCGG-3]. Primers were desigred to inroduce BamHl and Ndelsites (underlined) and an extra stop codon (in small leers)irto the cloned fragment. The PCR product was insertedinto pGEMTeasy (Promega), to generate pGEMT:Tm8-3. Weensured that no unintentional changes were inlroduced byPCR, by sequencing lhe 0.8 kb fragment, which includes theTm8-3 ORF gene. pGEMTTmS-S was digested with Ndeland BamHl and the o.8 kb fragmen was inserted betweenthe corresponding sies o pET_3a (Novagen, UK). Freshlyransformed E col BL21(DE3} cells carryng pET3a_Tm8-3were used to overproduce the corresponding protein. Cul-tures were grown at 37"C to an OD* of 0.5, and productionof the recombinanl protein was induced by the addition of1 mM IPTG. Afer cufure for 12 h at 28"A, the cells wereharvested by centrugation (500o9, 15min), suspendedin 50 mM sodium phosphate buffer {pH 8) and disruptedby sonicaion. Cell debri was removed by centrifugationat 15 o00 g or 15 min' and the protein was purified byion chromatography. The so|uble raction was applied to aMono_Q HR 5/5 column (Pharmacia Biotec}, equilibratedwith Tris_Hcl pH 8 buffer' The eluted fracions were collected,concentrated and further purified by size exclusion chroma-tography (Superdex 75 HFl; Pharmacia Biothech)' Fraconscontaining the puri{ied protein were finaly resolved bySDS-PAGE, in 12o/o polyacrylamide gels, and visualized byCoomassie Brilian Blue staining.We tested whether is oRF confened Tmp resistance toother bacterial species, such as Sinorhizabium mellot, byinseing the 0.8 kb fragment into a broad-host range cloningvector, pBBSyn. The Psyn promote; (Giacomini ea/.' 1994}was amplified with the PsyFw (5'-CAGGTAC0TATAAAAATAATTCTTGAC-3J wh a Kpnl site and PsynRv (5'_CGGGATCGTTAATGGCGCATATTATAC-3') wih a BamHlse primers and inserted no the pGEMT Easy Vector$ystem (Promega), to generate pGEMSyn. This plasmid wasdigested with Kpnl and BamHl. The fragment containingthe Psyn promoter was inlroduced into pBBRIMCS-2(Kovach e a'.' 1994) as a Kpnl-BamHl fragment, to generatepBBSyn.pBBSyn:Tm8-3 contains the O.8 l

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12 G. Tones-Corts eIal'et a'' 2ao1\, wilh adjustment of the final concentration of thestock oution to 'l00 mM. They were stored a _70'c, in 50 plaiquots, and their concenration was conirmed by readingthe absobance of a diluted solution at 282 and 340 nmreectively. Trimethoprim actae (sigma_Aldrich) solulionswere prepared in 0.1 M irnidazole-HCl bufer (pH 7), at con_Gentratons o 10 and 10o pM' and were stoed at -70'c' Ivalues for each substrate {DHF and NADPH) and 50% inhi-bltory concentrations {1C56) for Tmp were determined asdescribed by Baccanari and Joyner {1981). A ShimadzuUV-1 80o si spectrophotometer {Shimadzu Cop., Kyoto,Japan) wi a heated cuvette caniage and an inbuit programfor the integrated determinalion of initial reaction raeswas ued. Reactions were performed in semi_micro asticcuvettes in a final voume of 1 ml at 3o"C. For determinationsof l values fof each substrate, the concentration of the othersubstrate was held a a conslant at 't0opM. Non-specificNADPH-oxidase activity was determined for each enzymepreparation, by determining rates for each NADPH concen-traon in the absence of DHF, and then subracting thesevalues from lhe resuls obtained in the presence of DHF.nitia rales or each concentration o NADPH and DHF wereused to determine t and V.* values' according loMichaeli-Menten kinetics. The standard reacion mixture forthe determination of lCso values wa 10o pM NADPH,100 pM DHF, 75 mM 2-mercaptoethanol, 50 mM TES buffer(pH 7.0}' Tmp and enzyme extract in a final voume of 1 ml.lC5s values were determined by plotting initial reaction ratesagainst each Tmp concentration.Gen&ank acessionsThe nucleoide sequences described in this study havebeen deposited in the GenBank database under accessionnumbers: FN64046&FN64047 4.AcknorledgementsThis work was supported by the research projects BlO2003-a2473, Bo20o8_0o740 and CsD 20oHo06 of theConsolider-lngenio 2010 program from the Miniseio deCienca e lnnovacin. We would also like to thank he BBVAFoundaion (BBVA BlocoN 04-084 Pect). G.T.C andR.N.M. ere supported by a CSIC predoctora el|owship.V-M. was supported by a grant rom the abovemenonedBBVA Project. We are particularly grateful to M. Ferrer forinvaluable assitance in lhe construcon of the metagenomiclibrarie.RerencesAhmed, A.M., Nakagawa, T., Arakawa, E., Ramamurthy, T.,Shinoda, S., and Shimamoto, T. (2004) New aminoglyco-side aceltransferase gene, aac(3)-Id, in a class'l integronfrom a muftiresstant strain o Vbrio fuviales isolaled froman inant aged 6 months. J Antimicrob Chemother 53:.947_s5'.Allen, H.K., Moe, L.A., Rodbumrer, J., Gaarder, A., and Han-delsman, J. (2009) Functional metagenomics revealsdiverse p-lactamases in a remote Alaskan soil. ISME J 3:243-251.

A|tschul' S.F.' Gish, W., Miller' W., Myers, E.W., and pman,D.J. (1990) Basic loca alignment search tool. J MoI Bol215:40&-410.Ambler, R.P' (1980) The sructure o beta_lactamases. Pl,r7osTrans R Soc Lond B Bio Sci289: 321_331.Aminov, R.l. (2o09} The role of anbiocs and antibioic 'ess_ance in natJre. Enyiron Microbia 11:' 2970*2988.Aminov, R.1., and Mackie, R.l. {2007) Evolution and ecologyof antibiotic resisance genes. FEM$ Mcrobial Le 271:147-216.Azuena, E., and Mobashery, s. (2001) Aminoglycoside-modifying enzymes: mechanisms of catalytic pocsesand inhibtion. Drug Besst Updat4| 106_'17.Baccanari, D.P., and Joyner, .S. (1981} Dihydrofolate reduc-tae hysteresis and is efect on inhibitor binding analyses.Bochemistry 2o: 171 0-1 71 6.Clamp, M., Cuff, J., Searle, S.M., and Barton, G.J. (2004) TheJalview Java alignmenl editor. Bioinformatks 2O:, 428427.courois, S.' Cappellano' C.M.' Ball, M., Francou, F.X.,Nonnand, P., Helynck, G., et al- (2003) Recombinant envi-

ronmental libraries prode access to microbial diversity ordrug discovery from natura products. App Environ Mhro-pJ69:49-55.Da Cos1a, V.M., McGrann, K.M., Hughes, D.W, and Wight,G.D. {2006) Sampling the antibioic resistome. Science311:. 374-377.Da Costa, V.M., Griffithes, E-, and Wright, G.D. (2007)Expanding the soil antibiotic resitome: exporing environ_menla| diversi . Curr opn M'rcrabol 1a: 48I -489.Dale' G'E.' Broge1 C.' D'Arcy' A', Hartman, P.G-' DeHoogt,R.' Jolidon, S', e aL (1997) A single amino acid substtu_lion in Staphylacoccus aureus dihydrofolate reductaedetermines lrimelhoprim resistance. J Mol Biol266: 29-3O.Daniel, R. (2o04} The soi metagenome - a rich reource forthe discovery of novel natural products. Curr apin Botech-nol 15: 199-204.De la Torre, J.R., Chrisianson' L.M.' Beja, o., uzuki, M.T.,Karl, D.M., Heidelberg, J., and Delong, E.F. (2003) Prote-oodopsin genes are distributed among divergent mainebacteria taxa. P oc Nal Acad Scj USt lfi}: 1 2830_1 2835.Demaneche, S., Sanguin, H., Pot6, J., Navano, E., Bemillon,D., Mavingui, P-, et al. (2008) Antibiotic_resisant soil bac_teria in tansgenic plant fields. Proc Nal Acad Sci USA105: 3957-3962.Donato, J'J.' Moe, L.A., Converse, B.J., Sma, K.D.' Berk-lein, F.C., McManus, P.S., and Handelsman, J. (2010)Metagenomic analysis of apple orchard soil reveals an_biotic resistance genes encodng predicted bifunctionalproteins. Ap Envron Microbiol76: 4396-4401.Dyda, F., Klein, D.C., and Hickman, A.B. (20OO) cCNs-related N-acetyttransferases: a sructura ovewie'. AnnuRev Bophys Biomal Struct29 81-03-Fajardo, A., Linares, J.F., and Martinez, J.L. (2009) Towardsan ecological approach to antibiotics and antibiotic resis-ance genes. CIin Mcrobiol nfect15 (Suppl. 1): 1&16.Fierke, C.A., Johson, K.A., and Benkovic, S.J. ('1987)Conruction and evaluation of the kinetic scheme associ-ated with dihydrofolate reductase rom Escheichia coli.Biochemistry 26: 4O8S*4O92.Giacomini, A., Ollero, F.J., Squartini, A., and Nuti, M.P. {1994)construction o multipu4pose gene cartridges based on a

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