The pGRT1 plasmid of Pseudomonas putida DOT-T1Eencodes functions relevant for survival under harshconditions in the environmentemi_2492 2315..2327

Lázaro Molina,1 Estrella Duque,1 Manuel J. Gómez,2

Tino Krell,1 Jesus Lacal,1 Alicia García-Puente,1

Vanina García,1 Miguel A. Matilla,3

Juan-Luis Ramos1* and Ana Segura1

1Environmental Protection Department, EstaciónExperimental del Zaidín, CSIC, C/ Profesor Albareda1,18008-Granada, Spain.2Department of Molecular Evolution, Centro deAstrobiología INTA-CSIC, Ctra. Torrejón – Ajalvir, Km 4.Torrejón de Ardoz, 28850-Madrid, Spain.3Bio-Iliberis R&D, PTS, Avda. de la Innovación 1, 18100Armilla, Granada, Spain.

Summary

Pseudomonas putida DOT-T1E has the capacity togrow in the presence of high concentrations oftoluene. This ability is mainly conferred by an effluxpump encoded in a self-transmissible 133 kb plasmidnamed pGRT1. Sequence analysis of the pGRT1plasmid revealed several key features. Most of thegenes related to the plasmid maintenance functionsshow similarity with those encoded on pBVIE04from Burkholderia vietnamensis G4, and knock-outmutants in several of these genes confirmed theirroles. Two additional plasmid DNA fragments wereincorporated into the plasmid backbone by recombi-nation and/or transposition; in these DNA regions,apart from multiple recombinases and transposases,several stress-related and environmentally relevantfunctions are encoded. We report that plasmid pGRT1not only confers the cells with tolerance to toluene butalso resistance to ultraviolet light. We show here theimplication of a new protein in solvent tolerance whichcontrols the level of expression of the TtgGHI effluxpump, as well as the implication of a protein withhomology to the universal stress protein in solventtolerance and ultraviolet light resistance. Further-more, this plasmid encodes functions that allow thecells to chemotactically respond to toluene and par-ticipate in iron scavenging.

Introduction

Pseudomonas putida DOT-T1E has the extraordinarycapacity to withstand and even grow in the presence ofhigh concentrations of organic solvents such as toluene,styrene and xylenes (Ramos et al., 1995). The toxicity ofthese compounds derives from their preferential partition-ing in the cell membrane, which provokes the collapseof cell membrane potential and eventually leads to celldeath (Sikkema et al., 1995). Solvent tolerance in P.putida DOT-T1E is a multifactoral trait that involves chro-mosomal and plasmid encoded functions (Segura et al.,2001; 2005; Ramos et al., 2002; Rodríguez-Herva et al.,2007). The first mechanism of solvent tolerance involvesimpermeabilization of the cell membrane via a fast cisto trans isomerization of unsaturated fatty acids andmodification of phospholipid head groups (Keweloh andHeipieper, 1996; Junker and Ramos, 1999; Heipieperet al., 2001; 2003; Bernal et al., 2007; Pini et al., 2009).However, this impermeabilization does not prevent entryof toluene; the solvent denatures proteins and as a con-sequence a number of chaperones are induced (Seguraet al., 2005; Domínguez-Cuevas et al., 2006; Volkerset al., 2006). The main mechanism underlying solventtolerance lies in the action of RND (resistance-nodulation-cell division) (Kieboom et al., 1998; Kim et al., 1998;Ramos et al., 1998; Mosqueda and Ramos, 2000; Rojaset al., 2001) and ABC transporters (Kim et al., 1998;García et al., 2010), that in energy-dependent processesremove solvents from the cell to the outer medium. Thegenome of Pseudomonas putida DOT-T1E encodes over20 RND efflux pumps, three of which (TtgABC, TtgDEFand TtgGHI) have been described as responsible for thecooperative extrusion of solvents, although our resultshave shown that from a quantitative point of view the mainin vivo toluene efflux pump is TtgGHI (Rojas et al., 2001).The ttgABC and ttgDEF operons are chromosomallylocated (Mosqueda and Ramos, 2000; Duque et al.,2001), whereas the ttgGHI genes are located on the self-transmissible pGRT1 plasmid (Rodríguez-Herva et al.,2007). Acquisition of this plasmid by other bacteria of thegenus Pseudomonas suffices to increase the level ofsolvent tolerance for the recipient strain. Hosts recipientswith pGRT1 can be used to develop cell factories for

Received 16 March, 2011; accepted 22 March, 2011. *For correspon-dence. E-mail: jlramos@eez.csic.es; Tel. (+34) 958181608; Fax (+34)958129600.

Environmental Microbiology (2011) 13(8), 2315–2327 doi:10.1111/j.1462-2920.2011.02492.x

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd

biotransformation in two-phase systems (MacLeod andDaugulis, 2003; Rojas et al., 2004; Nijkamp et al., 2005),and to create strains which are more efficient in combat-ing pollutants when present at high concentrations. Inthis study we have sequenced the entire 133 kb pGRT1plasmid, and have generated a number of mutants toelucidate some of its encoded functions. Our resultsrevealed that the plasmid is based on a backbone ofgenes similar to genes of plasmid pBVIE04 of Burkhold-eria vietnamensis G4. Within this backbone, a series oftransposon and integration sequences bearing a varietyof genes are incorporated; many similar to Pseudomonasgenes. This plasmid encodes several stress-related deter-minants: (i) the ttgGHI operon and its corresponding regu-latory protein, TtgV, that confers a high level of solventtolerance to the host strain; (ii) an SdiA-regulated proteinthat is also involved in solvent tolerance through the regu-lation of the ttgV repressor protein; and (iii) a set of uvrgenes that enhance resistance to ultraviolet radiation.Other genes of environmental interest encoded in pGRT1are two mcpT genes that confer the host with positivechemotaxis towards toluene and a protein involved insiderophore release.

Results and discussion

Sequence features of pGRT1

The analysis of the 133451 bp of plasmid pGRT1 allowedus to annotate a total of 126 open reading frames (ORFs),which cover 86.3% of the nucleotide sequence (Fig. 1).The average G+C content of the entire pGRT1 plasmid is55.8%, a value that is lower than the average G+C contentof the P. putida DOT-T1E chromosome (61.3%). TheORFs are symmetrically distributed, with 66 ORFs inclockwise orientation and 60 in counter-clockwise orien-tation (Fig. 1). The 126 ORFs were classified into sixmain groups according to the predicted functions of thecorresponding proteins (Table 1): (1) DNA replication,modification, transfer and maintenance functions; (2)transposition; (3) regulatory proteins; (4) transport ofnutrients; (5) stress-related proteins, and (6) proteins ofunknown function.

A set of 41 ORFs (ª39.5% of the coding DNA with aG+C content of 55%) exhibits high similarity with ORFsof the pBVIE04 plasmid of Burkholderia vietnamensis G4and out of these, 31 encode proteins that are homologousto proteins related to DNA replication and modification,plasmid maintenance and mobilization and transfer ofplasmid DNA. This set of genes is clustered in two non-contiguous regions of 31 kb and 23 kb (from nucleotide121398–18863 and 41968–65351 respectively; Fig. 1),and can be considered the plasmid backbone. The inter-twined DNA between the two gene clusters that constitute

the plasmid backbone may have been recruited throughtransposition, insertion or recombination events asdescribed below.

The first DNA segment interrupting the proposedplasmid backbone (coordinates 18927–41331, in Fig. 1)is located between mobE and parB and encodes func-tions related with transposition and recombination andwith proteins involved in stress responses (Table S1). ThisDNA fragment exhibits a high level of identity with genesfound in other bacteria of the genus Pseudomonas (P.putida, P. aeruginosa, P. syringae and P. fluorescens), andit may have been acquired by means of genetic eventsthat occurred in previous hosts in which the primordialplasmid existed. The second piece of DNA interruptingthe plasmid backbone is flanked by traY and trbC (Fig. 1;Table S2), and comprises all the elements that character-ize a transposon (transposases, resolvases and resolu-tion sites [res], depicted in Fig. 2). Furthermore, flankingthis DNA sequence, there are 38 bp inverted repeats (IRs)identical to the left and right terminal IRs of Tn4653 (Tsudaet al., 1989). Upstream IR-1 and downstream IR-1R afive base pair duplication (TAACC) was found. Thesecharacteristic IRs are similar to those of transposonTn501 and contain the conserved EcoRI recognitionsequence typical of this family of transposons. Rodríguez-Herva and colleagues (2007) identified a pGRT1 deriva-tive, named pGRT1-VAN5 that had lost 5 ScaI fragmentscomprising around 50 kb. The current complete plasmidDNA sequence allowed us to determine that these frag-ments are contiguous and correspond with the loss of theTn4653-like transposon.

Among the genes encoded in this transposon are thoseof the toluene efflux pump (ttgGHI) (Table S2). The pres-ence of several transposases and integrases surroundingthe efflux pump genes suggests that this DNA fragmentmight have been incorporated into the pGRT1 plasmid (orinto the putative transposon) by insertion and integrationprocesses that brought in genes related to stress endur-ance. Flanking this DNA region there are two 7.6 kbinverted duplications (shown by black bars in Fig. 2).Among the five proteins encoded in each one of theseduplicated regions (Table S2) we identified two trans-posases, a resolvase and a putative methyl-acceptingchemotaxis protein (Fig. 2; Table S2). One of these dupli-cations is disrupted by the insertion of a DNA fragmentthat is almost identical to a region of the TOL pWW0plasmid and that includes ISPpu12 (Greated et al., 2002;Williams et al., 2002; Rodríguez-Herva et al., 2007). ThispWW0-derived fragment is inserted within the Tn4653Atransposase gene and the two resulting fragments havebeen named ORF100 and ORF113. Within these twotransposase gene fragments a duplication of 5 base pairshas been produced (CATCT). At the 3′ end of the trans-posases ORF73 and ORF96 two more 38 bp IRs are

2316 L. Molina et al.

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 2315–2327

found (positions 78705 and 102433). These IRs areidentical to IR-2 described in the pCAR1 plasmid ofPseudomonas resinovorans (Maeda et al., 2003) andshare 35 out of 38 nucleotides with the IRs of Tn4653.

Analysis of backbone genes: identification offunctions involved in plasmid replication,segregation and maintenance

The feature that best defines plasmids is that they repli-cate in an autonomous and self-controlled way (DelSolar et al., 1998). Plasmid replication is normally started

through the use of a replication initiation protein (Rep).Plasmid pGRT1 encodes two putative replicases, namely,ORF45 and ORF49 (Fig. 1). The ORF45 gene productbelongs to the RPA (replication initiation protein A) super-family (cl02339) that comprises single-stranded DNAbinding proteins that are involved in DNA replication,repair and recombination (pfam 10134). The pGRT1ORF45 protein is around 65% similar to replication initia-tion proteins of plasmids pEMT8 (Gstalder et al., 2003)and pBVIE04 of Burkholderia vietnamensis G4 (GenBankentry NC009228). The ORF49 protein shows the highestidentity (62–63%) with the replication initiation protein of

Fig. 1. Physical map of the pGRT1 plasmid. Arrows indicate deduced ORFs. Genes belonging to the main backbone are shown by brownarrows. Light brown arrows in the inner circle indicate the putative plasmid addiction systems. Red arrows represent transposition andintegration elements. Green arrows represent the ttgGHI efflux pump and the ttgV regulatory protein and other stress-related proteins. Plasmiddrawing has been done using CGView (Stothard and Wishart, 2005). The putative sequence of the origin of replication with eight iterons isdepicted in the inner circle. Numbers indicate the coordinates of the genes within the backbone.

Characterization of the pGRT1 plasmid 2317

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 2315–2327

plasmids pRSB105 (Schluter et al., 2007) and p2353B ofPseudomonas syringae pv. theae (Ma et al., 2007), andhas a conserved replicase domain (pfam 03090) at theN-terminal region, while it exhibits a primase domain atthe C-terminal region (pfam 08708) (Giraldo and Díaz-Orejas, 2001). Eight almost identical direct repeats of 15nucleotides (Fig. 1) were found upstream of ORF49, andthey are proposed to be the iterons that form the origin ofreplication for plasmid pGRT1.

To identify which one of the proteins, ORF45 or ORF49,was acting as a replication protein, we knocked-out eachof the ORFs by site-directed mutagenesis using mutantalleles marked with a kanamycin resistance gene asdescribed in Materials and Methods. If only one of thesetwo ORFs were involved in plasmid replication its inacti-vation would lead to plasmid loss, and no Kanamycin-resistant mutants associated with that ORF would beviable in the presence of the antibiotic. Otherwise, ifthe proteins were isoenzymes, both mutants could beobtained. Kanamycin-resistant mutants with inactivatedORF45 and ORF49 were obtained. The authenticity of themutations was confirmed by Southern blot, and we foundthat hybridization of plasmid DNA with ORF45 gene as aprobe yielded the expected pattern, while hybridization ofDNA extracted from different putative ORF49::WKmmutant cells yielded an unexpected hybridization pattern(Fig. S1A), which was best interpreted as correspondingto a mixture of the mutant and the wild-type alleles in thebacterial population. One single colony of ORF49::WKmmutant was re-streaked on Luria–Bertani (LB) plus kana-mycin, and several individual colonies were used toextract DNA and determine the hybridization pattern againby Southern blot using ORF49 gene as a probe. Thepattern seen with each of these individual colonies was asbefore, that is, it corresponded with the presence of thetwo alleles. This series of results suggests that ORF49is responsible for plasmid replication of pGRT1, whileORF45 is dispensable.

Other functions related with plasmid replication andmaintenance are encoded by pGRT1 (Table S3). Amongthem are homologous to ParA, ParB and KfrA, a setof proteins that participate in the segregation of plasmidcopies to daughter cells before division (Gerdes et al.,2000; Ebersbach and Gerdes, 2005; Adamczyk et al.,2006). In plasmid pGRT1 these proteins likely correspondto the type Ia partitioning loci as described by Gerdes andcolleagues (2000). Furthermore, plasmid pGRT1 bearsinformation for three putative toxin-antitoxin (TA) systemsrelated with plasmid maintenance (Gerdes et al., 2005;Kroll et al., 2010). The pGRT1 ORF66 and the threealmost identical direct repeats of 35 nucleotides thatare located upstream ORF66 (Fig. S2), constitute oneof these systems. These proteins belong to the post-segregational killing group (PSK) of antisense-RNATa

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© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 2315–2327

regulated loci (Fineran et al., 2009). The second putativeTA system found in pGRT1 (ORF126) belongs to theplasmid addiction system (Gerdes et al., 2005). ORF126has high identity with zeta toxin proteins; however, noORF with similarity to the antitoxin component was foundin pGRT1, so it is possible that this toxin is a remnant of anancestral TA system. The third putative TA system inpGRT1 (ORF1 and ORF81) belongs to the restrictionmodification class (Handa et al., 2000).

Genes involved in plasmid conjugation

Plasmid pGRT1 is a conjugative plasmid (Rodríguez-Herva et al., 2007). Three different sets of genes locatedat different positions within the plasmid sequence (Fig. 1)encode proteins putatively involved in the conjugativetransfer of pGRT1. The critical protein in the initiation ofconjugational transfer is a relaxase. BLAST analysis iden-tified ORF12 as a putative relaxase, encoded by themobA gene (Fig. 1). This enzyme initiates and terminates

conjugative DNA processing. The pGRT1 relaxasebelongs to the MOBp cluster, specifically to the cladeMOPp12 as defined by Garcillán-Barcia and colleagues(2009), which includes all the conjugative plasmids of theIncI complex. Interestingly, the subtype of secretionsystem (T4SS-IVB) that is encoded by pGRT1 is alsotypical of plasmids of the IncI group (Sexton and Vogel,2002).

To investigate the role of these three DNA regions in theconjugal transfer of pGRT1 we constructed knocked-outmutants in genes of the five/six putative operons relatedto conjugation functions (mobBA/traHIJK mobCDE,traCL, traMNOPQRTUWXY and trbAB-primase-trbC). Tothis end, a KmR cassette was used to interrupt genesmobA, traH, traC, traU, mobD and trbA. Mutants wereconfirmed by Southern blot (not shown) and then biparen-tal mating using strain P. putida KT2440 (crc-) (Aranda-Olmedo et al., 2005) as a recipient were performed withall the mutants. The frequency of transfer to P. putidaKT2440 (crc-) of pGRT1 mutants in traH, traC, traU and

Fig. 2. Schematic representation of the putative Tn4653-like transposon of pGRT1. The TtgGHI efflux pump and its regulatory gene ttgV aredepicted as grey stripped arrows. Dotted arrows indicated ORFs with homology to phage integrase and phage transcriptional regulators.Fused ISPsy20 transposase are drawn as vertically stripped arrows. Grey arrows indicate transposition functions, with the Tn4653 relatedtransposition functions in dark grey. The black bars under the arrows indicate the 7.6 kb inverted repetition on both sides of the putativetransposon. The downstream repetition is interrupted by sequences similar to those of pWW0 plasmid that includes the ISPpu12 that is shownas a grey bar. Black rectangles between the arrows indicate the presence of the inverted repeats whose sequences are displayed below. TheEcoRI sequence conserved in IR1 and IR-1R are shadowed in grey. In the upper part of the figure the sequence of the three resolution sites(res) are indicated.

Characterization of the pGRT1 plasmid 2319

© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 13, 2315–2327

trbA genes was below the limit of detection of the experi-ment (� 10-7), while the mobA mutant had a transferfrequency of about 10-5, much lower than that of themobD mutant or control strain pGRT1-ttgW::WKm (trans-fer frequency of around 10-2 transconjugant per donorcell. These results suggest that the mobD gene is notrequired for DNA transfer, while mobA, traH, traC,traU and trbA are all essential for pGRT1 plasmidconjugation.

pGRT1 plasmid is involved in the survival of P. putidaDOT-T1E to stress conditions

According to the annotation of the plasmid sequence,pGRT1 may not only confer the ability to thrive in highconcentrations of toluene, but also resistance to otherenvironmental threats; several ORFs, whose gene prod-ucts exhibit homology with putative proteins involved instress resistance, were annotated. We found three ORFs,ORF25, ORF36 and ORF95 that show identity with pro-teins involved in ultraviolet light protection. In addition weidentified the efflux pump genes (ttgGHI) that are criticalfor solvent tolerance; ORF32 has homology with an SdiA-regulated protein and ORF34 that is 96% identical tothe universal stress protein (UspA) of Pseudomonasaeruginosa.

To study the implication of these ORFs in stressresistance, we exposed P. putida DOT-T1E-100 (a strainlacking the entire pGRT1 plasmid; Rodríguez-Herva et al.,2007), to different stress conditions and compare its phe-notype with that of the wild-type strain. A series of knock-out mutants in some of the above mentioned ORFs weregenerated via site-directed mutagenesis and submittedto the same battery of tests, in order to be able to assignfunctions to these ORFs.

Resistance to ultraviolet light. Three different ORFs(ORF25 [ruvA], ORF36 [ruvB] and ORF95 [uvrD]) showedsequence identity with proteins related with resistanceto ultraviolet light. Cultures of the strain DOT-T1E-100exposed for 10 s to ultraviolet light showed a survival ratetwo orders of magnitude lower than that of the wild-type(Fig. S3). Although it is clear that plasmid pGRT1 encodesgenes relevant for survival to ultraviolet radiation expo-sure, our results suggested that ORF25, ORF36 andORF95 encode redundant DNA repair activities since forinstance, a mutant in ORF36 (ruvB) showed the samesurvival rate as the wild-type (not shown).

Because ORF34 encodes a protein with homology toUspA that is involved in response to different stresses inE. coli (Kvint et al., 2003; Nachin et al., 2005), a mutantin ORF34 was also assayed. Survival of this mutantstrain after exposure to ultraviolet light was one orderof magnitude lower than that of the wild-type strain

(Fig. S3). This result indicates that the ORF34 proteinis involved in the cells’ response to ultraviolet lightstress.

Solvent tolerance. We have previously identified that theTtgGHI efflux pump, belonging to the RND family, is themain player in the extrusion of toluene and relatedhydrocarbons (Rojas et al., 2001). It was also shownpreviously that transfer of the pGRT1 plasmid to solventsensitive strains (P. putida KT2440, P. aeruginosa PAO1)conferred the ability to survive a 0.3% (v/v) tolueneshock (Rodríguez-Herva et al., 2007). We have nowtested the influence of the presence/absence of plasmidpGRT1 on the growth of the host strain in the presenceof sub-lethal concentrations of toluene. Not significantdifferences in growth rate and yield were found betweenthe two strains growing on LB or M9 minimal mediumwith glucose. However, although 100% of DOT-T1E andDOT-T1E-100 cells were viable in culture media with0.075% (v/v) toluene, there was a significant differencein growth between both strains when this concentrationof toluene was present in the culture media. The wild-type cultures reached a turbidity of 2.56 � 0.52 after11 h whereas DOT-T1E-100 reached a turbidity of0.37 � 0.04. These results clearly reflect the beneficialrole of pGRT1 for optimal growth in the presence oftoluene.

To further dig in the plasmid functions related to solventtolerance, we generated a series of mutants in plasmidpGRT1. The protein sequence of ORF32 is almost iden-tical to a protein of unknown function (p032) from plasmidpBS228 of P. aeruginosa (Haines et al., 2007) and con-tains a domain identified in several SdiA-regulated pro-teins. In turn, SdiA has been involved in the control of theRND efflux pump, AcrAB, in E. coli through an unknownmechanism (Rahmati et al., 2002; Dyszel et al., 2010).For this reason we decided to explore the effect that amutation in ORF32 may exert over solvent tolerance and,in particular, on the expression of the ttgGHI operon andon ttgV its repressor gene. The ttgGHI operon is consti-tutively expressed at a moderate level, and its expressionincreases in the presence of organic solvents (Rojaset al., 2003). As shown in Fig. 3A, toluene tolerance of theORF32::WKm mutant strain was higher than that of thewild-type, suggesting that this gene might influencethe expression of the ttgGHI efflux pump. To test thishypothesis, plasmid pANA95 (ttgV::�lacZ) or pANA96(ttgG::�lacZ) (Rojas et al., 2003) were introduced byelectroporation into the corresponding wild-type andORF32::WKm mutant strain. In the ORF32::WKm mutantthe expression of the regulatory gene was lower than inthe wild-type, which was subsequently translated into ahigher expression of the efflux pump than in the wild-type(Fig. 3B). These results are in agreement with the physi-

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ological data; reduction of the expression of the ttgVrepressor enhances the expression of the efflux pumpand concomitantly increases solvent tolerance. Weconclude that ORF32 is a new modulator of the TtgGHIefflux pump and that this control is exerted throughmodulation of the expression of the regulatoryprotein TtgV.

We also tested the survival of the mutant in the putativeuniversal stress protein A (P. putida DOT-T1E-ORF34::WKm) after toluene shock. There were no significant dif-ferences in the survival rate between the wild-type andmutant strains when the cultures were not pre-induced;however, there was a two-orders-of-magnitude decreasein cell survival when the culture was pre-induced(Fig. 3A), suggesting that this protein has to be inducedby moderate toluene stress to carry out its protective role.

pGRT1 encodes other environmentallyrelevant functions

Genes involved in chemotaxis to toluene. Environmentalconditions can often be far from optimal for microbialgrowth and bacteria have evolved different strategies tooptimize the resources available in a particular niche.Bacteria have developed mechanisms to sense the pres-ence of carbon sources and to move towards them. Theanalysis of the ORFs encoded in pGRT1 revealed that twoof them, ORF72 and ORF97, exhibit homology to putativemethyl-accepting chemotaxis proteins (MCPs) and it wastherefore of interest to explore the phenotype associatedwith the loss of these two proteins. This was also moti-vated because plasmid encoded chemoreceptors thatrecognize aromatic compounds have been described, i.e.

Fig. 3. A. Survival of P. putida DOT-T1E, P. putida DOT-T1E ORF32::WKm and P. putida DOT-T1E ORF34::WKm after 0.3% (v/v) tolueneaddition. ‘+ toluene’ (v) indicates that the cultures have been pre-induced with toluene in the gas phase; ‘- toluene’ indicates that the cultureshave not been pre-induced. Open bars: cultures before toluene addition (t = 0); grey bars: control cultures after 30 min; black bars: cultures30 min after toluene addition.B. b-Galactosidase assays. Open bars: DOT-T1E (pMP220); black bars: ORF32::WKm (pMP220); dark grey bars: DOT-T1E (PttgG::′lacZ);light grey bars: ORF32::WKm (PttgG::′lacZ).

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NahY which is involved in the chemotactic response of P.putida (pNAH7) to naphthalene, and NbaY, a plasmidencoded chemoreceptor responsible for positive chemot-axis towards 2-nitrobenzoate (Grimm and Harwood,1999; Iwaki et al., 2007). We carried out agarose plugassays to determine if P. putida (pGRT1) reacts chemot-actically to toluene. In previous assays we found that thewild-type P. putida DOT-T1E (pGRT1) strain accumulatesin close proximity to the agarose plug containing 10%(v/v) toluene, indicating a positive chemotactic behaviour(J. Lacal et al., 2011). ORF72 and ORF97 encoded onpGRT1 are almost identical proteins, differing in a singleamino acid out of 552 residues. Using site-directedmutagenesis we inactivated the mcpT-1 (ORF72::WKm)and the mcpT-2 (ORF97::WKm) alleles (Fig. S1B). Quan-titative capillary assay with the wild-type, the mutants ineach of the mcpT alleles, and DOT-T1E-100, that lacksboth mcpT copies, were performed. Following exposurefor 5 min 2500 � 20 cells accumulated in the toluenecontaining capillary (value corrected using buffer control),while the mcpT-1 or mcpT-2 single mutants were barelyattracted and the chemotactic response was only margin-ally superior to the buffer control (capillary filled withagarose which contains buffer only). The chemotacticresponse of DOT-T1E-100 strain devoid of both alleles,towards toluene, was not distinguishable from thebuffer control. It then follows that pGRT1 confers positivechemotactic response to P. putida DOT-T1E and thatinactivation of one of the McpTs leads to partial lossof the chemotactic phenotype suggesting that toluene-chemotactic response in DOT-T1E is gene-dosedependent.

This positive chemotactic response to toxic compoundscombined with high resistance to solvents, encoded byttgGHI operon, makes pGRT1 the first plasmid to conferresistance and chemotactic response to organic solvents.Interestingly, pGRT1 did not carry any catabolic pathwayfor the degradation of organic compounds. It is thenplausible that these two properties have been selectedbecause they confer some advantages to the cellsbearing the plasmid, in a similar way as plasmids encod-ing antibiotic resistance have evolved.

Iron metabolism. Environmental bacteria have alsodeveloped several mechanisms to acquire nutrients andother essential growth factors. In Pseudomonas, produc-tion of fluorescent siderophores, low molecular weightcompounds synthesized in response to iron deficiency,is a common feature. In the ultraviolet light assays wedescribed above we observed that culture supernatants ofP. putida DOT-T1E-100 were less fluorescent than thoseof the wild-type strain while the turbidity of the cultureswere similar. This suggested that the presence of plasmidpGRT1 was required for production, secretion or regula-

tion of a fluorescent compound that was liberated to theculture media in response to iron deficiency. Since wehave a series of mutants in a large number of ORFs inpGRT1 we submitted the mutants to this rapid assay. Wefound that mutant ORF35::WKm exhibited a phenotypeidentical to that of DOT-T1E-100 (Fig. 4). This was sur-prising because BLAST of ORF35 scores highest with aputative sulfate permease (sulP) of Pseudomonas aerugi-nosa. To provide support to a potential role of ORF35in release of the fluorescent compound to the culturemedia, complementation of the ORF35::WKm mutant andP. putida DOT-T1E-100 strains with the wild-type genein trans was performed. We observed that the comple-mented strains exhibited culture supernatants withincreased fluorescence (Fig. 4), which links ORF35 withsecretion of the fluorescent compound. CAS-agar assays(Alexander and Zuberer, 1991) confirmed these results(Fig. S4A). Furthermore, DOT-T1E was able to grow andreach a turbidity of around 0.8 in M9 minimal mediumwithout added iron and supplied with 0.25 mM bypiridyl (apotent scavenger of iron); whereas no growth of the DOT-T1E-100 or ORF35::WKm took place in this medium. Tofurther characterize and quantify the production of thisfluorescent compound, the ultraviolet and visible absorp-tion spectrum (from 220 to 700 nm) of the culture super-natant of the different strains was recorded with aNanodrop. Two absorption peaks around 260 and 400 nmwere detected in the supernatants of the wild-type strain

Fig. 4. Production of siderophores by P. putida DOT-T1E and itsisogenic mutants. The wild-type strain P. putida DOT-T1E (blackbars), P. putida DOT-T1E-100 (open bars) and P. putidaDOT-T1E-ORF35::WKm (grey bars) were grown in M9 minimalmedium supplemented with glucose in the absence (- iron) and inthe presence (+ iron) of iron. Dotted bars correspond to the mutantstrain bearing the pSUL-COM plasmid and grown under the sameconditions. The relative fluorescence of the supernatants (recordedas OD260) was normalized with the turbidity of the cultures (OD660)and represented in the y-axis.

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grown in the absence of iron that were not present or weresignificantly reduced in the supernatant of cultures grownin the presence of iron (Fig. S4B). In culture supernatantsof P. putida DOT-T1E-100 and ORF35::WKm grown in theabsence of iron, the 400 nm peak was not detected andthe area of the 260 nm peak was smaller than in thewild-type and in the ORF35::WKm complemented strain(Fig. S4C). Although further analysis are needed to deter-mine the structure of the P. putida DOT-T1E siderophore,the adsorption spectrum is compatible with the DOT-T1Esiderophore containing one adenine-like aromatic residuein the peptide chain and the quinoline derivative typical ofall Pseudomonas pseudobactins (Boopathi and Rao,1990). The results indicate that ORF35 is likely involved inthe release of siderophores to the media.

Conclusions

The plasmid pGRT1 backbone encodes a number ofORFs related to genes from Burkholderia vietnamensisG4 pBVIE04 and most of these proteins are involved inplasmid replication, maintenance and conjugation. Inthis backbone, several insertion events have added ele-ments related with stress functions, mostly derived fromPseudomonas genes. The most striking phenotype con-ferred by this plasmid is the ability to resist high concen-trations of organic solvents. For this function, pGRT1plasmid encodes not only an efflux pump (TtgGHI) and itsregulatory protein TtgV, but also ORF32, a protein thatmodulates the efflux pump activity through the control ofthe levels of TtgV regulator. Linked to toluene resistanceis also the plasmid encoded, Usp protein. Interestingly,two almost identical genes encode methyl-acceptingchemotaxis proteins that are involved in chemotaxistoward toluene. This links the toluene dioxygenase deg-radation pathway, that is chromosomically encoded(Zylstra and Gibson, 1989, Mosqueda et al., 1999), withplasmid encoded functions related to the movement of thehost bacteria towards the substrate toluene. Other genesinvolved in stress resistance are ORF35, involved in sid-erophore release and three genes (ORF25, ORF36 andORF97) most likely involved in resistance to ultravioletlight exposure. All these plasmid encoded functions allowthe host strain, P. putida DOT-T1E, to thrive under harshenvironmental conditions.

Experimental procedures

Sequencing and annotation of pGRT1

Isolated pGRT1 was shotgun sequenced by Macrogen(Seoul, Korea), using the Sanger method. In addition,genomic DNA from strain P. putida DOT-T1E, containing boththe chromosome and plasmid pGRT1, was pyrosequenced

by Macrogen and assembled into 313 contigs. Assemblyof pGRT1 was performed with the package Phred-Phrad-Consed (Gordon et al., 1998) and was the result of combiningthe collection of 1344 Sanger reads with seven contigs fromthe pyrosequencing assembly. A single contig, representing acircular sequence, was assembled at a coverage of 25¥. Theassembled sequence was perfectly congruent with the physi-cal map of the plasmid generated by restriction endonucleasedigestion with ScaI. Plasmid pGRT1 was then automaticallyannotated using a pipeline that uses Glimmer 3.0 (Delcheret al., 1999) for gene prediction and BLAST and RPSBLASTfor functional assignment, by detecting sequence similaritiesin the NR, Swissprot, COG, Pfam, Smart and Prk databases(Altschul et al., 1997). Finally, automatic annotations weremanually curated.

Sequence was deposited in GenBank under accessionnumber HM626202.

Construction of mutants

For the construction of pGRT1 mutants (Table S4), gene frag-ments between 400 and 1000 bp were amplified with theappropriate oligonucleotides (Table S1), and cloned intopMBL1-T plasmid (Dominion MBL). Plasmids containingthe corresponding inserts were then cut with BamHI (thatcuts into the poly-linker of the vector but not in the insertsequence), and the WKm resistance cassette of plasmidpHP45W-Km (Prentki and Kirsch, 1984), previously excisedwith BamHI, was ligated into the BamHI restriction site.Approximately 600 ng of the resulting plasmid was electropo-rated into P. putida DOT-T1E (Enderle and Farwell, 1998) toallow a single recombination event between the clonedDNA fragment and its homologous sequence in the pGRT1plasmid. The resulting kanamycin (25 mg ml-1) resistantclones were selected and analysed by Southern blot. Clonesthat contained the insertion in the appropriate location werekept for further analysis.

Particularly, the mcpT-1 mutant (ORF72::WKm) wasgenerated by amplification of a 615 bp fragment fromP. putida DOT-T1E using oligonucleotides Mcp-1 and Mcp-2(Table S1). DNA was extracted from the gel (Qiagen gelextraction kit, Qiagen) and ligated into pMBL1-T plasmid(Dominion MBL). The resulting plasmid was cut with KpnI, theinsert was extracted from the gel and ligated into the pCHESIvector (Llamas et al., 2003), that had been previouslydigested with KpnI and dephosphorylated. The plasmid wassubsequently electroporated into P. putida DOT-T1E asabove. For construction of the mcpT-2 mutant (ORF97::WKm)oligonucleotides MCP-Eco y MCP-Bam were used for ampli-fication. The amplicon was cut with EcoRI and BamHI andligated into the pCHESI vector previously cut with the sameenzymes; and the resulting plasmid was electroporated intoP. putida DOT-T1E. Resulting clones were checked for theappropriate insertion by Southern blot.

For complementation of the DOT-T1E ORF35::WKmmutant, the gene was amplified with primers sulfF-com-F(5′-CTCTCTAAGCTTATGCTACAAACACTCAAACA-3′) andsulfF-com-R (5′-CTCTCTGGATCCCGTTGTTCTCCTTCTCAATG-3′). The 1.6 kb amplicon was extracted from anagarose gel, cut with BamHI and HindIII and ligated intoplasmid pBBR1MCS-5 (Kovach et al., 1995) that was previ-

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ously digested with the same restriction enzymes. Resultingplasmid, pSUL-COM was introduced into the DOT-T1EORF35::WKm by electroporation as described above. Posi-tive clones were selected on LB plates supplemented withkanamycin (Km) and gentamicin (Gm) (30 mg ml-1) andtested by Southern blot using a DNA probe labelled withdigoxigenin and obtained with oligonucleotides sulf-F andsulf-R (Table S1).

Conjugation experiments

Biparental matings between Pseudomonas strains wereperformed as previously described (Ramos-González et al.,1991). For the conjugal transfer, P. putida KT2440crc- (GmR)was used as recipient strain (Aranda-Olmedo et al., 2005),and P. putida DOT-T1E-58 (pGRT1-ttgW::WKm) as controlstrain (Rojas et al., 2003). Transconjugants were selected onplates supplemented with Gm and Km, donors on plates withKm and recipients on plates with Gm.

Growth of P. putida in the presence of sublethalconcentrations of toluene

We inoculated flasks containing 20 ml of LB media or M9minimal medium with glucose as carbon source with 6 ¥ 105

to 1 ¥ 106 cells of the P. putida DOT-T1E strain or its plasmid-less derivative P. putida DOT-T1E-100. Then we added or not0.075% v/v of toluene. Ten minutes after solvent addition,30 ml aliquots were taken, and serial dilutions were plated tocount the number of colony-forming unit (cfu) per ml. Growthof the culture was monitored with time.

Siderophore production

Strains were inoculated in M9 minimal medium supple-mented with glucose (25 mM). Iron citrate (6 mg l-1) wasadded to the control cultures (plus iron) and not to the iron-deprived cultures. When cultures reached an OD at 660 nmof around 1, they were illuminated with ultraviolet light in aLKB 2011-002 MacroVue transilluminator (LKB Bromma,Sweden) and photographed (GelDoc 2000, Bio-Rad). Torecord the UV-visible spectrum (220–700 nm) of culturesupernatant, 1 ml of culture was withdrawn, cells removed bycentrifugation (14 000 g ¥5 min) and the supernatant wasused to measure the absorption spectrum in a NanodropND-1000 spectrophotometer (modified from Boopathi andRao, 1990).

Survival following exposure to ultraviolet light

Cultures of the different strains were grown overnight in LBmedium containing the appropriate antibiotics. On the follow-ing day, serial dilutions of the culture were spotted ontoplates. Drops were allowed to dry on the plate for 30 min andthen subjected to ultraviolet exposure for 10 s on a LKB2011-002 MacroVue transilluminator, 254 nm wavelength(LKB-Bromma, Sweden) at a distance of 1.5 cm in a down-ward facing open plate. Control plates were prepared as

above but were not exposed to the ultraviolet light. Theassays were performed in duplicate in three independentassays.

Survival in response to toluene shock

Cells were grown overnight in 30 ml of LB medium in theabsence (non-induced cultures) and in the presence (inducedcultures) of toluene supplied in the vapour phase. On thefollowing day cultures were diluted to reach an OD660 of 0.05,and grown under the same conditions until they reached anOD660 of ~0.8. Then the cultures were divided in two halves;0.3% (v/v) toluene was added to one half, and the other wasused as a control. The number of viable cells was determined(before toluene addition and 10 or 30 min after exposure tothe solvent) by spreading suitable dilutions on LB plates. Theassays were performed at least three times.

Chemotaxis assays

Optimization of cell motility, cell cultures, and the qualitativeand quantitative capillary assay, were carried out as previ-ously described (Lacal et al., 2010). Briefly, capillaries werefilled with 2% low-melting agarose in chemotaxis buffer(40 mM K2HPO4/KH2PO4, 0.05% (v/v) glycerol, 10 mM EDTA,pH 7.0) and 10% of the attractant (toluene). Freshly grownmotile cells were resuspended in chemotaxis buffer to anOD660 of 0.1 and placed into an observation chamber formedby a Petri dish, a U shaped-tube and a coverslip. The capil-lary containing the attractant was then placed close to the cellsuspension and the number of cells that accumulated at thecapillary mouth 5 min later was determined by counting thenumber of cfu.

b-Galactosidase assays

Plasmids pANA95 (PttgV::′lacZ) and pANA96 (PttgG::′lacZ)(Rojas et al., 2003) were electroporated into the wild-type P.putida DOT-T1E and its isogenic mutant ORF32::WKm. Thecorresponding transformants were grown overnight on LBmedium with tetracycline. Transformants were grown over-night and the cultures were diluted 100-fold in the absence(not induced cultures) and in the presence (induced cultures)of toluene supplied through the gas phase (induced). After4 h of incubation at 30°C with shaking, b-galactosidase activ-ity was assayed in permeabilized whole cells according toMiller’s method (Miller, 1972). Assays were run in duplicateand were repeated for two independent rounds.

Acknowledgements

This study was supported by FEDER Grants: BIO2006-05668 and BIO2010-17227 from Ministry of Science andEducation and CVI-191 from Junta de Andalucía. Part of thisstudy was supported by the BACSIN project of the EuropeanCommission (FP7-KBBE-2007–1). We thank B. Pakuts forimproving the use of English in the manuscript and PalomaPizarro from Bio-Iliberis R&D for CAS assays.

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Supporting information

Additional Supporting Information may be found in the onlineversion of this article:

Fig. S1. A. Southern blot of the total DNA from the wild-typeand mutant ORF49::WKm. DNA was digested with BamHIand the blot was hybridized with a DIG-labelled probe syn-thesized by amplification using Rep1 and Rep2 primers.B. Southern blot analysis of the total DNA digested with ScaIfrom wild-type and mutants ORF72:WKm (mcpT-1) andORF97:WKm (mcpT-2). The blot was hybridized with a Dig-labelled mcp probed synthesized by PCR with the Mcp-1 andMcp-2 oligonucleotides.Fig. S2. Putative RNA regulatory element of the antisense-RNA regulated loci.

A. Basic RNA unit that acts as antitoxin in Erwinia carotovora.B. Upstream DNA of the toxN gene are the basic RNAunits; the first ATG of the ToxN protein is highlighted by acircle.Fig. S3. Survival of P. putida DOT-T1E, P. putida DOT-T1E-100 and DOT-T1E-ORF34::WKm after exposure to ultravioletlight. Serial dilutions of the cultures of the different strainswere drop-plated and allowed to dry. Then plates wereexposed to ultraviolet light (254 nm) for 10 s, and incubatedovernight at 30°C.Fig. S4. A. CAS assay. Siderophore production was deter-mined by the production of a color change in CAS plates fromblue to orange P. aeruginosa PAO1 and Bacillus subtillis wereused as positive and negative controls. The four P. putidastrains submitted to the assay were DOT-T1E, DOT-TIE100 –deficient in pGRT1; a pGRT1 mutant with a knock-out atORF35 and this mutant complemented with the orf35 genecloned in pBBRMCS5 plasmid.B. Absorption spectra of the supernatants of cultures of thewild-type strain grown in the absence and in the presence ofiron. C) Absorption spectra of supernatants of the differentcultures grown in absence of iron.Table S1. Set of genes interrupting the plasmid backbone.Listed are genes found between coordinates 18927 and41331 together with their relevant homologies. Other detailsare shown in Fig. 1.Table S2. Genes borne on the Tn4653-like transposon.Genes highlighted in dark grey are within the two 7.6 invertedrepeat sequences. Light grey indicates genes within theinsertion sequence ISPpu12.Table S3. Relevant genes involved in replication and main-tenance of plasmid pGRT1. Genes that are homologs to theTA system are highlighted in grey.Table S4. Oligonucleotides used to amplify DNA for con-struction of mutants. The sequences are given in the 5’-> 3’direction and the size of the amplicon is indicated in basepairs (bp).

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Characterization of the pGRT1 plasmid 2327

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