A New Large-DNA-Fragment Delivery System Based on IntegraseActivity from an Integrative and Conjugative Element

Ryo Miyazaki,* Jan Roelof van der Meer

Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and geneticengineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and liga-tion, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new genedelivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-baseddelivery is that it can stably introduce a large DNA fragment (at least 75 kb) into one or more specific sites (the gene for glycine-accepting tRNA) on a target chromosome. Integrase recombination activity in Escherichia coli is kept low by using a synthetichybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integra-tion, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vectorin E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged withthe integrase gene. We confirmed that the system could successfully introduce cosmid and bacterial artificial chromosome (BAC)DNAs from E. coli into the chromosome of Pseudomonas putida in a site-specific manner. The integrase delivery system worksin concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathwaysin a variety of host bacteria.

Progress in biology and biotechnology has been largelyachieved with the help of genetic tools that allowed the anal-

ysis and relatively simple engineering of gene functions. Increas-ingly, however, synthetic biology trends focus on designing, con-structing and implanting very large genetic constructions such asoperons, in microorganisms. The work horse for DNA manipula-tion is still the typical plasmid vector deployed in well-character-ized species for gene cloning such as Escherichia coli. In caseswhere plasmid stability or gene dosage is an issue, or where chro-mosomal placement is more appropriate for gene expression,chromosomal delivery vehicles (e.g., minitransposons) (1) andhomologous recombination are preferred options. However, withmore specialized and tailored designs, and with increased sizes ofDNA fragments to be placed, appropriate genetic tools becomescarce, in particular where E. coli is not the final host chassis.Specialized E. coli vectors, such as cosmids, fosmids, and BAC(bacterial artificial chromosomes), are capable of carrying largeDNA inserts (20 to 100 kb) but cannot be stably maintained with-out antibiotic selection. Furthermore, rarely can such large vectorsbe propagated outside E. coli.

The aim of this study was thus to develop a new useful vectorsystem to stably introduce very large DNA fragments cloned andassembled in E. coli into a variety of host species among the Beta-and Gammaproteobacteria. The system is based on the integrationcapacity of the IntB13 integrase, a P4-family tyrosine recombi-nase-type integrase from the Pseudomonas knackmussii B13 inte-grative and conjugative element ICEclc (2). ICEclc is a 103-kbelement which is inserted in the 3= ends of genes for glycine-ac-cepting tRNA (tRNAGly) in the host chromosome (3). At low fre-quencies, ICEclc can excise by recombination between short directrepeats at either end (within the so-called attL and attR attach-ment sites) (Fig. 1A). The excised ICEclc can transfer to a newrecipient cell through its own conjugation apparatus (4, 5) andsubsequently integrates into the recipient chromosome via site-specific recombination between an 18-bp sequence at the 3= end of

the tRNAGly gene on the chromosome (attB) and the identicalsequence on the excised ICEclc (attP), thus restoring the tRNAGly

gene (2). Both excision and integration are mediated by the IntB13integrase, but, importantly, the regulation of intB13 expression isquite different in the integrated than in the excised ICEclc form.Expression of intB13 in its integrated state is controlled by theweak promoter Pint, which is active in only �3% of cells duringstationary phase, in particular in cultures grown on 3-chloroben-zoate or fructose (6, 7). In the excised form, however, intB13 isexpressed from a constitutively active promoter which normallyfaces outwards from the ICEclc element (named Pcirc) but which isdisplaced through the recombination between attL and attR to aposition upstream of Pint (Fig. 1A) (8). Temporary constitutiveexpression of intB13 results in successful integration of ICEclc intothe attB site on the new host chromosome and its renewed silenc-ing because of disconnection of Pcirc and intB13. Probably as aresult of the tRNAGly gene sequence conservation, ICEclc is capa-ble of being integrated into the chromosomes of a variety of Beta-and Gammaproteobacteria (9).

The characteristics of ICEclc and in particular of the Pint/Pcirc-regulated expression of intB13 are thus potentially interesting forthe development of a chromosomal delivery system for large DNAfragments in species other than E. coli. The system would have the

Received 6 March 2013 Accepted 10 May 2013

Published ahead of print 17 May 2013

Address correspondence to Ryo Miyazaki, ryo.miyazaki@unil.ch.

* Present address: Ryo Miyazaki, Bioproduction Research Institute, NationalInstitute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00711-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.00711-13

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advantages of (i) targeting a relatively concise chromosomal site(the tRNAGly gene), which is restored upon integration, (ii) deliv-ering large DNA fragments, and (iii) achieving stable integrationbecause of very poor intB13 expression in the integrated form.Here we developed two vector systems that exploit the intB13properties, one functioning as a direct cloning vector and theother as a mini-transposable element that can be integrated into,e.g., BAC or other vector clones. Both vectors are designed to beemployed in E. coli, from where they can be delivered either viatransformation or via conjugation to their final host, where theconstruct is integrated in the chromosome. We show how intB13expression can be controlled in a manageable form for E. coli toachieve levels which are neither too high nor toxic (2) yet whichare sufficient to achieve site-specific chromosomal recombinationin hosts outside E. coli. We demonstrate by using a specific Pseu-domonas putida strain as the host with a conditional ICEclc-spe-cific enhanced green fluorescent protein (EGFP) trap (9) howlarge DNAs can be prepared in E. coli and screened for integrationinto the P. putida chromosome, from where they can be expressed.Several convenient functions were added to the delivery system,such as removal of antibiotic selection cassettes after integrationthrough FRT (Flp recombination target) recombination.

MATERIALS AND METHODSBacterial strains and culture conditions. Escherichia coli JM109 (10) orJM109�pir (11) was routinely used for plasmid propagation and cloningexperiments. E. coli HB101(pRK2013) was used as a helper strain forconjugative delivery of minitransposon constructs (12). Pseudomonas

putida UWC1-2756, which carries a conditional egfp trap fused to an attBsite for ICEclc on its chromosome (9), was used to detect the site-specificintegration of DNAs via IntB13 recombination. Bacteria were grown inLuria-Bertani medium (LB) (13) or type 21C mineral medium (MM)(14). When necessary, the following antibiotics were used at the followingconcentrations (�g per ml): ampicillin, 100; kanamycin, 25; chloram-phenicol, 12.5; tetracycline, 100 (for P. putida) or 10 (for E. coli); andgentamicin, 20 (for P. putida) or 10 (for E. coli). E. coli was grown at 37°C;P. putida was grown at 30°C.

DNA techniques. PCR, plasmid, cosmid, and BAC DNA isolations,DNA fragment recovery, DNA ligations, transformations into E. coli, andrestriction enzyme digestions were all carried out according to standardprocedures (13) or to specific recommendations by the suppliers of themolecular biology reagents (Qiagen GmbH, Promega, and Stratagene).Oligonucleotides used in this study are listed in Table S1 in the supple-mental material. Sanger-type DNA sequencing was performed on an au-tomated DNA sequencer using a 3.1 Big-Dye kit (ABI Prism 3100 DNAsequencer; Applied Biosystems). Sequences were aligned and verified withthe help of the Lasergene software package (version 9; DNASTAR Inc.,Madison, WI).

Integrase vector constructions. The plasmids used in this study arelisted in Table 1. In order to control integrase expression in E. coli but tohave sufficiently high expression in P. putida, we designed four promotervariants. In all variants, intB13 is expressed from Pcirc, which is repressedin E. coli producing LacI from inserted lac operators (lacO) at differentpositions up- and downstream of the Pcirc promoter (Fig. 1). Two syn-thetic oligonucleotide DNAs containing the Pcirc region with lacO in dif-ferent positions, designated Pcirc-lacO1 and Pcirc-lacO2, were digestedwith EcoRI and AatII and cloned into pRR171, which contains the ICEclcattP region of the excised form. This resulted in plasmids pRMPlacO1 and

FIG 1 Schematic representation of the promoter region of intB13 on ICEclc. (A) Configurations of excised and integrated ICEclc. Note that Pint is a tightlyregulated bistable promoter, whereas Pcirc is a strong constitutive promoter. Triangles represent the recombination sites of IntB13, an 18-bp sequence identicalto the 18-bp 3= end of the tRNAGly gene. (B) Nucleotide sequence of the 450-bp attP region containing Pcirc and Pint on the excised ICEclc form. The �35/�10boxes of Pcirc are enclosed by bold rectangles, and the putative �35/�10 boxes of Pint are bounded by thin rectangles. The four insertion positions of the lacOsequence are indicated. Note that the lacO2 insertion replaced the spacer sequence between �35 and �10 boxes of Pcirc. The 18-bp recombination site andputative IHF binding motives are indicated by triangles and pentagons in gray, respectively. Arrows point to inverted repeat structures. The transcription startposition from Pcirc is underlined, and the ribosome-binding site (rbs) for intB13 is underlined.

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pRMPlacO2, respectively. The other two synthetic oligonucleotides con-tained lacO downstream of Pcirc within the Pint region and were desig-nated Pint-lacO3 and Pint-lacO4. These fragments were digested with XbaIand NdeI and were first cloned into pRR169, which contains the Pint-intB13 fragment, thereby replacing the native Pint fragment. The resultantplasmids were digested with AatII and NotI to release 1.1-kb fragmentscontaining a partial intB13 gene with either Pint-lacO3 or Pint-lacO4.These were subsequently fused downstream of a fragment containingPcirc. This fragment was amplified by PCR using primers (090908 and090805) from a plasmid containing the attP site (pCK218-jim4) (15)and digested with EcoRI and AatII. Subsequently, the AatII-NotI (1.1 kb)and EcoRI-AatII (0.2 kb) fragments were ligated with EcoRI- and NotI-digested pRR171, resulting in plasmids pRMPlacO3 and pRMPlacO4,carrying the Pcirc-lacO3-intB13 and Pcirc-lacO4-intB13 modules, respec-tively (see Fig. S1A in the supplemental material).

In order to add more utility to the vector for cloning and delivery, wetook advantage of the exclusive replication of plasmids carrying the oriR6K

in hosts expressing the Pir protein (18). A 420-bp fragment containingoriR6K was amplified by PCR using primers (120301 and 120302) and theminitransposon delivery vector pBAM1 as a template (1). The PCR prod-uct was digested by EcoRI and NheI and ligated with pRMPlacO4 digestedwith EcoRI and XbaI to replace orip15A. The resultant plasmid carryingoriR6K was opened by NcoI and ScaI and ligated with a 67-bp fragmentcontaining a FRT site, which had been amplified with primers (120303and 120304) and pUC18miniTn7T-LAC as a template. A second FRTcopy was included in the primer to amplify the oriT of pBAM1 by PCRusing primers (120305 and 120306), and the oriT-FRT fragment was in-troduced into FseI-NsiI sites on the plasmid. The resultant plasmid wasfurther digested by EcoRI and ApaI and ligated with a 360-bp fragmentcontaining multiple cloning sites (MCS) and two transcriptional termi-nators (T0T1), which was amplified by using primers (120307 and 120308)and pUC18miniTn7T-LAC as a template. The final plasmid, pRMR6K-Tc, was digested by ScaI and FseI to replace the tetracycline resistance genewith kanamycin or gentamicin resistance genes, of which fragments wereamplified by PCR using primers 120504 and 120505 with pPROBE=-gfp

for kanamycin and primers 120506 and 120507 with pUC18miniTn7T-LAC for gentamicin. The two variants in antibiotic resistance were namedpRMR6K-Km and pRMR6K-Gm.

In order to produce a mini-transposable element carrying the Pcirc-intB13 cassettes, we digested pBAM1 with AatII and HindIII, and treatedthe linearized DNA with T4 polymerase (Fermentas). Then the bluntedfragment was self-ligated to remove the kanamycin resistance gene. Theresulting plasmid pBAM1nokana was digested with FseI and PshAI andtreated with T4 polymerase. Then the blunted fragment was again self-ligated to cure it of the indigenous oriT region (but this region was againreplaced later). The plasmid was subsequently opened by digestion withSalI and PstI and ligated with the XhoI-NsiI-digested fragment ofpRMR6K-Tc that includes the Pcirc-lacO4-intB13 module (and a neworiT), generating pRMTn-Tc. The same procedure was carried out togenerate pRMTn-Km and pRMTn-Gm from pRMR6K-Km andpRMR6K-Gm, respectively (see Fig. S1B in the supplemental material).

Reporter plasmid constructions. To measure expression from Pcirc inthe various constructs, the four Pcirc-lacO fragments with different lacOpositions were amplified by using primers (090908 and 090805) andpRMPlacO1, pRMPlacO2, pRMPlacO3, or pRMPlacO4 as the template.As a positive control, the original Pcirc was also amplified using the sameprimer set and pCK218-jim4. As a negative control, Pint was amplifiedusing primers 090803 and 090805 and plasmid pRR171 as the template.All PCR products were digested by EcoRI and BamHI and then clonedseparately in front of the promoterless egfp gene on the broad-host-rangeplasmid pPROBE=-gfp (17).

Fluorescence assays. E. coli JM109 or P. putida UWC1 carryingpPROBE=-derived plasmids were precultured in LB with kanamycin over-night. The cultures were diluted in triplicate at 1:1,000 into fresh mediawith or without IPTG (isopropyl-�-D-thiogalactopyranoside; 1 mM) andincubated until mid-exponential phase (optical density [OD] � 0.5). Avolume of 200 �l of the cultures was transferred into 96-well black micro-titer plates (Greiner Bio-one) with a flat transparent bottom, and bothculture turbidity (A600) and fluorescence emission (excitation at 480 nmand emission at 520 nm) were measured using a Fluostar fluorescencemicroplate reader (BMG Lab Technologies). Fluorescence intensitieswere normalized to culture density.

Nucleotide sequence accession numbers. The nucleotide sequencesof plasmids were submitted to GenBank under the following accessionnumbers: pRMR6K-Tc, AB777647; pRMR6K-Km, AB777648; pRMR6K-Gm, AB777649; pRMTn-Tc, AB777650; pRMTn-Km, AB777651;pRMTn-Gm, AB777652.

RESULTSConstructing a hybrid promoter to control integrase expres-sion. In order to diminish intB13 expression in E. coli but stillallow sufficient expression for chromosomal integration in hostsother than E. coli, we constructed and tested several promotervariants. Previously, we had observed that a pACYC-based plas-mid carrying intB13 downstream of the 450-bp region containingPcirc as in the attP region (Fig. 1A) of excised and recircularizedICEclc caused severe growth inhibition of E. coli (2). To control theexpression of intB13, we first tried to clone the gene either underthe control of the lac promoter (Plac) in E. coli lacIq or downstreamof the 2-hydroxybiphenyl-inducible hbpC promoter (PhbpC) in E.coli expressing the HbpR transcriptional regulator (19). Bothcases, however, did not produce any proper transformants in E.coli, suggesting that leaky expression of intB13 still caused lethaleffects on the host cells. Hence, we modified the 450-bp Pcirc-attPregion by introducing lacO sequences at different positions toblock transcription by binding of the LacI repressor (Fig. 1B). Thepositions of lacO insertions were chosen without disrupting the�35/�10 boxes of Pcirc and the 18-bp recombination site. In or-der to measure the relative promoter “strengths” in those variants,

TABLE 1 Plasmids used in this study

Plasmid Relevant characteristics Reference

pBAM1 mini-Tn5 vector, Kmr 1pRR169 pET3c derivative carrying intB13 2pRR171 pACYC184 derivative carrying Pint-intB13 2pCK218-jim4 pCK218 derivative carrying Pcirc 15pUC18miniTn7T-LAC pUC18 derivative carrying FRT and T0T1, Gmr 16pPROBE=-gfp Broad-host-range reporter vector derived from

pBBR1MCS-2, Kmr17

pPROBE=R pPROBE=-gfp derivative carrying Pint This studypPROBE=P pPROBE=-gfp derivative carrying Pcirc This studypPROBE=O1 pPROBE=-gfp derivative carrying Pcirc-lacO1 This studypPROBE=O2 pPROBE=-gfp derivative carrying Pcirc-lacO2 This studypPROBE=O3 pPROBE=-gfp derivative carrying Pcirc-lacO3 This studypPROBE=O4 pPROBE=-gfp derivative carrying Pcirc-lacO4 This studypRMPlacO1 pACYC184 derivative carrying

Pcirc-lacO1-intB13This study

pRMPlacO2 pACYC184 derivative carryingPcirc-lacO2-intB13

This study

pRMPlacO3 pACYC184 derivative carryingPcirc-lacO3-intB13

This study

pRMPlacO4 pACYC184 derivative carryingPcirc-lacO4-intB13

This study

pRMR6K-Tc R6K replicon, Pcirc-lacO4-intB13, oriT, FRT,MCS, Tcr

This study

pRMR6K-Km R6K replicon, Pcirc-lacO4-intB13, oriT, FRT,MCS, Kmr

This study

pRMR6K-Gm R6K replicon, Pcirc-lacO4-intB13, oriT, FRT,MCS, Gmr

This study

pRMTn-Tc pBAM1 derivative carrying Pcirc-lacO4-intB13,oriT, FRT, MCS, Tcr

This study

pRMTn-Km pBAM1 derivative carrying Pcirc-lacO4-intB13,oriT, FRT, MCS, Kmr

This study

pRMTn-Gm pBAM1 derivative carrying Pcirc-lacO4-intB13,oriT, FRT, MCS, Gmr

This study

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we cloned the modified promoter fragments (Pcirc-lacO1, -lacO2,-lacO3, and -lacO4) in the pBBR1MCS-based reporter plasmidpPROBE=-gfp and measured EGFP fluorescence in the presenceor absence of IPTG induction. In E. coli JM109 (lacIq), all fourfragments produced �10-fold-lower EGFP expression than wild-type Pcirc without lacO (Fig. 2B). EGFP expression increased morethan 4-fold after addition of 1 mM IPTG but was less than EGFPexpression from wild-type Pcirc. This result shows that Pcirc activitycan be repressed in E. coli by LacI binding at lacO and derepressedby addition of IPTG. Since all fragments also contain the weak Pint

promoter, we examined EGFP expression from Pint in the sameassay. As expected, EGFP expression from Pint alone was indistin-guishable from a negative control with only vector (Fig. 2B). Totest the resulting promoter activities outside E. coli, we introducedthe series of reporter plasmids into P. putida UWC1. Since UWC1does not carry lacIq, it cannot repress Pcirc, and EGFP expressionwill thus indicate the promoter strength in the host where theintegration by IntB13 is supposed to take place. Indeed, EGFP wasclearly produced in all UWC1 transformants, except those withthe plasmid carrying egfp under the control of Pint. The relativelevels of normalized EGFP fluorescence in UWC1 carrying thedifferent plasmids were similar to those in E. coli after addition ofIPTG, except for UWC1 carrying Pcirc-lacO4, which showed ac-tivity comparable to that obtained with wild-type Pcirc (Fig. 2C).

Site-specific integration of integrase plasmids. We decidedthat the LacIq-lacO variants might thus give sufficient intB13downregulation in E. coli while allowing high expression in P.

putida to ensure integration. The modified Pcirc promoters wereplaced upstream of the intB13 gene in its wild-type configurationon a pACYC-derived plasmid and then introduced into E. coliJM109. The transformants successfully grew on the selective me-dia, whereas cells transformed with a similar plasmid containingwild-type Pcirc did not. This indicated again that uncontrolledPcirc-intB13 expression is lethal in E. coli but also that we hadreduced intB13 expression sufficiently by adding lacO sequencesto protect the cells from this lethality. The plasmids, named pRM-PlacO1, pRMPlacO2, pRMPlacO3, and pRMPlacO4, were thuspurified from E. coli and then introduced by electroporation intoP. putida UWC1-2756. This strain carries four possible attB targetsites (at the 3= ends of the tRNAGly-3, tRNAGly-4, tRNAGly-5, andtRNAGly-6 genes) plus an artificial integration site fused to a con-ditional egfp trap (9). The pACYC-derived plasmids are incapableof replicating in UWC1-2756, and we thus expected integration ofthe plasmids in one of the five target sites. Intriguingly, transfor-mants resistant to tetracycline were obtained only in case ofusing pRMPlacO3 or pRMPlacO4, while no cells grew withpRMPlacO1, pRMPlacO2, or pRR171 (Table 2). Approximately20% of colonies showed green fluorescence, indicating that pRM-PlacO3 or pRMPlacO4 had specifically inserted into the egfp trapon the chromosome. Site-specific integrations of the plasmidsinto one of four original attB sites (i.e., genes for tRNAGly-3, -4 or-5) in the nonfluorescent transformants were confirmed by col-ony PCR to detect junctions of attB and attP (Fig. 3). To test thestability of insertions, we cultured one nongreen clone of P. putida

FIG 2 In vivo reporter assays of the promoter activities of the various integrase promoter constructs. (A) Schematic representation of Pint, wild-type Pcirc, andmodified Pcirc promoters, cloned in pPROBE=-gfp. Names of plasmids and promoters are shown on the right. The wild-type Pcirc and Pint fragments correspondto 450-bp and 303-bp (lacking 147 bp at the 5= end of Pcirc-wt) regions in Fig. 1B, respectively. Positions of lacO insertions are indicated by filled circles. (B)Normalized EGFP fluorescence expressed from the promoter constructs cloned in pPROBE=-gfp in E. coli JM109. Cells were grown in the presence (white bars)or absence (gray bars) of 1 mM IPTG. EGFP fluorescence values were normalized by culture density (A600). The bars represent the means; error bars indicatestandard deviations calculated from triplicates. (C) Same as panel B but expressed in P. putida UWC1 (without IPTG).

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with integrated pRMPlacO4 for 30 generations in liquid culturewithout antibiotic selection (three subsequent transfers) andscreened for the appearance of green colonies every tenth genera-tion, under the assumption that excision of the construct would befollowed at a detectable frequency by reintegration into the egfptrap. However, no green colony was detected among 300 coloniesscreened every tenth generation, suggesting that the integrationsremained stable. These results showed that pRMPlacO3 andpRMPlacO4 can function as site-specific integration vectors. Incontrast, pRMPlacO1 and pRMPlacO2 did not work, perhaps be-cause insertions of the lacO sequence at positions lacO1 and lacO2disrupt cis-acting sequences needed for efficient integrase activity,such as IHF binding sites or repeat structures. Most tyrosine-typerecombinases, to which IntB13 belongs, need additional factorsfor efficient recombination that interact with sequences sur-rounding the actual 18-bp recombination site.

Development of a site-specific integration system. PlasmidpRMPlacO3 and pRMPlacO4 thus functioned as vectors for site-specific integration of DNA in P. putida. To make them moreamenable to cloning and delivery, we added several genetic op-tions to pRMPlacO4. The origin of replication was replaced withoriR6K, which is low copy number but replicates only in �pirstrains, allowing to force chromosomal integration even in E. colistrains as hosts (data not shown). An origin-of-transfer sequence(oriT) was added in order to mobilize the plasmid from E. coli toits final host via bi- or triparental matings. A multiple cloning site(MCS) was added with transcriptional terminators (T0T1), andthree variants of plasmid were made with different antibiotic re-sistance genes. The antibiotic resistance genes are removable afterchromosomal integration through recombination at two FRTsites induced by a plasmid carrying the yeast FLP recombinase.The improved plasmids, named pRMR6K-Tc, pRMR6K-Km, andpRMR6K-Gm, thus function as suicide vectors and deliver DNAfragments (�10 kb) which can be cloned into the MCS by routinecloning techniques (Fig. 4A).

Development of a mini-transposable integrase vector forlarge fragment delivery. In order to obtain a system that wouldenable chromosomal integration of very large DNA moleculesproduced in E. coli, we decided to produce a mini-transposableintegrase fragment that can be randomly inserted into such largeDNA molecule in E. coli itself and then be delivered into its finalhost. To do this, we added a gene encoding a Tn5-family trans-posase (tnpA) and its cognate inverted repeat (IR) sequences tothe pRMR6K plasmids, generating pRMTn-Tc, pRMTn-Km, andpRMTn-Gm (Fig. 4B). The newer plasmids would in principle becapable of transposition of the module containing intB13, Pcirc-

lacO4, oriT, an antibiotic resistance gene, FRT sites, MCS, andT0T1 to other DNA substrates.

To illustrate the ability of the site-specific integration systemon minitransposons to introduce large DNA fragments into thehost chromosome, we tested a cosmid and two BAC in E. coli. Asan example, cosmid 1G3 contains an �25 kb fragment of ICEclc(3), including the genes for chlorocatechol degradation (clc). In-troduction of the clc genes in P. putida leads to complementationof a metabolic pathway which enables the host to use 3-chloro-benzoate as the sole carbon and energy source (20). pRMTn-Tcwas introduced by electroporation into an E. coli host without �pirthat already contained cosmid 1G3. Approximately 500 transfor-mants were obtained on LB agar with ampicillin and tetracycline,indicating that the transformants contained minitransposon in-sertions either in cosmid 1G3 or in the chromosome. To selectonly cosmids with minitransposon insertions, we picked 50 trans-formants at random and cultured them in pools of 10 transfor-mants each in LB containing both antibiotics. Cosmids were iso-lated from those cultures and used to retransform E. coli JM109 byelectroporation. In three out of five transformations, coloniesshowing resistance to both antibiotics were obtained, indicatingthat cells received cosmids with inserted minitransposons. Therestriction digestion pattern of cosmids isolated from the trans-formants indicated the insertion of the transposon (data notshown). One of such cosmids, designated 1G3-O4, was then in-troduced into P. putida UWC1-2756 by electroporation. From 2�g of cosmid 1G3-O4 a total of 30 P. putida transformants wasobtained, of which �20% were green fluorescent. All transfor-mants had a single-copy integration of 1G3-O4 at one of five tar-get sites on the chromosome. Moreover, the P. putida transfor-mants could utilize 3-chlorobenzoate as a sole carbon source,indicating successful expression of the clc genes in their insertedposition (Fig. 5A). These results demonstrated that the minitrans-poson from pRMTn-Tc conferred site-specific integration abilityto the cosmid which carries the clc genes and that transformantscan consequently grow on the specific carbon source.

To test the capacity for integrating even larger DNA frag-ments, pRMTn-Gm was introduced by electroporation into E.coli harboring BAC clones. We used two BACs, namedMED49C08 (insert, 75 kb) and MED66A03 (53 kb), containing

FIG 3 PCR analysis of site-specific integration of integrase plasmids into P.putida UWC1-2756. Amplification of the junctions between plasmids and tar-get sites of the chromosome were performed using two randomly chosentransformants as templates (16, 25) for each of the integrated plasmids.“Green” refers to P. putida transformants expressing EGFP, indicating inser-tion in the conditional trap; “nongreen” refers to P. putida transformants notexpressing EGFP, indicating insertion in one of the tRNAGly genes. Target sitesand expected amplicon sizes are shown on the right. M, Massruler DNA laddermix (Fermentas).

TABLE 2 Site-specific integration of plasmids into P. putida UWC1-2756

Plasmid Integration into attBa

Proportion of greentransformantsb

pRR171 NopRMPlacO1 NopRMPlacO2 NopRMPlacO3 Yes (6.7 102 2.2 102) 0.18 0.01pRMPlacO4 Yes (4.7 103 1.2 103) 0.19 0.07a Transformation efficiency (CFU/�g of DNA) is given in parentheses.b Proportion of the number of green fluorescent transformant colonies to the totalnumber of colonies.

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uncultured bacterium DNA with genes for photorhodopsinsfrom deep Mediterranean seawater (21). In both cases, approxi-mately 250 E. coli transformants showing resistance to both chlor-amphenicol (from the BAC) and gentamicin (from pRMTn-Gm)were obtained, indicating possible transposition of the integrasemodule into the BAC. The transformants were pooled as a mix-ture, regrown, and mixed with E. coli HB101/pRK2013 and P.putida UWC1-2756 to pass minitransposed integrase-BAC DNAsby triparental mating into P. putida. This resulted in �100UWC1-2756-derived transconjugant colonies for each of the BACclones tested that could grow on MM with succinate plus genta-micin. We confirmed that those transconjugants had a single spe-cific integration of a BAC fragment in one of the target attachmentsites (Fig. 5B). The proportion of EGFP-expressing transconju-gants was �20% in the case of MED66A03 integration, whereasalmost all transconjugants (�95%) obtained in the case ofMED49C08 were green fluorescent. This bias might be due tosome negative effects of MED49C08 integration into the othertarget attB sites. Unfortunately, photorhodopsin expression couldnot be detected in P. putida. We confirmed that in both cases offive randomly chosen transconjugants, all contained at least threeBAC-specific regions, suggesting the successful integration of theentire BAC into the chromosome of P. putida (Fig. 5C).

DISCUSSION

We showed here the successful development and usage of a chro-mosomal delivery system for large DNA fragments in host bacteria

outside E. coli (notably, P. putida). The system is based on theproperties of the IntB13 integrase from the ICEclc element of P.knackmussii B13 to integrate (at least) 75 kb DNA into the 3= 18-bpof the tRNAGly gene. The most crucial point for constructing theintegrase-based DNA delivery system was proper downregulationof intB13 expression in E. coli while maintaining sufficiently highexpression in the host cell where the DNA needs to be integrated.In previous work, even a low-copy-number plasmid carrying theintB13 gene downstream of the 450-bp Pcirc-attP fragment hadcaused severe growth inhibition of E. coli (2) and accumulatedDNA rearrangements (data not shown). Although we first tried toapply the Plac-LacI or PhbpC-HbpR regulatory system to controlintB13 expression in E. coli, no transformants with the correctconstructs were obtained, suggesting that some kind of detrimen-tal effect was still associated with leaky expression of intB13.Hence, we decided to use the original Pcirc-Pint promoters forintB13 control but repress Pcirc by adding lacO sites for LacIq re-pression. Under consideration of the positions of �35/�10 boxesof Pcirc and the 18-bp recombination sequence, we synthesizednew Pcirc promoters having lacO insertions at different positions.Two of the new promoters, those having lacO insertions at lacO3or lacO4, could integrate into the P. putida chromosomal targetsites, whereas those with lacO1 and lacO2 could not. As a typicalattP site for a tyrosine recombinase, similar to � integrase (22), thePcirc region contains two and one IHF binding motifs located up-and downstream of the 18-bp recombination site, respectively(Fig. 1B). IHF bends the DNA and assists the integrase to form a

FIG 4 Schematic representation of the integrase-based DNA delivery systems. (A) Suicide plasmid version for E. coli cloning of DNA fragments to be integratedin a final (different) host. Antibiotic resistance gene and oriT are removable by FRT/FLP recombination. Note that the situation represented here is for integrationinto the artificial conditional egfp trap, but would be similar for one of the cognate the tRNAGly gene integration sites. (B) Minitransposon version for tagginglarge DNA replicons in E. coli and subsequent chromosomal delivery to the final host. The minitransposon part is indicated by a magenta double arrow.

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Holliday junction (23). The Pcirc region also contains several re-peat sequences, which may act as binding sites for IntB13 or otheraccessory proteins for the recombination process. The insertionpositions of lacO1 and lacO2 appear to disrupt or touch one suchmotif (Fig. 1B), which is possibly the main reason for the failure ofintegration.

We further added several genetic options to both pRMR6K andpRMTn series of plasmids (Fig. 4). In particular, FRT sites may beuseful after integration to remove antibiotic resistance genesthrough recombination by a temporarily introduced FLP recom-binase (24). Although a few scars of the integrated plasmids stillremain on the chromosome even after FLP/FRT recombination,such as IRs, FRT, and oriR6K, they should not influence the cellgrowth or host genome structure. One may point out that theintB13 gene also remains after integration and may thus poten-tially lead to excision of the construct, although the requirementfor a separate excisionase has not been demonstrated. However,intB13 is completely silent in the integrated form because of itsphysical separation from Pcirc upon integration and because itlacks necessary other factors from ICEclc which are essential for itsexpression from Pint (6; S. Sulser, unpublished data; N. Prader-vand, unpublished data). Therefore, renewed excision should beminimal and rarely occur. Another unique possibility with thepRMTn plasmids is integrase tagging of DNA molecules via mini-transposition. This avoids specific cloning of large molecules inintegrase-bearing vectors. We showed that the Pcirc-lacO4-intB13module can transpose in vivo into cosmids and BACs. In addition,in vitro tagging by using purified Tn5 transposase (e.g., as in the

commercial EZ-Tn5 kit) can be imagined. After appropriate ver-ification of the integration, the tagged substrates are able to site-specifically integrate into target chromosomes without furthersubcloning steps.

Since the system is based on the IntB13 integrase from ICEclc,target chromosomes need to have a cognate attB site. The strainused as a target in this study, P. putida UWC1, has six tRNAGly

genes, and four of those six have perfect 18-bp sequence matchesto the ICEclc recombination sequence (i.e., tRNAGly-3, tRNAGly-4,tRNAGly-5, and tRNAGly-6 genes). Importantly, the tRNAGly genesequence is restored upon integration; therefore, there is no effectof integration on translation or codon efficiency. Most gamma-proteobacteria, including E. coli, Salmonella, Pseudomonas, Yer-sinia, and Erwinia, have 18-bp recombination sites in some oftheir tRNAGly genes that are completely identical to ICEclc, andseveral beta- and gammaproteobacteria are natural recipients forICEclc (9). Moreover, we have shown that IntB13 can recombinewith attB recombination sites carrying a few mismatches (i.e., atleast two mismatches in the 18 bp), albeit at lower frequencies (9).This should allow a wider variety of bacterial genomes as deliverytargets, such as Burkholderia xenovorans, Bordetella petri, Cupria-vidus necator, Xylella fastidiosa, and Xanthomonas campestris (9).To further broaden the target host range, one could first introducean artificial (but perfect) attB site with a promoterless egfp gene ona minitransposon such as plasmid pCK218-jimX, into which theIntB13 system should then be able to deliver its target.

Several vector systems that can introduce foreign DNA intotarget genomes have been developed and are used in molecular

FIG 5 Characterization of site-specific integration of large DNA molecules into P. putida UWC1-2756. (A) Cell growth on MM plates with 5 mM 3-chloro-benzoate as the sole carbon source. Note that the transformant with the integrase-tagged cosmid 1G3-O4 containing the clc genes (left) but not P. putidaUWC1-2756 (right) successfully grew. (B) PCR amplification of the junctions between integrase-tagged BACs (MED49C08 or MED66A03) and target sites of thechromosome. Randomly chosen transformants were used as templates. Note that only green fluorescent P. putida colonies were obtained from the transforma-tion with MED49C08. Target sites and expected amplicon sizes are shown on the right. M, Massruler DNA ladder mix (Fermentas). (C) PCR amplification ofBAC internal regions. Three regions named “start,” “middle,” and “end” in BACs, MED49C08 (left), and MED66A03 (right) were tested. Randomly chosentransformants were used as templates. Nucleotide positions of targets refer to the sequence available under accession number DQ077554 for MED49C08 andDQ065755 for MED66A03, and expected amplicon sizes (in parentheses) are shown on the right. N, P. putida UWC1-2756 (negative control); P, each BAC DNA(positive control); M, Massruler DNA ladder mix (Fermentas).

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genetics. The most sophisticated systems are perhaps the mini-Tn7 and mini-Tn5 vectors, which, however, are limited to deliv-ering relatively short DNA fragments (��10 kb) into the targetchromosome with and without site specificity, respectively (1, 16,25). Integration-proficient plasmids carrying a modified gene for�CTX integrase, which also belongs to the tyrosine recombinasefamily, have been developed for engineering P. aeruginosa PAO1(26). The plasmids contain the CTX cognate attP site and there-fore can site-specifically integrate into the native attB site onPAO1, which is formed by the 3= end of the gene for serine-accept-ing tRNA. An MCS is available for precise cloning, but the clon-able size is likely limited, as in the case of mini-Tn vectors. Tointroduce environmental DNA libraries into several host bacteria,a BAC shuttle vector system that carries a gene for �C31 integrase(serine recombinase) and its cognate attP has been reported (27).The vector could integrate at least a 38-kb fragment into both P.putida and Streptomyces lividans chromosomes, using a gene forapramycin resistance as a selective marker. The ICEclc-based sys-tem developed here differs from previous integration-delivery sys-tems in the size of the deliverable fragment and the configurationof the integrase control.

We can thus conclude that the new integrase-based DNA de-livery system developed here has a huge potential for moleculargenetics, and in particular for synthetic biology. The system worksin concert with existing vector systems and can introduce largeDNA fragments into chromosomes, forming a useful aid in theconstruction of new metabolic pathways. Since tyrosine recombi-nases also function in mammalian cells (23, 28), further modifi-cations to the system might make it possible to use it as a deliverysystem for eukaryotic organisms.

ACKNOWLEDGMENTS

We thank Oded Béjà for the kind gift of BAC DNAs.R.M. was supported by a postdoctoral fellowship from the Japan So-

ciety for the Promotion of Science for Research Abroad and by a grantfrom the Swiss National Science Foundation (31003A-124711/1). Thiswork was further supported by a grant from the FP7 project TARPOL(KBBE-212894).

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