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

Abstract

During the past few decades, numerous plasmid vectors have been developed for cloning, gene expression analysis, and genetic engineering. Cloning procedures typically rely on PCR amplification, DNA fragment restriction digestion, recovery, and ligation, but increasingly, procedures are being developed to assemble large synthetic DNAs. In this study, we developed a new gene delivery system using the integrase activity of an integrative and conjugative element (ICE). The advantage of the integrase-based delivery 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 synthetic hybrid promoter, which, however, is unleashed in the final target host, forcing the integration of the construct. Upon integration, the system is again silenced. Two variants with different genetic features were produced, one in the form of a cloning vector in E. coli and the other as a mini-transposable element by which large DNA constructs assembled in E. coli can be tagged with the 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 works in concert with existing vector systems and could thus be a powerful tool for synthetic constructions of new metabolic pathways in a variety of host bacteria.

INTRODUCTION

Progress in biology and biotechnology has been largely achieved with the help of genetic tools that allowed the analysis and relatively simple engineering of gene functions. Increasingly, however, synthetic biology trends focus on designing, constructing and implanting very large genetic constructions such as operons, in microorganisms. The work horse for DNA manipulation is still the typical plasmid vector deployed in well-characterized species for gene cloning such as Escherichia coli. In cases where plasmid stability or gene dosage is an issue, or where chromosomal placement is more appropriate for gene expression, chromosomal delivery vehicles (e.g., minitransposons) (1) and homologous recombination are preferred options. However, with more specialized and tailored designs, and with increased sizes of DNA fragments to be placed, appropriate genetic tools become scarce, 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 large DNA inserts (20 to 100 kb) but cannot be stably maintained without antibiotic selection. Furthermore, rarely can such large vectors be propagated outside E. coli.

The aim of this study was thus to develop a new useful vector system to stably introduce very large DNA fragments cloned and assembled in E. coli into a variety of host species among the Beta- and Gammaproteobacteria. The system is based on the integration capacity of the IntB13 integrase, a P4-family tyrosine recombinase-type integrase from the Pseudomonas knackmussii B13 integrative and conjugative element ICEclc (2). ICEclc is a 103-kb element which is inserted in the 3′ ends of genes for glycine-accepting tRNA (tRNA^Gly) in the host chromosome (3). At low frequencies, ICEclc can excise by recombination between short direct repeats at either end (within the so-called attL and attR attachment sites) (Fig. 1A). The excised ICEclc can transfer to a new recipient cell through its own conjugation apparatus (4, 5) and subsequently integrates into the recipient chromosome via site-specific recombination between an 18-bp sequence at the 3′ end of the tRNA^Gly gene on the chromosome (attB) and the identical sequence on the excised ICEclc (attP), thus restoring the tRNA^Gly gene (2). Both excision and integration are mediated by the IntB13 integrase, but, importantly, the regulation of intB13 expression is quite different in the integrated than in the excised ICEclc form. Expression of intB13 in its integrated state is controlled by the weak promoter P_int, which is active in only ∼3% of cells during stationary phase, in particular in cultures grown on 3-chlorobenzoate or fructose (6, 7). In the excised form, however, intB13 is expressed from a constitutively active promoter which normally faces outwards from the ICEclc element (named P_circ) but which is displaced through the recombination between attL and attR to a position upstream of P_int (Fig. 1A) (8). Temporary constitutive expression of intB13 results in successful integration of ICEclc into the attB site on the new host chromosome and its renewed silencing because of disconnection of P_circ and intB13. Probably as a result of the tRNA^Gly gene sequence conservation, ICEclc is capable of being integrated into the chromosomes of a variety of Beta- and Gammaproteobacteria (9).

Fig 1.

Schematic representation of the promoter region of intB13 on ICEclc. (A) Configurations of excised and integrated ICEclc. Note that P_int is a tightly regulated bistable promoter, whereas P_circ is a strong constitutive promoter. Triangles represent the recombination sites of IntB13, an 18-bp sequence identical to the 18-bp 3′ end of the tRNA^Gly gene. (B) Nucleotide sequence of the 450-bp attP region containing P_circ and P_int on the excised ICEclc form. The −35/−10 boxes of P_circ are enclosed by bold rectangles, and the putative −35/−10 boxes of P_int are bounded by thin rectangles. The four insertion positions of the lacO sequence are indicated. Note that the lacO2 insertion replaced the spacer sequence between −35 and −10 boxes of P_circ. The 18-bp recombination site and putative IHF binding motives are indicated by triangles and pentagons in gray, respectively. Arrows point to inverted repeat structures. The transcription start position from P_circ is underlined, and the ribosome-binding site (rbs) for intB13 is underlined.

The characteristics of ICEclc and in particular of the P_int/P_circ-regulated expression of intB13 are thus potentially interesting for the development of a chromosomal delivery system for large DNA fragments in species other than E. coli. The system would have the advantages of (i) targeting a relatively concise chromosomal site (the tRNA^Gly gene), which is restored upon integration, (ii) delivering large DNA fragments, and (iii) achieving stable integration because of very poor intB13 expression in the integrated form. Here we developed two vector systems that exploit the intB13 properties, one functioning as a direct cloning vector and the other as a mini-transposable element that can be integrated into, e.g., BAC or other vector clones. Both vectors are designed to be employed in E. coli, from where they can be delivered either via transformation or via conjugation to their final host, where the construct is integrated in the chromosome. We show how intB13 expression can be controlled in a manageable form for E. coli to achieve levels which are neither too high nor toxic (2) yet which are sufficient to achieve site-specific chromosomal recombination in hosts outside E. coli. We demonstrate by using a specific Pseudomonas putida strain as the host with a conditional ICEclc-specific enhanced green fluorescent protein (EGFP) trap (9) how large DNAs can be prepared in E. coli and screened for integration into 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 integration through FRT (Flp recombination target) recombination.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Escherichia coli JM109 (10) or JM109λpir (11) was routinely used for plasmid propagation and cloning experiments. E. coli HB101(pRK2013) was used as a helper strain for conjugative delivery of minitransposon constructs (12). Pseudomonas putida UWC1-2756, which carries a conditional egfp trap fused to an attB site for ICEclc on its chromosome (9), was used to detect the site-specific integration of DNAs via IntB13 recombination. Bacteria were grown in Luria-Bertani medium (LB) (13) or type 21C mineral medium (MM) (14). When necessary, the following antibiotics were used at the following concentrations (μg per ml): ampicillin, 100; kanamycin, 25; chloramphenicol, 12.5; tetracycline, 100 (for P. putida) or 10 (for E. coli); and gentamicin, 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, and restriction enzyme digestions were all carried out according to standard procedures (13) or to specific recommendations by the suppliers of the molecular biology reagents (Qiagen GmbH, Promega, and Stratagene). Oligonucleotides used in this study are listed in Table S1 in the supplemental material. Sanger-type DNA sequencing was performed on an automated DNA sequencer using a 3.1 Big-Dye kit (ABI Prism 3100 DNA sequencer; Applied Biosystems). Sequences were aligned and verified with the help of the Lasergene software package (version 9; DNASTAR Inc., Madison, WI).

Integrase vector constructions.

The plasmids used in this study are listed in Table 1. In order to control integrase expression in E. coli but to have sufficiently high expression in P. putida, we designed four promoter variants. In all variants, intB13 is expressed from P_circ, which is repressed in E. coli producing LacI from inserted lac operators (lacO) at different positions up- and downstream of the P_circ promoter (Fig. 1). Two synthetic oligonucleotide DNAs containing the P_circ region with lacO in different positions, designated P_circ-lacO1 and P_circ-lacO2, were digested with EcoRI and AatII and cloned into pRR171, which contains the ICEclc attP region of the excised form. This resulted in plasmids pRMPlacO1 and pRMPlacO2, respectively. The other two synthetic oligonucleotides contained lacO downstream of P_circ within the P_int region and were designated P_int-lacO3 and P_int-lacO4. These fragments were digested with XbaI and NdeI and were first cloned into pRR169, which contains the P_int-intB13 fragment, thereby replacing the native P_int fragment. The resultant plasmids were digested with AatII and NotI to release 1.1-kb fragments containing a partial intB13 gene with either P_int-lacO3 or P_int-lacO4. These were subsequently fused downstream of a fragment containing P_circ. This fragment was amplified by PCR using primers (090908 and 090805) 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 P_circ-lacO3-intB13 and P_circ-lacO4-intB13 modules, respectively (see Fig. S1A in the supplemental material).

Table 1.

Plasmids used in this study

Plasmid	Relevant characteristics	Reference
pBAM1	mini-Tn5 vector, Kmr	1
pRR169	pET3c derivative carrying intB13	2
pRR171	pACYC184 derivative carrying Pint-intB13	2
pCK218-jim4	pCK218 derivative carrying Pcirc	15
pUC18miniTn7T-LAC	pUC18 derivative carrying FRT and T0T1, Gmr	16
pPROBE′-gfp	Broad-host-range reporter vector derived from pBBR1MCS-2, Kmr	17
pPROBE′R	pPROBE′-gfp derivative carrying Pint	This study
pPROBE′P	pPROBE′-gfp derivative carrying Pcirc	This study
pPROBE′O1	pPROBE′-gfp derivative carrying Pcirc-lacO1	This study
pPROBE′O2	pPROBE′-gfp derivative carrying Pcirc-lacO2	This study
pPROBE′O3	pPROBE′-gfp derivative carrying Pcirc-lacO3	This study
pPROBE′O4	pPROBE′-gfp derivative carrying Pcirc-lacO4	This study
pRMPlacO1	pACYC184 derivative carrying Pcirc-lacO1-intB13	This study
pRMPlacO2	pACYC184 derivative carrying Pcirc-lacO2-intB13	This study
pRMPlacO3	pACYC184 derivative carrying Pcirc-lacO3-intB13	This study
pRMPlacO4	pACYC184 derivative carrying Pcirc-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

In order to add more utility to the vector for cloning and delivery, we took advantage of the exclusive replication of plasmids carrying the ori_R6K in hosts expressing the Pir protein (18). A 420-bp fragment containing ori_R6K was amplified by PCR using primers (120301 and 120302) and the minitransposon delivery vector pBAM1 as a template (1). The PCR product was digested by EcoRI and NheI and ligated with pRMPlacO4 digested with EcoRI and XbaI to replace ori_p15A. The resultant plasmid carrying ori_R6K was opened by NcoI and ScaI and ligated with a 67-bp fragment containing a FRT site, which had been amplified with primers (120303 and 120304) and pUC18miniTn7T-LAC as a template. A second FRT copy was included in the primer to amplify the oriT of pBAM1 by PCR using primers (120305 and 120306), and the oriT-FRT fragment was introduced into FseI-NsiI sites on the plasmid. The resultant plasmid was further digested by EcoRI and ApaI and ligated with a 360-bp fragment containing multiple cloning sites (MCS) and two transcriptional terminators (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 gene with kanamycin or gentamicin resistance genes, of which fragments were amplified 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 named pRMR6K-Km and pRMR6K-Gm.

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

Reporter plasmid constructions.

To measure expression from P_circ in the various constructs, the four P_circ-lacO fragments with different lacO positions were amplified by using primers (090908 and 090805) and pRMPlacO1, pRMPlacO2, pRMPlacO3, or pRMPlacO4 as the template. As a positive control, the original P_circ was also amplified using the same primer set and pCK218-jim4. As a negative control, P_int was amplified using primers 090803 and 090805 and plasmid pRR171 as the template. All PCR products were digested by EcoRI and BamHI and then cloned separately in front of the promoterless egfp gene on the broad-host-range plasmid pPROBE′-gfp (17).

Fluorescence assays.

E. coli JM109 or P. putida UWC1 carrying pPROBE′-derived plasmids were precultured in LB with kanamycin overnight. The cultures were diluted in triplicate at 1:1,000 into fresh media with or without IPTG (isopropyl-β-d-thiogalactopyranoside; 1 mM) and incubated until mid-exponential phase (optical density [OD] = 0.5). A volume of 200 μl of the cultures was transferred into 96-well black microtiter plates (Greiner Bio-one) with a flat transparent bottom, and both culture turbidity (A₆₀₀) and fluorescence emission (excitation at 480 nm and emission at 520 nm) were measured using a Fluostar fluorescence microplate reader (BMG Lab Technologies). Fluorescence intensities were normalized to culture density.

Nucleotide sequence accession numbers.

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

RESULTS

Constructing a hybrid promoter to control integrase expression.

In order to diminish intB13 expression in E. coli but still allow sufficient expression for chromosomal integration in hosts other than E. coli, we constructed and tested several promoter variants. Previously, we had observed that a pACYC-based plasmid carrying intB13 downstream of the 450-bp region containing P_circ as in the attP region (Fig. 1A) of excised and recircularized ICEclc caused severe growth inhibition of E. coli (2). To control the expression of intB13, we first tried to clone the gene either under the control of the lac promoter (P_lac) in E. coli lacI^q or downstream of the 2-hydroxybiphenyl-inducible hbpC promoter (P_hbpC) in E. coli expressing the HbpR transcriptional regulator (19). Both cases, however, did not produce any proper transformants in E. coli, suggesting that leaky expression of intB13 still caused lethal effects on the host cells. Hence, we modified the 450-bp P_circ-attP region by introducing lacO sequences at different positions to block transcription by binding of the LacI repressor (Fig. 1B). The positions of lacO insertions were chosen without disrupting the −35/−10 boxes of P_circ and the 18-bp recombination site. In order to measure the relative promoter “strengths” in those variants, we cloned the modified promoter fragments (P_circ-lacO1, -lacO2, -lacO3, and -lacO4) in the pBBR1MCS-based reporter plasmid pPROBE′-gfp and measured EGFP fluorescence in the presence or absence of IPTG induction. In E. coli JM109 (lacI^q), all four fragments produced >10-fold-lower EGFP expression than wild-type P_circ without lacO (Fig. 2B). EGFP expression increased more than 4-fold after addition of 1 mM IPTG but was less than EGFP expression from wild-type P_circ. This result shows that P_circ activity can be repressed in E. coli by LacI binding at lacO and derepressed by addition of IPTG. Since all fragments also contain the weak P_int promoter, we examined EGFP expression from P_int in the same assay. As expected, EGFP expression from P_int alone was indistinguishable from a negative control with only vector (Fig. 2B). To test the resulting promoter activities outside E. coli, we introduced the series of reporter plasmids into P. putida UWC1. Since UWC1 does not carry lacI^q, it cannot repress P_circ, and EGFP expression will thus indicate the promoter strength in the host where the integration by IntB13 is supposed to take place. Indeed, EGFP was clearly produced in all UWC1 transformants, except those with the plasmid carrying egfp under the control of P_int. The relative levels of normalized EGFP fluorescence in UWC1 carrying the different plasmids were similar to those in E. coli after addition of IPTG, except for UWC1 carrying P_circ-lacO4, which showed activity comparable to that obtained with wild-type P_circ (Fig. 2C).

Fig 2.

In vivo reporter assays of the promoter activities of the various integrase promoter constructs. (A) Schematic representation of P_int, wild-type P_circ, and modified P_circ promoters, cloned in pPROBE′-gfp. Names of plasmids and promoters are shown on the right. The wild-type P_circ and P_int fragments correspond to 450-bp and 303-bp (lacking 147 bp at the 5′ end of P_circ-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 indicate standard deviations calculated from triplicates. (C) Same as panel B but expressed in P. putida UWC1 (without IPTG).

Site-specific integration of integrase plasmids.

We decided that the LacI^q-lacO variants might thus give sufficient intB13 downregulation in E. coli while allowing high expression in P. putida to ensure integration. The modified P_circ promoters were placed upstream of the intB13 gene in its wild-type configuration on a pACYC-derived plasmid and then introduced into E. coli JM109. The transformants successfully grew on the selective media, whereas cells transformed with a similar plasmid containing wild-type P_circ did not. This indicated again that uncontrolled P_circ-intB13 expression is lethal in E. coli but also that we had reduced intB13 expression sufficiently by adding lacO sequences to protect the cells from this lethality. The plasmids, named pRMPlacO1, pRMPlacO2, pRMPlacO3, and pRMPlacO4, were thus purified from E. coli and then introduced by electroporation into P. putida UWC1-2756. This strain carries four possible attB target sites (at the 3′ ends of the tRNA^Gly-3, tRNA^Gly-4, tRNA^Gly-5, and tRNA^Gly-6 genes) plus an artificial integration site fused to a conditional egfp trap (9). The pACYC-derived plasmids are incapable of replicating in UWC1-2756, and we thus expected integration of the plasmids in one of the five target sites. Intriguingly, transformants resistant to tetracycline were obtained only in case of using pRMPlacO3 or pRMPlacO4, while no cells grew with pRMPlacO1, pRMPlacO2, or pRR171 (Table 2). Approximately 20% of colonies showed green fluorescence, indicating that pRMPlacO3 or pRMPlacO4 had specifically inserted into the egfp trap on the chromosome. Site-specific integrations of the plasmids into one of four original attB sites (i.e., genes for tRNA^Gly-3, -4 or -5) in the nonfluorescent transformants were confirmed by colony PCR to detect junctions of attB and attP (Fig. 3). To test the stability of insertions, we cultured one nongreen clone of P. putida with integrated pRMPlacO4 for 30 generations in liquid culture without antibiotic selection (three subsequent transfers) and screened for the appearance of green colonies every tenth generation, under the assumption that excision of the construct would be followed at a detectable frequency by reintegration into the egfp trap. However, no green colony was detected among 300 colonies screened every tenth generation, suggesting that the integrations remained stable. These results showed that pRMPlacO3 and pRMPlacO4 can function as site-specific integration vectors. In contrast, pRMPlacO1 and pRMPlacO2 did not work, perhaps because insertions of the lacO sequence at positions lacO1 and lacO2 disrupt cis-acting sequences needed for efficient integrase activity, such as IHF binding sites or repeat structures. Most tyrosine-type recombinases, to which IntB13 belongs, need additional factors for efficient recombination that interact with sequences surrounding the actual 18-bp recombination site.

Table 2.

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

Plasmid	Integration into attBa	Proportion of green transformantsb
pRR171	No
pRMPlacO1	No
pRMPlacO2	No
pRMPlacO3	Yes (6.7 × 102 ± 2.2 × 102)	0.18 ± 0.01
pRMPlacO4	Yes (4.7 × 103 ± 1.2 × 103)	0.19 ± 0.07

^aTransformation efficiency (CFU/μg of DNA) is given in parentheses.

^bProportion of the number of green fluorescent transformant colonies to the total number of colonies.

Fig 3.

PCR analysis of site-specific integration of integrase plasmids into P. putida UWC1-2756. Amplification of the junctions between plasmids and target sites of the chromosome were performed using two randomly chosen transformants as templates (16, 25) for each of the integrated plasmids. “Green” refers to P. putida transformants expressing EGFP, indicating insertion in the conditional trap; “nongreen” refers to P. putida transformants not expressing EGFP, indicating insertion in one of the tRNA^Gly genes. Target sites and expected amplicon sizes are shown on the right. M, Massruler DNA ladder mix (Fermentas).

Development of a site-specific integration system.

Plasmid pRMPlacO3 and pRMPlacO4 thus functioned as vectors for site-specific integration of DNA in P. putida. To make them more amenable to cloning and delivery, we added several genetic options to pRMPlacO4. The origin of replication was replaced with ori_R6K, which is low copy number but replicates only in λpir strains, allowing to force chromosomal integration even in E. coli strains as hosts (data not shown). An origin-of-transfer sequence (oriT) was added in order to mobilize the plasmid from E. coli to its final host via bi- or triparental matings. A multiple cloning site (MCS) was added with transcriptional terminators (T₀T₁), and three variants of plasmid were made with different antibiotic resistance genes. The antibiotic resistance genes are removable after chromosomal integration through recombination at two FRT sites induced by a plasmid carrying the yeast FLP recombinase. The improved plasmids, named pRMR6K-Tc, pRMR6K-Km, and pRMR6K-Gm, thus function as suicide vectors and deliver DNA fragments (<10 kb) which can be cloned into the MCS by routine cloning techniques (Fig. 4A).

Fig 4.

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

Development of a mini-transposable integrase vector for large fragment delivery.

In order to obtain a system that would enable chromosomal integration of very large DNA molecules produced in E. coli, we decided to produce a mini-transposable integrase fragment that can be randomly inserted into such large DNA molecule in E. coli itself and then be delivered into its final host. To do this, we added a gene encoding a Tn5-family transposase (tnpA) and its cognate inverted repeat (IR) sequences to the pRMR6K plasmids, generating pRMTn-Tc, pRMTn-Km, and pRMTn-Gm (Fig. 4B). The newer plasmids would in principle be capable of transposition of the module containing intB13, P_circ-lacO4, oriT, an antibiotic resistance gene, FRT sites, MCS, and T₀T₁ to other DNA substrates.

To illustrate the ability of the site-specific integration system on minitransposons to introduce large DNA fragments into the host chromosome, we tested a cosmid and two BAC in E. coli. As an example, cosmid 1G3 contains an ∼25 kb fragment of ICEclc (3), including the genes for chlorocatechol degradation (clc). Introduction of the clc genes in P. putida leads to complementation of a metabolic pathway which enables the host to use 3-chlorobenzoate as the sole carbon and energy source (20). pRMTn-Tc was introduced by electroporation into an E. coli host without λpir that already contained cosmid 1G3. Approximately 500 transformants were obtained on LB agar with ampicillin and tetracycline, indicating that the transformants contained minitransposon insertions either in cosmid 1G3 or in the chromosome. To select only cosmids with minitransposon insertions, we picked 50 transformants at random and cultured them in pools of 10 transformants each in LB containing both antibiotics. Cosmids were isolated from those cultures and used to retransform E. coli JM109 by electroporation. In three out of five transformations, colonies showing resistance to both antibiotics were obtained, indicating that cells received cosmids with inserted minitransposons. The restriction digestion pattern of cosmids isolated from the transformants indicated the insertion of the transposon (data not shown). One of such cosmids, designated 1G3-O4, was then introduced into P. putida UWC1-2756 by electroporation. From 2 μg of cosmid 1G3-O4 a total of 30 P. putida transformants was obtained, of which ∼20% were green fluorescent. All transformants had a single-copy integration of 1G3-O4 at one of five target sites on the chromosome. Moreover, the P. putida transformants could utilize 3-chlorobenzoate as a sole carbon source, indicating successful expression of the clc genes in their inserted position (Fig. 5A). These results demonstrated that the minitransposon from pRMTn-Tc conferred site-specific integration ability to the cosmid which carries the clc genes and that transformants can consequently grow on the specific carbon source.

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-chlorobenzoate as the sole carbon source. Note that the transformant with the integrase-tagged cosmid 1G3-O4 containing the clc genes (left) but not P. putida UWC1-2756 (right) successfully grew. (B) PCR amplification of the junctions between integrase-tagged BACs (MED49C08 or MED66A03) and target sites of the chromosome. Randomly chosen transformants were used as templates. Note that only green fluorescent P. putida colonies were obtained from the transformation with MED49C08. Target sites and expected amplicon sizes are shown on the right. M, Massruler DNA ladder mix (Fermentas). (C) PCR amplification of BAC internal regions. Three regions named “start,” “middle,” and “end” in BACs, MED49C08 (left), and MED66A03 (right) were tested. Randomly chosen transformants were used as templates. Nucleotide positions of targets refer to the sequence available under accession number DQ077554 for MED49C08 and DQ065755 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).

To test the capacity for integrating even larger DNA fragments, pRMTn-Gm was introduced by electroporation into E. coli harboring BAC clones. We used two BACs, named MED49C08 (insert, 75 kb) and MED66A03 (53 kb), containing uncultured bacterium DNA with genes for photorhodopsins from deep Mediterranean seawater (21). In both cases, approximately 250 E. coli transformants showing resistance to both chloramphenicol (from the BAC) and gentamicin (from pRMTn-Gm) were obtained, indicating possible transposition of the integrase module into the BAC. The transformants were pooled as a mixture, regrown, and mixed with E. coli HB101/pRK2013 and P. putida UWC1-2756 to pass minitransposed integrase-BAC DNAs by triparental mating into P. putida. This resulted in ∼100 UWC1-2756-derived transconjugant colonies for each of the BAC clones tested that could grow on MM with succinate plus gentamicin. We confirmed that those transconjugants had a single specific integration of a BAC fragment in one of the target attachment sites (Fig. 5B). The proportion of EGFP-expressing transconjugants was ∼20% in the case of MED66A03 integration, whereas almost all transconjugants (>95%) obtained in the case of MED49C08 were green fluorescent. This bias might be due to some negative effects of MED49C08 integration into the other target attB sites. Unfortunately, photorhodopsin expression could not be detected in P. putida. We confirmed that in both cases of five randomly chosen transconjugants, all contained at least three BAC-specific regions, suggesting the successful integration of the entire BAC into the chromosome of P. putida (Fig. 5C).

DISCUSSION

We showed here the successful development and usage of a chromosomal delivery system for large DNA fragments in host bacteria outside E. coli (notably, P. putida). The system is based on the properties of the IntB13 integrase from the ICEclc element of P. knackmussii B13 to integrate (at least) 75 kb DNA into the 3′ 18-bp of the tRNA^Gly gene. The most crucial point for constructing the integrase-based DNA delivery system was proper downregulation of intB13 expression in E. coli while maintaining sufficiently high expression in the host cell where the DNA needs to be integrated. In previous work, even a low-copy-number plasmid carrying the intB13 gene downstream of the 450-bp P_circ-attP fragment had caused severe growth inhibition of E. coli (2) and accumulated DNA rearrangements (data not shown). Although we first tried to apply the P_lac-LacI or P_hbpC-HbpR regulatory system to control intB13 expression in E. coli, no transformants with the correct constructs were obtained, suggesting that some kind of detrimental effect was still associated with leaky expression of intB13. Hence, we decided to use the original P_circ-P_int promoters for intB13 control but repress P_circ by adding lacO sites for LacI^q repression. Under consideration of the positions of −35/−10 boxes of P_circ and the 18-bp recombination sequence, we synthesized new P_circ promoters having lacO insertions at different positions. Two of the new promoters, those having lacO insertions at lacO3 or lacO4, could integrate into the P. putida chromosomal target sites, whereas those with lacO1 and lacO2 could not. As a typical attP site for a tyrosine recombinase, similar to λ integrase (22), the P_circ 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 Holliday junction (23). The P_circ region also contains several repeat sequences, which may act as binding sites for IntB13 or other accessory proteins for the recombination process. The insertion positions of lacO1 and lacO2 appear to disrupt or touch one such motif (Fig. 1B), which is possibly the main reason for the failure of integration.

We further added several genetic options to both pRMR6K and pRMTn series of plasmids (Fig. 4). In particular, FRT sites may be useful after integration to remove antibiotic resistance genes through recombination by a temporarily introduced FLP recombinase (24). Although a few scars of the integrated plasmids still remain on the chromosome even after FLP/FRT recombination, such as IRs, FRT, and ori_R6K, they should not influence the cell growth or host genome structure. One may point out that the intB13 gene also remains after integration and may thus potentially lead to excision of the construct, although the requirement for a separate excisionase has not been demonstrated. However, intB13 is completely silent in the integrated form because of its physical separation from P_circ upon integration and because it lacks necessary other factors from ICEclc which are essential for its expression from P_int (6; S. Sulser, unpublished data; N. Pradervand, unpublished data). Therefore, renewed excision should be minimal and rarely occur. Another unique possibility with the pRMTn plasmids is integrase tagging of DNA molecules via minitransposition. This avoids specific cloning of large molecules in integrase-bearing vectors. We showed that the P_circ-lacO4-intB13 module 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 verification of the integration, the tagged substrates are able to site-specifically integrate into target chromosomes without further subcloning steps.

Since the system is based on the IntB13 integrase from ICEclc, target chromosomes need to have a cognate attB site. The strain used as a target in this study, P. putida UWC1, has six tRNA^Gly genes, and four of those six have perfect 18-bp sequence matches to the ICEclc recombination sequence (i.e., tRNA^Gly-3, tRNA^Gly-4, tRNA^Gly-5, and tRNA^Gly-6 genes). Importantly, the tRNA^Gly gene sequence is restored upon integration; therefore, there is no effect of integration on translation or codon efficiency. Most gammaproteobacteria, including E. coli, Salmonella, Pseudomonas, Yersinia, and Erwinia, have 18-bp recombination sites in some of their tRNA^Gly genes that are completely identical to ICEclc, and several beta- and gammaproteobacteria are natural recipients for ICEclc (9). Moreover, we have shown that IntB13 can recombine with attB recombination sites carrying a few mismatches (i.e., at least two mismatches in the 18 bp), albeit at lower frequencies (9). This should allow a wider variety of bacterial genomes as delivery targets, such as Burkholderia xenovorans, Bordetella petri, Cupriavidus necator, Xylella fastidiosa, and Xanthomonas campestris (9). To further broaden the target host range, one could first introduce an artificial (but perfect) attB site with a promoterless egfp gene on a minitransposon such as plasmid pCK218-jimX, into which the IntB13 system should then be able to deliver its target.

Several vector systems that can introduce foreign DNA into target genomes have been developed and are used in molecular genetics. The most sophisticated systems are perhaps the mini-Tn7 and mini-Tn5 vectors, which, however, are limited to delivering relatively short DNA fragments (<∼10 kb) into the target chromosome with and without site specificity, respectively (1, 16, 25). Integration-proficient plasmids carrying a modified gene for ΦCTX integrase, which also belongs to the tyrosine recombinase family, have been developed for engineering P. aeruginosa PAO1 (26). The plasmids contain the CTX cognate attP site and therefore can site-specifically integrate into the native attB site on PAO1, which is formed by the 3′ end of the gene for serine-accepting tRNA. An MCS is available for precise cloning, but the clonable size is likely limited, as in the case of mini-Tn vectors. To introduce environmental DNA libraries into several host bacteria, a BAC shuttle vector system that carries a gene for ΦC31 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 for apramycin resistance as a selective marker. The ICEclc-based system developed here differs from previous integration-delivery systems in the size of the deliverable fragment and the configuration of the integrase control.

We can thus conclude that the new integrase-based DNA delivery system developed here has a huge potential for molecular genetics, and in particular for synthetic biology. The system works in concert with existing vector systems and can introduce large DNA fragments into chromosomes, forming a useful aid in the construction of new metabolic pathways. Since tyrosine recombinases also function in mammalian cells (23, 28), further modifications to the system might make it possible to use it as a delivery system for eukaryotic organisms.