Site-specific bacterial chromosome engineering mediated by IntA integrase from Rhizobium etli
© The Author(s). 2016
Received: 16 March 2016
Accepted: 28 June 2016
Published: 29 June 2016
The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, stability, incompatibility, and copy number variance. The site-specific integrase IntA from Rhizobium etli CFN42 catalyzes a direct recombination between two specific DNA sites: attA and attD (23 bp). This recombination is stable. The aim of this work was to develop a R. etli derivative that may be used as recipient for the integration of foreign DNA in the chromosome, adapting the IntA catalyzed site-specific recombination system.
To fulfill our aim, we designed a Rhizobium etli CFN42 derivative, containing a “landing pad” (LP) integrated into the chromosome. The LP sector consists of a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene. Between the lacZ promoter and the GFP gene we inserted an IntA attachment site, which does not affect transcription from the lac promoter. Also, a mobilizable donor vector was generated, containing an attA site and a kanamycin resistance gene; to facilitate insertion of foreign DNA, this vector also contains a multicloning site. There are no promoters flanking the multicloning site. A biparental mating protocol was used to transfer the donor vector into the landing pad strain; insertion of the donor vector into the landing pad sector via IntA-mediated attA X attA recombination thereby interrupted the expression of the green fluorescent protein, generating site-specific cointegrants. Cointegrants were easily recognized by screening for antibiotic sensitivity and lack of GFP expression, and were obtained with an efficiency of 6.18 %.
Integration of foreign DNA in Rhizobium, lacking any similarity with the genome, can be easily achieved by IntA-mediated recombination. This protocol contains the mating and selection procedures for creating and isolating integrants.
A critical factor for genome engineering of complex, novel characteristics in bacteria, is the efficient introduction and stabilization of large segments of DNA lacking similarity to the host genome. Plasmid-based strategies are of limited use in this regard, due to problems of plasmid instability and unforeseen consequences generated by copy number variations. Site-specific recombination mediated by integrases can neatly circumvent these problems, providing a means for effective integration of large DNA fragments in specific genomic locations. Tyrosine recombinases are one of the two classes of proteins that catalyze site-specific recombination . Most of the members of this family are highly efficient in promoting recombination, leading to excision, integration or both events, although in the case of bidirectional tyrosine recombinases, with contrasting efficiencies. Unlike homologous recombination, which requires extensive similarity between recombining sequences, tyrosine recombinases catalyze recombination between substrates sharing limited sequence identity. Sequence identity normally spans a short region (8–15 bp) where DNA exchange occurs. The properties of high efficiency and reduced size of target sequences for recombination, make integrases excellent tools for the manipulation of DNA. Integrases are also amenable to a variety of genetic modifications that may enable the production of custom integrases with selectivity for novel target sequences .
Systems for chromosomal integration of foreign DNA mediated by tyrosine integrases were devised as early as the 1990s for Escherichia coli , Staphylococcus aureus  and Actinobacteria [5, 6]. All of them employ special plasmid vectors harboring the corresponding recombinase recognition sequence (att or attachment site), where foreign DNA can be cloned. Upon introduction into target cells expressing the cognate integrase, site-specific integration occurs via the endogenous attachment site.
The two most widely used systems for in vivo recombination based on tyrosine recombinases are Cre-loxP and Flp-frt . These systems have a proven efficacy in a variety of biological systems. However, both promote excision more readily than integration. Moreover, given the complex manipulations needed for generation for strains with new features, there is a growing demand of novel systems that employ different recombination systems.
In a previous study we characterized the function of the Rhizobium etli IntA site-specific recombinase, through a combination of in vivo and in vitro assays . IntA belongs to the tyrosine-recombinase family. It allows cointegration of plasmids p42a and the symbiotic plasmid via site-specific recombination between attachment regions in R. etli. The results described previously revealed that IntA is able to catalyze both integration (2.4×10−5) and excision (2.1×10−6) events in vivo . The recombination requires the presence of a characteristic 23 bp sequence (the att sector) harboring two arms of a palindrome plus a divergent central region . The sequence of attA (TCCGATAAGCATTACTTATCGGA) differs by a single nucleotide (shown in italics) from attD; this difference does not affect recombination efficiency with IntA, since attA X attA, attA X attD and attD X attD recombination are all equally likely .
In the present study, we took advantage of the high efficiency and specificity of integration afforded by this system, to construct an integration system for R. etli based on site-specific recombination via IntA integrase. This system allows integration of large DNA segments, in a manner independent of homologous recombination, into predefined sectors in the genome of R. etli.
Results and discussion
Oligonucleotides used in this work
Genome location b
Source or reference
To evaluate if the LP sector can be used as an acceptor of IntA-mediated in vivo recombination, we employed plasmid pK18 mob att Δplac (see Methods and Fig. 1b). This plasmid is a modification of the suicide vector pK18 mob containing an attA site, a kanamycin resistance gene, and lacking the placZ promoter; it also harbors a MCS, where defined segments can be easily introduced. In all our experiments the IntA activity was provided from the intA gene encoded in plasmid pRetCFN42a, present in the LP strain. Plasmid pK18 mob att Δplac was mobilized by biparental matings, from an E. coli donor to the R. etli LP strain. Upon entrance of the plasmid into the LP strain, IntA-mediated recombination may occur with any of three targets: the attA located in plasmid pRetCFN42a, the attD from plasmid pRetCFN42d, or the attA present in the chromosomal LP sector. Insertions of pK18 mob att Δplac in the chromosomal LP sector disrupt transcription of the GFP gene coming from the lacZ promoter (Fig. 1c). Thus, insertions in the LP sector can be easily detected by a simple screen for kanamycin and spectinomycin resistant, nonfluorescent derivatives.
To evaluate the stability of integrants of pK18 mob att Δplac in the LP sector, one integrant was serially subcultured in the absence of antibiotics, for a period corresponding to 21 generations. Dilutions were plated on solid media without antibiotics. A total of 10 000 colonies were screened for GFP expression, interestingly, all of them remained nonfluorescent.
After demonstrating that integration may occur on the LP sector, we then asked if integration on the LP sector still leaves it susceptible to further integration events. Integrants on the LP sector possess two att sites separated by the inserted DNA. These integrants are nonfluorescent due to the integration of sequences lacking an active promoter for transcription of the GFP gene. We reasoned that further integration of a sequence harboring an active promoter on the rightmost att site (i. e. the one closer to the GFP gene) should restore GFP production.
To verify the location of additional insertions within the LP sector, specific PCR assays were set up. These PCR assays comprised two sets of primers; the first one included a primer complementary to the left side of the LP sector (ChrleftD) and the other matched the sequence of pG18 mob att (G18lw) while the second pair had a primer complementary to the right side of the LP sector (ChrightD) and another to pG18 mob att (G18up). Amplification with the first set of primers revealed integration on the left att sequence, while PCR products obtained with the second set reveals integration on the right att sequence (Fig. 6c). As expected, four of the integrants characterized revealed amplification with the second set of primers but not with the first set, indicating that integration indeed occurred on the rightmost att site (Fig. 6d). Interestingly, one of the fluorescent derivatives gave positive amplification with both primer sets, indicating the possibility of multiple integration events on the LP sector (Fig. 6d).
As shown in this work, site-specific bacterial chromosome engineering mediated by IntA allows the efficient integration of sequences lacking extensive homology to the host genome, other than the att sequence. Use of this approach requires a specially-constructed strain, harboring the so-called Landing Pad, containing a single att region, as well as GFP to allow easy identification of strains harboring the desired integration. The size of the construct to be integrated is limited only by the size of DNA that can be successfully cloned into the donor vector via ligation. Transfer of foreign DNA into the LP strain is done by conjugation, thus circumventing the need for DNA purification and transformation, making it a very robust approach. Under appropriate conditions, integrants were derived at a high frequency (10−4) compared to the frequency of double crossover recombination (10−6). The integrated DNA is flanked by copies of the att sequences, which are potential targets for IntA-mediated excision. Nevertheless, integrants are very stable, due to the low frequency of excision, evaluated previously at 2.1×10−6 . In fact, in the experiments reported here, no IntA-mediated excision events were detected. The finding that the att sequences added by integration are still active for recombination, open up the possibility of larger, more complex alternatives of strain construction in a sequential way.
Although this is not the first system described that allows integration mediated by site-specific recombination, it offers some advantages compared to other systems. For instance, recombineering via λ-red, a phage based system, requires larger homologous segments, and has limited effectiveness in bacteria other than E. coli  The ΦC31 integrase has a different recombination target than IntA, and it has demonstrated efficient activity in disparate hosts, including Sinorhizobium meliloti . IntA has the advantages of efficiency and modularity, given that any donor cassette can potentially be integrated into any LP-strain. In this regard, several rhizobial species possess IntA-homologs, with conceivably different specificities. Ideally, the IntA system may be combined with other site-specific systems with different specificity, to facilitate bacterial genome engineering.
Bacterial strains, plasmids and growth conditions
Rhizobium strains were grown at 30 °C in PY (peptone-yeast extract) medium . Escherichia coli strains were grown at 37 °C in LB (Luria-Bertani) medium . When needed, antibiotics were added at the following concentrations (in micrograms per milliliter): nalidixic acid, 20; spectinomycin, 100; kanamycin, 30.
Plasmid transfer from E. coli to Rhizobium was done by biparental mating, using E. coli S17.1 harboring the suicide vector (pK18 mob att Δplac) as a donor and R. etli 557 (LP-strain) as recipient, the strains were grown in liquid medium to stationary phase, washed twice with PY medium, mixed in a donor/recipient ratio of 1:2 on PY plates, and incubated at 30 °C overnight. After incubation, cells were resuspended in MgSO4 10 mM-Tween 0.01 %, serially diluted and plated on solid PY medium containing nalidixic acid, spectinomycin and kanamycin. Cointegration frequencies were evaluated independently at least thrice, and are expressed as number of transconjugants per recipient cell. For evaluation of GFP production, colonies were visualized under ultraviolet light (425 nm).
Molecular and microbiological procedures
Basic DNA manipulations and molecular techniques were done using established procedures . Extraction of DNA from agarose gels was done with a GeneJET extraction kit (Fermentas); plasmids were isolated with high pure plasmid isolation kit (Roche). All oligonucleotides used were synthesized at the Unidad de Síntesis of the Instituto de Biotecnología, Universidad Nacional Autónoma de México; all PCR amplifications were carried out using High Fidelity Taq polymerase (Invitrogen). Amplification protocols consisted of 30 cycles of 1 min at 94 °C, 1 min at variable temperature (depending on the primer combination), and 1 to 3 min at 68 °C. After amplification, PCR products were extracted with phenol and precipitated with ethanol. The DNAs were resuspended in Tris-EDTA buffer and digested with the appropriate restriction enzyme(s) to generate the required ends in the fragments. The DNA fragments were purified before cloning by isolating them from the agarose gel. For ligations, T4 polynucleotide ligase (Fermentas) was used. Plasmid transformation of E. coli was done using CaCl2-competent cells. All plasmid constructions were verified by restriction analysis and PCR and, in most of the cases, by DNA sequencing.
To generate the LP sector, a cassette containing a green fluorescent protein gene under the control of the lacZ promoter and a spectinomycin resistance gene  was modified by the introduction of an att site. To do that, the attA site was amplified from R. etli genomic DNA using primers UpattA1 and LwattA1, gel-purified and restricted with EcoRI and BamHI and ligated into similarly restricted pCR2.1®-TOPO® TA vector (Thermo Fisher Scientific) From the resulting plasmid, the attA sequence was excised by digestion with XbaI and ligated into XbaI-digested pGX534 , giving rise to plasmid pRG10. In this plasmid, the lacZ promoter and the GFP gene are separated by an att sequence. To facilitate insertion of the LP sector in the chromosome, a 477 bp chromosomal segment (from nucleotide 1108084 to nucleotide 1108560, corresponding to the non-coding region) was amplified using primers chr_left_in_hind and chr_right_in_eco (Table 1). This PCR fragment was digested with HindIII and EcoRI and cloned into similarly digested pK18mob-sacB  yielding pGX117. To substitute a single PstI site present within the non-coding region with a NotI site, pGX117 was digested with PstI and ligated to oligonucleotide Link-PstxNot, giving rise to plasmid pGX191.
To generate a chromosomal entry vector for the LP sector, plasmid pRG10 was digested with NotI (thus excising a NotI cassette harboring the LP sector), and the resulting fragment was cloned into NotI digested pGX191. The resulting chromosomal entry vector for the LP sector was called pRG11.
To generate a plasmid useful for integration in the LP sector, a derivative of plasmid pK18 mob , harboring an attD sequence and lacking the native plac promoter was constructed. To do that, two complementary oligonucleotides (Lw23bp and Up23bp, Table 1), containing the attD sequence, were heat-denatured and reannealed, and the resulting double-stranded fragment was restricted with BamHI. This product was ligated into similarly restricted pK18 mob, giving rise to pK18 mob att. To remove a 160 bp segment containing the lac promoter, pK18 mob att was restricted with AseI and EcoRI, ends were filled-in with Klenow polymerase and the plasmid was religated in a blunt-end ligation, resulting in plasmid pK18 mob att Δplac (3.66 kb).
A separate vector, useful to investigate the possibility of repeated insertions into an already occupied LP sector (plasmid pG18 mob att) was also constructed. To this end, the attA site was amplified from R. etli genomic DNA using primers UpattA1 and LwattA1, gel-purified and restricted with EcoRI and BamHI and ligated into similarly restricted pG18 mob , thus generating pG18 mob att (2.88 kb).
Plasmid profiles and hybridization
Rhizobium plasmids were visualized by the Eckhardt procedure . Gels were transferred onto Hybond N+ membranes (Amersham) using the manufacturer’s protocol and cross-linked using a UV cross linker unit (Stratagene). Hybridizations were performed overnight using α32P-dCTP-labelled probes (Megaprime kit; Amersham) under high-stringency conditions (65 °C in rapid-Hyb buffer, Amersham). Hybridization signals were detected with a PhosphorImager (Molecular Dynamics).
For determination of stability of the integrated DNA by flow cytometry, cells from the desired strain were subjected to three serial overnight cultures in the absence of antibiotics (representing about 21 generations) and analyzed by flow cytometry. Cells were analyzed in the ImageStream X imaging flow cytometer (Amnis Corporation, Seattle, WA) using the 488 nm solid-state laser with appropriate compensation controls and settings. Data were acquired using INSPIRE acquisition software and the 60X objective lens on a low speed setting. Data from a minimum of 107 cells were collected for each sample and analyzed using IDEAS 5.0 software. The instrument and INSPIRE software were set up as follows: Channel 01 (bright field), channel 02 (green fluorescence) and channel 12 (scattering channel). Magnification was 60X, providing a pixel size of 0.3 μm2 and the lasers 488 and 745 activated for fluorescence and side-scatter, respectively. The flow rate was set to low speed/high sensitivity and stream alignment was adjusted where necessary. Focused images of single cells were initially identified from the brightfield images taken by the imaging flow cytometer by using the manufacturer’s recommended analysis algorithm, the gradient root mean square feature. To reduce artifacts and background noise, we used the default mask of the IDEAS 5.0 software called M01 and an investigator-defined mask, which was composed of a combination of mask features (system 50, erode 2, range 300–2,000). Areas of interest smaller than 300 pixels were deselected. Images that still had a spot count of two or more, were excluded and designated as “doublets.”
°C, celsius degree; att, attachment site; attA, attachment site plasmid a; attD, attachment site plasmid d; Bp, base pairs; Chr, chromosome; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; GFP, green fluorescent protein; Gmr, gentamicin resistant; LB, luria-bertani; LP, landing pad; MCS, multi cloning site; Nm, nanometer; PCR, polymerase chain reaction; PY, peptone-yeast extract; UV, ultraviolet.
We gratefully acknowledge Laura Cervantes and Araceli Dávalos for skillful technical assistance, Paul Gaytán and Eugenio López (Unidad de Síntesis de Oligonucleótidos, Instituto de Biotecnología, UNAM) for help with oligonucleotide synthesis and Andres Saralegui Amaro (Laboratorio Nacional de Microscopía Avanzada, Instituto de Biotecnología, UNAM) for help with flow cytometry experiments. G.T-T is member of the Research Career of CONICET, Argentina.
The study was supported with internal funds from the Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México.
Availability of data and materials
The data that support the results of the present study are included within the journal article.
RH-T, GT-T, SB and DR conceived and designed the experiments; RH-T and GT-T carried out the experiments; RH-T, GT-T, SB and DR analyzed the data; RH-T and DR wrote the paper. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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