Catalogue of reagents for recombinase/conjugation cloning. Please note that the template plasmids are suicide plasmids that require either AS11 or EKA260 for replication and that the λ Red plasmids and pCP20 are temperature-sensitive for replication (requiring 30 degrees C). The pJW plasmids are derived from either pKD3 (pJW101 and pJW102) or pKD46 (pJW103, pJW104, pJW105, and pJW106) (Quick, Shah, and Wilson 2010).
The ability to obtain DNA clones of genes that normally reside in microbial genomes was a huge technical advance in molecular biology. At first, cloning genes utilized approaches involving the complementation of mutants or the screening of genomic libraries to find sequences that hybridized to homologous DNA probes. Typically, this involved using restriction enzymes to clone random genomic fragments followed by subcloning of a smaller piece of the original clone. Then the development of PCR and genomic sequencing allowed specific genomic sequences to be amplified and cloned with more convenience. Now genes are able to be synthesized “from scratch” and ordered from various companies or institutions. However, if many genes contained on a contiguous large genomic segment are required to be cloned, significant technical barriers exist. For the purposes of this discussion, we will establish that a “large” genomic segment constitutes greater than 10 kilobases, since PCR and man-made DNA synthesis become technically challenging and/or costly above this DNA size. Therefore, a convenient, reproducible, and cost-efficient technique to clone large sections of microbial genomes would be highly advantageous.
Frequently bacteria organize genes that work together for a common function as a continuous, physically-linked series across a genome. Large genomic fragments containing many genes that work together for a specific function are very useful for the following reasons: (1) bacteria are able to be engineered for specific purposes in a “quantum leap” using such DNA clones; and (2) basic evolutionary questions are able to be answered using large genomic clones, such as: “Can the cloned gene set be expressed and functional outside of the context of the original genome/species?” These approaches extend the study of genomics by identifying potentially interesting parts of genomes identified via sequencing and studying them in different strain backgrounds. A clear example of this approach is the cloning of protein secretion systems and the subsequent study of these clones (Blondel et al. 2010; Ham et al. 1998; Hansen-Wester, Chakravortty, and Hensel 2004; McDaniel and Kaper 1997; Wilson, Coleman, and Nickerson 2007; Wilson and Nickerson 2006). However, many other gene systems can be studied in this way, with examples including polysaccharide secretion pathways (for capsule and LPS synthesis) and metabolic pathways (anabolism and/or catabolism of key molecules, such as those used in bioremediation). Our ability to extend genomics beyond sequencing to the utilization of newly-identified multi-gene pathways to engineer bacteria will depend upon our ability to clone, manipulate, and transfer large genomic fragments.
A recent strategy that exploits recombineering and conjugation provides a convenient approach to cloning large bacterial genomic fragments (Blondel et al. 2010; Santiago, Quick, and Wilson 2011; Wilson, Figurski, and Nickerson 2004; Wilson and Nickerson 2007). This approach involves insertion of recombinase sites (e.g., FRT, loxP) at positions flanking a targeted genomic region, followed by subsequent recombinase-mediated excision of the region as a non-replicating circular molecule (Fig. 1). Then the excised region is “captured” via either site-specific or homologous recombination onto a conjugative plasmid (such as the broad-host-range IncP plasmid R995) that allows the transfer and isolation of the desired construct in a fresh recipient strain (Fig. 1). The advantages of this approach are (1) the highly specific targeting of exact cloning endpoints using recombineering and (2) the use of conjugation to allow the desired construct to be isolated away from the donor strain (in which the recombination events take place). In addition, except for the synthesis of recombineering PCR products, this protocol takes place entirely in bacterial cells, using basic, low-cost microbiological techniques. Though early approaches used subcloned DNA fragments to allow homologous recombination, the use of recombineering for both the introduction of target flanking sites and the capture on R995 alleviates the need for this subcloning.
2. Targeted cloning of large bacterial genomic fragments
2.1. The VEX-Capture technique
The original technique using this approach is termed VEX-Capture (Wilson, Coleman, and Nickerson 2007; Wilson, Figurski, and Nickerson 2004; Wilson and Nickerson 2006, 2006, 2007). The pVEX series of suicide plasmids was used to introduce loxP sites into regions flanking targeted genomic regions via homologous recombination (Fig. 2) (Ayres et al. 1993). Cre recombinase (expressed from a plasmid) was used to excise the targeted region and homologous recombination was used to capture the excised circle (Fig. 2). Note that the homologous recombination is driven by the endogenous bacterial RecA-mediated mechanism. A series of Salmonella typhimurium genomic islands ranging from 26 to 50 kilobases in size were targeted for cloning using this technique (Wilson, Coleman, and Nickerson 2007; Wilson, Figurski, and Nickerson 2004; Wilson and Nickerson 2006, 2006). Since these islands contain genes that are unique to S. typhimurium, one of the initial basic applications of these clones was to study their gene expression patterns in different bacteria (Wilson, Figurski, and Nickerson 2004; Wilson and Nickerson 2006). Though some S. typhimurium genes on the tested genomic island were expressed in all bacteria, several genes displayed genus-specific expression patterns (Fig. 3). This indicated that the mechanisms used to express these genes are absent or function differently in certain bacterial genera. These mechanisms could be the focus of study to understand gene expression functions that work only in certain bacterial groups, such as pathogens or environmental bacteria.
Two separate S. typhimurium type III secretion systems were cloned using the VEX-Capture approach (Wilson, Coleman, and Nickerson 2007; Wilson and Nickerson 2006). These systems are encoded at the Salmonella pathogenicity island 1 and 2 regions (SPI-1 and SPI-2, respectively) of the S. typhimurium genome (McClelland et al. 2001). Both clones are
functional and serve to complement protein secretion defects in S. typhimurium mutants that are deleted for each SPI-1 and SPI-2 island (Fig. 4). However, the authors found remarkably different results between R995 + SPI-1 and R995 + SPI-2 when tested for expression in other Gram-negative bacteria (Fig. 5). The R995 + SPI-2 clone readily displays expression of SPI-2 (indicated using Western blot analysis of the SseB protein) in other Gram-negative genera, while the R995 + SPI-1 clone displays an expression defect outside of S. typhimurium (assayed using Western blot analysis of the SipA and SipC proteins). This result suggests that the regulatory mechanisms controlling SPI-1 and SPI-2 expression have evolved differently and in such a way that manifests itself upon transfer to new bacterial backgrounds.
2.2. VEX-Capture modified
A modification of VEX-Capture was used to clone the type VI secretion system encoded at Salmonella pathogenicity island 19 (SPI-19) in the S. gallinarum genome (Blondel et al. 2010). In this approach, the loxP sites and markers (for chloramphenicol and spectinomycin resistance) were PCR-amplified from the pVEX vectors and inserted into flanking positions using phage λ Red recombination (Fig. 6). The SPI-19 region was excised via Cre recombinase and captured onto R995 using homologous recombination (Fig. 6). The resulting R995 + SPI-19 clone was used to complement the colonization defect of the S. gallinarum SPI-19 deletion strain in a chicken infection model (Blondel et al. 2010). In addition, the authors transferred the R995 + SPI-19 clone into S. enteriditis, a species that contains significant sequence deviation in SPI-19 relative to S. gallinarum, to test if the presence of the S. gallinarum SPI-19 would increase S. enteriditis chicken colonization.
Interestingly, the presence of SPI-19 decreased the ability of S. enteriditis to colonize in this infection model (Blondel et al. 2010). This is consistent with the observations described above that demonstrate genomic island phenotypes can differ greatly, depending on the bacterial background.
2.3. New R995 derivatives allow an “all recombinase” approach
Recently an entirely recombinase-based approach for this techninque has been described using modified R995 plasmids (Santiago, Quick, and Wilson 2011). The new series of R995 derivatives encode a range of different marker combinations to increase utility in situations where several markers are used or are already present in the strain background. In addition, these R995 derivatives contain FRT sites that can facilitate the capture of genomic regions that have been excised using the Flp/FRT system (Fig. 7). A major advantage to this approach is that no regions of homology are needed to be cloned into any plasmids. Thus, the only step that takes place outside of cells is the amplification of the PCR products used for λ Red insertion of FRT sites into the flanking positions in the genome. This technique was demonstrated by cloning 20-kilobase regions from the S. typhimurium and Escherichia coli genomes (Santiago, Quick, and Wilson 2011).
2.4. Catalogue of reagents
Table 1 serves as a summary list of reagents used for the recombinase/conjugation-based cloning of genomic fragments. The PCR template plasmids are suicide plasmids and can
only replicate in corresponding strains that encode either the R6K Pir protein or P1 RepA protein (Ayres et al. 1993; Datsenko and Wanner 2000). This allows the PCR reaction to be directly electroporated into target cells with no background problems caused by the replication of the templates. It is worthwhile to note the PCR template plasmids with FRT sites contain two such sites flanking a given antibiotic resistance marker. Thus, care must be taken to amplify products containing only one FRT site for the second flanking insertion into the genome to avoid marker loss problems upon Flp expression (please refer to Fig. 7 for more details). It is also worthwhile to note that the self-transmissible IncP plasmid R995 displays a remarkably broad-host-range for both its conjugation and replication system (Adamczyk and Jagura-Burdzy 2003; Pansegrau et al. 1994; Thorsted et al. 1998). This facilitates R995 conjugative transfer to a wide variety of Gram-negative and Gram-positive bacteria and replication in almost all Gram-negative bacteria. Any other conjugative plasmid could be used for this procedure. However, IncP plasmid R995 and related plasmids are excellent options due to their broad-host-range, fully sequenced genomes, and high degree of characterization (especially for the IncPα plasmids R995, RK2, RP4, etc.).
Recombineering and conjugation can be exploited to provide a convenient, reproducible, and cost-effective technique for cloning large bacterial genomic fragments. This technique can be performed using easily obtained PCR products, readily available plasmids and strains, and simple, basic microbiology protocols. One question regarding the use of this system is: how large a genomic fragment can be accommodated by R995? So far, the biggest fragment cloned using this technique has been about 50 kilobases, but the upper limits of size have not yet been tested in any systematic way. To make genomic clones more amenable to medical or environmental applications, removal of antibiotic resistance markers and the conjugative transfer system would need to be accomplished. We are currently pursuing the development of alternative selection schemes and removable conjugation systems to address this issue. Overall, the use of the recombinase/conjugation cloning approach is currently underdeveloped as a technique and could expand the field of genomics by providing experiment-based strategies to answer important evolutionary questions.
We acknowledge the advice, technical help, and overall support of Dr. David Figurski, Dr. Cheryl Nickerson, and the Villanova University Biology Department.