A simple method for construction of pir+ Enterobacterial hosts for maintenance of R6K replicon plasmids

Background The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (oriR6Kγ) is dependent on the pir gene-encoded π protein. Originally encoded by R6K, this protein is usually provided in trans in hosts engineered to support replication of plasmids harboring oriR6Kγ. In Escherichia coli this is commonly achieved by chromosomal integration of pir either via lysogenization with a λpir phage or homologous recombination at a pre-determined locus. Findings Current methods for construction of host strains for oriR6Kγ-containing plasmids involve procedures that do not allow selection for presence of the pir gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn7-based method for rapid and reliable construction of pir+ host strains. Using a curable mini-Tn7 delivery plasmid, pir expressing derivatives of several commonly used E. coli cloning and mobilizer strains were isolated using both the wild-type pir+ gene as well as the copy-up pir-116 allele. In addition, we isolated pir+ and pir-116 expressing derivatives of a clinical isolate of Salmonella enterica serovar Typhimurium. In both E. coli and S. enterica serovar Typhimurium, the presence of the pir+ wild-type or pir-116 alleles allowed the replication of oriR6Kγ-containing plasmids. Conclusions A mini-Tn7 system was employed for rapid and reliable engineering of E. coli and S. enterica serovar Typhimurium host strains for plasmids containing oriR6Kγ. Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with oriR6Kγ.


Background
The γ origin of replication of the broad-host-range plasmid R6K (ori R6Kγ ) has been used to construct conditionally replicative cloning and transposon delivery vectors too numerous to cite them all, with some of the most well-known vectors described in the late 1980s and early 1990s [1][2][3][4][5][6]. Replication of these vectors requires the π protein encoded by the pir gene which on R6K is located next to the γ origin of replication [7,8]. For maintenance of conditionally replicative plasmids that contain ori R6Kγ but lack pir, the π protein is expressed in trans from pir located on a compatible plasmid or, most frequently, on a λ phage or a gene inserted into the chromosome via homologous recombination at a predetermined locus [9]. In cells harboring a wild-type pir + gene, ori R6Kγ containing plasmids are maintained at 15 copies or less depending on size of the ori R6Kγ plasmid and pir gene source. A number of pir mutations have been identified that alter plasmid copy number, for example the pir-116 allele [10]. In cells harboring this allele integrated into the chromosome, ori R6Kγ plasmids are maintained at a copy number of about 250 per cell which compares to 15 copies per cell when the pir + allele is integrated at the same chromosomal locus [9]. The host range for ori R6Kγ -containing plasmids is limited because construction of strains supporting their replication involves methods that do not allow selection for presence of the pir gene and require cumbersome and time-consuming screening steps. To allow expansion of plasmid host range to customized genetic strain backgrounds we therefore developed a mini-Tn7-based method for rapid and reliable construction of enterobacterial pir + host strains.

Results and discussion
Development of a mini-Tn7 based ori R6Kg chromosomal insertion system We sought to employ the mini-Tn7 method described by McKenzie and Craig [11] for chromosomal insertion of pir alleles in the absence of selection. For this purpose, the pir + and pir-116 genes were cloned into the mini-Tn7 delivery vector pGRG36 ( Figure 1) and published procedures [11] followed in an attempt to transpose the cloned pir genes into the chromosomes of various E. coli strains. However, in some strains, despite repetition and exhaustive PCR screening, this method proved ineffective for this purpose as the majority of colonies obtained after completion of the procedure did not contain the desired mini-Tn7 insertions or did not result in any insertions, for unexplained reasons. We therefore designed a method that allows positive selection of strains containing chromosomally inserted pir alleles ( Figure 2). The rationale for this method is to establish the delivery vector with a temperature-sensitive replicon (ts), here pSC101ori ts , at permissive temperature (30°C), then introduce an ori R6Kγ reporter plasmid at 37°C, creating conditions at which the mini-Tn7 delivery vector is cured and replication of the reporter plasmid is dependent on the presence of a chromosomally-integrated pir gene. After verification of the desired mini-Tn7-pir insertions the reporter plasmid is then cured using sucrose counter-selection.
Mini-Tn7 insertion of pir genes in E. coli and S. enterica serovar Typhimurium Following the procedure outlined in Figure 2, we readily obtained mini-Tn7-pir insertions in several commonly used E. coli laboratory cloning and mobilizer strains-DH5α, JM108, MC4100, SM10, RHO3-and a clinical S. enterica serovar Typhimurium isolate. Exconjugants examined by PCR contained the desired insertion, either mini-Tn7-pir + or mini-Tn7-pir-116 ( Figure 3A). The plasmid copy number of pR6KT2 was greatly elevated in pir-116-containing E. coli DH5α and S. enterica serovar Typhimurium 14028S host strains when compared to the same strains containing chromosomally inserted wildtype pir + ( Figure 3B). An alternative to employing an ori R6Kγ reporter plasmid is to use mini-Tn7-pir elements that contain a Km r selection marker that after verification of desired inserts can be removed using Saccharomyces cerevisiae Flp recombinase-mediated site-specific excision, followed by curing of the Flp recombinase expression plasmid. We have successfully used this strategy in E. coli. Both strategies require equal time and effort.
These strategies allow extension of the host range of ori R6Kγ containing plasmids to virtually any enterobacterial strain, something that was, to date, only possible using relatively cumbersome and time-consuming methods, e.g. isolation of λpir lysogens or chromosomal insertion of cloned pir alleles via site-specific recombination at a predetermined locus [9]. Mini-Tn7 elements insert at naturally evolved attTn7 sites that are usually located in intergenic regions downstream of conserved glmS genes [12][13][14][15][16]. This alleviates the need for selecting potential insertion sites not affecting bacterial fitness when choosing recombinant DNA strategies for pir allele insertion into a bacterial genome. Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with ori R6Kγ . As described, the procedure relies on availability of ts replicons which may limit its applicability to bacteria that can tolerate the non-permissive temperatures needed Figure 2 Overview of steps involved in chromosomal mini-Tn7pir insertion. A) Introduce the mini-Tn7-pir + delivery vector by conjugation. Select ampicillin resistant (Amp r ) colonies at 30°C to establish the delivery vector. B) Grow Amp r cells at 30°C in presence of arabinose to induce the genes encoding the Tn7 site-specific transposition pathway. C) Introduce the ori R6Kγ reporter pR6KT2 by conjugation and grow at 37°C in the presence of gentamicin (Gm) to cure the mini-Tn7-pir + delivery vector and report integrants based on pir-dependent replication of pR6KT2. Establish Gm r and Amp susceptible (Amp s ) phenotype. D) Cure pR6KT2 reporter plasmid by plating on sucrose-containing medium. Verify Gm s and Amp s phenotype, and confirm pir + integrants by PCR (using primer pair 2372 and 2373 for E. coli or 2374 and 2375 for S. enterica serovar Typhimurium). It must be noted that PCR-based insertion site verification strategies are limited to bacteria for which sequence information about the glmS flanking sequences is available. Methods for identifying Tn7 insertion sites in bacteria for which genome sequences are unknown have been described [12]. for plasmid curing. The methods described here were developed for Enterobacteriaceae which, like most bacteria, can tolerate 37°C, a temperature at which most plasmids with ts replicons, including pSC101ori ts , are readily cured. For bacteria with growth temperature optima less than 37°C, the described strategy will not work and require appropriate modifications, i.e. inclusion of different counter-selection markers, for example sacB [17]. Lastly, though many manipulations described in this paper use conjugations as means for introduction of plasmids into cells, some them could also be done by plasmid transformation which would alleviate the need for counter-selection strategies required for bacterial matings. We, however, consistently find that conjugations are equally convenient and more efficient means of plasmid transfer than transformation.

Bacterial strains, media and growth conditions
Bacterial strains used in this study are listed in Table 1. Bacteria were routinely grown at 37°C in Luria Bertani broth Lennox (LB) [18] or on LB agar purchased from MO BIO Laboratories, Carlsbad, CA. The sacB-containing ori R6Kγ reporter pR6KT2 was cured by plating plasmid-containing cells on yeast extract-tryptone (YT) sucrose medium containing 10 g/l yeast extract (Difco, Detroit, MI), 16 g/l tryptone (Fisher Scientific, Fairlawn, NJ), 16 g/l Bacto agar (Becton, Dickinson and Company, Sparks, MD) and 15% sucrose (w/v) [19]. Strains containing temperature-sensitive (TS) plasmid derivatives were grown at 30°C (permissive temperature) for plasmid maintenance and 37°C or 42°C (non-permissive temperature) for plasmid curing. Antibiotics were added at the following concentrations: 100 μg/ml ampicillin (Amp), 10-15 μg/ml gentamicin (Gm) and 35 μg/ml kanamycin (Km) for E. coli and S. enterica serovar Typhimurium harboring plasmids or for selection of chromosomally-integrated mini-Tn7 elements. Antibiotics were purchased from EMD Biosciences, San Diego, CA (Gm) and Sigma, St. Louis, MO (Amp and Km). For E. coli strain RHO3, media were supplemented with diaminopimelic acid (DAP; LL-, DD-, and meso-isomers; The pir gene carried by λpir has a truncation that removes the coding region for the carboxy-terminal 30 amino acids of the π protein. Despite this truncation a λpir lysogen maintains plasmids with at the same copy number as cells carrying a wild-type pir + gene [9]. 2 The pir-116 allele is dominant over the pir gene carried by the lysogenic λ phage and leads to increased copy number of ori R6Kγ containing plasmids. Sigma) which was added at a final concentration of 400 μg/ml for agar plates and 200 μg/ml for broth cultures. 5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid (XGluc; Gold Biotechnology, St. Louis, MO) was added to media at a final concentration of 40 μg/ml. Induction of gene expression from the arabinose operon promoter (P BAD ) was achieved by addition of L-arabinose to media at a final concentration of 0.5% (w/v).

Isolation of plasmid DNA
Plasmid DNAs were isolated from E. coli and S. enterica serovar Typhimurium by using a Fermentas GeneJET Plasmid MiniPrep Kit (Fermentas, Glen Burnie, MD).

Transposition of mini-Tn7
The respective mini-Tn7 delivery vectors were transformed into E. coli mobilizer strain RHO3 [19]. Conjugation of delivery plasmid into E. coli and S. enterica serovar Typhimurium strains was achieved by biparental mating using previously described methods [19] with some minor modifications. Briefly, RHO3 donor and E. coli and S. enterica serovar Typhimurium recipient cultures were grown overnight at 30°C (pGRG36-based donor strains) or 37°C (recipient strains) in LB medium with the appropriate nutritional (DAP) and antibiotic (Amp) supplements for RHO3 with the mini-Tn7 delivery vector. One ml of donor and recipient were placed into separate 1.7 ml microcentrifuge tubes and harvested by centrifugation in a microcentrifuge for 30 s at 13,400×g and room temperature. Cells were washed twice in 1 ml LB medium and then re-suspended in 200 μl of LB medium. Equal volumes (25 μl) of each cell suspension were transferred to a cellulose acetate membrane (13 mm diameter; 0.45 μM pore size; Sartorius Stedim, Bohemia, NY) sitting on an LB agar plate containing 400 μg/ml DAP and 0.5% arabinose. After overnight incubation at 30°C, the membrane was transferred to a microcentrifuge tube containing 1 ml of LB and cells dislodged by centrifugation in a microfuge for 30 s at 13,400×g and room temperature. After removing the membrane, cells were washed twice in 1 mL LB and then re-suspended in 200 μl of LB medium. The entire sample was placed on an LB-agar plate with 100 μg/ml Amp and 0.5% arabinose, and a portion streaked for single colonies with an inoculating loop. The plates were incubated at 30°C overnight or until single colonies were clearly discernable.
A single purified colony was then used as recipient for the ori R6Kγ reporter pR6KT2. This plasmid was introduced via biparental mating from RHO3 as described above, except that recipient cells were grown in the presence of Amp and arabinose and RHO3/pR6KT2 cultures were grown in the presence of Gm and DAP.
After overnight incubation at 30°C mating mixtures were recovered and plated on LB with 15 μg/ml Gm at 37°C to cure the temperature-sensitive mini-Tn7 delivery vector and select for pR6KT2. Purified colonies were patched on LB, LB + Gm and LB + Amp to confirm the loss of the mini-Tn7 delivery vector (Amp susceptibility) and presence of pR6KT2 (Gm resistance). After verification of mini-Tn7 insertions by PCR, pR6KT2 was cured by streaking a single colony on YT medium with sucrose and XGluc, and incubating overnight at 37°C. Single colonies were patched on LB and LB + Gm plates to confirm the loss of the plasmid. The pir gene insertions in the resulting strains were then re-confirmed by PCR and sequencing of the resulting DNA fragments.
When using the mini-Tn7-pir-FKm vectors, the protocol for conjugation was as described above for mini-Tn7 delivery without antibiotic selection. Exconjugants were grown overnight in LB + DAP and arabinose at 30°C, and Km r transposon insertions were selected by plating conjugation mixtures on LB plates with 35 μg/ml Km followed by incubation at 42°C to cure the delivery plasmid. The Km r marker can optionally be deleted from the strain with the mini-Tn7-pir-FKm insertion by transformation with pFLP2 (or any other Flp recombinase-expressing plasmid such as pCP20 [25]), testing transformants for Km susceptibility and then curing pFLP2 by plating on sucrose-containing media following previously described protocols [17].

Confirmation of mini-Tn7-pir insertions
Insertions of mini-Tn7-pir at attTn7 in E. coli and S. enterica serovar Typhimurium were performed by colony PCR using DNA in boiling preparation lysates as templates. These lysates were obtained by transferring separate colonies to individual sterile microcentrifuge tubes containing 30 μl of sterile water and boiling for 10 min. Using 6 μl of these boiling preparations and Taq DNA polymerase from New England Biolabs, PCR reactions were performed in a total volume of 50 μl. Primer pairs 2372 (5'-GATGCTGGTGGCGAAGCTGTC) & 2373 (5'-GATGACGGTTTGTCACATGGAG) and 2374 (5'-CAG CAACGACAACATGCACA) & 2375 (5'-AAACCAT CAGCGCGGAACAA) were used for E. coli and S. enterica serovar Typhimurium, respectively. In each case, the entire PCR reaction was analyzed on a 1% agarose gel. Expected PCR fragment sizes are 678 bp for E. coli without a mini-Tn7 insertion and 2,539 bp for derivatives containing mini-Tn7-pir + and mini-Tn7-pir-116. Fragment sizes for S. enterica serovar Typhimurium without and with mini-Tn7-pir insertions are 485 bp and 2,345 bp, respectively. When utilizing mini-Tn7-pir-FKm, the fragment sizes obtained with strains containing pir + or pir-116 insertions change by +1,470 bp when the Km r marker is present or by +145 bp after its Flp-mediated excision.

Plasmid construction
Plasmids used in this study are listed in Table 2. The Gateway-compatible mini-Tn7 delivery vector pGRG36GW was constructed by cloning of a 1,770-bp StuI-XhoI fragment from pUC18-mini-Tn7T-Gm-GW [16] between the SmaI and XhoI sites of pGRG36 [11], followed by transformation into the gyrA462 strain DB3.1.
All plasmid constructions were verified by restriction digest and DNA sequence analysis.