Refactoring the Conjugation Machinery of Promiscuous Plasmid RP4 into a Device for Conversion of Gram-Negative Isolates to Hfr Strains

Chromosomal exchange and subsequent recombination of the cognate DNA between bacteria was one of the most useful genetic tools (e.g., Hfr strains) for genetic analyses of E. coli before the genomic era. In this paper, yeast assembly has been used to recruit the conjugation machinery of environmentally promiscuous RP4 plasmid into a minimized, synthetic construct that enables transfer of chromosomal segments between donor/recipient strains of P. putida KT2440 and potentially many other Gram-negative bacteria. The synthetic device features [i] a R6K suicidal plasmid backbone, [ii] a mini-Tn5 transposon vector, and [iii] the minimal set of genes necessary for active conjugation (RP4 Tra1 and Tra2 clusters) loaded as cargo in the mini-Tn5 mobile element. Upon insertion of the transposon in different genomic locations, the ability of P. putida-TRANS (transference of RP4-activated nucleotide segments) donor strains to mobilize genomic stretches of DNA into neighboring bacteria was tested. To this end, a P. putida double mutant ΔpyrF (uracil auxotroph) Δedd (unable to grow on glucose) was used as recipient in mating experiments, and the restoration of the pyrF+/edd+ phenotypes allowed for estimation of chromosomal transfer efficiency. Cells with the inserted transposon behaved in a manner similar to Hfr-like strains and were able to transfer up to 23% of their genome at frequencies close to 10–6 exconjugants per recipient cell. The hereby described TRANS device not only expands the molecular toolbox for P. putida, but it also enables a suite of genomic manipulations which were thus far only possible with domesticated laboratory strains and species.

NucleospinÒ Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). Localization of mini-Tn5 insertions was performed via the arbitrary PCR amplification protocol outlined by 2 . DNA sequencing was outsourced to Macrogen (Spain). Special protocols were used for plasmid purification and colony PCR in S. cerevisiae (see "Yeast Assembly Methods" below).
Transformation of E. coli laboratory strains was carried out with chemically competent cells using the CaCl2 method 1 . P. putida was transformed with plasmids via tripartite mating as described in 3 and selected in solid M9 minimal media supplemented with 0.2% w/v citrate and appropriate antibiotics. Other mating protocols used in this work are explained elsewhere in the Experimental Procedures Section. Construction of P. putida strains for Genome transfer assays. Donor strains P. putida-TRANS#X are derivatives of P. putida EM42 4 with the TRANS device inserted in the genome via a mini-Tn5 transposon. They also display three mutations conferring Sm, Rif and Nal resistance and were constructed as follows: P. putida EM42 harboring the plasmid pSEVA2514-rec2 was first subjected to one cycle of recombineering with mutagenic oligos SR, RR and NR using the procedure outlined in 5 . Mutations introduced by these oligos in target genes rpsL (PP_0449), rpoB (PP_0447) and gyrA (PP_1767) are translated into resistance to Sm, Rif, and Nal, respectively (see Table S3 for details). After selection on LB-agar plates supplemented with the three antibiotics, triple resistant colonies were streaked in the same media and the target genes were amplified with oligo pairs rpsL-Fw/rpsL-Rv, rpoB-F/rpoB-R, and gyrA-F/gyrA-R. The correct sequence changes were verified by sequencing the amplicons. One selected clone was cured of pSEVA2514-rec2 by iterative cultivation in LB without Km, obtaining the strain P. putida TA280. In order to insert the TRANS device into this strain, a bi-parental mating was performed as follows: P. putida TA280 and E. coli TransforMaxä EC100Dä pir + (pTRANS) were inoculated, respectively, in 3 ml LB-Sm and 3 ml LB-ApKm. After overnight growth, cultures were adjusted to OD600 ~1.0 and 1 ml of each was centrifuged 1 min/11000 rpm and resuspended in 1 ml of 10 mM MgSO4. Aliquots of 200 µl were mixed in an Eppendorf tube, 1 ml of 10 mM MgSO4 was added, and the aliquots were then spun down as before and resuspended in 10 µl of the same solution. This sample was placed on top of a LB-agar plate and incubated 18 h at 30 ºC. The cellular patch was scraped out with an inoculation loop and resuspended in 1 ml of 10 mM MgSO4. The bacterial suspension was plated in M9-Citrate-Km agar plates to select the P. putida TA280 trans-conjugants with mini-Tn5 insertions in the genome. After overnight incubation at 30 ºC, around 200 colonies were obtained. Mini-Tn5 localization of 32 clones was performed as described in 2 by two rounds of arbitrary PCRs with oligo pairs MEO-Km-extF/ARB6 and MEO-Km-intF/ARB2. Eighteen representative clones of P.
putida-TRANS#X with insertions throughout the genome were selected (Fig. 3) and two of them (#9 and #18) were assayed in this work.
The recipient strain was constructed by tagging P. putida EM42 ΔpyrFΔedd 6 with msfGFP via a modification of the delivery system described by 7 . Briefly: a tetra-parental mating was set up with receptor P. putida EM42 ΔpyrFΔedd, donor E. coli (pTn7-M-PEM7-GFP) and the helper strains E.
coli HB101 (pRK600) -for conjugation-and E. coli (pTNS2) -for transposition. The four strains were grown overnight in 3 ml LB supplemented with appropriate antibiotics (Table S1) and mated applying the protocol explained in the previous paragraph. Selection was made in M9-Citrate-Gm-Ura solid media and after incubation at 30 ºC for 18 hours, colonies showed green fluorescence.
Some of them were streaked in the same media and subjected to diagnostic PCRs (primer pairs PS2/PP5408-F and PEM7-F/Tn7-GlmS) to confirm the correct integration of the transposon in the attTn7 locus. PCR analysis yielded the expected bands of 2.2 Kb and 1.2 Kb, respectively (data not shown). The double deletion DpyrF Dedd makes the obtained P. putida JS40 both uracil auxotrophic and unable to grow on media utilizing glucose as the sole carbon source, while the Tn7 insertion confers constitutive fluorescence and resistance to Gm. To properly check such a phenotype, this strain and donor P. putida-TRANS#9 were grown in different culture media (Fig.   S2).
Yeast assembly methods and plasmid construction. The procedure for assembling plasmid constructs via homologous recombination in the yeast Saccharomyces cerevisiae followed the pipeline described in 8  [iii] as PCR linkers produced ad-hoc for non-adjacent fragments (see details below). DNA fragments designed for a given plasmid construct were PCR amplified and the products were purified and quantified. Equimolar mixtures of PCR fragments were used for yeast transformation (see individual assemblies for details). Yeast were transformed via a modified version of the lithium acetate 9 : 2 mL YPD were inoculated with S. cerevisiae CRY1-2 and incubated overnight at 30 ºC with shaking. The next day, 25 ml of fresh YPD were inoculated with 1 ml of the overnight culture and grown at 30 ºC/170 rpm. Culture was monitored until OD600 reached ~1.0 (around 5 hours).