Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange

Abstract

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, as well as single-nucleotide insertions, deletions and substitutions, in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selections are enabled solely by suicide vector–encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic-resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires 2 weeks.

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Figure 1: Two-step allelic exchange.
Figure 2: Workflow for making precision-engineered mutant strains of P. aeruginosa.
Figure 3: How to design and build synthetic mutant alleles.
Figure 4
Figure 5: Systematic disruption of type IV pilus gene loci in P. aeruginosa PAO1.
Figure 6: Outcomes of merodiploid selection and sucrose counter-selection using the two-step allelic exchange protocol.

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Accessions

NCBI Reference Sequence

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Acknowledgements

The authors thank M.F. Hynes, M. Wilton, D. van Ditmarsch, J.D. Rich and G. Winsor for useful discussions and feedback on this manuscript. J.J.H. is supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) and a Canada Research Chair (Tier II) from the Canadian Institutes for Health Research (CIHR). Infrastructure has been provided to J.J.H. through support from the Canada Foundation for Innovation (CFI). M.R.P. was supported by a National Institute for Allergy and Infectious Disease Grant (2R01AI077628-05A1).

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L.R.H. and J.J.H. wrote the manuscript; L.R.H., B.R.B., H.A., Y.I., T.E.R., M.E.L., J.J.Y., B.S.T., K.M.S., R.J.S., P.L.H., P.K.S., T.T.-N., M.R.P., H.P.S. and J.J.H. designed, tested and/or troubleshot the protocol; L.R.H., H.A., T.E.R., M.E.L., C.L. and J.J.H. created strains and plasmids; T.E.R., M.E.L., C.L. and J.J.H. generated data presented in ANTICIPATED RESULTS and Supplementary Information. All authors revised and proofread the manuscript.

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Correspondence to Joe J Harrison.

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Integrated supplementary information

Supplementary Figure 1 Irgasan (Triclosan®) and chlroamphenicol select against Pseudomonas aeruginosa, which may lead to protocol failure.

Bacteria from replicate biparental bacterial matings were suspended in 1.0 ml phosphate buffered saline (Step 36A.ix), then divided into three equal aliquots and plated on Vogel-Bonner minimal medium (VBMM) agar, or lysogeny broth (LB) agar containing 25 μg/ml irgasan (Irg) or 10 μg/ml chloramphenicol (Cm). All agar contained 60 μg/ml gentamicin (Gm) to select for merodiploids. Solid lines and error bars represent the mean number and standard deviation of P. aeruginosa merodiploids recovered after conjugation. By comparison to nutritional selection on VBMM agar, the use of Irg and Cm in selective LB agar media reduced the number of merodiploids recovered during selection.

Supplementary Figure 2 Step-by-step outcomes for building an allelic exchange vector and genome engineering.

This example focuses on building a deletion allele for pelF and creating a knockout in P. aeruginosa PAO1. (A) Vector selection and primer design (Step 1, see Supplementary Tutorial). (B) Results of PCR to synthesize mutant allele (Steps 7 and 12). Each lane for a target amplicon corresponds to a different temperature on a thermal gradient (from left to right: 70.8, 68.7, 65.6 and 61.7 °C). (C) Screening for insertion of the ΔpelF allele into pDONRPEX18Gm (Step 27). Each lane corresponds to a different E. coli clone isolated at Step 20. The band at 1253 bp corresponds to the anticipated size of the cloned ΔpelF allele (VC, vector control with no insert). (D) PCR to detect the ΔpelF mutation in the PAO1 cell line (Step 44 and 45). Each lane corresponds to a different P. aeruginosa colony isolated from NSLB + 15% sucrose agar (Step 38). The wild type pelF and ΔpelF alleles are anticipated to produce bands at 2476 and 1009 bp, respectively. This PCR step is used to identify the desired mutants, which are picked off of agar plates made at Step 39. (E) Growth of the chosen colonies on selective media (Step 39). Arrows indicate the P. aeruginosa PAO1 ΔpelF mutants that were selected based on PCR results in the previous panel and subsequently verified by sequencing (Step 47). Due to cloning efficiency in E. coli (Steps 13-19) and the theoretical 50% probability that a second crossover will fix a mutant allele in the chromosome, 4 colonies have been screened in Panels C, D and E.

Supplementary Figure 3 Proof-of-principle: A genetic dissection of biofilm matrix usage in P. aeruginosa PAO1.

Bacteria were grown in 3 ml LB overnight in 16 mm culture tubes at 37 °C at 250 rpm. The spent medium and planktonic cells were removed by aspiration. The adherent biomass was stained with crystal violet and rinsed to remove residual stain and loose material. A ΔwspF strain overproduces biofilm. Biofilm production requires the extracellular polymers Pel and Psl. Strains with ΔpelF or ΔpslD mutations produce less biofilm, whereas a strain with ΔpelF and ΔpslD mutations produces no biofilm.

Supplementary Figure 4 E. coli S17.1 and SM10 strains are functionally equivalent donor strains.

Each datum point represents an independent mating with P. aeruginosa PAO1 and either E. coli S17.1 or SM10 bearing one of four different deletion alleles (in pDONRPEX18Gm). Solid lines and error bars represent the mean and standard deviation of P. aeruginosa merodiploids recovered after conjugation with each of the E. coli donor strains, respectively.

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Supplementary Figures 1–4, Supplementary Tutorial, Supplementary Tables 1–3 (PDF 2359 kb)

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Hmelo, L., Borlee, B., Almblad, H. et al. Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange. Nat Protoc 10, 1820–1841 (2015). https://doi.org/10.1038/nprot.2015.115

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