Genome modifications and cloning using a conjugally transferable recombineering system

Abstract

The genetic modification of primary bacterial disease isolates is challenging due to the lack of highly efficient genetic tools. Herein we describe the development of a modified PCR-based, λ Red-mediated recombineering system for efficient deletion of genes in Gram-negative bacteria. A series of conjugally transferrable plasmids were constructed by cloning an oriT sequence and different antibiotic resistance genes into recombinogenic plasmid pKD46. Using this system we deleted ten different genes from the genomes of Edwardsiella ictaluri and Aeromonas hydrophila. A temperature sensitive and conjugally transferable flp recombinase plasmid was developed to generate markerless gene deletion mutants. We also developed an efficient cloning system to capture larger bacterial genetic elements and clone them into a conjugally transferrable plasmid for facile transferring to Gram-negative bacteria. This system should be applicable in diverse Gram-negative bacteria to modify and complement genomic elements in bacteria that cannot be manipulated using available genetic tools.

Keywords: Bacterial pathogens; Genetic modification; Recombineering.

Fig. 1

Schematic maps of conjugally transferable recombinogenic and flp recombinase plasmids constructed in this study. The oriT sequence cloned into these plasmids facilitates the conjugal transfer of these plasmids using appropriate donor E. coli strain. Red recombinogenic plasmids pMJH46, pMJH65 and flp recombinase plasmid pCMT-flp are easily cured after heat induction at 37 °C due to temperature sensitive repA101 gene. Plasmid maps were generated by CLC Genomics Workbench (version 4.9).

Fig. 2

Targeted deletion of E. ictaluri genes ompLC, dtrA and eihA by recombineering. (Panel A) Colonies gown on 2 × YT plates supplemented with kanamycin were selected for PCR screening of ompLC gene deleted mutants. Lanes 1, 3–9 and 11 represent the PCR products of ompLC gene mutants disrupted with the kanR gene (ompLC::kanR) and lanes 2, 10 and 12 represents the PCR product of wild type ompLC gene of E. ictaluri strain Alg-08-183. (Panel B) Removal of the kanamycin resistance marker using the Flp recombinase of plasmid pCP20. PCR screening of E. ictaluri mutants plated after temperature induction showed that all tested mutants had lost the antibiotic resistance marker. (Panel C) PCR confirmation of deletion of the ompLC and drtA genes from E. ictaluri strain Alg-08-183 and eihA from E. ictaluri strain R4383.

Fig. 3

Determination of recombination frequency in A. hydrophila. (Panel A) The effect of dsDNA substrate concentration on recombination frequency in A. hydrophila was determined using four different dsDNA substrate concentration ranging from 0.75 μg to 5.0 μg per recombineering experiment. (Panel B) Four different primer combinations were generated using modified and unmodified primers. Modified primers included four consecutive phosphorothioate bonds at the 5′ end of the primers. Type “−/−” used unmodified primers as a negative control, type “+/−” included modification of the forward primer but not the reverse primers, type “−/+” included modification to the reverse but not forward primer, and type “+/+” included phosphorothioate bonds in both primers. The latter condition in which both primers were modified provided significantly more mutants than any other types of dsDNA substrates used for recombineering (***p-value = 0.0026). (Panel C) The effect of varying the length of the homologous regions of the dsDNA substrate to the targeted chromosomal site on the recombination frequency was determined using approximately 60 bp, 250 bp and 500 bp of homologous sequence at both the 5′ and 3′ ends. The average number of mutants obtained was derived from three independent recombineering experiments.

Fig. 4

Strategy for PCR-free cloning of large bacterial genetic regions. The major steps of cloning large genetic inserts are indicated. The catR-oriT-oriR (pMJH97) cassette was PCR amplified using primer pairs with 50–60 bp homologous sequence at their 5′-ends specific to the targetred site. Depending on the choice of restriction enzymes, the resulting dsDNA substrate can be integrated upstream or downstream of the targeted site of the genome using the recombineering system. Once the catR-oriT-oriR (pMJH97) cassette integration into the genome was confirmed by PCR and sequencing using primers P1 and P2, the genomic DNA of integrants was restriction digested with an appropriate restriction enzyme to clone into E. coli after self-ligation using T4 DNA ligase. The cloning of the correct insert into the plasmid pMJH97 was verified by PCR and sequencing using vector and insert specific primers P3 and P4, respectively. The plasmids with cloned inserts were then readily transfered to other Gram-negative bacterial strain by oriT sequence-mediated conjugal transfer using an appropriate donor strain.