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. 2020 May 24;20(1):129.
doi: 10.1186/s12866-020-01819-2.

Efficient dual-negative selection for bacterial genome editing

Francesca Romana Cianfanelli  1 , Olivier Cunrath  1 , Dirk Bumann  2
Affiliations

Efficient dual-negative selection for bacterial genome editing

Francesca Romana Cianfanelli et al. BMC Microbiol. .

Abstract

Background: Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering.

Results: Here, we established a time-effective variant of consecutive single-crossovers. This method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in-frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Adapted versions enabled efficient gene editing also in Pseudomonas aeruginosa and multi-drug resistant (MDR) Escherichia coli clinical isolates.

Conclusions: Our method is time-effective and allows facile manipulation of multiple bacterial species including MDR clinical isolates. We anticipate that this method might be broadly applicable to additional bacterial species, including those for which recombineering has been difficult to implement.

Keywords: Gene manipulation; Homologous recombination; MDR; Mutagenesis; Salmonella.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
An optimized method for genome editing in Salmonella enterica. a Map of suicide plasmid pFOK (aphA, aminoglycoside phosphotransferase gene conferring resistance to kanamycin; I-sceI gene encoding meganuclease; oriT, origin of conjugational transfer; PtetA, tetA promoter; R6K γ ori, pi-dependent origin of replication; sacB, levansucrase gene; tetR, tetracycline repressor gene; traJ, transcriptional activator for conjugational transfer genes; MCR, multi cloning region containing EcoRI and BamHI recognition sites). b Mechanisms of negative selection for SacB and I-SceI, c Efficiency of negative selection for various chromosomal loci (sitABCD deletion - orange, foxA deletion - yellow, ssrB point mutation – green, and phoQ chimeric insertion - magenta [30]) using either SacB or I-SceI, or a combination of both. Fifty colonies were screened for each mutation. d Schematic representation of the consecutive single crossover procedure. Recombination can occur in one of the two homologous sequences (routes 1 and 2). Only alternate single crossover events involving both homologous sequences lead to the desired mutation, while two consecutive single crossovers in the same regions lead to reversion to wild-type (WT) e Overview of the entire procedure. Ideally, each step can be completed in one working day. f Schematic representation of preferential recombination in the right flanking region. External primers 1 and 2 together with plasmid-specific primers oOPC-614 and oOPC-615 can be used to screen co-integrant clones to reveal such bias and to identify rare variants for promoting mutant generation in the second single crossover. g Recombination bias for foxA deletion. PCR results of ex-conjugant screening using external primer 1 (oOPC-396) / oOPC-614 or external primer 2 (oOPC-397) / oOPC-615. Rare ex-conjugants (clones 5, 10) with recombination in the non-preferred flanking region were used for subsequent counter-selection
Fig. 2
Fig. 2
Maps of alternative suicide plasmids. a pFOG carrying aac (3)-I which confers resistance to gentamicin. b pFOKT carrying tpm coding for thiopurine-S-methyltranferase which confers resistance to tellurite
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