FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica

Abstract

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of "scar" DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria.

Figure 1. Schematic of FRUIT method.

(A) Schematic of FRUIT for introducing point mutations or deletions. PCR product is amplified from the recombineering template plasmid (pAMD001), incorporating flanking sequence with identity to the desired site of recombination. This PCR product is introduced into cells expressing λ recombinase proteins and recombinants are selected using the thyA marker (growth on media lacking thymine). A mutation can then be introduced by recombineering a second PCR product, selecting for recombinants using counter-selection of thyA (growth in the presence of trimethoprim). (B) Schematic of FRUIT for introducing FLAG tags. As above, except that loss of thyA occurs spontaneously due to homologous recombination of duplicate sets of FLAG tags.

Figure 2. FRUIT mutagenesis of MG1655 (E. coli K-12) lacZ, H10407 (ETEC) eslA, and 14028s (S. enterica serovar Typhimurium) oafA.

(A) Schematic indicating the mutation within lacZ. (B) β-galactosidase assay in wild-type MG1655 and mutant MG1655 with a stop codon introduced within lacZ. (C) Schematic inidicating the mutation in the putative LexA site. (D) ChIP/qPCR assay to measure association of LexA with the region upstream of sulA (known LexA site) and the region upstream of eslA in wild-type and mutant strains. Relative occupancy values represent background-subtracted enrichment relative to that upstream of sulA. (E) Schematic indicating the deletion of oafA. (F) Soft agar motility assay of wild-type or mutant strains in the presence or absence of Sal4 antibody. Values indicate the diameter of the halo of motile cells after the indicated time.

Figure 3. FRUIT epitope-tagging of MG1655 (E. coli K-12) allR, H10407 (ETEC) allR, and 14028s (S. enterica serovar Typhimurium) HilD.

(A) Schematic indicating C-terminal tagging with three FLAG tags. (B) ChIP/qPCR assay to measure association of MG1655 AllR-FLAG₃ with the region upstream of allA (known AllR site in E. coli K-12) . Values are also shown for a control ChIP with an untagged strain. Occupancy unit values represent background-subtracted enrichment relative to a control region. (C) ChIP/qPCR assay to measure association of H10407 AllR-FLAG₃ with the region upstream of allA, or with predicted non-target regions upstream of galE and purR. Occupancy unit values represent background-subtracted enrichment relative to a control region. (D) ChIP/qPCR assay to measure association of 14028s HilD-FLAG₃ with the regions upstream of prgH and invH (known HilD targets) . Values are also shown for a control ChIP with an untagged strain. Occupancy unit values represent background-subtracted enrichment relative to a control region. (E) Western blot probing extracts from untagged and FLAG-tagged strains for MG1655 (K-12), H10407 (ETEC) and 14028s (S. enterica). Note that the anti-FLAG antibody cross-reacts with a protein expressed E. coli K-12.

Figure 4. FRUIT promoter swaps in MG1655 (E. coli K-12).

(A) Schematic indicating the plasmid templates used for FRUIT. (B) Schematic indicating replacement of the lacZYA promoter with P_high, P_med, P_low or P_rha promoters. (C) β-galactosidase assay in ΔlacZ MG1655 and mutant strains with P_high, P_med or P_low driving expression of lacZYA (cells were grown without IPTG). (D) β-galactosidase assay in ΔlacZ MG1655 and a mutant strain with P_rha driving expression of lacZYA. Assays were performed ± rhamnose.