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.

Identification of HilD-Regulated Genes in Salmonella enterica Serovar Typhimurium

“…HilD was C-terminally epitope tagged with three FLAG tags, as described previously (31). We confirmed that FLAG-tagged HilD is fully active by measuring expression from the hilA promoter (see Fig.…”

Genome-Scale Analyses of Escherichia coli and Salmonella enterica AraC Reveal Noncanonical Targets and an Expanded Core Regulon

“…Note that SAC001 and AMD115 also contain the ⌬(araDaraB)567 mutation that lacks the araBAD operon. AMD187 (E. coli MG1655 araC-3ϫFLAG), JTW010 (E. coli MG1655 with ytfQ AraC site mutation, araC-3ϫFLAG), and CB005 (S. enterica serovar Typhimurium 14028s araC-3ϫFLAG) were constructed using the FRUIT recombineering system (18). The PCR product used to generate the initial tagged strains was made using oligonucleotides JW1141 and JW1142 for E. coli and JW2895 and JW2901 for S. enterica, with pAMD135 as the template.…”

Exposure of Salmonella enterica Serovar Typhimurium to a Protective Monoclonal IgA Triggers Exopolysaccharide Production via a Diguanylate Cyclase-Dependent Pathway

“…The murine hybridoma secreting Sal4, the monoclonal polymeric IgA antibody, was obtained from Marian Neutra (Children's Hospital, Boston, MA) (23). Hybridomas secreting the IgA antibodies (34). The primer sequences used to construct the mutant strains used in this study are listed in Table S1 in the supplemental material.…”