Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli

Abstract

Background: The lambda Red recombineering technology has been used extensively in Escherichia coli and Salmonella typhimurium for easy PCR-mediated generation of deletion mutants, but less so in pathogenic species of E. coli such as EHEC and EPEC. Our early experiments with the use of lambda Red in EHEC and EPEC have led to sporadic results, leading to the present study to identify factors that might improve the efficiency of Red recombineering in these pathogenic strains of E. coli.

Results: In this report, we have identified conditions that optimize the use of lambda Red for recombineering in EHEC and EPEC. Using plasmids that contain a Ptac-red-gam operon and a temperature-sensitive origin of replication, we have generated multiple mutations (both marked and unmarked) in known virulence genes. In addition, we have easily deleted five O157-specific islands (O-islands) of EHEC suspected of containing virulence factors. We have examined the use of both PCR-generated substrates (40 bp of flanking homology) and plasmid-derived substrates (approximately 1 kb of flanking homology); both work well and each have their own advantages. The establishment of the hyper-rec phenotype requires only a 20 minute IPTG induction period of red and gam. This recombinogenic window is important as constitutive expression of red and gam induces a 10-fold increase in spontaneous resistance to rifampicin. Other factors such as the orientation of the drug marker in recombination substrates and heat shock effects also play roles in the success of Red-mediated recombination in EHEC and EPEC.

Conclusions: The lambda Red recombineering technology has been optimized for use in pathogenic species of E. coli, namely EHEC and EPEC. As demonstration of this technology, five O-islands of EHEC were easily and precisely deleted from the chromosome by electroporation with PCR-generated substrates containing drug markers flanked with 40 bp of target DNA. These results should encourage the use of lambda Red recombineering in these and other strains of pathogenic bacteria for faster identification of virulence factors and the speedy generation of bacterial mutants for vaccine development.

Figure 1

Schematic of λ Red-promoted PCR-mediated recombination. PCR products, containing a drug marker flanked by 40–60 bp of target DNA, are generated by primers designated 5KO and 3KO (see Table 6 in Additional file #2) and electroporated into EHEC containing pKM201 or pKM208 (or EPEC containing pTP223). Chromosomal replacements were verified by PCR using primers upstream (U), downstream (D) and within the drug marker (M) (data not shown).

Figure 2

Uncontrolled λ Red expression is mutagenic. A single fresh colony of EHEC strain TUV93-0 (with indicated plasmids) was suspended in 1 ml LB containing 100 μg/ml ampicillin. The cell suspension was diluted with LB-ampicillin to a final concentration of 5 × 10⁴ cells/ml and aliquoted to 24 culture tubes. Overnight cultures (0.3 ml each) of EHEC with control plasmid (circles), pKM201 (squares), pKM208 (triangles) and pKM208 with IPTG added (diamonds) were plated on LB plates containing 100 μg/ml rifampicin. Plates were incubated overnight at 37 degrees and rifampicin resistant colonies were counted 24 hours later. Average number of Rif^R colonies (with standard errors) for each culture are as follows: control plasmid, 14.1 (2.7); pKM201, 173 (33.3); pKM208, 40.4 (9.9); pKM208 + IPTG, 194 (21.9).

Figure 3

Time course for promotion of hyper-rec phenotype. EHEC strain TUV93-0 containing pKM208 (five cultures, 20 ml each) was grown for electrocompetence as described in the Methods section. At various times prior to collection, IPTG was added to four of the cultures to a final concentration of 1 mM; the fifth culture received no IPTG. The cells were heat shocked for the final 15 minutes, prepared for electroporation and electroporated with DNA (~0.25 μg) containing the kan gene flanked by 40 bp of EHEC DNA (resulting in a deletion of O-islands #130 and #131). After suspension in LB, the cells were grown for 90 minutes at 37°C and plated on LB plates containing 20 μg/ml kanamycin. The number of Kan^R transformants per 10⁸ survivor and total number of Kan^R transformants are plotted as a function of IPTG concentration. The data points are averages of two experiments (ranges are shown). A random check of 160 colonies showed that 95% re-struck successfully to fresh LB plates containing 20 μg/ml kanamycin; 10 of 10 of these colonies were verified by PCR analysis to be true recombinants (data not shown). Insert: 0.1 ml of electrocompetent cells, prepared with and without 1 hour IPTG induction, were spread on LB plates containing 100 μg/ml rifampicin to determine total number of Rif^R mutants. Dilutions of the cells were titered on LB plates to determine total cell density; experiments done in triplicate (+/- standard error).