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. 2022 Aug 23;10(9):1689.
doi: 10.3390/microorganisms10091689.

A Robust One-Step Recombineering System for Enterohemorrhagic Escherichia coli

Lang Peng  1 , Rexford Mawunyo Dumevi  1 , Marco Chitto  1 , Nadja Haarmann  2 , Petya Berger  1   3 , Gerald Koudelka  4 , Herbert Schmidt  2 , Alexander Mellmann  1   3 , Ulrich Dobrindt  1 , Michael Berger  1
Affiliations

A Robust One-Step Recombineering System for Enterohemorrhagic Escherichia coli

Lang Peng et al. Microorganisms. .

Abstract

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3-4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.

Keywords: SOS response; enterohemorrhagic Escherichia coli; recombineering.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Schematic representation of the positions of chromosomal homology (CH) between EHEC O157:H7 EDL933 and E. coli K-12 MG1655 that were used to design the primers for the recombination substrates. (A) CH for the deletion of the lacZYA operon. (B) CH for the deletion of the regulatory gene lacI. (C) CH for the insertion upstream of the phage lambda attachment site attB.
Figure 2
Figure 2
Numbers of chloramphenicol-resistant colonies obtained for the indicated genetic locus. (A) Number of chloramphenicol-resistant colonies in EHEC O157:H7 EDL933 Δstx1/2. (B) Number of chloramphenicol-resistant colonies in E. coli K-12 MG1655. Shown are the average values of three technical replicates of three independent experiments. Statistically significant t-test comparisons are indicated by * (p < 0.05), ** (p < 0.01) and “ns” if not statistically significant.
Figure 3
Figure 3
Sparfloxacin-dependent induction of the SOS response in E. coli MG1655 PrecA SENSOR. The promoter activity of PrecA as the increase of fluorescence over time in the presence of the indicated plasmid and the indicated sugar is shown. The black arrow indicates the time point of the addition of sparfloxacin (f. c. 15 µg/mL).
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