A Robust One-Step Recombineering System for Enterohemorrhagic Escherichia coli

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 lambd...

Verfasser: Peng, Lang
Dumevi, Rexford Mawunyo
Chittò, Marco
Haarmann, Nadja
Berger, Petya
Koudelka, Gerald
Schmidt, Herbert
Mellmann, Alexander
Dobrindt, Ulrich
Berger, Michael
FB/Einrichtung:FB 05: Medizinische Fakultät
Dokumenttypen:Artikel
Medientypen:Text
Erscheinungsdatum:2022
Publikation in MIAMI:16.06.2023
Datum der letzten Änderung:16.06.2023
Angaben zur Ausgabe:[Electronic ed.]
Quelle:Microorganisms 10 (2022) 9, 1689, 1-12
Schlagwörter:enterohemorrhagic Escherichia coli; recombineering; SOS response
Fachgebiet (DDC):610: Medizin und Gesundheit
Lizenz:CC BY 4.0
Sprache:English
Förderung:Finanziert durch den Open-Access-Publikationsfonds der Westfälischen Wilhelms-Universität Münster (WWU Münster).
Förderer: Deutsche Forschungsgemeinschaft / Projektnummer: 276606594
Förderer: Deutsche Forschungsgemeinschaft / Projektnummer: SFB1009 B04
Förderer: Deutsche Forschungsgemeinschaft / Projektnummer: SFB1009 B05
Format:PDF-Dokument
URN:urn:nbn:de:hbz:6-30009535465
Weitere Identifikatoren:DOI: 10.17879/80019472879
Permalink:https://nbn-resolving.de/urn:nbn:de:hbz:6-30009535465
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  • Onlinezugriff:10.3390_microorganisms10091689.pdf

    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.