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...
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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.