一种用于肠出血性大肠杆菌的强大一步重组工程系统

A Robust One-Step Recombineering System for Enterohemorrhagic .

作者信息

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

机构信息

Institute of Hygiene, University of Münster, 48149 Münster, Germany.

Institute of Food Science and Biotechnology, University of Hohenheim, 70599 Stuttgart, Germany.

出版信息

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

Abstract

Enterohemorrhagic (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 together with , , and . We demonstrate that the genetic manipulation is 3-4 times more efficient in EHEC O157:H7 EDL933 Δ/ with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in K-12 MG1655. Coexpression of 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 isolates.

摘要

肠出血性大肠杆菌(EHEC)可导致人类严重腹泻。为了改善治疗方案,深入了解EHEC的致病性至关重要。使用经典的一步法对EHEC进行基因操作,如噬菌体λ(λ)Red功能的瞬时过表达,效率并不高。在此,我们基于与 、 和 的瞬时共表达,提供了一种在EHEC中提高重组工程效率的强大且可靠的方法。我们证明,与单独过表达λ Red功能相比,使用我们的方法在EHEC O157:H7 EDL933 Δ/ 中进行基因操作的效率提高了3至4倍。两种重组工程系统在K-12 MG1655中表现出相似的效率。 的共表达并未增强Gam对司帕沙星介导的SOS反应的抑制作用。因此,对RecFOR途径的额外抑制而非对SOS反应诱导的RecBCD途径的更强抑制可能通过间接阻断噬菌体诱导导致重组工程效率提高。尽管需要更多实验来阐明重组工程效率提高的确切机制细节,但我们建议使用我们的方法对EHEC和其他携带前噬菌体的 分离株进行强大的基因操作。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/21f9/9504302/0464fd412fc6/microorganisms-10-01689-g001.jpg

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