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A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide

The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, ch...

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Autores principales: Qin, Jilong, Hong, Yaoqin, Pullela, Karthik, Morona, Renato, Henderson, Ian R., Totsika, Makrina
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9270391/
https://www.ncbi.nlm.nih.gov/pubmed/35804085
http://dx.doi.org/10.1038/s41598-022-15997-8
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author Qin, Jilong
Hong, Yaoqin
Pullela, Karthik
Morona, Renato
Henderson, Ian R.
Totsika, Makrina
author_facet Qin, Jilong
Hong, Yaoqin
Pullela, Karthik
Morona, Renato
Henderson, Ian R.
Totsika, Makrina
author_sort Qin, Jilong
collection PubMed
description The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a fivefold increase in the yield of engineered mutant colonies obtained in S. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by eightfold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA–protein complexes into clinically important bacteria.
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spelling pubmed-92703912022-07-10 A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide Qin, Jilong Hong, Yaoqin Pullela, Karthik Morona, Renato Henderson, Ian R. Totsika, Makrina Sci Rep Article The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches. However, the generally low transformation frequency among natural isolates poses technical hurdles to widely applying common methods in molecular biology, including transformation of large constructs, chromosomal genetic manipulation, and dense mutant library construction. Here we demonstrate that culturing clinical isolates in the presence of polymyxin B nonapeptide (PMBN) improves their transformation frequency via electroporation by up to 100-fold in a dose-dependent and reversible manner. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. Moreover, we show a fivefold increase in the yield of engineered mutant colonies obtained in S. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Lastly, we demonstrate that PMBN treatment can enhance the delivery of DNA-transposase complexes into UPEC and increase transposon mutant yield by eightfold when constructing Transposon Insertion Sequencing (TIS) libraries. Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA–protein complexes into clinically important bacteria. Nature Publishing Group UK 2022-07-08 /pmc/articles/PMC9270391/ /pubmed/35804085 http://dx.doi.org/10.1038/s41598-022-15997-8 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Qin, Jilong
Hong, Yaoqin
Pullela, Karthik
Morona, Renato
Henderson, Ian R.
Totsika, Makrina
A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide
title A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide
title_full A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide
title_fullStr A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide
title_full_unstemmed A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide
title_short A method for increasing electroporation competence of Gram-negative clinical isolates by polymyxin B nonapeptide
title_sort method for increasing electroporation competence of gram-negative clinical isolates by polymyxin b nonapeptide
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9270391/
https://www.ncbi.nlm.nih.gov/pubmed/35804085
http://dx.doi.org/10.1038/s41598-022-15997-8
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