In particular, high-efficiency transformation of Gram-negative pathogenic bacteria with either large DNA constructs (> 10 kb) or with DNA–protein complexes (e.g. However, electrotransformation efficiency can remain low due to the presence of surface polysaccharides in some pathogenic bacteria, which act as a major barrier in successfully employing current DNA delivery methods 7. For this reason, almost all DNA delivery approaches for pathogenic bacteria solely rely on electrotransformation which involves washing bacterial cells with 10% (v/v) glycerol water followed by electro-permeabilisation at high voltages 5, 6. Despite the high efficiency of chemical transformation for delivering DNA into laboratory adapted Gram-negative bacteria, especially Escherichia coli K-12 strains, it has limited success with natural and pathogenic isolates 4. The most commonly used method for DNA delivery into bacterial cells is chemical transformation which involves cell washes with Mg 2+ and/or Ca 2+ under ice-cold conditions followed by heat-shock treatment 1, 2, 3.
Therefore, PMBN can be used as a powerful electropermeabilisation adjuvant to aid the delivery of DNA and DNA–protein complexes into clinically important bacteria.ĭelivery of foreign DNA into Gram-negative bacteria is a routine procedure in molecular microbiology and is essential for gene cloning, mutagenesis, and construction of mutant libraries. 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. enterica with the widely used lambda-Red recombineering method, when cells are cultured in the presence of PMBN. Moreover, we show a fivefold increase in the yield of engineered mutant colonies obtained in S. Using our PMBN electroporation method we show efficient delivery of large plasmid constructs into UPEC, which otherwise failed using a conventional electroporation protocol. The effect was observed for PMBN-binding uropathogenic Escherichia coli (UPEC) and Salmonella enterica strains but not naturally polymyxin resistant Proteus mirabilis. 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. 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. The study of clinically relevant bacterial pathogens relies on molecular and genetic approaches.
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