Genome modifications and cloning using a conjugally transferable recombineering system

Abstract

The genetic modification of primary bacterial disease isolates is challenging due to the lack of highly efficient genetic tools. Herein we describe the development of a modified PCR-based, λ Red-mediated recombineering system for efficient deletion of genes in Gram-negative bacteria. A series of conjugally transferrable plasmids were constructed by cloning an oriT sequence and different antibiotic resistance genes into recombinogenic plasmid pKD46. Using this system we deleted ten different genes from the genomes of Edwardsiella ictaluri and Aeromonas hydrophila. A temperature sensitive and conjugally transferable flp recombinase plasmid was developed to generate markerless gene deletion mutants. We also developed an efficient cloning system to capture larger bacterial genetic elements and clone them into a conjugally transferrable plasmid for facile transferring to Gram-negative bacteria. This system should be applicable in diverse Gram-negative bacteria to modify and complement genomic elements in bacteria that cannot be manipulated using available genetic tools.

Type II Secretion Is Essential for Virulence of the Emerging Fish Pathogen, Hypervirulent Aeromonas hydrophila

“…Fifty microliter of freshly-prepared electrocompetent vAh Ml09-119 + pMJH65 was transferred to pre-chilled 1.5 ml tubes. One thousand five hundred nanogram exeC-FRTcat-exeE linear dsDNA (2.5 μl) was added to electrocompetent vAh ML09-119 cells, prepared as previously described ( 25 ), gently mixed, and transferred to chilled, 0.1 cm electroporation cuvettes. Cells were subjected to a single exponential decay pulse of 1.2 kV, 25 μF, and 200 Ω using an Eppendorf Eporator (Hamburg, Germany), followed by the immediate addition of 1 ml SOC recovery media, and incubated overnight with shaking at 30°C.…”

“…The pMJH65 plasmid, which confers Tet resistance, was cured from exeD :FRT cat mutants by heat induction as previously described ( 25 ). Following heat induction, cells were streaked for isolation on non-selective TSA plates, then isolated colonies were streaked onto TSA+Tet selective agar.…”

“…Bacterial strains and plasmids used in this study are presented in Table 1. Hypervirulent A. hydrophila ML09-119 containing the recombinogenic plasmid, pMJH65 (25), was used as the wild-type vAh in which deletion mutations were generated.…”

Phage Therapy: What Have We Learned?

“…Results of studies on certain model or industrially-applicable bacteria that were depleted of prophages and certain other mobile elements as well as certain determinants of mutability indicate that such strains have a better genomic stability and are more efficient producers of certain compounds than their wild-type parents [ 89 , 199 , 219 , 252 , 254 , 255 ]. Engineering of their genomes does not need to be associated with the permanent presence of heterologous DNA, as markerless gene knock-out or gene replacement systems have been developed for a number of pathogenic bacterial species and are in constant further development [ 254 , 255 , 256 , 257 , 258 , 259 , 260 , 261 , 262 , 263 , 264 , 265 , 266 , 267 , 268 , 269 , 270 , 271 , 272 , 273 , 274 , 275 , 276 ].…”

Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection

“…Adapting recombineering for a new species is challenging; however, because the λ Red and RecET systems do not necessarily maintain high efficiency across different bacteria (Huang et al 2017;Hossain et al 2015;Hu et al 2014), there may be a dependence on host-specific machinery (Datta et al 2008;Muyrers et al 2000). Since recombineering was first applied in E. coli (Datsenko & Wanner 2000;Baba et al 2006), a few other phage homologous recombination systems have been found to promote recombination in Pseudomonas, Vibrio, Lactobacillus, Mycobacteria, Photorhabdus and Staphylococcus (van Kessel & Hatfull 2007;Yin et al 2015;Yang et al 2015;Swingle, Bao, et al 2010;Oh & Van Pijkeren 2014;Aparicio et al 2018;Penewit et al 2018;Lee et al 2017;Datta et al 2008), and we have explored whether this technology can be established in Shewanella.…”