Abstract
Shewanella oneidensis MR-1 is an invaluable host for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals and understanding extracellular electron transfer. However, genetic manipulation is challenging due to the lack of genetic tools. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. Here, we describe development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of ~4.0 x 106 transformants/µg DNA. High transformation efficiency is maintained when cells are frozen for long term storage. In addition, we report a new prophage-mediated genome engineering (recombineering) system using a λ Red Beta homolog from Shewanella sp. W3-18-1. By targeting two different chromosomal alleles, we demonstrate its application for precise genome editing using single strand DNA oligonucleotides and show that an efficiency of ~5% recombinants among total cells can be obtained. This is the first effective and simple strategy for recombination with markerless mutations in S. oneidensis. Continued development of this recombinant technology will advance high-throughput and genome modification efforts to engineer and investigate S. oneidensis and other environmental bacteria.
Highlights
Shewanella oneidensis strain MR-1 is a widely used Shewanella strain but displays a low efficiency of electrotransformation for heterologous plasmids derived from different bacterial species, due to its native restriction-modification system[11]
The latter contains a mutation in the DNA-cytosine methyltransferase that prevents methylation of cytosine residues, which is useful for plasmid purification and transformation into other bacteria because of host modification-dependent restriction systems[43]
Conjugation has been widely used as the means to transfer plasmid DNA in bacteria, electroporation is more convenient for large-scale studies and allows transfer of both circular and linear DNA molecules
Summary
Shewanella oneidensis strain MR-1 is a widely used Shewanella strain but displays a low efficiency of electrotransformation for heterologous plasmids derived from different bacterial species, due to its native restriction-modification system[11]. We found that using cells grown to late exponential phase increases electroporation efficiency by nearly 400-fold compared to those from early exponential phase, as conventional methods describe This surprising finding allowed us to make electrocompetent cells by using overnight cultures which, when frozen at −80 °C, still maintain the same high transformation efficiency. Developing this high efficiency method for DNA uptake in S. oneidensis has enabled us to introduce single-stranded DNA oligonucleotides (oligos) to perform in vivo homologous recombination–mediated genetic engineering, known as recombineering, a powerful tool for precise DNA editing developed in E. coli. In contrast to other mutagenesis methods, this technology allows precise editing at the single nucleotide level in a few days
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