Trans-Kingdom Conjugation within Solid Media from Escherichia coli to Saccharomyces cerevisiae.

Maximillian P M Soltysiak,Samir Hamadache,Rebecca S Meaney,David R Edgell,Preetam Janakirama,Bogumil J Karas

doi:10.3390/ijms20205212

Maximillian P M Soltysiak, Samir Hamadache + Show 4 more

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https://doi.org/10.3390/ijms20205212

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Abstract

Conjugation is a bacterial mechanism for DNA transfer from a donor cell to a wide range of recipients, including both prokaryotic and eukaryotic cells. In contrast to conventional DNA delivery techniques, such as electroporation and chemical transformation, conjugation eliminates the need for DNA extraction, thereby preventing DNA damage during isolation. While most established conjugation protocols allow for DNA transfer in liquid media or on a solid surface, we developed a procedure for conjugation within solid media. Such a protocol may expand conjugation as a tool for DNA transfer to species that require semi-solid or solid media for growth. Conjugation within solid media could also provide a more stable microenvironment in which the conjugative pilus can establish and maintain contact with recipient cells for the successful delivery of plasmid DNA. Furthermore, transfer in solid media may enhance the ability to transfer plasmids and chromosomes greater than 100 kbp. Using our optimized method, plasmids of varying sizes were tested for transfer from Escherichia coli to Saccharomyces cerevisiae. We demonstrated that there was no significant change in conjugation frequency when plasmid size increased from 56.5 to 138.6 kbp in length. Finally, we established an efficient PCR-based synthesis protocol to generate custom conjugative plasmids.

Highlights

Conjugation is a widespread bacterial mechanism for DNA transfer and a major contributor to the spread of antibiotic resistance and virulence factors [1]
The pTA-Mob plasmid encodes the machinery required for conjugal transfer of plasmids that contain an origin of transfer [35]
For this study and future applications, we designed a method to build an alternative version of pTA-Mob, named pTA-Mob 2.0, that can self-mobilize and replicate in both E. coli and S. cerevisiae

Summary

Introduction

Conjugation is a widespread bacterial mechanism for DNA transfer and a major contributor to the spread of antibiotic resistance and virulence factors [1]. Various bacterial donor species have been used to deliver DNA to eukaryotic recipients such as the yeast Saccharomyces cerevisiae [4,5,6,7,8,9], algal diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana [9,10,11,12,13], and mammalian cells [14,15,16,17] Conventional transformation techniques, such as electroporation and chemical transformation [18], have been developed for many species, yet suffer from some drawbacks [19]. While it has been previously demonstrated that plasmids up to 875 kbp in size can be transferred to prokaryotic recipients, the upper size limit of conjugal transfer to eukaryotes has yet to be determined [25]

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