Abstract
Purpose
We have previously shown that DNA constructs can be introduced into isolated mitochondria through the process of conjugative transfer from an E. coli host. We set out to generate a conjugative E. coli strain that would be able to introduce itself into the cytoplasm of a mammalian cell for the purpose of transferring DNA into the mitochondria in the cell.
Methods
We have now developed a method for making E. coli strains from which nonreplicating populations of daughter cells can be generated. We used this approach to modify a facultative intracellular enteroinvasive E. coli (EIEC) and introduced conjugative functions to this new strain.
Results
We demonstrate that this new strain can generate large populations of nonreplicating cells that are capable of conjugative transfer to other cells and can readily invade mammalian tissue culture cells, live in the cytoplasm of the cell for several days, and that do not kill the invaded mammalian cell.
Conclusions
We successfully constructed an E. coli host suitable for intracellular conjugative transfer but, due to the lack of suitable mitochondrial screening or selectable markers, we have not yet been able to determine if these bacterial vectors can in fact transfer DNA into intracelluar mitochondria.
Similar content being viewed by others
Abbreviations
- eGFP:
-
enhanced green fluorescent protein
- EIEC:
-
enteroinvasive Escherichia coli
- PCR:
-
Polymerase Chain Reaction
References
Waters VL. Conjugative transfer in the dissemination of beta-lactam and aminoglycoside resistance. Front Biosci. 1999;4:D433–56.
Heinemann JA, Sprague Jr GF. Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature. 1989;340:205–9.
Waters VL. Conjugation between bacterial and mammalian cells. Nature Genet. 2001;29:375–6.
Yoon YG, Koob MD. Transformation of isolated mammalian mitochondria by bacterial conjugation. Nucleic Acids Res. 2005;33:e139.
Murphy KC. Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol. 1998;180:2063–71.
Hashimoto-Gotoh T, Sekiguchi M. Mutations of temperature sensitivity in r plasmid psc101. J Bacteriol. 1977;131:405–12.
Koob M, Szybalski W. Cleaving yeast and Escherichia coli genomes at a single site. Science. 1990;250:271–3.
Koob MD, Shaw AJ, Cameron DC. Minimizing the genome of Escherichia coli. Motivation and strategy. Ann N Y Acad Sci. 1994;745:1–3.
Oaks EV, Wingfield ME, Formal SB. Plaque formation by virulent shigella flexneri. Infect Immun. 1985;48:124–9.
Parsot C. Shigella spp. And enteroinvasive Escherichia coli pathogenicity factors. FEMS Microbiol Lett. 2005;252:11–8. Epub 2005 Sep 2015.
Galan JE, Curtiss 3rd R. Distribution of the inva, -b, -c, and -d genes of salmonella typhimurium among other salmonella serovars: Inva mutants of salmonella typhi are deficient for entry into mammalian cells. Infect Immun. 1991;59:2901–8.
Wechsler JA, Gross JD. Escherichia coli mutants temperature-sensitive for DNA synthesis. Mol Gen Genet. 1971;113:273–84.
Waxman DJ, Strominger JL. Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics. Annu Rev Biochem. 1983;52:825–69.
Figurski DH, Helinski DR. Replication of an origin-containing derivative of plasmid rk2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA. 1979;76:1648–52.
Frey J, Chandler M, Caro L. The effects of an Escherichia coli dnaats mutation on the replication of the plasmids cole1 psc101, r100.1 and rtf-tc. Mol Gen Genet. 1979;174:117–26.
Yoon YG, Koob MD. Toward genetic transformation of mitochondria in mammalian cells using a recoded drug-resistant selection marker. J Genet Genomics. 2011;38:173–9.
Acknowledgments & Disclosures
We thank Michael Sadowsky for the gifts of plasmids and E.coli strains. This work was funded by the National Institutes of Health (NS052612) and by the Minnesota Partnership for Biotechnology and Medical Genomics.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yoon, Y.G., Koob, M.D. Nonreplicating Intracellular Bacterial Vector for Conjugative DNA Transfer into Mitochondria. Pharm Res 29, 1040–1045 (2012). https://doi.org/10.1007/s11095-012-0701-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11095-012-0701-0