RRC ID 27879
Author Moriguchi K, Edahiro N, Yamamoto S, Tanaka K, Kurata N, Suzuki K.
Title Transkingdom genetic transfer from Escherichia coli to Saccharomyces cerevisiae as a simple gene introduction tool.
Journal Appl. Environ. Microbiol.
Abstract Transkingdom conjugation (TKC) permits transfer of DNA from bacteria to eukaryotic cells using a bacterial conjugal transfer system. However, it is not clear whether the process of DNA acceptance in a recipient eukaryote is homologous to the process of conjugation between bacteria. TKC transfer requires mobilizable shuttle vectors that are capable of conjugal transfer and replication in the donor and recipient strains. Here, we developed TKC vectors derived from plasmids belonging to the IncP and IncQ groups. We also investigated forms of transfer of these vectors from Escherichia coli into Saccharomyces cerevisiae to develop TKC as a simple gene introduction method. Both types of vectors were transferred precisely, conserving the origin of transfer (oriT) sequences, but IncP-based vectors appeared to be more efficient than an IncQ-based vector. Interestingly, unlike in agrobacterial T-DNA (transfer DNA) transfer, the efficiency of TKC transfer was similar between a wild-type yeast strain and DNA repair mutants defective in homologous recombination (rad51Δ and rad52Δ) or nonhomologous end joining (rad50Δ, yku70Δ, and lig4Δ). Lastly, a shuttle vector with two repeats of IncP-type oriT (oriT(P)) sequences flanking a marker gene was constructed. TKC transfer of this vector resulted in precise excision of both the oriT(P) loci as well as the marker gene, albeit at a low frequency of 17% of all transconjugants. This feature would be attractive in biotechnological applications of TKC. Taken together, these results strongly suggest that in contrast to agrobacterial T-DNA transfer, the circularization of vector single-stranded DNA occurs either before or after transfer but requires a factor(s) from the donor. TKC is a simple method of gene transfer with possible applications in yeast genetics and biotechnology.
Volume 79(14)
Pages 4393-400
Published 2013-7
DOI 10.1128/AEM.00770-13
PII AEM.00770-13
PMID 23666333
PMC PMC3697487
MeSH Base Sequence DNA, Bacterial / genetics* DNA, Bacterial / metabolism DNA, Fungal / genetics* DNA, Fungal / metabolism DNA, Single-Stranded / genetics* DNA, Single-Stranded / metabolism Escherichia coli / genetics* Genetic Vectors / genetics Genetic Vectors / metabolism Plasmids / genetics Plasmids / metabolism Polymerase Chain Reaction Saccharomyces cerevisiae / genetics* Transduction, Genetic / methods*
IF 3.633
Times Cited 4
WOS Category BIOTECHNOLOGY & APPLIED MICROBIOLOGY MICROBIOLOGY
Resource
Yeast BYP7570 BYP7573