Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier
Abstract
Progress towards understanding the molecular basis of virulence in Clostridium difficile has been hindered by the lack of effective gene transfer systems. We have now, for the first time, developed procedures that may be used to introduce autonomously replicating vectors into this organism through their conjugative, oriT-based mobilization from Escherichia coli donors. Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous C. difficile plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems. The characterized replicon is the first C. difficile plasmid replicon to be sequenced and encodes a large replication protein (RepA) and a repetitive region composed of a 35 bp iteron sequence repeated seven times. Strain CD6 has two RM systems, CdiCD6I/M.CdiCD6I and CdiCD6II/M. CdiCD6II, with equivalent specificities to Sau96I/M. Sau96I (5'-GGNMCC-3') and MboI/M. MboI (5'-GMATC-3') respectively. A second strain (CD3) possesses a type IIs restriction enzyme, Cdi I, which cleaves the sequence 5'-CATCG-3' between the fourth and fifth nucleotide to give a blunt-ended fragment. This is the first time that an enzyme with this specificity has been reported. The sequential addition of this site to vectors showed that each site caused between a five- and 16-fold reduction in transfer efficiency. The transfer efficiencies achieved with both strains equated to between 1.0 x 10-6 and 5.5 x 10-5 transconjugants per donor.
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Transfer of DNA via conjugation has been reported to occur in single strand, by which means the incoming DNA can evade the recipient's restriction endonucleases.
“However, transfer of DNA via conjugation has been reported to occur in single strand, by which means the incoming DNA can evade the recipient's restriction endonucleases (Jennert et al., 2000; Purdy et al., 2002).”
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- Authors
- Purdy, D., O'Keeffe, T. A. T., Elmore, M., Herbert, M., McLeod, A., Bokori‐Brown, M., Ostrowski, A., & Minton, N. P.
- Affiliations
- University of Nottingham (GB)
- Corresponding author
- Minton, Nigel P.
- Venue
- Molecular Microbiology
- Publisher
- Wiley
- Published
- 2002-10
- Volume
- 46
- Issue
- 2
- Pages
- 439-452
- ISSN
- 0950-382X (ISSN-L)
- 1365-2958
- OpenAlex
- W1837590098
- Cited by
- 255
- References
- 31
- Citations by year
- 2026: 7 · 2025: 11 · 2024: 13 · 2023: 12 · 2022: 14 · 2021: 13 · 2020: 10 · 2019: 19 · 2018: 17 · 2017: 9 · 2016: 16 · 2015: 20 · 2014: 18 · 2013: 10 · 2012: 12
- FWCI
- 2.47
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Molecular Microbiology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Health Sciences › Medicine › Infectious Diseases › Clostridium difficile and Clostridium perfringens research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Keywords
- Clostridium difficile, conjugative transfer, shuttle vector, restriction-modification systems, plasmid replicons, type IIS restriction enzymes, MboI, transconjugants
- MeSH terms
- Amino Acid Sequence, Base Sequence, Conjugation, Genetic, DNA Replication, Escherichia coli, Genetic Vectors, Molecular Sequence Data, Plasmids, Replicon, DNA Restriction-Modification Enzymes, Clostridioides difficile, Replication Origin
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