Cited by 255Bacterial Genetics and BiotechnologyClostridium difficile and Clostridium perfringens researchCRISPR and Genetic Engineering

Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier

Purdy, Des, O'Keeffe, Triona A. T., Elmore, Michael, Herbert, Mike, McLeod, Anne, Bokori‐Brown, Monika, Ostrowski, Anna, Minton, Nigel P.·2002·Molecular Microbiology

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.

Integrity signals

Registry

Crossref · 10.1046/j.1365-2958.2002.03134.x

Cites

31 of 31 references verified0 citation errors · 0 not checkable

Statistics

Recomputing p-values needs the paper’s full text.

Claims the paper makes

This paper’s full text isn’t openly available, so its claims and self-check weren’t run. The registry and citation checks still apply.

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Cites

Flagged first, then by citations

Cited by

Sorted by citations

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

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review