Sie können Bookmarks mittels Listen verwalten, loggen Sie sich dafür bitte in Ihr SLUB Benutzerkonto ein.
Medientyp:
E-Artikel
Titel:
Conjugative transfer of clostridial shuttle vectors from Escherichia coli to Clostridium difficile through circumvention of the restriction barrier
Beteiligte:
Purdy, Des;
O'Keeffe, Triona A. T.;
Elmore, Michael;
Herbert, Mike;
McLeod, Anne;
Bokori‐Brown, Monika;
Ostrowski, Anna;
Minton, Nigel P.
Erschienen:
Wiley, 2002
Erschienen in:Molecular Microbiology
Sprache:
Englisch
DOI:
10.1046/j.1365-2958.2002.03134.x
ISSN:
0950-382X;
1365-2958
Entstehung:
Anmerkungen:
Beschreibung:
<jats:title>Summary</jats:title><jats:p>Progress towards understanding the molecular basis of virulence in <jats:italic>Clostridium difficile</jats:italic> 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, <jats:italic>oriT</jats:italic>‐based mobilization from <jats:italic>Escherichia coli</jats:italic> donors. Successful transfer was achieved through the use of a plasmid replicon isolated from an indigenous <jats:italic>C. difficile</jats:italic> plasmid, pCD6, and through the characterization and subsequent circumvention of host restriction/modification (RM) systems. The characterized replicon is the first <jats:italic>C. difficile</jats:italic> 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, <jats:italic>Cdi</jats:italic>CD6I/M<jats:italic>.Cdi</jats:italic>CD6I and <jats:italic>Cdi</jats:italic>CD6II/M. <jats:italic>Cdi</jats:italic>CD6II, with equivalent specificities to <jats:italic>Sau</jats:italic>96I/M. <jats:italic>Sau</jats:italic>96I (5′‐GGN<jats:sup>M</jats:sup>CC‐3′) and <jats:italic>Mbo</jats:italic>I/M. <jats:italic>Mbo</jats:italic>I (5′‐G<jats:sup>M</jats:sup>ATC‐3′) respectively. A second strain (CD3) possesses a type IIs restriction enzyme, <jats:italic>Cdi</jats:italic> 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 × 10<jats:sup>−6</jats:sup> and 5.5 × 10<jats:sup>−5</jats:sup> transconjugants per donor.</jats:p>