Precise manipulation (in-frame deletions and substitutions) of the Clostridium difficile genome is possible through a two-stage process of single-crossover integration and subsequent isolation of double-crossover excision events using replication-defective plasmids that carry a counterselection marker. Use of a codA (cytosine deaminase) or pyrE (orotate phosphoribosyltransferase) as counter selection markers appears equally effective, but there is considerable merit in using a pyrE mutant as the host as, through the use of allele-coupled exchange (ACE) vectors, mutants created (by whatever means) can be rapidly complemented concomitant with restoration of the pyrE allele. This avoids the phenotypic effects frequently observed with high-copy-number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention.
Keywords: Allele-coupled exchange (ACE); Allelic exchange; Clostridium difficile; Complementation; Counterselection marker; Overexpression; Pseudo-suicide; codA; pyrE.