Figure 2. Strategies for mutation complementation in S. pyogenes.

Complementation of a mutation (obtained using strategies described in Basic Protocols 3, 4, 5 and/or 6) is required to confirm the role of a gene of interest. The GAS strain with a mutated allele (depicted in black, with a Δ sign) of the gene of interest is manipulated to express a wild-type copy of that gene (depicted in blue) and additional tests are performed to see if the wild-type phenotype can be restored. (A) The wild-type allele of the gene of interest can be expressed in trans from a plasmid replicating in GAS. (B) The wild-type allele of the gene of interest can be re-introduced by ectopic integration at the location of mutation using single crossover integration of a conditionally replicative plasmid or a suicide vector. (C) The mutated allele of the gene of interest can be replaced by its wild-type gene using allelic exchange. (D) If the mutation of the gene of interest was carried out by single crossover using a conditionally replicating plasmid, wild-type allele of the gene of interest can be restored by rescuing the mutation by allowing the plasmid to excise from the chromosome and be lost in the absence of selection. (E) The wild-type allele of the gene of interest can be introduced by phage transduction (Cho and Caparon, 2006).