Allelic-Exchange Procedure inStaphylococcus aureus

This protocol continues a series of methods for the construction of an in-frame gene deletion in Staphylococcus aureus strain RN4220. To this end, we describe in this protocol an allelic-exchange procedure for S. aureus . We have previously described how an allelic-exchange plasmid containing a desired gene deletion (in this case, pIMAY*-Δ tagO ) can be constructed and isolated from Escherichia coli , then introduced into electrocompetent S. aureus cells by electroporation. This plasmid contains a temperature-sensitive origin of replication, a counterselectable marker ( pheS * gene) and confers chloramphenicol resistance to S. aureus . As a specific example, we present the construction of strain RN4220*Δ tagO from strain RN4220 carrying the pIMAY*-Δ tagO plasmid. The protocol can be easily adapted for the construction of other gene deletions and/or allelic-exchange plasmids.