Allelic-Exchange Procedure inStaphylococcus aureus

Abstract

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