OBJECTIVE: To identify novel protein-protein interactions in S. aureus and to assess these interactions for their suitability in the development of novel antibiotic chemotherapies. METHODS: Gene candidates involved in the essential cellular processes of transcription, cell division, and DNA replication will be isolated from the genome of S. aureus RN4220 and fused with Tandem Affinity Purification (TAP) tags. Tagged genes will be crossed back into the host chromosome via homologous recombination, or, alternatively, TAP-tagged fusion proteins will be expressed from a P_xyl promoter harboured on specially constructed S. aureus-E.coli shuttle plasmids. TAP-tagged macromolecular complexes formed in vivo will be isolated via affinity purification, and the protein composition of each complex will be determined by peptide mass fingerprinting. RESULTS: During the construction of relevant staphylococcal plasmids, a proof-of-concept model for TAP-tagging experiments was established in the naturally transformable and highly recombinogenic Gram-negative coccobacillus Acinetobacter baylyi ADP1. The rpoC gene, which codes for the beta prime subunit of RNA polymerase (RNAP), was selected for initial analysis via TAP-tagging. Preliminary tandem mass-spectrometric analyses indicated that the majority of expected protein-protein interactions were detected in affinity purified eluates of tagged strains, along with a suite of previously unknown putative interactions. TAP-tagging isolations and mass spectrometric analyses will be optimised prior to the commencement of staphylococcus-based experiments. CONCLUSIONS: TAP-tagging coupled with tandem mass spectrometry is an efficient means of identifying novel protein-protein interactions. Methodologies developed in the genetically amenable bacterium Acinetobacter baylyi ADP1 are expected to be directly transferable to analyses in S. aureus.