Staphylococcus aureus continues to be a clinical burden globally due to its ability to rapidly adapt to antibiotic stress. The overwhelming majority of clinical MRSA (methicillin-resistant Staphylococcus aureus) isolates exhibit a low-level ß-lactam resistance (oxacillin MIC 2-4 µg/ml). Yet, these are capable of developing high-level resistance to oxacillin (MIC =256 µg/ml) by an unknown mechanism(s). Therefore, an experimental system to explore underlying molecular basis of high-level resistance was developed here. The aim of the project was to construct genetically amenable MRSA strains by introducing plasmid-borne and single copy chromosomal mecA into wellcharacterised, methicillin sensitive genetic background SH1000 which allowed to mimic the resistance phenotype identical to the naturally occurring MRSA clinical isolates. The progression of resistance and whole genome sequencing data revealed single nucleotide polymorphisms in c-di-AMP phosphodiesterase (gdpP) to be responsible for high-level resistance when plasmid-borne mecA was used. When, single copy mecA was introduced mutations in either rpoB (RNA polymerase ß subunit) or rpoC (RNA polymerase ß' subunit) were found associated with increased resistance properties. The impact of the genetic mutations (rpoB and rpoC) responsible for highlevel resistance were examined at the transcriptional level using RNA-seq. Introduction of mecA induces metabolic stress resulting in substantial gene expression compared to SH1000 but is reversed upon acquisition of rpoB/rpoC mutations. These findings suggested that expression of high-level resistance requires not only elevated amounts of cellular PBP2A but also normalised gene expression. Collectively, this study offers some important insights into physiological aspects of S. aureus.