## 2 Results
### Complementation Studies to Confirm the Role of Purine Pathways in Persistence  
To confirm that _purB_ and _purM_ are responsible for defective persistence, we complemented the _purB_ and _purM_ mutants with the wildtype _purB_ and _purM_ gene, respectively, using the _S. aureus-E.coli_ shuttle vector pRAB11 (Table 1). The shuttle vector pRAB11 harbors a _tet_ operator that is induced by anhydrotetracycline (ATc) [18]. Our findings suggest that both _purB_ and _purM_ are important for rifampicin persistence (Figure 4). Under prolonged rifampicin exposure, the complemented _purB_ mutant restored partial persistence to rifampicin on day 5 (Figure 4A) with an average of \(1\times 10^{4}\) CFU/mL compared to the parental strain that harbored \(1\times 10^{7}\) CFU/mL. The _purB_ mutant transformed with the pRAB11 vector control showed a defect in persistence, and no difference was observed among any of the strains under non-stressed conditions (Figure 4A). Similarly, the complemented _purM_ mutant restored rifampicin persistence at a level similar to the parental strain. On day 5, the complemented _purM_ mutant had an average of \(1\times 10^{6}\) CFU/mL compared to the parental strain that had \(1\times 10^{7}\) CFU/mL (Figure 4B). The _purM_ mutant transformed with the pRAB11 vector control had no colonies by day 5, and no phenotypic difference appeared under non-stressed conditions (Figure 4B). The complemented _purB_ mutant and _purM_ mutant both restored persistence in heat exposure (\(5\lx@math@degree\)C) (Figure 4C,D). After forty minutes of heat exposure, the _purB_-complemented mutant and _purM_-complemented mutant had roughly \(7.8\times 10^{4}\) CFU/mL and \(1.64\times 10^{4}\) CFU/mL, respectively, which is comparable to USA300, which had \(7.5\times 10^{4}\) CFU/mL. Additionally, the complemented _purB_ mutant and _purM_ mutant restored persistence when exposed to a low pH of 4 (Figure 4E,F). After 9 h of exposure to a pH of 4.0, the _purB_ complemented and _purM_ complemented mutants had roughly \(0.4\times 10^{4}\) CFU/mL and \(2.5\times 10^{4}\) CFU/mL, respectively, comparable to USA300, which had \(2.1\times 10^{4}\) CFU/mL. In contrast, the mutants harboring the empty vector control had a significantly lower number of cells under heat and acid stress conditions compared to the respective complemented strains. Figure 3: (**A**, **B**) Parental strain USA300 and _pur_ mutants showed no defects in log phase or stationary phases under non-stressed conditions. _Pur_ mutants had defective persistence in exposure to (**C**) rifampicin (\(2\)\(\mathrm{\SIUnitSymbolMicro m}\), \(>\)\(10\times\) MIC), (**D**) gentamicin (\(60\)\(\mathrm{\SIUnitSymbolMicro m}\), \(>\)\(10\times\) MIC), (**E**) heat (\(58\)\(\mathrm{\SIUnitSymbolMicro C}\)), and (**F**) a low pH of 4.0. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Primer Name** & **Sequence** & **Source or Reference** \\ \hline purBF & 5’-GCAAGATCTATGATTGAAACGCCTATTCTAG-3’ & This study \\ purBR & 5’-ACGGAATCTTATGCTAATCCAGGCGTTTG-3’ & This study \\ purMF & 5’-GCTAGATCTATGCTAAAAGCATTAGAACAATC-3’ & This study \\ purMR & 5’-ACGGAATCTTATCCATCCCCAACAATTCAAT-3’ & This study \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Primers used in this study. The underlined sequences AGATCT and GAATTC represent the BgIII and EcoRI restriction sites incorporated for cloning the wildtype gene into shuttle vector pRAB11 for complementation. Figure 4: Restoration of persistence phenotype in complemented mutants. Survival of stationary phase cultures of _S. aureus purB_ and _purM_ mutants transformed with vector pRAB11 or pRAB11-wildtype _purB_ and _purM_ and the parent strain USA300 upon (**A**, **B**) rifampicin exposure (2 ug/mL, \(>\)10\(\times\) MIC) at 5 days post-exposure; (**C**, **D**) heat exposure (58 \({}^{\circ}\)C for 40 min) and (**E**, **F**) low pH of 4.0 for 9 h. The _S.