## 3 Discussion  
Despite the discovery of _S. aureus_ persisters in 1944 and the prevalence of persistent infections caused by _S. aureus_, the molecular mechanisms underlying _S. aureus_ persister survival has remained largely unknown [19]. The convenience of a comprehensive transposon-mutant library has allowed us to perform the first whole genome-wide mutant analysis and to provide insight into the molecular basis of _S. aureus_ persisters. This study represents the first systemic analysis of persister genes and pathways in a circulating clinical isolate of _S. aureus_. We identified 124 different mutants that showed defects in rifampicin persistence. A majority of the mutants belongs to metabolite production and regulation pathways such as purine biosynthesis. Our findings revealed that five purine biosynthesis genes (_purB_, _purF_, _purH_, _purM_, and SAUSA300_0147) are involved in _S. aureus_ persistence. In particular, we chose _purB_ and _purM_, which encode adenylosuccinate lyase and phosphoribosylaminoimidazole synthetase respectively, for confirmation, and, indeed, complementation studies show that genes involved in purine biosynthesis are important for antibiotic and stress tolerance in _S. aureus_. Although the molecular mechanisms by which purine pathway mediate persistence remain to be determined, our findings are consistent with previous research suggesting that defective purine synthesis has decreased biofilm formation and attenuated virulence in persistent infections such as endocarditis [20]. In addition, purine biosynthesis mutants (_purL_ and _purM_) of _Burkholderia_ fail to colonize in the host symbiotic organ and exhibit decreased biofilm formation [21]. The role of _pur_ genes in Staphylococcus persistence is novel, and future animal studies are needed to confirm the virulence and persistence of the _S. aureus pur_ mutants _in vivo_. Several mechanisms may be underlying the defect in persistence seen in purine biosynthesis mutants. For example, a defect in the purine biosynthesis pathway may lead to decreased downstream energy production, amino acid biosynthesis and urea cycle activation, which may be responsible for the defect in persistence observed in this study to rifampicin and other antibiotics and stresses (Figure 5). Purine biosynthesis has also been shown to be associated with survival in stressed conditions such as vancomycincin and daptomycin in other strains of _S. aureus_[22, 23]. The final step in purine nucleotide synthesis leads to AMP formation, increased AMP, and thus ATP energy levels, which were observed in antibiotic resistant strains [23]. It is hypothesized that increased purine biosynthesis would allow for more energy used in generating polymers, one of the most energy demanding process in bacteria. In _S. aureus_ and other Gram-positive bacteria, the peptidoglycan layer is the most abundantly large polymer [23]. While mechanisms regarding purine biosynthesis in persistence remain to be further elucidated, our hypothesis is consistent with the participation of other genes in persistence from our data. Mutants such as _accC_, _budA_, _Cap5J_, _gltA_, _gntK_, _leuD_, _murA_, _mgo_, each of which play a role in carbohydrate metabolism, SAUSA300_0918, which plays a role in lipid metabolism, _fmtC_, SAUSA300_0729, and SAUSA300_1677 in cell wall and membrane synthesis all showed defects in rifampicin persistence, which suggests a possible role of purine metabolism and of cell envelope formation in persister formation. Other general mechanisms involved in persistence may help explain the role of purines in persister biology. Studies have shown that pathways involved in DNA repair mechanisms, SOS response, and energy production affect persister survival [7,24]. Purines are important substrates for both DNA synthesis and hence will alter DNA repair processes that make it difficult for the bacteria to repair their genetic material upon stress damage.