## 3 Discussion
Additionally, purines are starting compounds for GTP synthesis and thus play an important role for energy production in regulating cell growth [25]. Bacterial cells can be  
Figure 5: The schematic diagram of the _pur_ pathway. The disruption of genes (_purB_, \(F\), \(H\), \(M\)) involved in the purine biosynthesis pathway may cause defects in downstream energy production, amino acid biosynthesis and urea cycle, which may be responsible for the defect in persistence observed in this study to rifampicin and other antibiotics and stresses. sent into a dormant-like stage through modulation of energy and alarmone (p)ppGpp production [26]. However, imbalance GTP synthesis and metabolism is associated with decreased (p)ppGpp synthesis. Since purines play a crucial role in producing (p)ppGpp substrates, our _pur_ mutants may experience the lack of (p)ppGpp modulation into a persistence state. This phenomenon has been shown in _Pseudomonas_, where (p)ppGpp levels affect the persistence in starvation, biofilm formation, and oxidative stress [27]. Similar mechanisms may explain why different _pur_ mutants were unable to persist in our screen. While our studies focused primarily on genes involved in purine biosynthesis, it is important to note that, based on our screen, metabolic processes showed the largest role in rifampicin tolerance and should be further explored in future studies. For example, arginine biosynthesis has recently been proposed as contributing to successful _S. aureus_ infection [28]. Most research focused on genes encoded by the arginine catabolic mobile element (ACME), which does not include _argJ_, an acetyltransferase [15, 29] that was identified as important to persistence based on our screen. Nonetheless, studies suggested that arginine synthesis might be pivotal in allowing _S. aureus_ to colonize the skin and survive in abscesses [28]. The bacterial cells use arginine to increase the pH by ammonia production of the extracellular milieu on the skin, which may aid the survival of _S. aureus_[28, 29]. As another example, _gltS_, which encodes a sodium/glutamate symporter, has been shown to be upregulated during biofilm formation [30]. Glutamate is required for the development of arginine and produce byproducts such as ornithine. Ornithine is a pivotal compound in cell metabolism in making prolines, polyamines, antibiotics, proteins, and peptidoglycan. The products driven by ornithine synthesis enhance bacterial growth and pathogenesis [31]. As for the _gltS_ transporter, acquisition of amino acid glutamate allows for the production of NH\({}_{3}\) in the periplasm of the cell to allow for the bacteria to adapt to the acidic environment, similar to the skin [32]. The role of glutamate in regulating bacterial stress includes both the upregulation of _GcrR_[33], which controls the acid tolerance resistance in _Streptococcus mutans_, and the downregulation of _MarR_, which regulates virulence factors, in response to antibiotics and oxidative stresses [34]. Despite the significant findings of this study, there are some limitations. First, due to _S. aureus_'s ability to cause wound infections, endocarditis and systemic disease [6, 7], different animal models (e.g., skin model, intraperitoneal injection, and murine model for urinary tract) should be considered to validate our findings [35, 36, 37]. These will be addressed in future studies. Secondly, a mutant screen only explores persister formation at the DNA and RNA level. Further proteomic, metabolomic and even epigenetic analyses (as our screen indicated genes with acetyl-, and methyl-transferase activities to be important) would offer more comprehensive insight into the effects of gene mutations on persistence. Thirdly, while this screen is comprehensive in the sense that all the non-essential genes of the clinical isolate of MRSA are included, their effects on the persistence of these mutants are determined by the conditions of our assays.