## 2 Results  
### Identification of New Persister Genes  
To better understand the mechanisms of persister formation, we performed a genetic screen using the Network on Antimicrobial Resistance in _Staphylococcus aureus_ (NARSA) mutant library, which contained mutations in all the 1952 non-essential genes in the genome of the USA300 strain [12]. We exposed all mutants in the library to rifampicin, which is a bactericidal agent for _S. aureus_ that has been used to treat serious MRSA [13,14]. Persister isolation and analysis is shown schematically in Figure 1. The stationary phase culture was exposed to rifampicin (2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, \(>\)10\(\times\) MIC). Using a 384-well pin replicator, bacteria were stamped onto tryptic soy agar (TSA) plates and mutants with defective persistence, as indicated by the lack of growth, were recorded (Figure 1B). Figure 1: (**A**) Work flow for isolation of mutants with defect in persistence. The _S. aureus_ USA300 transposon library from NARSA was grown in tryptic soy broth for 16 h to reach stationary phase. Rifampicin was added to the stationary phase culture at 2 \(\mathrm{\SIUnitSymbolMicro g}\)/mL (\(>\)10\(\times\) MIC). Before rifampicin exposure, bacteria were stamped onto tryptic soy agar plates (TSA) using a 384-pin replicator as the input control. Over the course of a 4-day antibiotic exposure, bacteria were washed and plated onto TSA plates daily and all mutants that showed defective growth was recorded and tallied until Day 4; (**B**) Representative agar plate images with mutant library after the 4-day rifampicin exposure where some mutants show defect in growth. The screen identified 124 mutants that failed to grow on TSA plates after exposure with rifampicin (Figure 2). Of the mutants that showed defective persistence after rifampicin exposure, 29% of them had mutated genes that play a role in the metabolism in pathways such as carbohydrate metabolism, amino acid, and purine biosynthesis. Among the rest of the mutants, 14% of the candidates represented pathways that play a role in genetic processes such as DNA replication and repair, transcription and translation, 11% of the mutants were involved in environmental signaling processes (e.g., transporters and sensor kinases), 9% were enzymes (e.g., proteases, hydrolases, _etc._), and a minority of the gene hits played a role in bacterial pathogenesis such as toxin production (5%), originated from phage derived proteins (4%), or were involved in cell envelope development and drug efflux pathways (both 2%) (Figure 2A,B). Figure 2: _Cont._  
Mutants Involved in Regulating Purine Biosynthesis are More Susceptible to a Variety of Stresses Including Antibiotics, Heat and Low pH  
Metabolic genes account for 29% of the gene hits that our screen identified. Our data imply that metabolic pathways and genes play a crucial role in persistence. Out of the metabolic genes, a prominent group of mutants, which includes _purB_, _purF_, _purH_, _purM_, and SAUSA300_0147, all play a role in purine biosynthesis (Figure 2C,D). To further validate the role of purine biosynthesis and its possible involvement in persistence and tolerance to antibiotics and stress, we chose two genes, _purB_ and _purM_, adenylosuccinate lyase and phosphoribosylaminoimidazole synthetase, respectively, for further confirmation [15]. The _purB_ and _purM_ mutants not only showed defective persistence to rifampicin (Figure 2) but were also identified as playing a role in persistence to gentamicin using a similarly high throughput method [16]. We further evaluated _purB_ and _purM_, since these two genes could potentially be core regulators in persistence. We first performed a growth curve study to exclude the possibility that these mutants have growth defects compared to the parental strain.