## 3 Discussion  
Although no common mutated genes were found in the H*R derivatives (representing the three genetic backgrounds used in the study), in all of them, we were able to detect the presence of mutated genes related to guanine metabolism:  
- In LGA251 H*R derivatives, both mutations found in _hpt_ (Figure 2) gave rise to truncated proteins: in LGA251 HRC2, the frameshift mutation gave rise to a protein only 14% of the normal size (25 aa versus 179 aa), whereas in LGA251 HRC5, the SNP mutation led to the formation of a premature STOP codon at the amino acid 164. Mutations in _hpt_ were already described in MRSA populations with an H*R-associated phenotype belongingto different genetic backgrounds and carrying \(mecA\) as the methicillin genetic determinant [17; 18]. The production of function-deficient Hpt proteins in these two LGA251 H*R derivatives will impact GTP availability in the cell due to its role in guanine metabolism, namely on the availability of GMP, which is then converted to GTP by the Gmk enzyme. Decreased intracellular levels of GTP is a phenomenon that also occurs when the stringent stress response is activated [36]. Low levels of GTP are thought to impair transcription and translation processes leading to slow growth and--as a consequence--to an increase in the tolerance of bacterial cells to antimicrobials [36], which may explain the H*R phenotype presented by strains LGA251 HRC2 and LGA251 HRC5 and their slower growth in comparison with the parental strain LGA251, as depicted in Figure 3 and Table S3. Figure 2: _Cont._  
Figure 3: _Cont._  
Figure 2: Overview of the mutations found in genes related to guanine metabolism: (**A**) _lpt_ in LGA251 HRC2 and LGA251 HRC5; (**B**) _relA2_ in RUSA239 HRC4; (**C**) _guaB_ in SS37 HRC2, SS37 HRC5, SS37 HRC8, and SS37 HRC9. Mutations are shown as grey bars. - In the derivative RUSA239 HRC4, an SNP mutation in _relA2_ has been detected (Figure 2), which gave rise to a premature STOP codon after the hydrolase domain of the encoded protein. The gene _relA2_ encodes for a GTP pyrophosphokinase that promotes the synthesis of (p)ppGpp, an effector of the stringent stress response. Mutations in _relA2_ have been linked with oxacillin H*R phenotypes in MRSA isolates [17; 18; 24]. However, in most cases, the synthetase domain of the enzyme remained active, and the H*R phenotypes have been associated with the synthesis of higher amounts of (p)ppGpp, which, in turn, would activate the stringent stress response. In contrast, the mutated _relA2_ found in RUSA239 HRC4 produces a smaller enzyme lacking the synthetase and regulatory domains (TGS and ACT). Another MRSA isolate showing an oxacillin H*R phenotype--BB8, de  
Figure 3: Growth curves of (**A**) LGA251, (**B**) RUSA239, and (**C**) SS37 parental heterosexual populations and the respective H*R sub-populations. Bacterial cultures were grown in TSB at 37 \({}^{\circ}\)C with aeration. All points represent the geometric mean of three technical plus three biological triplicates. Error bars represent 95% confidence intervals. scribed by [PERSON] et al. [17]--was characterized by the presence of a mutation that impaired the synthetase domain of ReIA due to a premature STOP codon at the beginning of this domain. Intriguingly, in both cases, those mutations were the only ones obtained in the WGS analysis performed, which could indicate that these isolates may have additional undetected mutations. This may be due to the sequencing technology used (short-reads) or the minimum criteria defined to call SNPs in both studies. Additional studies including the use of long-reads sequencing technology are needed to better understand the mechanism responsible for the H*R phenotype in these two isolates. - All except one SS37 H*R derivatives showed the presence of a mutation in the \(guaB\) gene (Figure 2).