## 3 Discussion
Despite the different nature of the mutations found in \(guaB\), which were scattered all over the gene, all of them led to the formation of truncated proteins due to the presence of premature STOP codons in the new coding sequences. \(guaB\) codes for IMP dehydrogenase, which catalyzes the conversion of inosine 5'-phosphate (IMP) to xanthosine 5'-phosphate (XMP), an essential step in the de novo synthesis of guanine nucleotides [32]; therefore, it plays an important role in the regulation of cell growth, as depicted in Figure 3 and Table S3, which could explain the H*R phenotype seen in these H*R derivatives. Of further note are other mutations that are not involved in guanine metabolism but may potentially influence the H*R phenotype:  
- LGA251 HRC4 and LGA251 HRC10 each carry the same single SNP mutation at nucleotide 1934923, leading to the substitution of a guanine for an adenine in the acceptor stem of a tRNA annotated as tRNA_48 in the LGA251 genome and as tRNA-Met in several \(S\). \(aureus\) genomes. This mutation is close to the CCA 3'-terminal group used to attach amino acids to tRNAs, which can impair protein translation and consequently lead to slow growth. In fact, these two H*R derivatives showed a slower growth in comparison with their parental strain, as illustrated in Figure 3 and in Table S3. Since \(\beta\)-lactam antibiotics are effective on actively growing cells, slower growing cells would benefit under these conditions and would support higher concentrations of the antibiotic than when actively growing. Although these two derivatives share the same SNP, their growth is somewhat different, as seen in Figure 3. As this study was performed with short-read sequencing technology, and the genomes were not closed, one of these derivatives may have additional mutations that could not be detected here with the criteria used to call SNPs, which may eventually explain this difference. - RUSA239 HRC2 carries an SNP mutation that led to the substitution of phenylalanine residue by a serine at amino acid 254 of PheS--the alpha subunit of the phenylalanine-tRNA synthase, a residue that has different chemical properties. Phe254 is part of the large hydrophobic pocket that forms the active site of this essential enzyme for protein biosynthesis [37]. Thus, mutations in this location can influence cellular growth. Indeed, RUSA239 HRC2 clearly showed slower growth in comparison with the parental strain RUSA239 (Figure 3 and Table S3). RUSA239 HRC3 and RUSA239 HRC6 showed a unique SNP in the same intergenic region, around 300 bp upstream of \(glmS\) gene, coding for a glutamine-fructose-6-phosphate aminotransferase (isomerizing) that [PERSON] et al. proposed to be involved in glucosamine-6-P formation from fructose-6-P to be then incorporated in peptidoglycan biosynthesis [38]. Although being near the 5' end of the \(glmS\), the mutations found in RUSA239 HRC3 and RUSA239 HRC6 are not present in typical locations for regulatory processes. Moreover, the growth differences of these two derivatives in comparison to the reference strain were not statistically significant (Table S3), pointing to a need for further studies to ascertain the importance of these unique mutations in the expression of this gene and in the H*R phenotypes of these strains. In conclusion, we could not detect mutations affecting the same genetic determinants in the 15 oxacillin H*R derivative populations in this study, which were isolated from three parental strains carrying different methicillin resistance determinants--_mecA_, _mecC_, and _mecA1_. However, we did find mutations in three genetic determinants involved in guanine metabolism (and consequently in the stringent stress response in derivatives of the three parental populations).