Inactivation of a purine biosynthesis repressor promotes ribosome synthesis to overcome antibiotic stress

Macrolide, lincosamide, and streptogramin (MLS) antibiotics are structurally distinct molecules that inhibit protein synthesis by binding overlapping sites within the 23S rRNA of bacterial ribosomes. The clinical utility of MLS antibiotics has diminished due to the dissemination of <u>e</u>rythromycin <u>r</u>esistance rRNA <u>m</u>ethyltransferase (Erm) genes. <i>Staphylococcus aureus</i> ErmB methylates the universally conserved A2058 nucleotide of the 23S rRNA (m⁶A2058), resulting in cross-resistance to all MLS antibiotics by reducing drug-binding affinity. The operonic upstream ribosome stalling peptide ErmBL was thought to be the sole regulatory element controlling ErmB synthesis and the extent of MLS resistance. Unexpectedly, our laboratory evolution experiments revealed that numerous loss-of-function mutations outside the bicistronic <i>ermBL-ermB</i> operon amplify ErmB-mediated MLS resistance. Among these are mutations in genes critical for purine <i>de novo</i> biosynthesis and the salvage pathway. Specifically, inactivation of the gene encoding the purine biosynthesis transcriptional repressor (PurR) converts an otherwise moderately resistant <i>ermBL-ermB (ermB⁺</i>) strain into one exhibiting pronounced hyper-resistance to MLS antibiotics. This cooperative resistance phenotype is not attributable to increased <i>ermB</i> expression or elevated m⁶A2058 modification. Instead, <i>purR</i> inactivation leads to derepression of purine and pyrimidine biosynthesis, accompanied by increased expression of ribosome components. We find that the elevated ribosome abundance and translational capacity of the <i>ermB⁺</i>∆<i>purR</i> are directly proportional to its accelerated growth rate, thereby priming <i>S. aureus</i> for survival under high MLS concentrations. These findings support a model in which expanded nucleotide metabolites and a surplus of antibiotic-free ribosomes drive global translation to buffer the inhibitory effects of MLS antibiotics.IMPORTANCEThe Erm rRNA methyltransferase superfamily represents the most prevalent determinant of MLS resistance in nosocomial Gram-negative and Gram-positive bacteria. Previous studies have established that <i>erm</i> expression is primarily governed by upstream ribosome stalling peptide and the 5' untranslated regions. Using the widespread <i>S. aureus ermBL-ermB</i> (<i>ermB</i>⁺) operon as a model system, we unexpectedly identified second-site mutations in <i>purR</i> that synergistically enhance MLS resistance in an <i>ermB</i>⁺ background. Loss of <i>purR</i> function derepresses nucleotide biosynthesis and ribosome production, thereby promoting bacterial growth under antibiotic stress. While numerous <i>purR</i> single-nucleotide polymorphisms across multiple species have been associated with antibiotic resistance, no study has directly linked these sequence polymorphisms to their regulatory function. Our results highlight the critical role of ribosome abundance and nucleotide metabolism in shaping antibiotic efficacy.