Proteostasis modulates gene dosage evolution in antibiotic-resistant bacteria

Evolution of gene expression frequently drives antibiotic resistance in bacteria. We had previously (Patel and Matange, <i>eLife</i>, 2021) shown that, in <i>Escherichia coli</i>, mutations at the <i>mgrB</i> locus were beneficial under trimethoprim exposure and led to overexpression of dihydrofolate reductase (DHFR), encoded by the <i>folA</i> gene. Here, we show that DHFR levels are further enhanced by spontaneous duplication of a genomic segment encompassing <i>folA</i> and spanning hundreds of kilobases. This duplication was rare in wild-type <i>E. coli</i>. However, its frequency was elevated in a <i>lon</i>-knockout strain, altering the mutational landscape early during trimethoprim adaptation. We then exploit this system to investigate the relationship between trimethoprim pressure and <i>folA</i> copy number. During long-term evolution, <i>folA</i> duplications were frequently reversed. Reversal was slower under antibiotic pressure, first requiring the acquisition of point mutations in DHFR or its promoter. Unexpectedly, despite resistance-conferring point mutations, some populations under high trimethoprim pressure maintained <i>folA</i> duplication to compensate for low abundance DHFR mutants. We find that evolution of gene dosage depends on expression demand, which is generated by antibiotic and exacerbated by proteolysis of drug-resistant mutants of DHFR. We propose a novel role for proteostasis as a determinant of copy number evolution in antibiotic-resistant bacteria.