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, eLife , 2021) shown that, in Escherichia coli , mutations at the mgrB locus were beneficial under trimethoprim exposure and led to overexpression of dihydrofolate reductase (DHFR), encoded by the folA gene. Here, we show that DHFR levels are further enhanced by spontaneous duplication of a genomic segment encompassing folA and spanning hundreds of kilobases. This duplication was rare in wild-type E. coli . However, its frequency was elevated in a lon -knockout strain, altering the mutational landscape early during trimethoprim adaptation. We then exploit this system to investigate the relationship between trimethoprim pressure and folA copy number. During long-term evolution, folA 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 folA 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.