Restoration of Nitrate Respiration in <i>Pseudomonas aeruginosa</i> under Growth Limitations

Gene mutations are a fundamental survival strategy in bacteria and often accompany cell growth. We herein demonstrate that Pseudomonas aeruginosa recovered growth under anoxic conditions through a mutation in an essential regulatory gene required for nitrate respiration, overcoming initial growth limitations. Although the open-ocean isolate P. aeruginosa Ocean-1187 possesses denitrification-related genes, it was initially unable to grow under nitrate-respiring conditions due to a defect in the transcriptional regulator NarL, part of the NarX-NarL two-component system that controls the expression of denitrification genes. The growth of this strain was limited by the defective NarL; however, a prolonged incubation under anoxic conditions led to the emergence of spontaneous mutants that regained the ability to grow. Whole-genome sequencing and functional complementation assays revealed that a single amino acid substitution in NarL was sufficient to restore nitrate respiration. Additional variants independently isolated from separate cultures also carried substitutions at the same residue, underscoring its functional importance. The present study shows that a single amino acid substitution in an essential regulatory protein restored metabolic function. These results offer novel insights into the adaptive strategies of bacteria, highlighting the emergence of critical mutations even under restricted growth conditions.