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We previously reported that magnesium sequestration by\n                  <jats:italic>Candida albicans<\/jats:italic>\n                  enables\n                  <jats:italic>Pseudomonas aeruginosa<\/jats:italic>\n                  to become colistin-resistant. Here, we show that Mg\u00b2\u207a depletion drives\n                  <jats:italic>P. aeruginosa<\/jats:italic>\n                  to evolve greater colistin resistance through genetic changes in lipid A biosynthesis-modification pathways and a putative magnesium transporter. These mutations synergize with the Mg\n                  <jats:sup>2+<\/jats:sup>\n                  -sensing PhoPQ two-component signaling system to remodel lipid A structures of the bacterial outer membrane in previously uncharacterized ways. One predominant mutational pathway relies on early mutations in\n                  <jats:italic>htrB2<\/jats:italic>\n                  , a non-essential gene involved in lipid A biosynthesis, which enhances resistance but compromises outer membrane integrity, resulting in fitness costs and increased susceptibility to other antibiotics. A second pathway achieves increased colistin resistance independently of\n                  <jats:italic>htrB2<\/jats:italic>\n                  mutations without compromising membrane integrity. In both cases, reduced binding of colistin to the bacterial membrane underlies resistance. Our findings reveal that Mg\n                  <jats:sup>2+<\/jats:sup>\n                  scarcity unleashes two novel trajectories of colistin resistance evolution in\n                  <jats:italic>P. aeruginosa<\/jats:italic>\n                  . 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