Identifying and targeting the Mg-Fe-PmrAB regulatory circuit reverses phosphate starvation-driven polymyxin tolerance in Enterobacteriaceae

Phosphate (Pi) scarcity, a pervasive stressor prevalent in bacterial infections and dysbiotic host environments, potently alters antimicrobial susceptibility against cationic antibiotics like polymyxins across diverse bacterial taxa. While established Pi depletion-induced antimicrobial tolerance mechanisms often involve membrane phospholipid remodelling, Enterobacteriaceae largely lack these pathways, leaving the mechanism underlying their adaptation to Pi scarcity unresolved. Here, we reveal that Pi limitation robustly induces polymyxin tolerance in Enterobacteriaceae through 4-amino-4-deoxy-L-arabinose (L-Ara4N) modification of the Gram-negative outer membrane component lipid A, reflecting a distinct adaptive strategy. This modification is driven by strong arn operon induction, mediated by the PmrAB two-component system. We discover that Pi depletion triggers cellular Mg²⁺ release, prompting compensatory Fe³⁺ mobilization to the cell envelope that directly activates PmrAB. Crucially, this metal-dependent signaling axis offers a directly targetable mechanism for reversing polymyxin tolerance, a significant deviation from the less accessible, PhoBR-regulated phospholipid remodelling strategies in other bacteria. We demonstrate that Mg²⁺ supplementation or Fe³⁺ chelation effectively suppresses PmrAB activation and restores polymyxin susceptibility, thereby establishing a novel, metal-centric paradigm for understanding and pharmacological intervention in stress-induced antimicrobial tolerance.