Siderophore sharing protects clonal <i>Pseudomonas aeruginosa</i> biofilms from colistin

ABSTRACT Siderophores mediate cooperative iron acquisition and stress tolerance in polymicrobial communities. However, whether and how such social roles emerge within clonal populations of a single bacterial strain remain unclear. Here, we investigate whether and how such social roles emerge within clonal populations of a single bacterial strain. Using single-cell transcriptomics, we analyzed the response of clonal Pseudomonas aeruginosa PAO1 biofilms to colistin and identified 13 transcriptionally distinct subpopulations, with siderophore biosynthesis genes enriched in one subpopulation. Upon colistin exposure, these genes were specifically upregulated in this subpopulation, whereas their cognate receptor genes were ubiquitously expressed across all subpopulations. Through genetic and physiological experiments, we demonstrate that siderophore sharing between the subpopulations confers cross-protection against colistin by enhancing resistance in a growth-independent manner, as well as by promoting biofilm formation and stability. Therefore, the spontaneous emergence of siderophore producers and non-producer subpopulations, along with their intercellular interaction mediated by siderophore sharing, constitutes a strategy for colistin protection. IMPORTANCE This work provides a single-cell transcriptomic atlas of P. aeruginosa biofilms and reveals transcriptional heterogeneity within the clonal population. Colistin treatment markedly reshaped the subpopulation structure, with a siderophore biosynthesis-related subpopulation being significantly enriched, while siderophore receptor genes were broadly expressed across all subpopulations. This pattern of “production specialization” and “reception universalization” extends the applicability of public goods theory to clonal bacterial populations at the single-cell level and further reveals the important role of siderophore sharing in mediating cross-protection among genetically identical subpopulations. These findings deepen our understanding of the complexity of bacterial behaviors and provide potential therapeutic targets for combating biofilm-associated infections.