Sulfur starvation induces an Fe-replete response and attenuates virulence pathways in Pseudomonas aeruginosa PAO1

Abstract Background Understanding bacterial responses to nutrient limitation is critical for developing targeted antimicrobial strategies. Sulfur starvation uniquely induces not only genes responsible for sulfur scavenging but also prominent antioxidant defenses. However, the biological rationale behind the simultaneous induction of antioxidants during sulfur limitation remains largely unexplored. Our study addresses this gap by integrating transcriptomic, proteomic, and targeted metabolomic data from Pseudomonas aeruginosa PAO1 grown under sulfur-free conditions. Results As anticipated, transcripts and proteins involved in sulfur assimilation and metabolism—including members of the msu, ssu, and cys operons—were upregulated, along with key antioxidant enzymes such as Ohr, LsfA, and SodB. Unexpectedly, however, genes encoding iron uptake systems (pyoverdine, pyochelin, and heme metabolism operons) were markedly downregulated, while iron storage proteins (BfrB, Dps, and PA4880) were elevated, indicating an iron-replete metabolic state. Further targeted metabolic profiling and iron quantification assays confirmed reduced Fe acquisition and diminished extracellular levels of siderophore and phenazine metabolites. This shift in iron homeostasis correlated with the repression of multiple virulence factors regulated by Fur and PrrF, including quorum-sensing components, efflux pumps, and phenazine biosynthesis enzymes. Conclusion Our integrative analysis reveals that sulfur starvation critically regulates iron homeostasis by linking reduced Fe uptake to the induction of antioxidant defenses. This iron-buffering response likely mitigates oxidative damage from unincorporated Fe, representing a protective metabolic adaptation. Additionally, the concurrent attenuation of virulence pathways suggests that targeting sulfur metabolism could disrupt iron-dependent virulence gene regulation, offering therapeutic insights into nutritional immunity strategies. Collectively, our findings uncover a novel sulfur-iron axis that plays a central role in oxidative stress management and pathogenicity modulation in bacteria. Graphical abstract Using high-throughput RNA-sequencing and proteomics techniques, we identified genes and metabolites differentially expressed during sulfur starvation in P. aeruginosa grown in minimal media. Additional experiments using a range of biophysical techniques were used to quantify select metabolites and Fe. Overall, we found that sulfur starvation induced an Fe-replete response, characterized by the repression of Fe uptake pathways and the upregulation of Fe storage genes.