Omics Analyses of Sulfur Starvation Response in Pseudomonas aeruginosa: Linking Oxidative Stress to Iron Homeostasis

Abstract

Inorganic sulfur, primarily in the form of sulfate, is the preferred sulfur source utilized by bacteria. However, sulfate availability in terrestrial environments is severely limited, comprising less than 1–5% of the total available sulfur, with the majority existing in organic (organosulfur) forms. Consequently, soil bacteria have evolved strategies to utilize a diverse range of organosulfur compounds. Previous nutritional studies have established that sulfate limitation triggers extensive metabolic reprogramming characterized predominantly by the upregulation of sulfur-scavenging genes. Particularly, sulfur deprivation also consistently induces the expression of antioxidant genes, yet the underlying mechanisms remain poorly understood. To address this knowledge gap, we employed an integrative omics approach combining RNA sequencing (RNA-seq), label-free proteomics, fluorometric assays, and computational network inference analyses, using the opportunistic pathogen Pseudomonas aeruginosa PAO1 cultured in sulfur-free media (SFM) as our experimental model. As expected, RNA-seq and proteomics datasets revealed pronounced overexpression of genes and proteins associated with scavenging and assimilation of organosulfur compounds (ssu, msu, and sfn operons), alongside antioxidant genes encoding organic hydroperoxide resistance protein (Ohr), 1-Cys peroxiredoxin (LsfA), and iron-dependent superoxide dismutase (SodB). Surprisingly, we uncovered a previously unreported response: sulfur starvation significantly downregulated numerous iron-acquisition pathways, including siderophore biosynthesis (pyoverdine, pyochelin operons), heme uptake clusters (has, phu), and genes related to xenosiderophore transport. Concomitantly, sulfur-deprived cells displayed significant upregulation of iron-storage proteins (BfrB, Dps, PA4880), indicative of an Fe-replete cellular state. Quantitative Fe measurements using inductively coupled plasma optical emission spectroscopy (ICP-OES) demonstrated a significantly diminished iron uptake capacity in sulfur-starved cells, aligning with our omics findings. Complementary quantitative HPLC analyses further corroborated these results by revealing decreased extracellular concentrations of pyoverdine siderophore. To understand the oxidative stress dynamics under sulfur deprivation, we employed fluorometric reactive oxygen species (ROS) assays using 2',7'-dichlorofluorescein diacetate (DCFH-DA), revealing a sustained, time-dependent increase in ROS levels in sulfur-starved cells. Additionally, the assessment of intracellular labile Fe using Calcein-AM fluorescence demonstrated a biphasic response, with an initial rise in intracellular Fe at early starvation (2.5 h), followed by a notable decline below control levels at later time points (5–7.5 h). While initially perplexing, this biphasic iron response is consistent with the physiological adaptations observed, namely downregulated iron uptake and increased iron sequestration, which collectively diminish intracellular unincorporated Fe levels over time. Time-course proteomics provided additional insights, demonstrating a progressive, sustained elevation of antioxidant proteins (Ohr, LsfA, SodB), and Fe-storage proteins (BfrB, PA4880, PA0962), underscoring their crucial roles in mitigating oxidative damage through prolonged sulfur limitation. To complement our experimental findings, we conducted a computational analysis to elucidate the regulatory circuitry underpinning the sulfur starvation response. This approach was necessitated by the observed differential expression of key regulatory elements, including siderophore biosynthesis regulators (pvdS), and the iron-responsive small RNAs (PrrF). Leveraging our transcriptomic dataset, we employed GENIE3, a machine-learning-based network inference tool, to construct gene regulatory networks aimed at identifying key transcription factors and regulatory hubs involved in mediating the sulfur deprivation response. Our computational analyses identified MexL as a putative regulatory hub, potentially orchestrating gene expression changes associated with sulfur limitation. Although the predictive accuracy of our inferred network was constrained by the limited sample size and suboptimal precision-recall metrics, MexL emerged as a biologically relevant candidate due to its well-established regulatory roles. Specifically, MexL is known to influence phenazine biosynthesis pathways and the expression of the MexJK-OprM efflux pump system, both significantly altered under sulfur-starved conditions in our omics analyses. Therefore, despite methodological limitations, the identification of MexL as a central regulator is both plausible and biologically compelling. Nonetheless, these computational predictions should be interpreted cautiously and require rigorous experimental validation. Overall, our integrative analyses support a model wherein sulfur deprivation triggers Fe dyshomeostasis in Pseudomonas aeruginosa, initially elevating intracellular Fe pools and subsequently activating Fe-storage mechanisms to mitigate Fe-induced oxidative stress. This adaptive response offers a mechanistic explanation for the induction of antioxidant genes consistently observed during sulfate limitation, shedding light on a longstanding puzzle regarding oxidative stress responses under sulfur scarcity. Additionally, our study also reveals that sulfur starvation significantly suppresses multiple virulence determinants, including quorum-sensing regulators, RND-type efflux pumps, phenazine biosynthetic enzymes, and hydrolytic proteases, many of which are established targets of the ferric uptake regulator (Fur) and iron-responsive PrrF small RNAs. In conclusion, our findings position sulfur availability as a central modulator of Fe metabolism, oxidative stress responses, and virulence gene expression in P. aeruginosa. These mechanistic insights significantly advance our understanding of bacterial adaptation to sulfur-limiting environments and highlight sulfur metabolism as a promising therapeutic target. Exploiting bacterial sulfur dependency could represent an innovative nutritional immunity strategy, potentially addressing limitations associated with traditional Fe-targeted interventions that inadvertently risk enhancing bacterial virulence.