In the age of rising antibiotic resistance among various pathogens, the search for alternative antimicrobial strategies has become a pressing scientific challenge. Metal ions are indispensable to life, participating in a vast range of structural and catalytic roles across all forms of biology. The Biological Inorganic Chemistry Group explores the fundamental chemistry of metal ions in biological systems, focusing on their role in host-pathogen interactions. In this article we aim to focus on four aspects, representing the main scientific interests of our group. Metal homeostasis and assimilation chapter explores the coordination chemistry of biologically essential Mn(II) and Fe(II) ions using model peptides and fragments of metal transport proteins. By varying the number and position of His, Asp, and Glu residues, the study revealed how sequence composition governs metal-binding strength and selectivity. Studies on bacterial Fe(II) transporters, such as FeoB, identified potential metal-binding regions and mechanisms of ion transfer, deepening our understanding of metal recognition and transport in biological systems. To better understand iron uptake in pathogens, we explored synthetic siderophore analogs as tools for studying this process. Ferrichrome mimics effectively chelated Fe(III) and were taken up by Pseudomonas putida and Escherichia coli. Fluorescent labeling allowed visualization of these complexes in cells. Ferrioxamine E analogs showed promise as 68Ga-labeled imaging agents. The ability of various siderophores to bind metal ions such as Cu(II), Zn(II), Ni(II), Bi(III), and Zr(IV) has also been studied. One of the chapters focuses on chaperonins and metalloproteinases as bacterial virulence factors. GroEL1, a chaperonin present e.g.in Mycobacterium, binds metal ions like Cu(II) via its unstructured His-rich C-terminal tail, helping bacteria survive under toxic metal conditions. Peptide studies showed that specific His residues position affects metal complex stability, and various metals create distinctly different complex geometries. Bacterial metalloproteinases need Zn(II) for activity. Research on peptide models revealed how Cu(II) and Ni(II) can inhibit these enzymes by displacing Zn(II). Studies on human MMP-14 enabled the identification and detailed characterization of the metal-binding site, as well as the elucidation of the interactions between peptide-based inhibitors, the catalytic metal ion, and the enzyme active site. Detailed knowledge of virulence proteins may enable their use as a potential target for novel drugs. Membrane proteins are vital for transport, signaling, and maintaining membrane integrity. However, their studies are challenging due to poor solubility and detergent sensitivity. Lipoprotein nanodiscs (NDs) has revolutionized research on these proteins, by providing a soluble, stable, and physiologically relevant environment for membrane protein reconstitution. Their defined size, high stability, and compatibility 34 W. WOŹNIAK-LASZCZYŃSKA, K. GŁADYSZ, P. POTOK, M. ZAWADA, B. ORZEŁ, K.SZCZERBA… with structural and functional studies make NDs a powerful tool for investigating membrane transporters, ion channels, and receptors. This article highlights the key aspects of metal interactions with siderophores, transport and chaperone protein fragments, and metalloproteinases, and demonstrates how nanodiscs can advance the study of membrane proteins.

Heat stress is a major abiotic constraint that severely limits maize (Zea mays L.) productivity under changing climate conditions. This study explored a novel integrative strategy to enhance thermotolerance in maize through the combined application of methyl jasmonate-loaded chitosan nanoparticles (MJNPs) and eucalyptus-derived biochar (EBB). Methyl jasmonate was nano-encapsulated using the ionic gelation method and characterized by SEM, TEM, and FTIR analyses, which confirmed uniform spherical nanoparticles and effective surface functionalization. A greenhouse experiment was conducted under controlled heat stress (40°C) to evaluate physiological, biochemical, nutrient uptake, yield, and gene expression responses across eight treatments. Relative to non-stressed control plants, heat stress alone reduced plant height by 37%, photosynthetic rate (PN) by 46%, relative water content (RWC) by 25%, and grain number and grain weight by 25% and 6%, respectively. However, the combined MJNPs + biochar treatment under heat stress (HEMN) markedly alleviated these adverse effects. Compared with heat-stressed plants without amendments, HEMN increased plant height by 39%, RWC by 8%, membrane stability index (MSI) by 14%, and PN by 21%. In addition, grain number and seed weight increased by 10% and 6%, respectively, relative to heat-stressed plants, while water-use efficiency (WUE) improved by 13% under the same comparison. Nutrient uptake of phosphorus, magnesium, and iron increased by 15-22% in HEMN-treated plants compared with heat-stressed controls. Gene expression analysis revealed pronounced upregulation of stress-responsive genes, including HSP70, DHN3, and LEA-1, as well as auxin biosynthesis-related genes (TAA1, ZmYUC1, CYP79B2) and aquaporins in HEMN-treated plants relative to heat stress alone, indicating activation of coordinated molecular defense mechanisms. Furthermore, principal component analysis (PCA) and hierarchical clustering of gene-expression heatmaps confirmed strong multivariate associations between enhanced physiological performance and transcriptional activation, supporting the integrated nature of thermotolerance regulation. These findings demonstrate that the synergistic application of MJNPs and biochar significantly enhances maize thermotolerance relative to heat stress alone by improving water relations, nutrient homeostasis, photosynthetic performance, and molecular stress responses. This integrated nano-biochar strategy represents a scalable and environmentally sustainable approach for mitigating climate-induced heat stress and improving crop resilience in future agricultural systems.

The role of cyclic AMP–protein kinase A (PKA) signaling in siderophore-mediated iron uptake and its connection to virulence remains poorly understood in phytopathogenic fungi. Genetic studies demonstrate that the A. alternata adenylate cyclase (AaAC) regulates diverse cellular processes, including growth, conidiation, iron homeostasis, autophagy, siderophore biosynthesis, and toxin production. Deletion of AaAC results in impaired siderophore secretion, disrupted expression of iron-responsive genes, and a complete loss of ACT toxin biosynthesis, leading to markedly reduced virulence. Transcriptomic analysis under iron-deficient conditions reveals that AaAC deletion induces widespread changes in gene expression, notably the downregulation of genes involved in siderophore biosynthesis and ACT toxin production. These findings indicate that AaAC regulates metabolic pathways essential for fungal survival and pathogenicity. Mutants lacking the GTP-binding protein alpha subunit (Gα), the PKA catalytic subunit, or its regulatory subunit also reduce siderophore production. The findings suggest that environmental cues influencing siderophore biosynthesis are transmitted via a signaling cascade from Gα to AaAC and then to PKA. Additionally, AaAC negatively affects autophagy under nutrient-rich conditions. Gene ontology analysis reveals upregulation of autophagy-related genes, suggesting that AaAC may contribute to cellular energy preservation and physiological stability. These results indicate that AaAC is a key integrator of environmental signals, vital for maintaining iron homeostasis, controlling toxin biosynthesis, and driving virulence in A. alternata .

Exploring the molecular recognition between FpvA and colicin E9 immunity protein for disrupting FpvA-mediated Iron uptake in Pseudomonas aeruginosa: an advanced computational study

Iron (Fe) is an essential micronutrient for plant development and productivity. Nevertheless, the role of gibberellins (GAs) in the control of iron homeostasis is less studied compared to other growth regulators. We found that GAs modulate iron homeostasis in maize by inducing deficiency-like responses independent of rhizosphere iron availability. Plant phenotyping demonstrated that exogenous GA3 application under iron-sufficient conditions phenocopied iron deprivation, while inhibiting GA biosynthesis with mepiquat chloride prevented the development of typical symptoms of Fe deficiency (–Fe). Gibberellins positively control strategy II Fe uptake genes, albeit indirectly, as opposed to the direct negative transcriptional regulation of phytosiderophore biosynthesis. Additionally, gibberellins disrupt iron partitioning by suppressing root-to-shoot Fe translocation, causing iron overaccumulation in roots of GA3 treated plants. A functional ferrous iron uptake pathway was identified and was found to operate in conjunction with the strategy II uptake pathway via the differentially regulated Zea mays Iron-Regulated Transporter (IRT) paralogs ZmIRT1 and ZmIRT2. Root responses are spatially organized: gene expression in the lateral root sector reflects the shoot iron status, while transcriptional responses in the root apex correlate with local Fe demands. This study demonstrates that maize leverages a hybrid ferric/ferrous iron uptake strategy and establishes novel roles of GAs as pivotal regulators of iron homeostasis.

Microalgae have recently gained attention for their ability to incorporate iron into organic molecules, potentially enhancing bioavailability and mitigating associated risks, suggesting a promising role in the field of iron supplementation. Among microalgal species, nitrogen-fixing organisms present a relatively high iron demand due to their unique metabolic requirements, positioning them as promising candidates for addressing iron deficiencies. However, information on the dynamics and extent of iron accumulation in diazotrophic cyanobacteria remains limited. Understanding how these organisms regulate iron uptake and storage under different cultivation conditions is essential to evaluate their potential for nutritional and biotechnological applications. This work investigates the iron accumulation dynamics of the diazotrophic species Nostoc sp. PCC 7120 under both batch and continuous systems. The results highlight its ability to adapt and survive across a wide range of iron concentration and demonstrate a strong correlation between iron availability and the internal quota of iron in biomass. High-density batch cultivation revealed that the iron content in Nostoc sp. can span a remarkably wide range, up to tens of thousands of mgFe kgX-1, far exceeding typical values reported in the literature. This high accumulation mainly results from consistent external biosorption of the metal onto the cell surface. In continuous steady-state culture, the externally biosorbed fraction was lower but still detectable, suggesting that adsorption substantially contributes to overall iron accumulation. Under strong iron limitation (below 0.5 mgFe L-1), a marked increase in exopolysaccharide production was observed, suggesting that the extracellular matrix plays a functional role in the biosorption of the metal. Nostoc sp. PCC 7120 exhibits an exceptional capacity to tolerate and accumulate iron over a broad concentration range, mediated by both intracellular uptake and extracellular adsorption. These findings reveal that diazotrophic cyanobacteria can effectively modulate their iron management strategies under different regimes and highlight their potential as biofactories for iron-enriched biomass and as model systems for studying biologically driven metal sequestration.

Abstract The bioavailability of iron from different sources to phytoplankton, driving substantial carbon dioxide uptake of the large blooms downstream of South Georgia Island, remains unknown. Although geochemical characterization suggests that iron from glacial meltwater and groundwater is bioavailable, phytoplankton iron uptake measurements are lacking. In this study, additional to assessing iron chemical speciation and weathering processes, iron-55 uptake by a natural phytoplankton community was quantified in seawater sampled from low and high chlorophyll waters around South Georgia, to which iron from nearshore sources (glacial meltwater and groundwater) was added. Iron bioavailability depended on the chemistry of the fertilized seawater and the chemical composition of the source itself. Aggregation of dissolved organic matter in high chlorophyll water scavenged dissolved iron, making it unavailable to phytoplankton. In low chlorophyll water, as opposed to iron from groundwater, iron from glacial meltwater was bioavailable to phytoplankton and would increase carbon dioxide fixation by 80-100%.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, resulting in significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids from plasma membranes also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

Abstract Purpose Organic amendments (OAs) from agricultural and livestock residues are usually used to improve soil fertility in crop systems, however, the knowledge of the banana agroecosystem remains limited. The aimed of this study was to evaluate and compare the effect of four OAs commonly used in Canary Islands, on soil health and plant performance. Methods A two-year greenhouse experiment was carried out over a complete crop cycle. The treatments included cow manure, chicken manure, compost, pelletised compost, and a non-amended control. Soil chemical properties, microbial activity, plant growth, and plant nutrient content were monitored across the experiment. Results All OAs improved soil parameters relative to the control, with differing impacts among treatments. Chicken manure markedly enhanced soil microbial activity (increasing induced respiration and the abundance of bacterial, actinobacterial, and fungal populations), increased phosphorus and electrical conductivity, and reduced pH (by up to 1.5 units). Cow manure increased soil nutrient availability (especially nitrogen and phosphorus) and stimulated basal microbial respiration. Compost promoted fungal abundance and increased total and oxidisable organic matter throughout the crop cycle, whereas pelletised compost showed variable effects. In terms of plant development, chicken and cow manure significantly improved height, biomass, pseudostem circumference, and leaf area. Chicken manure outperformed cow manure in several yield-related parameters, including bunch and hand weight, number of fingers/hand, finger size, and rachis diameter. At harvest, chicken manure also showed the highest uptake of calcium, iron, manganese, nitrogen, potassium, sodium, and zinc. Conclusions These results highlight the different effects of each OAs and their specific potential to improve soil biological parameters, nutrient availability, and banana yield as effective tools to enhance agroecosystem sustainability. Graphical Abstract

The feeding rhythm is a major temporal regulator of metabolic physiology, yet its impact on microbiome-derived functional traits relevant to cardiometabolic disease remains insufficiently understood. Our previous work demonstrated that ad libitum, daytime-restricted, and nighttime-restricted feeding produce markedly different atherosclerotic outcomes in Apoe-/- mice, indicating that the feeding rhythm acts as a modifiable determinant of atherogenic susceptibility. Here, we used shotgun metagenomics to profile risk-associated microbial functional modules-including Type III and Type VI secretion systems (T3SS/T6SS), siderophore-based iron acquisition pathways, quorum-sensing (QS) regulators, and antimicrobial resistance determinants-across feeding regimens. The feeding rhythm induced pronounced functional segregation independent of α-diversity, which was consistent with selective functional reprogramming rather than taxonomic restructuring. Daytime feeding, which is misaligned with the murine active phase, is associated with coordinated enrichment of the T3SS/T6SS, iron uptake, and QS pathways, forming a tightly interconnected "virulence-iron-QS-ARG" functional consortium. In contrast, circadian-aligned nighttime feeding resulted in attenuated virulence orientation and enhanced metabolic-cooperative signatures. Network inference further revealed strong coactivation of virulence secretion, iron mobilization, and QS modules under circadian misalignment. These findings show that the feeding rhythm modulates atherogenic susceptibility not only through host metabolism but also by remodeling gut microbial functional capacities, highlighting microbial functional ecology as an integral component of diet-host interactions.

Halogenated phenazine (HP) compounds have shown promise as antimicrobial agents, particularly against biofilm-associated Gram-positive pathogens. Among these compounds, HP-29 demonstrates potent activity against methicillin-resistant Staphylococcus aureus by inducing rapid iron starvation. As maintenance of trace metals homeostasis is critical for the survival of Streptococcus mutans, this study investigated the antimicrobial efficacy of HP-29 and the impact of metal supplementation on this major oral and occasional systemic pathogen. As anticipated, HP-29 inhibited S. mutans growth in a dose-dependent manner, with iron supplementation alleviating the antimicrobial effect. Cobalt, manganese, or nickel supplementation also mitigated the inhibitory activity of HP-29, but, unexpectedly, the addition of zinc greatly enhanced HP-29 antimicrobial activity. This zinc-driven potentiation of HP-29 extended to other Gram-positive pathogens, including Enterococcus faecalis and S. aureus. Inductively coupled plasma mass spectrometry analysis revealed that intracellular iron content decreased significantly following exposure to HP-29. When combined with zinc, HP-29 triggered a 5-fold increase in intracellular zinc and reduced manganese levels by ~50%. Transcriptome analysis showed that HP-29 treatment, with or without zinc, altered expression of genes linked to iron and manganese uptake as well as zinc efflux, suggesting broad disruption of metal ion regulation. These findings highlight HP-29 as a potent antimicrobial that broadly impairs metal homeostasis. The unexpected synergy of HP-29 with zinc points toward a promising dual-agent therapeutic strategy against Gram-positive pathogens.IMPORTANCEWidespread development of antibiotic resistance has created a constantly moving target when combating infectious microbes. Here, we further explore an antimicrobial halogenated phenazine, HP-29, which is effective against Gram-positive bacteria through disruption of intracellular trace metal equilibrium. We showed that HP-29 inhibits growth of the oral and systemic pathogen Streptococcus mutans and that its antimicrobial effect is greatly potentiated by the addition of zinc. The zinc-mediated enhancement of HP-29's efficacy was also observed in other Gram-positive pathogens, including Enterococcus faecalis and Staphylococcus aureus. Intracellular trace metal quantifications and transcriptome analysis confirmed that HP-29 treatment impairs trace metal homeostasis, an outcome that is exacerbated when S. mutans is treated with both HP-29 and zinc. The observed synergy of HP-29 with zinc supports the development of a dual-agent therapeutic strategy against Gram-positive pathogens.

Neisseria gonorrhoeae (Gc) is the gram-negative bacterium that causes gonorrhea, a prevalent sexually transmitted infection that can have life-threatening clinical sequelae. Gc requires iron for human infection and uses the iron-responsive, iron-binding transcriptional repressor Fur to maintain iron homeostasis. Gc infects mucosal sites, where the neuroendocrine hormone norepinephrine (NE) is produced by the autonomic nervous system and various epithelial and immune cell types. Here, we show that NE derepresses the Fur regulon to alter bacterial iron homeostasis and increase Gc survival. By RNA-seq, we determined that NE rewires gonococcal gene expression to increase capacity for iron uptake while enabling increased intracellular iron availability. Of the 30 genes that were differentially expressed in NE-treated compared to untreated bacteria, 27 have Fur box-containing promoters. A possible mechanism for how NE derepresses the Fur regulon is through direct demetalation of Fur, as NE directly decreased binding of Fur in vitro to a DNA amplicon containing the Fur-binding sequence from Gc tbpB. NE increased the labile intracellular iron pool in Gc, evidenced by increased streptonigrin sensitivity, without significantly increasing the total bacterial iron content, suggesting that NE leads to the redistribution of cellular iron. The work presented here provides a novel mechanism for how Gc survives iron limitation within its obligate human host.IMPORTANCENeisseria gonorrhoeae (Gc) is the bacterial pathogen that causes gonorrhea, a sexually transmitted infection with an estimated global annual incidence of 87 million individuals. During infection, Gc must overcome iron limitation imposed by nutritional immunity. Here, we show that the host neuroendocrine hormone norepinephrine, which is present at the mucosal surfaces Gc infects, promotes the survival of iron-limited Gc. Our results support a novel mechanism by which norepinephrine works through the ferric uptake regulator, Fur, to enhance the capacity of Gc to take up iron and make it bioavailable. Our findings show that Gc responds to host-derived cues that enable it to resist iron limitation.

Novel duck orthoreovirus (NDRV) infection induces severe splenic necrosis in ducks, resulting in a cascade of detrimental consequences, including immunosuppression, secondary infections, and diminished vaccine efficacy. Avian orthoreovirus (ARV) exhibits high tropism for macrophages, with splenic macrophages being identified as the primary target cells of NDRV. Although ferroptosis has been implicated in this pathological process, the molecular mechanism underlying NDRV‐induced cellular damage remains poorly elucidated. In this study, an in vitro model of NDRV infection was established using HD11 cells to systematically investigate its effect on ferroptosis and the associated mechanisms. Our results indicate that NDRV infection triggers ferroptosis and markedly elevates intracellular Fe2+ levels. Mechanistically, NDRV upregulates transferrin receptor 1 (TfR1), thereby enhancing iron uptake, promoting iron accumulation, and ultimately inducing ferroptosis. This study is the first to reveal that NDRV induces macrophage ferroptosis by hijacking cellular iron metabolism, providing a theoretical foundation for understanding the mechanism through which NDRV infection mediates splenic necrosis and immune cell injury.

Overexpression of the mRNA binding protein Ssd1 extends the yeast replicative lifespan. Using microfluidics to trap and image single cells throughout their lifespans, we find that lifespan extension by Ssd1 overexpression is accompanied by formation of cytoplasmic Ssd1 foci. The age-dependent Ssd1 foci are condensates that appear dynamically in a cell-cycle-dependent manner, and their failure to resolve during mitosis coincided with the end of lifespan. Ssd1 overexpression was epistatic with calorie restriction (CR) for lifespan extension, and yeast overexpressing Ssd1 or undergoing CR were resistant to iron supplementation-induced lifespan shortening, while their lifespans were reduced by iron chelation. The nuclear translocation of the Aft1 transcriptional regulator of the iron regulon occurred during aging in a manner that predicted remaining lifespan but was prevented by CR. Accordingly, age-dependent induction of the Fit2 and Arn1 high-affinity iron transporters within the iron regulon was reduced by CR and Ssd1 overexpression. Consistent with age-dependent activation of the iron regulon, intracellular iron accumulated during aging but was prevented by CR and Ssd1 overexpression. Moreover, lifespan extension by Ssd1 overexpression or CR was epistatic to inactivation of the iron regulon. These studies reveal that CR and Ssd1 overexpression extend the yeast replicative lifespan by blocking deleterious age-dependent iron uptake, identifying novel therapeutic targets for lifespan extension and providing insight into how CR may extend the lifespan and healthspan in humans.

Rhizosphere microbiomes produce iron chelators or siderophores to capture ferric iron, an essential nutrient for growth that is not always bioavailable. Plants with siderophore activity aid in phytoremediation by removing heavy metals and other pollutants. The Cyperus genus of plants has a high affinity for iron uptake in the phytoremediation of wastewater. Herein, we isolated a siderophore-producing microbe from the rhizosphere of Cyperus virens, an understudied member of this genus, using the Chrome Azurol S (CAS) assay. While this isolate has siderophore activity, it did not exhibit antimicrobial activity when plated on Aspergillus niger, A. flavus, or Bacillus subtilis. The isolate was identified as Streptomyces sp. PD-S100-1 through genomic sequencing and de novo assembly with a draft genome size of 8.2 Mbp and 73% GC content. antiSMASH analysis of the genome identified several siderophore biosynthetic gene clusters, including those involved in the production of mirubactin A and bacillibactin. Liquid chromatography-mass spectrometry (LC-MS) detected several siderophores, including mirubactin A–D, enterobactin, and other catecholates from these siderophore families. The positive CAS assay, siderophore gene cluster identification, and LC-MS/MS analyses show that the rhizosphere of C. virens contains siderophore-producing bacteria. Most detected metabolites, including enterobactin, increased in the presence of cerium, a lanthanide involved in the expression of secondary metabolites, whereas mirubactin production was reduced. The presence of rhizosphere siderophore-producing bacteria suggests that C. virens may have potential applications in environmental phytoremediation, targeting pollutants from wastewater, mining, agriculture, and energy production. KEYWORDS: Siderophore; Mirubactin A; Enterobactin; Bacillibactin; Rhizosphere; Cyperus virens; Streptomyces; CAS Assay

Comparative Iron biofortification in Hericium erinaceus: A study of different ionic forms and their uptake efficiency.

Targeting STK17B kinase activates ferroptosis and suppresses drug resistance in multiple myeloma.

Excess aldosterone and cortisol promote myocardial iron deficiency: A potential pathway to cardiac injury.

Fermentative growth decreases the iron demand of Staphylococcus aureus.

Burkholderia pseudomallei (Bp), an environmental bacterium and opportunistic pathogen endemic to tropical regions, is highly adaptive and thrives in diverse environments, from soil to human hosts. Bacterial adaptation is critical for survival, virulence modulation, and persistence during infection and can manifest as colony morphotype variation (CMV). Although Bp adaptation has been studied, CMV remains poorly understood. Here, we characterized five clinical Bp isolates exhibiting heterogeneous populations with rough and smooth colony morphologies. We used phenotypic assays, whole-genome sequencing, and proteomics to investigate the molecular pathways reflecting CMV, by comparing smooth and rough morphotypes. Although phenotypic differences in protease activity, hemolysis, mucoidy, iron uptake, and antibiotic sensitivity-including to antimicrobial agents commonly used to treat infections-were rare, these traits alone could not distinguish morphotypes or groups of isolates. Genomic comparisons revealed either no differences or limited isolate-specific mutations, which do not explain the overall difference in phenotypes. In contrast, proteomic analysis uncovered consistent shifts in protein abundance related to virulence, including quorum sensing, DNA methylation, and secretion systems. Rough variants showed higher abundance of EPS-associated proteins, the BpsI3/R3 quorum-sensing system, and the global regulator ScmR, whereas smooth variants displayed higher abundances of proteins belonging to type III/VI secretion and siderophore biosynthesis pathways. These findings suggest that CMV is driven by phase variation and regulatory mechanisms rather than punctual genomic modifications. Our study underscores the limitations of phenotype or genome-based classification alone in the context of CMV and highlights the value of integrated multi-omics approaches to uncover CMV-associated biomarkers, with potential applications in diagnostics and the development of targeted therapies against persistent and drug-resistant Bp infections.IMPORTANCEBurkholderia pseudomallei (Bp), the causative agent of melioidosis, is endemic to Australia, Asia, Africa, and the Americas. It predominantly affects Indigenous populations and individuals suffering from diabetes, chronic lung or kidney disease, or alcoholism. Bp is known for its exceptional genomic and phenotypic plasticity, enabling rapid adaptation to diverse environments. This adaptability is reflected by colony morphotype variation (CMV), including reversible phase variation between smooth and rough colonies. In this study, we report rough and smooth colonies from clinical samples and emphasize the importance of characterizing CMV through multi-omics approaches rather than relying solely on genomics and phenotypic traits. By integrating genomic, phenotypic, and proteomic data, we identified that a limited number of mutations, including one in a putative regulatory element, likely drive major molecular changes between morphotypes. These affect the expression of quorum-sensing systems, the transcriptional regulator ScmR, DNA methyltransferase, and virulence-associated genes.