Project description:Background Streptomyces are key contributors to soil microbiome function, known for their biosynthetic diversity. While advances in -omics technologies have improved our understanding of microbiome composition and metabolic potential, the mechanisms underpinning interspecies interactions remain poorly resolved. Here, we investigate the molecular basis of interactions among four sympatric Streptomyces soil microbiome isolates, focusing on phenotypic, metabolomic and transcriptomic responses. Results Co-culture experiments revealed that one isolate, strain A, exhibited pronounced phenotypic changes when grown alongside each of the other three strains. Untargeted metabolomics and RNA-seq analyses showed that strain A undergoes distinct metabolic and transcriptional shifts depending on its partner, with the strongest response elicited by strain C. Despite all four strains possessing a conserved desferrioxamine biosynthetic gene cluster, only strain C constitutively produced desferrioxamine B (DFO-B), a hydroxymate siderophore, indicating a role of iron bioavailability in the interaction. Supplementation with DFO-B or iron mimicked the growth stimulation of strain A observed in co-culture with strain C, and CRISPR base editing of desD in strain C abolished both DFO production and the phenotypic induction of strain A. However, transcriptomic profiles of strain A varied significantly depending on the partner strain, with distinct sets of biosynthetic gene clusters and metabolic pathways activated in response to strains B and C, suggesting additional cues beyond DFO-B. In contrast, strain D did not elicit growth stimulation in its partners, and itself showed downregulation of amino acid and carbon metabolism when co-cultured with strain C. These findings indicate that Streptomyces interactions are not only mediated by siderophore piracy but also involve complex, strain-specific molecular responses. Conclusions Our findings demonstrate that Streptomyces interactions are highly strain-specific and only partly mediated by siderophore piracy, with DFO-B acting as a potent interspecies cue. The divergent molecular responses to different partners suggest nuanced mechanisms of microbial sensing and competition. These insights advance our understanding of microbial crosstalk and highlight the ecological and evolutionary complexity of siderophore-mediated interactions. By integrating transcriptomics, metabolomics, and biochemical assays, we present a robust framework for dissecting microbial interactions, with implications for microbiome engineering and synthetic community design.
Project description:Serratia marcescens is a bacterium frequently found in the environment, but over the last several decades it has evolved into a concerning clinical pathogen, causing fatal bacteremia. To establish such infections, pathogens require specific nutrients; one very limited but essential nutrient is iron. We sought to characterize the iron acquisition systems in S. marcescens isolate UMH9, which was recovered from a clinical bloodstream infection. Using RNA sequencing (RNA-seq), we identified two predicted siderophore gene clusters (cbs and sch) that were regulated by iron. Mutants were constructed to delete each iron acquisition locus individually and in conjunction, generating both single and double mutants for the putative siderophore systems. Mutants lacking the sch gene cluster lost their iron-chelating ability as quantified by the chrome azurol S (CAS) assay, whereas the cbs mutant retained wild-type activity. Mass spectrometry-based analysis identified the chelating siderophore to be serratiochelin, a siderophore previously identified in Serratia plymuthica. Serratiochelin-producing mutants also displayed a decreased growth rate under iron-limited conditions created by dipyridyl added to LB medium. Additionally, mutants lacking serratiochelin were significantly outcompeted during cochallenge with wild-type UMH9 in the kidneys and spleen after inoculation via the tail vein in a bacteremia mouse model. This result was further confirmed by an independent challenge, suggesting that serratiochelin is required for full S. marcescens pathogenesis in the bloodstream. Nine other clinical isolates have at least 90% protein identity to the UMH9 serratiochelin system; therefore, our results are broadly applicable to emerging clinical isolates of S. marcescens causing bacteremia.
Project description:We investigated the function of the SNX/H-type regulator of G-protein signaling (RGS) protein RGS4 and found alterations in enzyme regulation, stress response, siderophore production and metabolism of several carbon sources in light and darkness
Project description:Therapeutic angiogenesis based on gene therapies is a potential peripheral artery disease (PAD) treatment approach. Here, we developed a graphene nanoparticle-based IL-4 plasmid delivery system (GNPs-pIL-4) to reprogram macrophage polarization and activate the OSM/GSNOR/ENG axis to improve angiogenesis and tissue repair in ischemic limbs. Single-cell RNA sequencing analysis revealed that GNPs-pIL-4 treatment significantly enhanced the number and strength of intercellular communications in ischemic tissues, with enrichment of pathways associated with endothelial sprouting and neovascularization.
Project description:The aim of the experiment was to identify genes differentially expressed between the susceptible wild type strain P. aeruginosa PAO1 (PT5) and a mutant resistant to a drug-siderophore conjugate, in order to obtain information on the resistance mechanism(s). A mutant of PT5 able to grow at 4 mg/l BAL30072, a drug-siderophore conjugate, was selected in vitro . The susceptible wild type strain PT5 and the mutant (BAL6) were grown in LB medium and the mutant also in the presence of 4 mg/l BAL30072 to mid-exponential growth phase (OD600 =2) in triplicate cultures. RNA was extracted using the RNeasy Kit (Qiagen). A total of nine Affymterix P. aeruginosa arrays were hybridized (one for each replicate) under standard conditions.
Project description:Pseudomonas aeruginosa is a common nosocomial pathogen which produces siderophores to solubilize and transport chelated Fe3+ to aid its survival in both the environment and the host. However, there is a lack of comprehensive understanding regarding the molecular mechanisms underlying siderophore synthesis, uptake, and regulation within various ecological niches. In this study, we demonstrated that the BfmRS two-component system, part of the core genome of P. aeruginosa, plays a crucial role in siderophore metabolism. We have identified BfmS as an osmosensing histidine kinase that responds to external osmolytes, then modulates the activation of the response regulator BfmR. Under high osmolality, BfmR could directly bind to the promoters of pvd, fpv, and femARI gene clusters, thereby enhancing their expression and promoting siderophore metabolism. The proteomic and phenotypic analyses confirmed that deletion of bfmRS results in reduced expression levels of siderophore-related proteins as well as siderophore production. Importantly, loss of bfmR or bfmS significantly impaired bacterial survival in both iron deficiency medium and mouse lung infection models. Furthermore, phylogenetic analysis revealed that BfmRS is highly conserved and widely distributed across Pseudomonas species, evidences also proved that the BfmR of P. putida KT2440 and P. sp. MRSN12121 activated siderophore genes in response to high osmolality. Overall, this study sheds light on the previously unexplored signal transduction pathway, BfmRS, which governs the siderophore regulation in Pseudomonas species through perceiving an osmotic upshift. Considering that siderophores serve as unique social mediators, our findings contribute to a better understanding of how siderophores facilitate bacterial interactions with their eukaryotic hosts and contribute to the establishment of stable communities.
Project description:Activating the cryptic secondary metabolic gene clusters is a vital research field in Streptomyces. The marine Streptomyces sp. FJNU027 strain which could produce tirandamycins was cultured in the oligotrophic medium. Compared with normal medium, a differential product in oligotrophic culture was found by HPLC assay. After mass fermentation, 2 mg of the differential product was obtained from 30 L fermentation broth by solvent extraction, column chromatography over sephadex LH-20 and reverse phase C18, and other methods. It was identified as 4,4',5,5'-tetramethyl-[1,1'- diphenyl]-2,2'-diol by NMR and MS data. The production of this compound was enhanced with the increment of cultural time. Transcriptome sequencing analysis showed that the highest upregulated genes under oligotrophic condition were glycosidase, TraR/DksA C4-type zinc finger protein and ribonuclease encoding genes, while the expression of a MarR family transcriptional regulator was most significantly decreased under oligotrophic condition. The results indicate that oligotrophic culture is an effective method for altering the secondary metabolism of Streptomyces.