Project description:Bacteria in the genus Streptomyces are soil dwelling oligotrophs and important producers of secondary metabolites. Previously we showed that global mRNA expression was subject to a series of metabolic and regulatory switches during the life time of a fermentor batch culture of S. coelicolor M145. Here we analyse the proteome from eight time points from the same fermentor culture and, as phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (ΔphoP), defective in the control of phosphate utilisation. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index which also revealed the importance of stress pathway proteins in both cultures. As expected the ΔphoP mutant was deficient in expression of PhoP-dependent genes and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that co-ordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures and these were not confined to the stationary phase.
Project description:Bacteria in the genus Streptomyces are soil dwelling oligotrophs and important producers of secondary metabolites. Previously we showed that global mRNA expression was subject to a series of metabolic and regulatory switches during the life time of a fermentor batch culture of S. coelicolor M145. Here we analyse the proteome from eight time points from the same fermentor culture and, as phosphate availability is an important regulator of secondary metabolite production, compare this to the proteome of a similar time course from an S. coelicolor mutant, INB201 (M-NM-^TphoP), defective in the control of phosphate utilisation. The proteomes provide a detailed view of enzymes involved in central carbon and nitrogen metabolism. Trends in protein expression over the time courses were deduced from a protein abundance index which also revealed the importance of stress pathway proteins in both cultures. As expected the M-NM-^TphoP mutant was deficient in expression of PhoP-dependent genes and several putatively compensatory metabolic and regulatory pathways for phosphate scavenging were detected. Notably there is a succession of switches that co-ordinately induce the production of enzymes for five different secondary metabolite biosynthesis pathways over the course of the batch cultures and these were not confined to the stationary phase. 36 samples, no replicates; one hour resolution from 23-36h and 41-48h; half hour resolution from 36-41h; two hour resolution 48-60h; sample missing for 34 h
Project description:The rhizosphere is a small region surrounding plant roots that is enriched in biochemicals from root exudates and populated with fungi, nematode, and bacteria. Interaction of rhizosphere organisms with plants is mainly promoted by exudates from the roots. Root exudates contain biochemicals that come from primary and secondary metabolisms of plants. These biochemicals attract microbes, which influence plant nutrition. The rhizosphere bacteria (microbiome) are vital to plant nutrient uptake and influence biotic and abiotic stress and pathogenesis. Pseudomonas is a genus of gammaproteobacteria known for its ubiquitous presence in natural habitats and its striking ecological, metabolic, and biochemical diversity. Within the genus, members of the Pseudomonas fluorescens group are common inhabitants of soil and plant surfaces, and certain strains function in the biological control of plant disease, protecting plants from infection by soilborne and aerial plant pathogens. The soil bacterium Pseudomonas protegens Pf-5 (also known as Pseudomonas fluorescens Pf-5) is a well-characterized biological strain, which is distinguished by its prolific production of the secondary metabolite, pyoverdine. Knowledge of the distribution of P. fluorescens secretory activity around plant roots is very important for understanding the interaction between P. fluorescens and plants and can be achieved by real time tracking of pyoverdine. To achieve the capability of real-time tracking in soil, we have used a structure-switching SELEX strategy to select high affinity ssDNA aptamers with specificity for pyoverdine over other siderophores. Two DNA aptamers were isolated, and their features compared. The aptamers were applied to a nanoporous aluminum oxide biosensor and demonstrated to successfully detect PYO-Pf5. This sensor provides a future opportunity to track the locations around plant roots of P. protegens and to monitor PYO-Pf5 production and movement through the soil.
Project description:Histone modifications have been shown to be crucial for secondary metabolism in various filamentous fungi. Here we studied the influence of histone acetylation on secondary metabolite production in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites including pigments and mycotoxins. Deletion of the classical HDACs FfHdF1, FfHdF2 and FfHdF3 indicated that FfHdF1 and FfHdF2 are major regulators of secondary metabolism, whereas FfHdF3 is involved in developmental processes but dispensable for secondary metabolite production in F. fujikuroi. Microarray analysis with the major HDAC FfHdF2 revealed differential regulation of several secondary metabolite gene clusters, subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are responsible for gene silencing but also gene activation. Chromatin immunoprecipitation assays with M-NM-^TffhdF2 revealed significant alterations regarding the acetylation state in the landscape of secondary metabolite gene clusters thereby providing insights into the regulatory mechanism. In addition, the class I HDAC FfHdF1 also has major impact on secondary metabolism in F. fujikuroi. Furthermore, deletion of both ffhdF1 and ffhdF2 resulted in de-repression of secondary metabolites under normally repressing conditions. Thus, manipulation of HDAC encoding genes might provide a powerful tool for the activation of cryptic secondary metabolites. Investigation of whole genome gene expression of the Fusarium fujikuroi wild type IMI58289, M-NM-^TffhdF2 mutant under nitrogen starvation and nitrogen sufficient conditions. In this study we hybridized in total 12 microarrays using total RNA recovered from a wild-type culture of F. fujikuroi IMI58289 and M-NM-^TffhdF2 mutant culture. All cultures were grown on a 6 mM Gln (10%) and a 60 mM Gln medium (100%). For each combination of culture and medium a biological replicate was created. Each chip measures the expression level of 14,397 genes from F. fujikuroi IMI58289 with eight 60-mer probes.
Project description:We investigated the metabolism of six secondary metabolite producing fungi of the Penicillium genus, during nutrient depletion in the stationary phase of batch fermentations and assessed conserved metabolic responses across species using genome-wide transcriptional profiling. Coexpression analysis revealed that expression of secondary metabolite biosynthetic genes correlates with expression of genes associated with pathways responsible for generation of precursor metabolites for secondary metabolism. Our results highlight the main metabolic routes for precursor supply of the secondary metabolism during nutrient depletion, and suggests that regulation of fungal metabolism is tailored to meet the demands for secondary metabolite production. These findings can aid in identifying wild type species, which are optimized for production of specific secondary metabolites, and therefore can be utilized as high yielding cell factories.
Project description:Genome-wide microarray analysis was performed using RNA extracted from soil cultures of Streptomyces coelicolor A3(2) in the presence or absence of chitin. The vast majority of genes in chitin and amino sugar metabolism, as well as many other genes for carbon and energy, nitrogen and sulfur metabolism, were differentially expressed in response to addition of chitin. Moreover, the gene expressions of eight gene clusters for secondary metabolites were also significantly up-regulated in the chitin amended soil. To reveal the role of a pleiotropic transcriptional regulator, DasR, which has been reported to be involved in regulation of chitin metabolism, antibiotic production and morphological differentiation, the gene expression patterns of wild type and dasR mutant in soil amended with chitin were compared by microarray analysis. The dasR mutation resulted in up-regulation of four antibiotic gene clusters and down-regulation of chitin metabolism.