Project description:Pseudomonas aeruginosa produces the toxic secondary metabolite hydrogen cyanide (HCN) at high cell population densities and low aeration. We have used Affimetrix microarrays to investigate the impact of HCN as a signal in cell-cell communication by comparing the transcriptome of the wild-type strain PAO1 to that of an HCN-negative mutant (PAO6344) under cyanogenic conditions. Total RNA was extracted from cultures of the wild-type strain PAO1 as well as from the isogenic HCN negative mutant (PAO6344), when HCN production was maximal (i.e. end of exponential growth phase). Each RNA pool derived from 3 technical replicates. 1 biological replicate per strain was performed on a different day.
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:Extremely slow growth imposed by energy limitation is a ubiquitous but poorly understood physiological state for microbes. We used oxygen limitation to impose this state on Pseudomonas aeruginosa and measured newly synthesized proteins using a time-selective proteome labeling method (BONCAT) to identify relevant regulators and metabolic pathways. We further characterized one upregulated protein that has no homology to any known protein domains. This small, acidic protein is post-transcriptionally regulated and physically interacts with RNA polymerase, binding near the secondary channel during transcription elongation, and leading to widespread effects on gene expression. For some genes, the impacts on transcript and protein levels are different, suggesting possible modulation of translation as well. These effects have phenotypic consequences, as deletion of the gene affects biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. Based on these phenotypes, we have designated the protein SutA (survival under transitions).
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: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 ΔffhdF2 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, ΔffhdF2 mutant under nitrogen starvation and nitrogen sufficient conditions.
Project description:Pseudomonas aeruginosa produces the toxic secondary metabolite hydrogen cyanide (HCN) at high cell population densities and low aeration. We have used Affimetrix microarrays to investigate the impact of HCN as a signal in cell-cell communication by comparing the transcriptome of the wild-type strain PAO1 to that of an HCN-negative mutant (PAO6344) under cyanogenic conditions.
Project description:Extremely slow growth imposed by energy limitation is a ubiquitous but poorly understood physiological state for microbes. We used oxygen limitation to impose this state on Pseudomonas aeruginosa and measured newly synthesized proteins using a time-selective proteome labeling method (BONCAT) to identify relevant regulators and metabolic pathways. We further characterized one upregulated protein that has no homology to any known protein domains. This small, acidic protein is post-transcriptionally regulated and physically interacts with RNA polymerase, binding near the secondary channel during transcription elongation, and leading to widespread effects on gene expression. For some genes, the impacts on transcript and protein levels are different, suggesting possible modulation of translation as well. These effects have phenotypic consequences, as deletion of the gene affects biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. Based on these phenotypes, we have designated the protein SutA (survival under transitions).
Project description:The fungal pathogen Aspergillus fumigatus is frequently cultured from the sputum of cystic fibrosis (CF) patients along with the bacterium, Pseudomonas aeruginosa. A. fumigatus secretes a range of secondary metabolites, and one of these, gliotoxin, has inhibitory effects on the host immune response. In this study, the effect of P. aeruginosa culture filtrate (CuF) on fungal growth and gliotoxin production was investigated. Exposure of A. fumigatus hyphae to P. aeruginosa cells induced increased production of gliotoxin and a decrease in fungal growth. In contrast exposure of A. fumigatus hyphae to P. aeruginosa CuF lead to increased growth and decreased gliotoxin production. Quantitative proteomic analysis was employed to characterize the proteomic response of A. fumigatus upon exposure to P. aeruginosa CuF. Changes in the profile of proteins involved with secondary metabolite biosynthesis (gliotoxin, fumagillin, pseurotin A), and changes to the abundance of proteins involved in oxidative stress (e.g. formate dehydrogenase) and detoxification (e.g. thioredoxin reductase) were observed, suggesting that the bacterial secretome has a profound effect on the fungal proteome. Alterations in the abundance of proteins involved in detoxification and oxidative stress, highlight the ability of A. fumigatus to differentially regulate protein synthesis in response to environmental stresses imposed by competitors such as P. aeruginosa. Such responses may ultimately have serious detrimental effects on the host.
Project description:The fungal pathogen Aspergillus fumigatus is frequently cultured from the sputum of cystic fibrosis (CF) patients along with the bacterium, Pseudomonas aeruginosa. A. fumigatus secretes a range of secondary metabolites, and one of these, gliotoxin, has inhibitory effects on the host immune response. In this study, the effect of P. aeruginosa culture filtrate (CuF) on fungal growth and gliotoxin production was investigated. Exposure of A. fumigatus hyphae to P. aeruginosa cells induced increased production of gliotoxin and a decrease in fungal growth. In contrast exposure of A. fumigatus hyphae to P. aeruginosa CuF lead to increased growth and decreased gliotoxin production. Quantitative proteomic analysis was employed to characterize the proteomic response of A. fumigatus upon exposure to P. aeruginosa CuF. Changes in the profile of proteins involved with secondary metabolite biosynthesis (gliotoxin, fumagillin, pseurotin A), and changes to the abundance of proteins involved in oxidative stress (e.g. formate dehydrogenase) and detoxification (e.g. thioredoxin reductase) were observed, suggesting that the bacterial secretome has a profound effect on the fungal proteome. Alterations in the abundance of proteins involved in detoxification and oxidative stress, highlight the ability of A. fumigatus to differentially regulate protein synthesis in response to environmental stresses imposed by competitors such as P. aeruginosa. Such responses may ultimately have serious detrimental effects on the host.