Project description:Pantoea sp. YR343, isolated from the Populus deltoides rhizosphere, is a robust plant root colonizer that produces indole-3-acetic acid (IAA). Genomic and metabolomic analyses predicted that Pantoea sp. YR343 synthesizes IAA primarily using the indole-3-pyruvate pathway. Pantoea sp. YR343 proteomes showed upregulation of IpdC for growth in the presence of tryptophan or IAA versus controls.
Project description:The irreversible decarboxylation step, which commits 2-oxo acids to the Ehrlich pathway, was initially attributed to pyruvate decarboxylase. However, the yeast genome was shown to harbour no fewer than 5 genes that show sequence similarity with thiamine-diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases { while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases. Transcriptome analysis and decarboxylase activity measurements on an S. cerevisiae aro10 strain, a double aro10 thi3 deletion strain and a quadruple pdc1,5,6,aro10 mutant strains grown in carbonââ¬âlimited chemostat with phenylalanine as nitrogen source indicated that:; i) PDC5 is strongly upregulated in an aro10 background (Fig. 2) and also encodes a broad-substrate ñ-keto acid decarboxylase. ii) PDC5 expression depends on the presence of THI3 (Fig. 2), and; iii) in contrast to cell extracts from a strain expressing ARO10 only (pdc1,5,6, thi3), cell extract from a strain that only contains THI3 (pdc1,5,6,aro10) did not produce any ñ-keto acid decarboxylase activity . THI3 has recently been demonstrated to be involved in regulation of thiamine homeostasis in S. cerevisiae, which further suggests that its role in the Ehrlich pathway may be regulatory rather than catalytic. A systematic investigation of the catalytic properties of all five (putative) TPP-dependent decarboxylases (Aro10p, Thi3p, Pdc1p, Pdc5p, Pdc6p) is essential for a final resolution of the substrate specificity of these key enzymes in the Ehrlich pathway. Experiment Overall Design: Comparison of glucose limited chemostat cultivations with phenylalanine as sole nitrogen sources of the following strains: Experiment Overall Design: - CENPK113-7D MATa MAL2-8c SUC2 Experiment Overall Design: - CENPK555-4A MATa MAL2-8c SUC2 ydr380w delta Experiment Overall Design: - CENPK5632-3B MATalpha MAL2-8c SUC2 ydr380w delta ydl080c delta Experiment Overall Design: - CENPK609-11A MATa MAL2-8c SUC2 pdc1, 5, 6 delta ydr380w delta
Project description:The irreversible decarboxylation step, which commits 2-oxo acids to the Ehrlich pathway, was initially attributed to pyruvate decarboxylase. However, the yeast genome was shown to harbour no fewer than 5 genes that show sequence similarity with thiamine-diphosphate dependent decarboxylase genes. Three of these (PDC1, PDC5 and PDC6) encode pyruvate decarboxylases { while ARO10 and THI3 represent alternative candidates for Ehrlich-pathway decarboxylases. Transcriptome analysis and decarboxylase activity measurements on an S. cerevisiae aro10 strain, a double aro10 thi3 deletion strain and a quadruple pdc1,5,6,aro10 mutant strains grown in carbon–limited chemostat with phenylalanine as nitrogen source indicated that: i) PDC5 is strongly upregulated in an aro10 background (Fig. 2) and also encodes a broad-substrate α-keto acid decarboxylase. ii) PDC5 expression depends on the presence of THI3 (Fig. 2), and iii) in contrast to cell extracts from a strain expressing ARO10 only (pdc1,5,6, thi3), cell extract from a strain that only contains THI3 (pdc1,5,6,aro10) did not produce any α-keto acid decarboxylase activity . THI3 has recently been demonstrated to be involved in regulation of thiamine homeostasis in S. cerevisiae, which further suggests that its role in the Ehrlich pathway may be regulatory rather than catalytic. A systematic investigation of the catalytic properties of all five (putative) TPP-dependent decarboxylases (Aro10p, Thi3p, Pdc1p, Pdc5p, Pdc6p) is essential for a final resolution of the substrate specificity of these key enzymes in the Ehrlich pathway. Keywords: Strain comparison
Project description:Differential gene expression analysis of five different strains of Crabtree-negative Saccharomyces cerevisiae that lack pyruvate decarboxylase activity. The three different acids studied were lactic, malic, and 3-hydroxypropionic acid.
Project description:Auxin amino acid conjugates are considered storage forms of auxins available as a source of active auxins on the plant demand. We treated Brassica rapa seedlings with 0.01 mM indole-3-acetyl-L-alanine (IAA-Ala), indole-3-propionyl-L-alanine (IPA-Ala), and indole- 3-butyryl-L-alanine (IBA-Ala) and examined their effects on the transcriptome. All auxin conjugate treatments caused similar patterns in transcription profiles compared to the control, but with different intensities of over- and under-expression depending on the treatment. Most auxin-related DEGs were identified after IBA-Ala treatment, followed by IPA-Ala and IAA-Ala, respectively.
Project description:Transcriptional profiling of Arabidopsis thaliana seedlings treated with auxin (indole-3-acetic acid), highlighting to the physiological function of auxin by observing early response of gene expressions in Arabidopsis seedlings. Two-condition experiment, auxin-treated seedlings vs. control seedlings. Biological replicates:2 control replicates, 2 auxin-treated.
Project description:The root cap-specific conversion of the auxin precursor indole-3-butyric acid (IBA) into the main auxin indole-3-acetic acid (IAA) generates a local auxin source which subsequently modulates both the periodicity and intensity of auxin response oscillations in the root tip of Arabidopsis, and consequently fine-tunes the spatiotemporal patterning of lateral roots. To explore downstream components of this signaling process, we investigated the early transcriptional regulations happening in the root tip during IBA-to-IAA conversion in Col-0 and ibr1 ibr3 ibr10 triple mutant after 6 hours of IBA treatment.
Project description:Indole is ubiquitously synthesized by plants and bacteria and functions as an inter species signaling molecule to modulate a wide variety of cellular activities. However, it is not clear how the indole signal is perceived and responded by plant growth promoting rhizobacteria (PGPR) at the rhizosphere. Here, we demonstrated that indole enhanced antibiotic tolerance of Pseudomonas fluorescens 2P24, a PGPR well known for its biocontrol capacity. By conducting quantitative proteomic analysis, we showed that indole influences the expression of multiple genes including the emhABC operon encoding the major multidrug efflux pump in P. fluorescens 2P24. The indole-induced antibiotic tolerance was not related to bacterial dormancy or slow growth, but depended on the emhABC operon and the divergently transcribed TetR-like regulator emhR. By binding to the semi-palindromic operator sequence, EmhR repressed the expression of emhABC. It was further revealed that indole bound to EmhR and weakened the interaction between EmhR and the operator. This is consistent with our finding that indole-induced expression of the EmhABC efflux pump is dependent on EmhR. Using homology modeling and molecular dynamics simulation, we found that indole binding resulted in significantly decreased distance between the two DNA-recognizing α3 helices within the EmhR dimer, which would possibly account for its compromised DNA binding capacity. EmhR was further shown to globally influence protein expressions, especially transporters and proteins involved in the denitrification pathway. This EmhR-dependent, indole-induced antibiotic tolerance is likely to be prevalent in the Pseudomonas species, as the EmhR homologue in Pseudomonas syringae was also shown to be responsible for the indole-induced antibiotic tolerance. Taken together, our results revealed an indole-sensing transcription factor EmhR responsible for indole-induced antibiotic tolerance in Pseudomonas species and have important implications on the general mechanism for the indole sensing and responses in rhizobacteria.
Project description:Root exudates are composed of primary and secondary metabolites known to modulate the rhizosphere microbiota. Glucosinolates are defense compounds present in the Brassicaceae family capable of deterring pathogens, herbivores and biotic stressors in the phyllosphere. In addition, traces of glucosinolates and their hydrolyzed byproducts have been found in the soil, suggesting that these secondary metabolites could play a role in the modulation and establishment of the rhizosphere microbial community associated with this family. We used Arabidopsis thaliana mutant lines with disruptions in the indole glucosinolate pathway, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 16S rRNA amplicon sequencing to evaluate how disrupting this pathway affects the root exudate profile of Arabidopsis thaliana, and in turn, impacts the rhizosphere microbial community. Chemical analysis of the root exudates from the wild type Columbia (Col-0), a mutant plant line overexpressing the MYB transcription factor ATR1 (atr1D) which increases glucosinolate production, and the loss-of-function cyp79B2cyp79B3 double mutant line with low levels of glucosinolates confirmed that alterations to the indole glucosinolate biosynthetic pathway shifts the root exudate profile of the plant. We observed changes in the relative abundance of exuded metabolites. Moreover, 16S rRNA amplicon sequencing results provided evidence that the rhizobacterial communities associated with the plant lines used were directly impacted in diversity and community composition. This work provides further information on the involvement of secondary metabolites and their role in modulating the rhizobacterial community. Root metabolites dictate the presence of different bacterial species, including plant growth-promoting rhizobacteria. Our results suggest that alterations in the indole glucosinolate pathway cause disruptions beyond the endogenous levels of the plant, significantly changing the abundance and presence of different metabolites in the root exudates of the plants as well as the microbial rhizosphere community.