Project description:Plants endo-beta-1,4-glucanases, belonging to Glycoside Hydrolase Family 9, have functional roles in cell wall biosynthesis and remodeling via endohydrolysis of (1-4)-beta-D-glucosidic linkages. Modification of cell wall chemistry via RNAi-mediated downregulation of PdKOR1, a endo-beta-1,4-glucanase gene, in Populus deltoides, has been shown to have functional consequences to the composition of secondary metabolome and ability of modified roots to interact with microbes. The molecular remodeling that underlies the observable differences at metabolic, physiological and morphological levels in roots is not well understood. Here we used a LC-MS/MS-based proteome profiling approach to survey the molecular remodeling in root tissues of PdKOR and control plants. A total of 14316 peptides were identified and these mapped to 7139 P. deltoides. Based on 90% sequence identity, the measured protein accessions represent 1187 functional protein groups. Analysis of GO categories and specific individual proteins shows differential expression of proteins relevant to plant-microbe interactions, cell wall chemistry and metabolism. This proteome dataset can serve as a useful resource for deriving new hypotheses and empirical testing pertaining to functional roles of proteins and pathways in differential priming of plant roots to interactions with microbes.
Project description:Cellulases and related β-1,4-glucanases are essential components of lignocellulose-degrading enzyme mixtures. The detection of β-1,4-glucanase activity typically relies on monitoring the breakdown of purified lignocellulose-derived substrates or synthetic chromogenic substrates, limiting the activities which can be detected and complicating the tracing of activity back to specific components within complex enzyme mixtures. As a tool for the rapid detection and identification of β-1,4-glucanases, a series of glycosylated cyclophellitol inhibitors mimicking β-1,4-glucan oligosaccharides have been synthesised. These compounds are highly efficient inhibitors of HiCel7B, a well-known GH7 endo-β-1,4-glucanase. An elaborated activity-based probe facilitated the direct detection of β-1,4-glucanases within a complex fungal secretome without any detectable cross-reactivity with β-D-glucosidases. These probes and inhibitors add valuable new capacity to the growing toolbox of cyclophellitol-derived probes for the activity-based profiling of biomass-degrading enzymes.
Project description:Characterization of a metagenomic regulatory sequence library derived from M. xanthus, E. coli, and O. urethralis genomes in strains expressing different RpoD ortholog variants. Targeted DNA and RNA seq used to profile relative DNA and RNA abundances, respectively of each regulatory sequence construct in the library.
Project description:We examined the effect of high-level expression of the commercially important endo-1,4-β-xylanase XynA on the B. subtilis transcriptome using RNA-seq. Rather unexpectedly, we found a reduced expression of several protein chaperones, including ClpC, ClpE and ClpX, was downregulated when XynA was overproduced. Expression of these proteins is controlled by the transcriptional repressor CtsR. CtsR levels are directly controlled by regulated proteolysis, involving ClpC and its cognate protease ClpP. Preventing this downregulation by knocking out the involved transcriptional repressor CtsR resulted in increased XynA production by more than 25 %.
Project description:Characterization of new bacterial catabolic genes and mobile genetic elements by high throughput genetic screening of a soil metagenomic library
Project description:Massively parallel reporter assays (MPRA) are widely used to discover functional enhancers but have largely been limited to transfected cell models, which are confounded by vector-induced innate immune responses and lack the physiologically relevant cellular and endogenous hormonal context and chromatin environment of complex mammalian tissues. Here, we combine hydrodynamic injection (HDI) with a modified STARR-seq-based MPRA, designated HDI-STARR-seq (PMID:39716078) to determine condition-specific enhancer activity in mouse liver at scale. Analysis of a synthetic STARR-seq library, comprised of 23,912 reporters regions spanning 1839 ATAC regions enabled functional assessment of mouse liver enhancer activity in vivo across three distinct biological conditions: male iver, feamle liver and continuous growth hormone (GH)-infused male liver, which is substantially feminized with regards to gene expression. HDI-STARR-seq reporters representing 305 ATAC regions showed sex-biased and GH-responsive enhancer activity that mirrored the regulation of their chromatin accessibility in hepatocytes, validating these sites as functional, physiologically regulated enhancers. These enhancers were enriched for activating histone marks (H3K4me1, H3K27ac) and for binding sites for GH-activated transcriptional regulator STAT5 and for the STAT5-dependent, sex-specific repressors BCL6 and CUX2, consistent with a cohesive regulatory network controlling hepatocyte lipid and xenobiotic metabolism. Further, de novo motif analysis identified binding sites for HNF4A and for several novel factors specifically enriched at the regulated enhancers. Importantly, sex-biased and GH-regulated functional enhancers were linked to both MASLD (metabolic dysfunction-associated steatotic liver disease)-enabling and MASLD-protective genes, indicating that GH-dependent chromatin remodeling at these loci is a key determinant of sex-differential liver metabolic disease susceptibility. This integrated in vivo approach defines a validated set of GH-regulated hepatocyte enhancers through which chromatin accessibility and transcription factor binding drive sexual dimorphism in hepatic metabolism and sex-specific MASLD risk.