Transcriptomic impact of PrsA over-expression during fed-batch Bacillus subtilis alpha-amylase fermentation
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ABSTRACT:
ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168
PROVIDER: GSE189556 | GEO | 2022/07/06
REPOSITORIES: GEO
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Project description:Transcriptome analysis was used to investigate the global stress response of the Gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon that responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild-type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example citM, ylxF, yloA, ykoJ and several genes of the flgB operon. However, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction. Secretion stress was applied by overproducing the well-secreted AmyQ α-amylase (pKTH10 vector) from B. amyloliquefaciens. Besides examining secretion stress in wild-type cells, we compared transcriptome profiles of a cssS mutant strain under conditions of high-level AmyQ production. Samples for transcriptome analyses were collected at the late exponential growth stage (one hour before the transition point) and 3 hours upon entry in the stationary growth phase. Three independent cultures of each strain were used and cells were sampled for macroarray experiments. Duplicate spots were averaged in Array-Pro software (Media Cybernetics, Inc.) and the signal was normalized after background subtraction by calculation of the percentage of total signal per gene using Microsoft Excel.
2007-10-01 | E-GEOD-8014 | biostudies-arrayexpress
Project description:Transcriptome analysis was used to investigate the global stress response of the Gram-positive bacterium Bacillus subtilis caused by overproduction of the well-secreted AmyQ α-amylase from Bacillus amyloliquefaciens. Analyses of the control and overproducing strains were carried out at the end of exponential growth and in stationary phase, when protein secretion from B. subtilis is optimal. Among the genes that showed increased expression were htrA and htrB, which are part of the CssRS regulon that responds to high-level protein secretion and heat stress. The analysis of the transcriptome profiles of a cssS mutant compared to the wild-type, under identical secretion stress conditions, revealed several genes with altered transcription in a CssRS-dependent manner, for example citM, ylxF, yloA, ykoJ and several genes of the flgB operon. However, a high affinity CssR-binding was only observed for htrA and htrB, and possibly for citM. In addition, the DNA macroarray approach reveal that several genes of the sporulation pathway are downregulated by AmyQ overexpression, and a group of motility-specific (σD-dependent) transcripts were clearly upregulated. Subsequent flow cytometric analyses demonstrate that upon overproduction of AmyQ as well as a non-secretable variant of the α-amylase, the process of sporulation is severely inhibited. The same experiments were implemented to investigate the expression levels of the hag promoter, a well-established reporter for σD-dependent gene expression. This approach confirmed the observations based on our DNA macroarray analyses and led us to conclude that expression levels of several genes involved in motility are maintained at high levels under all conditions of α-amylase overproduction. Keywords: secretion stress response
2007-10-01 | GSE8014 | GEO
Project description:In a previous study we used RNA-seq to identify cellular stresses related to the overexpression of xylanase XynA, and found that upregulation of the CtsR regulon improves the yield of XynA production in B. subtilis. In this study, we compared the transcriptomes of B. subtilis cells overexpressing either the xylanase XynA or the heterologous amylase AmyM, to identify general and enzyme-specific stress responses. In addition, we further compared the translational profiles of cells overexpressing XynA and AmyM using ribosome profiling.
2024-08-30 | GSE270692 | GEO
Project description:Prolonged stress has adverse consequences for neurons in the hippocampus (HIPP). The amygdala (AMY) is a brain region that is similar to HIPP across many measures, suggesting that chronic stress might modulate AMY and HIPP function in similar ways. However, studies addressing this issue have produced surprising results. For example, a regimen of chronic stress shown to produce atrophy in HIPP neurons caused dendritic branching in AMY neurons. These and other data have revealed that excessive stress induces fundamentally opposing processes in the AMY and HIPP, such that AMY function is facilitated by levels of stress that produce deleterious effects in HIPP. The mechanisms that contribute to the opposing responses of these brain regions to chronic stress are unknown. Such knowledge may suggest novel directions for pharmacological interventions to protect the HIPP from stress-mediated damage.,We will determine gene expression patterns in the hippocampus and amygdala under basal and stressful conditions, with a particular interest in those genes that are differentially regulated across the two regions. ,We hypothesize that genes that are oppositely regulated in the amygdala and hippocampus after stress mediate the opposing functional consequences of chronic stress in these two brain regions. Moreover, genes which are differentially expressed in these regions under basal conditions may underlie the distinct responses of these regions to stress.,Rats will receive either 30sec of handling (Control group; n=15) or 3hr of immobilization stress (Stress group; n=15) each day for 14 consecutive days. This stress paradigm has previously been shown to produce opposing effects on dendritic morphology and region-dependent behaviors for the hippocampus versus amygdala. Twenty-four hours after the last stress or handling session, the rats will be overanaesthetized, and the brain will be removed and placed in ice-cold buffer. Tissue will be rapidly dissected from the basolateral complex of the amygdala and the CA3 region of the hippocampus of both hemispheres, flash frozen in liquid nitrogen, and stored at -80C until further processing. The tissue from each brain region will be pooled across groups, yielding four samples (Control-Hippocampus, Control-Amygdala, Stress-Hippocampus, and Stress-Amygdala). Total RNA will be extracted using standard methods, and sent to the centers. Each sample will be run in triplicate, utilizing a total of 12 Affymetrix Rat Genome U34A gene chips. A triplicate array assay of pooled samples has previously been shown to both substantially increase the sensitivity of detecting genes of interest and reduce the variability associated with individual microarrays. In addition, the large number of rats used in each group will reduce the variability in gene expression associated with individual responses to chronic stress, as well as variability due to slight anatomical differences in the tissue extracted from each rat.
2007-07-31 | E-GEOD-2870 | biostudies-arrayexpress
Project description:Our study showed that optimizing ncRNA expression can increase or lower the yield of alpha-amylase enzyme production in Bacillus subtilis while revealing a range of potentially novel ncRNAs.
2023-02-22 | GSE179570 | GEO
Project description:Prolonged stress has adverse consequences for neurons in the hippocampus (HIPP). The amygdala (AMY) is a brain region that is similar to HIPP across many measures, suggesting that chronic stress might modulate AMY and HIPP function in similar ways. However, studies addressing this issue have produced surprising results. For example, a regimen of chronic stress shown to produce atrophy in HIPP neurons caused dendritic branching in AMY neurons. These and other data have revealed that excessive stress induces fundamentally opposing processes in the AMY and HIPP, such that AMY function is facilitated by levels of stress that produce deleterious effects in HIPP. The mechanisms that contribute to the opposing responses of these brain regions to chronic stress are unknown. Such knowledge may suggest novel directions for pharmacological interventions to protect the HIPP from stress-mediated damage. We will determine gene expression patterns in the hippocampus and amygdala under basal and stressful conditions, with a particular interest in those genes that are differentially regulated across the two regions. We hypothesize that genes that are oppositely regulated in the amygdala and hippocampus after stress mediate the opposing functional consequences of chronic stress in these two brain regions. Moreover, genes which are differentially expressed in these regions under basal conditions may underlie the distinct responses of these regions to stress. Rats will receive either 30sec of handling (Control group; n=15) or 3hr of immobilization stress (Stress group; n=15) each day for 14 consecutive days. This stress paradigm has previously been shown to produce opposing effects on dendritic morphology and region-dependent behaviors for the hippocampus versus amygdala. Twenty-four hours after the last stress or handling session, the rats will be overanaesthetized, and the brain will be removed and placed in ice-cold buffer. Tissue will be rapidly dissected from the basolateral complex of the amygdala and the CA3 region of the hippocampus of both hemispheres, flash frozen in liquid nitrogen, and stored at -80C until further processing. The tissue from each brain region will be pooled across groups, yielding four samples (Control-Hippocampus, Control-Amygdala, Stress-Hippocampus, and Stress-Amygdala). Total RNA will be extracted using standard methods, and sent to the centers. Each sample will be run in triplicate, utilizing a total of 12 Affymetrix Rat Genome U34A gene chips. A triplicate array assay of pooled samples has previously been shown to both substantially increase the sensitivity of detecting genes of interest and reduce the variability associated with individual microarrays. In addition, the large number of rats used in each group will reduce the variability in gene expression associated with individual responses to chronic stress, as well as variability due to slight anatomical differences in the tissue extracted from each rat. Keywords: dose response
2005-07-06 | GSE2870 | GEO
Project description:We recently identified the nonreceptor tyrosine kinase syk as a tumor suppressor in pancreatic ductal adenocarcinoma cells. Reintroduction of syk into Panc1 cells promoted a more differentiated phenotype and retarded invasion and tumorigenic growth. Gene array analysis identified over 2,000 transcripts differentially expressed at FDR<0.01. Among these were members of the MMP2 axis, which were subsequently shown to regulate Panc1 invasion. Keywords: Genetic manipulation (stable transgene expression)
2009-09-09 | GSE18000 | GEO
Project description:Canonical Wnt signaling controls proliferation and differentiation of osteogenic progenitor cells, and tumor-derived secretion of the Wnt antagonist Dickkopf-1 (Dkk1) is correlated with osteolyses and metastasis in many bone malignancies. However, the role of Dkk1 in the oncogenesis of primary osteosarcoma (OS) remains unexplored. Here, we over-expressed Dkk1 in the OS cell line MOS-J. Contrary to expectations, Dkk1 had autocrine effects on MOSJ cells in that it increased proliferation and resistance to metabolic stress in vitro. In vivo, Dkk1 expressing MOS-J cells formed larger and more destructive tumors than controls. These effects were attributed in part to up-regulation of the stress response enzyme and cancer stem cell marker aldehyde-dehydrogenase-1 (ALDH1) through Jun-N-terminal kinase signaling. This is the first report linking Dkk1 to tumor stress resistance, further supporting the targeting of Dkk1 not only to prevent and treat osteolytic bone lesions but also to reduce numbers of stress-resistant tumor cells. Two samples were analyzed, one human DKK1 transfected MOS-J cell sample and one control vector transfected MOS-J cell sample.
2012-12-22 | E-GEOD-43112 | biostudies-arrayexpress
Project description:Methylation of lysine 4 at histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multi-subunit (SET1/MLL) complexes have been described that contain methyltransferase activity towards H3K4, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. Peptides were applied to online nanoLC-MS/MS, using a 120 min acetonitrile gradient (5.6 - 76%). Mass spectra were recorded on an LTQ-Orbitrap-Velos mass spectrometer (Thermo) selecting the 15 most intense precursor ions of every full scan for fragmentation. Raw data were analyzed using MaxQuant 1.3.0.5, with label-free quantification (LFQ), match between runs (between triplicates) and the iBAQ algorithm enabled. MaxQuant default settings were used for peptide identification; Enzyme: Trypsin/P, MS tolerance (FTMS): 6 ppm, max missed cleavages: 2, max charge: 7, MS/MS tolerance (ITMS): 0.5 Da, Peptide FDR: 0.01 and Protein FDR: 0.01. Normalized mass spectrometric intensities (LFQ intensities) were compared between the GFP-tagged and control sample, using an adapted permutation-based false discovery rate (FDR) t-test in Perseus (MaxQuant package).
2013-09-13 | PXD000172 | Pride
Project description:The gene aml encoding alpha-amylase in Streptomyces lividans was cloned in the multicopy plasmid pIJ486, generating plasmid pAMI11. Plasmid pAMI11 and pIJ486 were propagated in S. lividans TK21 to obtain S. lividans TK21(pAMI11) and its isogenic strain S. lividans TK21(pIJ486). Transcriptional profiling of the bacterium that overproduces alpha-amylase mainly resulted in the upregulation of genes involved in the biogenesis and function of ribosomes, together with the upregulation of the genes involved in the redox processes, the ABC transporters, the central carbon, aminoacid and purine /pyrimidine metabolism. Moreover, some genes involved in oxidative stress were upregulated. The number of genes downregulated was much lower than the upregulated ones. Therefore, bacteria respond by favouring alpha-amylase overproduction that apparently does not cause damage to the cell.
2015-07-23 | GSE61448 | GEO