CRISPRi screen for a-amylase yield improvements in Bacillus subtilis
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ABSTRACT:
ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168
PROVIDER: GSE179570 | GEO | 2023/02/22
REPOSITORIES: GEO
Similar Datasets
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: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:The production of alpha-amylase (AMY) enzyme in Bacillus subtilis at a high rate leads to accumulation of unfolded AMY, which causes secretion stress. The over-expression of the PrsA chaperon aids the enzyme folding and thus reduces stress. Stress pathways are complex and intertwined in regulation of a variety of cellular mechanism; for instance, PrsA over-expression leads to reduced cell growth. To capture an overview over the impacted mechanisms, we analyze the transcriptomic changes during fed-batch AMY fermentation between a PrsA over-expressing strain and a control in a time-series RNA-seq experiment (n=3, 6 timepoints). We found evidence of transcription in 542 previously un-annotated regions, of which 234 had significant changes in transcription levels (Overall FDR 0.01). The pairwise comparison of up-/downregulated genes and their over-representation in biological processes (FDR 0.01) suggest that the PrsA over-expression might alter ATP biosynthesis activity, amino acid metabolism, and cell wall stability. An additional investigation of the protein-protein association network highlights potential cell motility signaling. Overall, these highlighted mechanisms could potentially further reduce secretion stress or detrimental aspects of PrsA over-expression during AMY production.
2022-07-06 | GSE189556 | GEO
Project description: Not available
| S-EPMC9250202 | biostudies-literature
Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes.
2015-01-26 | GSE65272 | GEO
Project description:Initiation of bacterial DNA replication takes place at the origin of replication (oriC), a region characterized by the presence of multiple DnaA boxes that serve as the binding sites for the master initiator protein DnaA. The absence or failure of DNA replication can result in bacterial cell growth arrest or death. Here, we aimed to uncover the physiological and molecular consequences of stopping replication in the model bacterium Bacillus subtilis. For this purpose, DNA replication was blocked using a CRISPRi approach specifically targeting DnaA boxes 6 and 7, which are essential for replication initiation. We characterized the phenotype of these cells and analyzed the overall changes in the proteome using quantitative mass spectrometry. Cells with arrested replication were elongating and not dividing but showed no evidence of DNA damage response (DDR). Moreover, these cells did not cease translation over time. This study sets the ground for future research on non-replicating but translationally active B. subtilis, which might be valuable for biotechnological applications.
2024-03-26 | PXD036876 | Pride
Project description: Not available
| S-EPMC6749698 | biostudies-literature
Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes. For each sample analyzed in this study three biological replicates were performed. Three different samples were taken from a strain expressing the WalR-SPA protein as well as from wild-type (168) without a tagged WalR. Samples were taken from exponentially growing cells in low phosphate medium (LPDM) as well as from phosphate-limited cells (T2). Each sample compares ChIP DNA vs. Total DNA from the same cells.
2015-01-26 | E-GEOD-65272 | biostudies-arrayexpress
Project description:Transcriptional response of Bacillus subtilis KS002 to targocil
2014-02-01 | GSE54577 | GEO
Project description:Transcriptional response of Bacillus subtilis to ramoplanin in wild-type CU1065.
2011-06-17 | GSE30002 | GEO