Membrane protein overproduction stress in Bacillus subtilis
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ABSTRACT:
ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168
PROVIDER: GSE35916 | GEO | 2018/12/30
REPOSITORIES: GEO
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Project description:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host.
2012-05-25 | GSE34505 | GEO
Project description:Background. Bacillus subtilis is a favorable host for the production of industrially relevant proteins because of its capacity of secreting proteins into the medium to high levels, its GRAS (Generally Recognized As Safe) status, its genetic accessibility and its capacity to grow in large fermentations. However, production of heterologous proteins still faces limitations. Results. This study aimed at the identification of bottlenecks in secretory protein production by analyzing the response of B. subtilis at the transcriptome level to overproduction of eight secretory proteins of endogenous and heterologous origin and with different subcellular or extracellular destination: secreted proteins (NprE and XynA of B. subtilis, Usp45 of Lactococcus lactis, TEM-1beta -lactamase of Escherichia coli), membrane proteins (LmrA of L. lactis and XylP of Lactobacillus pentosus) and lipoproteins (MntA and YcdH of B. subtilis). Responses specific for proteins with a common localization as well as more general stress responses were observed. The latter include upregulation of genes encoding intracellular stress proteins (groES/EL, CtsR regulated genes). Specific responses include upregulation of the liaIHGFSR operon under Usp45 and TEM-1 beta-lactamase overproduction; cssRS, htrA and htrB under all secreted proteins overproduction; sigW and SigW-regulated genes mainly under membrane proteins overproduction; and ykrL (encoding an HtpX homologue) specifically under membrane proteins overproduction. Conclusions. The results give better insights into B. subtilis responses to protein overproduction stress and provide potential targets for genetic engineering in order to further improve B. subtilis as a protein production host. Samples for transcriptome analyses were induced at the exponential-growth phase (OD600 = 0.7) with 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). Cells were harvested 30 min after induction. Three or four independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment.
2012-05-24 | E-GEOD-34505 | biostudies-arrayexpress
Project description:We systematically investigated the impact of genetic variation and the environment on protein overproduction costs in Saccharomyces cerevisiae To identify genes mediating protein overproduction costs, genetic perturbations were applied. The yEVenus overexpression plasmid was crossed into gene deletion backgound. The generation of double mutant strains enabled us to investigate the transcriptional response of the cells under two different internal stress: deletion of a given gene, plus the overexpression of a gratuitous protein To explore the cellular response under gratuitous protein overproduction, a sensitized background was used (sse1 deletion). Our aim was to identify differentially expressed genes upon protein overproduction. 3-3 biological replicates were used for the control (single deletion strain with an empty plasmid) and for the overexpression strains (single deletion strain with yEVenus multi-copy plasmid) as well. The cellular response to overexpression was normalized to the control sse1 deletion strain, without overexpression.
2016-09-01 | E-GEOD-68753 | biostudies-arrayexpress
Project description:We systematically investigated the impact of genetic variation and the environment on protein overproduction costs in Saccharomyces cerevisiae To identify genes mediating protein overproduction costs, genetic perturbations were applied. The yEVenus overexpression plasmid was crossed into gene deletion backgound. The generation of double mutant strains enabled us to investigate the transcriptional response of the cells under two different internal stress: deletion of a given gene, plus the overexpression of a gratuitous protein
2016-09-01 | GSE68753 | GEO
Project description:Comprison of the transcriptome of wild type Bacillus subtilis to the mutants sigW, rasP and prsW to select all candidate sigW regulated genes. We identified 89 genes as being sigW regulated, including several non-coding RNA's. The effects of rasP and prsW mutations on sigW regulated genes were relatively mild, implying that RasP or PrsW are not strictly required for sigW activation. The rasP mutant has a peliotropic phenotype, affecting competence development, protein secretion and membrane protein production. We show that these are not mirrored in the transcriptome, suggesting that RasP exerts its effects at the post transcriptional level.
2013-01-02 | GSE35236 | GEO
Project description:Expression of ykrL of Bacillus subtilis, encoding a close homologue of the Escherichia coli membrane protein quality control protease HtpX, was shown to be upregulated under membrane protein overproduction stress. Using DNA affinity chromatography, two proteins were found to bind to the promoter region of ykrL: Rok, known as a repressor of competence and genes for extracytoplasmic functions, and YkrK, a novel type of regulator encoded by the gene adjacent to ykrL, but divergently transcribed. Electrophoretic mobility shift assays showed Rok and YkrK binding to the ykrL promoter region as well as YkrK binding to the ykrK promoter region. Comparative bioinformatic analysis of the ykrL promoter regions in related Bacillus species revealed a consensus motif, which was demonstrated to be the binding site of YkrK. Deletion of rok and ykrK in a PykrL-gfp reporter strain showed that both proteins are repressors of ykrL expression. In addition, conditions which activated PykrL (membrane protein overproduction, dissipation of the membrane potential, salt- and phenol stress) point to the involvement of YkrL in membrane protein quality control.
2012-04-04 | GSE33456 | GEO
Project description:Comprison of the transcriptome of wild type Bacillus subtilis to the mutants sigW, rasP and prsW to select all candidate sigW regulated genes. We identified 89 genes as being sigW regulated, including several non-coding RNA's. The effects of rasP and prsW mutations on sigW regulated genes were relatively mild, implying that RasP or PrsW are not strictly required for sigW activation. The rasP mutant has a peliotropic phenotype, affecting competence development, protein secretion and membrane protein production. We show that these are not mirrored in the transcriptome, suggesting that RasP exerts its effects at the post transcriptional level. RNA samples prepared from B. subtilis 168 and mutants sigW, rasP, and prsW were grown in Luria Bertani broth to O.D 600nm 1.0 were reverse transcribed, labeled and hybridized to tiling arrays. All hybridizations (except prsW which was in duplicate) were performed in triplicate using RNA isolated from independent cultures.
2013-01-02 | E-GEOD-35236 | biostudies-arrayexpress
Project description:Toxin-antitoxin (TA) systems are ubiquitous throughout bacterial and archaeal genomes. TA systems consist of a stable toxin that inhibits growth and a labile antitoxin that prevents toxicity of the toxin. Here we made an artificial TA system (arT/arA) and performed a DNA microarray study for overproduction of the toxin.
2014-05-13 | GSE54271 | GEO
Project description:Expression of ykrL of Bacillus subtilis, encoding a close homologue of the Escherichia coli membrane protein quality control protease HtpX, was shown to be upregulated under membrane protein overproduction stress. Using DNA affinity chromatography, two proteins were found to bind to the promoter region of ykrL: Rok, known as a repressor of competence and genes for extracytoplasmic functions, and YkrK, a novel type of regulator encoded by the gene adjacent to ykrL, but divergently transcribed. Electrophoretic mobility shift assays showed Rok and YkrK binding to the ykrL promoter region as well as YkrK binding to the ykrK promoter region. Comparative bioinformatic analysis of the ykrL promoter regions in related Bacillus species revealed a consensus motif, which was demonstrated to be the binding site of YkrK. Deletion of rok and ykrK in a PykrL-gfp reporter strain showed that both proteins are repressors of ykrL expression. In addition, conditions which activated PykrL (membrane protein overproduction, dissipation of the membrane potential, salt- and phenol stress) point to the involvement of YkrL in membrane protein quality control. Samples for transcriptome analyses were induced at the exponential-growth phase (OD600 = 0.8) with 0.1% subtilin (subtilin containing supernatant of subtilin producing B. subtilis strain ATCC 6633). Cells were harvested 30 min after induction. Three independent cultures of each strain (target strains and controls) were used, and cells were sampled for microarray experiment. One of each strain was collected in double volume for technical replicates.
2012-04-03 | E-GEOD-33456 | biostudies-arrayexpress
Project description:Toxin-antitoxin (TA) systems are ubiquitous throughout bacterial and archaeal genomes. TA systems consist of a stable toxin that inhibits growth and a labile antitoxin that prevents toxicity of the toxin. Here we made an artificial TA system (arT/arA) and performed a DNA microarray study for overproduction of the toxin. arT was overexpressed in Escherichia coli BW25113 and compared to the empty vector.
2014-05-13 | E-GEOD-54271 | biostudies-arrayexpress