Project description:In Bacillus cereus the catabolite control protein CcpA was shown to be involved in optimizing the efficiency of glucose catabolism by activating genes encoding glycolytic enzymes including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, and by repressing genes encoding the citric acid cycle and gluconeogenic enzymes. Two B. cereus-specific CcpA-regulated operons were identified, encoding enzymes involved in the catabolism of fuculose/arabinose and aspartate. In addition, a genome search using the CRE-site consensus predicted the B. cereus CcpA regulon to include 10 PTS-system gene clusters as well as genes coding for overflow metabolic enzymes leading to acetoin and acetate. Notably, catabolite repression of the genes encoding non-hemolytic enterotoxin (Nhe) and hemolytic (Hbl) enterotoxin appeared CcpA-dependent, and for the corresponding enterotoxin operons, putative CRE-sites were identified. This points to metabolic control of enterotoxin gene expression and suggests that CcpA-mediated glucose sensing provides an additional mode of control to PlcR activated expression of nhe and hbl genes in B. cereus. Keywords: Time course analysis by comparing transriptomes of the wildtype and the ccpA deleton strain. The wildtype (B. cereus ATCC 14579) and ccpA deletion strain were sampled during aerobic growth in Brain heart infusion broth. Samples of wildtype and ccpA deletion strain were compared at 4 time points, i.e. early exponential (OD600 0.2), mid-exponential (OD600 0.8), transition phase (OD600 4), and stationary phase (OD600 8). For each time point biological duplo's were obtained, which were subsequently differently labelled to perform a dye swap.
Project description:In Bacillus cereus the catabolite control protein CcpA was shown to be involved in optimizing the efficiency of glucose catabolism by activating genes encoding glycolytic enzymes including a non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase that mediates conversion of D-glyceraldehyde 3-phosphate to 3-phospho-D-glycerate in one single step, and by repressing genes encoding the citric acid cycle and gluconeogenic enzymes. Two B. cereus-specific CcpA-regulated operons were identified, encoding enzymes involved in the catabolism of fuculose/arabinose and aspartate. In addition, a genome search using the CRE-site consensus predicted the B. cereus CcpA regulon to include 10 PTS-system gene clusters as well as genes coding for overflow metabolic enzymes leading to acetoin and acetate. Notably, catabolite repression of the genes encoding non-hemolytic enterotoxin (Nhe) and hemolytic (Hbl) enterotoxin appeared CcpA-dependent, and for the corresponding enterotoxin operons, putative CRE-sites were identified. This points to metabolic control of enterotoxin gene expression and suggests that CcpA-mediated glucose sensing provides an additional mode of control to PlcR activated expression of nhe and hbl genes in B. cereus. Keywords: Time course analysis by comparing transriptomes of the wildtype and the ccpA deleton strain.
Project description:The influence of the presence of glucose in the Y1 growth medium of Bacillus cereus strain ATCC 14579 was studied by transcriptional analysis. Furthermore, the role of CcpA in glucose induction or repression of gene expression was assessed by use of a ccpA deletion strain. In total, 300 genes were glucose repressed and 173 genes glucose activated. For 212 genes the glucose repression was clearly CcpA dependent, whereas for 116 genes CcpA dependent glucose induction was observed. Functional analysis of the glucose regulated genes showed these genes mainly to be involve in energy production and conversion and in metabolism. Furthermore, genes that were glucose repressed were shown to be involved in cell motility.
Project description:A comparative transcriptome approach was used to assess genes involved in metabolism and pathogenesis that are specifically activated during anaerobic growth of the spore-forming food-borne human pathogen Bacillus cereus ATCC 14579. Growth under anaerobic conditions in Brain Heart Infusion broth revealed a reduced growth rate and a lower yield as compared to that under aerobic conditions. Comparative transcriptome analysis of cells harvested at early- and mid-exponential growth phase, transition phase and stationary phase, subsequently showed hundreds of genes to be induced under anaerobic condition. These included novel genes identified for anaerobic growth of B. cereus, encoding metabolic pathways, such as the arginine deiminase pathway (ArcABDC), a formate dehydrogenase (FdhF) and a pyruvate fomate lyase (Pfl), and alternative respiratory proteins, such as arsenate reductases. Furthermore, the nitrosative stress response was induced in the anaerobic transition phase of growth, conceivably due to the production of nitric oxide as a by-product of nitrite and nitrate respiration. Notably, both hemolytic enzyme and enterotoxin encoding genes were activated in different oxygen limiting conditions, i.e. hemolytic enzyme encoding genes were induced during anaerobic growth, whereas enterotoxin encoding genes were induced in the transition and stationary phase of aerobic cultures reaching a high cell density. These data point to metabolic rearrangements, stress adaptation and activation of the virulent status of B. cereus under anaerobic conditions, such as encountered in the human GI-tract. Keywords: time course, anaerobic growth
Project description:Here, the role of σM and its regulon in stress response and survival of B. cereus ATCC 14579 was assessed by comparative transciptome and phenotypic analysis of this strain and its sigM deletion strain. Exposure of B. cereus ATCC 14579 to a wide range of stresses revealed expression of sigM, encoding σM, to be up-regulated mainly in the presence of ethanol and after alkaline pH-shock. Next to this, disc diffusion tests showed the sigM deletion strain to be more sensitive to oxidizing agents and to be more resistant to cell-wall targeting antibiotics than the wild-type strain. The σM regulon was subsequently determined by comparative transcriptional analyses of the wild-type and its sigM-deletion strain after exposure to ethanol. The putative σM-regulon was shown to consist of 29 genes, several of these genes are predicted to be involved in counteracting oxidative stress, such as an NADH oxidase, a ferredoxin, and a lysine decarboxylase or could encode enzymes involved in methionine metabolism, leading toward L-cysteine production, including luxS. Screening of promoter upstream regions allowed for the assessment of a B. cereus consensus promoter binding site for σM. Since the consensus promoter binding site for B. cereus ATCC 14579 σM, its regulon and the predicted functionalities are different from the corresponding features in B. subtilis, it can be concluded that σM plays a unique role in B. cereus stress response and survival. Keywords: Stress response, comparative transcriptome study
Project description:A comparative transcriptome approach was used to assess genes involved in metabolism and pathogenesis that are specifically activated during anaerobic growth of the spore-forming food-borne human pathogen Bacillus cereus ATCC 14579. Growth under anaerobic conditions in Brain Heart Infusion broth revealed a reduced growth rate and a lower yield as compared to that under aerobic conditions. Comparative transcriptome analysis of cells harvested at early- and mid-exponential growth phase, transition phase and stationary phase, subsequently showed hundreds of genes to be induced under anaerobic condition. These included novel genes identified for anaerobic growth of B. cereus, encoding metabolic pathways, such as the arginine deiminase pathway (ArcABDC), a formate dehydrogenase (FdhF) and a pyruvate fomate lyase (Pfl), and alternative respiratory proteins, such as arsenate reductases. Furthermore, the nitrosative stress response was induced in the anaerobic transition phase of growth, conceivably due to the production of nitric oxide as a by-product of nitrite and nitrate respiration. Notably, both hemolytic enzyme and enterotoxin encoding genes were activated in different oxygen limiting conditions, i.e. hemolytic enzyme encoding genes were induced during anaerobic growth, whereas enterotoxin encoding genes were induced in the transition and stationary phase of aerobic cultures reaching a high cell density. These data point to metabolic rearrangements, stress adaptation and activation of the virulent status of B. cereus under anaerobic conditions, such as encountered in the human GI-tract. B. cereus ATCC 14579 was grown in BHI in 50 ml. Aerobic in a Erlenmeyer flask, shaking at 200 rpm. Anaerobic in a closed flask, flushed with Nitrogen-gas for 30 min, also shaking at 200 rpm. Transcriptome analyses Phase compared to mid-exponential phase Anaerobic (OD600) 0.2 compared to 0.4 Early-exponential 1.0 compared to 0.4 Transition 1.1 compared to 0.4 Stationary Aerobic (OD600) 0.2 compared to 0.8 Early-exponential 4.0 compared to 0.8 Transition 8.0 compared to 0.8 Stationary Aerobic to anaerobic (OD600) Anaerobic 0.6 to aerobic 0.6
Project description:The stress response of B. cereus ATCC 14579 is monitored true time, showing an enormous response in gene expression. Keywords: Stress response, comparative transcriptome analysis.
Project description:The Bacillus cereus ATCC 14579 alternative σ factor σZ and its putative regulon have been characterized. σZ shows overall similarity with ECF σ factors and sigZ constitutes an operon together with asfZ encoding its putative anti-σ factor. Expression analysis revealed sigZ to be induced by an array of stresses, including exposure to ethanol, alkaline pH and heat shock, and a typical promoter binding site for the sigZ-operon was identified by 5’RACE. Phenotypic characterization of B. cereus ATCC 14579 and its sigZ-deletion strain revealed diminished growth performance and sporulation capacity. The σZ-regulon was successfully established by transcriptome analysis of a nisin inducible sigZ-overexpression strain. Overexpression of sigZ was shown to affect expression of 42 genes, including 33 genes encoding proteins located in the extracytoplasm. The identified σZ regulon contained genes encoding proteins situated in the extracytoplasm involved in cell surface modifications and transport. The regulation of genes encoding cell surface modification proteins implies σZ to be involved in the regulation of interaction of B. cereus ATCC 14579 with its environments, which includes human intestinal cells, possibly influencing its virulence status. Keywords: Comparative transcriptome study
Project description:Here, the role of σM and its regulon in stress response and survival of B. cereus ATCC 14579 was assessed by comparative transciptome and phenotypic analysis of this strain and its sigM deletion strain. Exposure of B. cereus ATCC 14579 to a wide range of stresses revealed expression of sigM, encoding σM, to be up-regulated mainly in the presence of ethanol and after alkaline pH-shock. Next to this, disc diffusion tests showed the sigM deletion strain to be more sensitive to oxidizing agents and to be more resistant to cell-wall targeting antibiotics than the wild-type strain. The σM regulon was subsequently determined by comparative transcriptional analyses of the wild-type and its sigM-deletion strain after exposure to ethanol. The putative σM-regulon was shown to consist of 29 genes, several of these genes are predicted to be involved in counteracting oxidative stress, such as an NADH oxidase, a ferredoxin, and a lysine decarboxylase or could encode enzymes involved in methionine metabolism, leading toward L-cysteine production, including luxS. Screening of promoter upstream regions allowed for the assessment of a B. cereus consensus promoter binding site for σM. Since the consensus promoter binding site for B. cereus ATCC 14579 σM, its regulon and the predicted functionalities are different from the corresponding features in B. subtilis, it can be concluded that σM plays a unique role in B. cereus stress response and survival. The sigM deletion strain of B. cereus ATCC 14579 was cultured to an OD600 of ~0.6. Here the first RNA sample was taken, 0 min. After sampling 4% of ethanol was added (v/v), and samples were taken after 10, 30 and 60 minutes of exposure. Comparisons performed were 0 min - 10 min, 0 min - 30 min, and 0 min - 60 min.