Gene induction by ethanol stress in B. cereus ATCC 14579
ABSTRACT: The stress response of B. cereus ATCC 14579 is monitored true time, showing an enormous response in gene expression. The wild-type 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.
Project description:The stress response of as sigZ deletion strain of B. cereus ATCC 14579 is monitored true time by use of microarrays. The sigZ regulon in ethanol stress response was determined and compared with the regulon determined by micorarray analysis of overexpression of sigZ. The sigZ 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.
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.
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. Both an empty vector strain and an sigZ overexpressing strain were grown to an OD600 of ~0.5, after which 0.2 ng/ml nisin was added to both cultures. After 90 minutes from both cultures a sample was taken for RNA isolation. Comparisons performed were in duplicate with dye swap, the empty vector strain after 90 min nisin with the overexpressing strain after 90 min nisin.
Project description:This SuperSeries is composed of the following subset Series: GSE13711: Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strain ATCC 14579 GSE13729: Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strain ATCC 10987 Refer to individual Series
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 food-borne human pathogen Bacillus cereus is found in environments that often have a low pH, such as food and soil. The physiological response upon exposure to several levels of acidity were investigated of B. cereus model strain ATCC 14579, to elucidate the response of B. cereus to acid stress. pH 5.4, pH 5.0, pH 4.8 and pH 4.5 were selected to conduct microarray analyses, based on the differences in physiological response upon exposure to the acid conditions. The transcriptome data revealed response specific profiles. Showing mechanisms induced upon all the different acid down-shocks, such as nitrate reductase and energy production genes, and several genes specifically expressed differentially in mild or lethal levels of acidity, such as F1F0-ATPase and cydAB. Furthermore, mechanisms involved in oxidative stress response were found highly up-regulated in response to both mild and lethal acid stress. The induction of oxidative stress related genes may be a response to the formation of reactive oxygen species by a perturbation of the electron transport chain. Therefore, the formation of hydroxyl radicals and/ or peroxynitrite was monitored upon exposure to the different levels of acidity with a fluorescent probe in a flow cytometer. The formation of these oxidative compounds was shown to be specific for lethal pHs and a model to relate radical formation with the observed transcriptome profiles was proposed. Per acid down-shock three exposure times (i.e., 10, 30 and 60 min) were each compared with non-exposed cells (i.e., t0). In total 4 different acid down-shocks were applied, pH 5.4, pH 5.0, pH 4.8 and pH 4.5. The experiments were performed in duplicate and the duplicate samples were hybridised with 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. 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:Bacteria are able to cope with the challenges of sudden increase of salinity by activating adaptation mechanisms. In this study, exponentially growing cells of the food-borne pathogen Bacillus cereus ATCC 14579 were exposed to both mild (2.5% NaCl w/v) and severe (5% NaCl w/v) salt stress conditions. B. cereus continued growth at a reduced rate when shifted to mild salt stress. Exposure to severe salt stress resulted in a lag period, and after 60 min cellular growth was resumed filamentously. Whole-genome expression analyses of cells exposed to 2.5% salt stress revealed an overlap with that of cells exposed to 5% salt stress, suggesting that the corresponding genes (n = 147) were involved in a general salt stress response. Up-regulation of osmolyte, Na+/H+ and di-/tripeptide transporters and activation of an oxidative stress response were important aspects of this general salt stress response. Activation of this response may confer cross-protection towards other stresses, and increased resistance to heat and H2O2 was indeed observed. Notably, a temporal shift was observed between the observed transcriptome and phenotype responses of severely salt-stressed cells including cellular filamentation, reduced chemotaxis performance, catalase activity and optimal oxidative stress resistance. The linkage of transcriptomes and phenotypic characteristics can contribute to a better understanding of cellular stress adaptation strategies and possible cross protection mechanisms. ATCC 14579 was grown to an OD600 of ~0.5, after that 2.5% or 5% NaCl (w/v) was added. Before addition of NaCl and after 10, 30 and 60 minutes of NaCl-exposure a sample was taken for RNA isolation. Comparisons (untreated versus salt-treated) performed were in duplicate with dye swap.
Project description:Comparative phenotype and transcriptome analyses were performed with Bacillus cereus ATCC 14579 exposed to acid down-shock to pH 5.5 set with different acidulants. When acidified with hydrochloric acid (HCl), growth was diminished, whereas 2 mM undissociated lactic acid (HL) or acetic acid (HAc) stopped growth without inactivation (bacteriostatic condition), and 15 mM undissociated HAc caused growth arrest and, finally, cell death, as reflected by a 3 to 4 log inactivation (bactericidal condition). Within the first 60 min after pH down-shock, the intracellular ATP levels of cultures shocked with HCl were increased. The bacteriostatic pH shocks did not result in increased nor decreased intracellular ATP levels, indicating that the high energy status within the stressed aerobically grown B. cereus cells could be maintained. In contrast, exposure to 15 mM undissociated HAc resulted in significant lower ATP levels, which was in accordance with the observed inactivation. The transcriptomic responses pH down-shocked cultures were studied in the same time frame. The analyses revealed general and specific responses coupled to the different phenotypes and the acidulant used. The general acid stress response, shown in all different pH shocks, involves modulation of pyruvate metabolism and an oxidative stress response. The shifts in pyruvate metabolism include induction dehydrogenases of a butanediol fermentation pathway under non-lethal acid stress conditions and of lactate, formate, and ethanol fermentation pathways under 15 mM HAc stress. Other 15 mM HAc-specific responses were induction of the alternative electron-transport systems, including cydAB, and fatty acid biosynthesis genes. Differences in gene expression for the bacteriostatic organic acid stress conditions compared to the growth-retarded inorganic stress condition indicated a more stringent oxidative stress response, including induction of an additional catalase gene and a gene encoding a Dps-like protein. Moreover, modulations in amino acid and oligopeptide transport were also found for the 2 mM HAc and HL shocks. HL-specific and HAc-specific responses both involve amino acid metabolism. Our study on the genome-wide responses of aerobically grown B. cereus pH 5.5 shocks provides a unique overview of the different responses induced by three acidulants relevant for food preservation. Per acid down-shock three exposure times (i.e., 10, 30 and 60 min) were each compared with non-exposed cells (i.e., t0). In total 4 different pH 5.5 acid down-shocks were applied. pH 5.5 was reached by adding different acidulants i.e., hydrochloric acid (HCl), lactic acid (HL) resulting in 2 mM undissociated HL, acetic acid (HAc) resulting in 15 mM undissociated HAc, and a combination of acetic acid and hydrochloric acid (HAc/HCl) resulting in 2 mM undissociated HAc. The experiments were performed in duplicate and the duplicate samples were hybridised with a dye-swap.
Project description:Sorbic acid (SA) is widely used as a preservative, but the effect of SA on spore germination and outgrowth has gained limited attention up to now. Therefore, the effect of sorbic acid on germination of spores of B. cereus strain ATCC 14579 was analyzed both at phenotype and transcriptome level. Spore germination and outgrowth was assessed at pH 5.5 without and with 0.75, 1.5 and 3.0mM (final concentrations) undissociated sorbic acid (HSA). This resulted in distinct HSA concentration-dependent phenotypes, varying from delays in germination and outgrowth to complete blockage of germination at 3.0mM HSA. The phenotypes reflecting different stages in the germination process could be confirmed using flow cytometry and could be recognized at transcriptome level by distinct expression profiles. In the absence and presence of 0.75 and 1.5mM HSA, similar cellular ATP levels were found up to the initial stage of outgrowth, suggesting that HSA-induced inhibition of outgrowth is not caused by depletion of ATP. Transcriptome analysis revealed the presence of a limited number of transcripts in dormant spores, outgrowth related expression, and genes specifically associated with sorbic acid stress, including alterations in cell envelope and multi-drug resistance. The potential role of these HSA-stress associated genes in spore outgrowth is discussed. Per concentration of undissociated sorbic acid (0, 0.75, and 1.5mM) four exposure times (10, 30, 60, and 120 minutes) were each compared with dormant spores (i.e., t0). The experiments were performed in duplicate and the duplicate samples were hybridized with a dye-swap