Unknown,Transcriptomics,Genomics,Proteomics

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Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strain ATCC 14579

ABSTRACT: 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.

ORGANISM(S): Bacillus cereus

SUBMITTER: Richard van Kranenburg

PROVIDER: E-GEOD-13711 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Analysis of acid-stressed Bacillus cereus reveals a major oxidative response and inactivation-associated radical formation.

Mols Maarten M van Kranenburg Richard R van Melis Clint C J CC Moezelaar Roy R Abee Tjakko T

Environmental microbiology 20100113 4

Acid stress resistance of the food-borne human pathogen Bacillus cereus may contribute to its survival in acidic environments, such as encountered in soil, food and the human gastrointestinal tract. The acid stress responses of B. cereus strains ATCC 14579 and ATCC 10987 were analysed in aerobically grown cultures acidified to pH values ranging from pH 5.4 to pH 4.4 with HCl. Comparative phenotype and transcriptome analyses revealed three acid stress-induced responses in this pH range: growth ra ...[more]

PMID: 20074238

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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: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

Dataset Information

Comparative transcriptome and phenotype analysis of acid-stressed Bacillus cereus strain ATCC 14579

Publications

Analysis of acid-stressed Bacillus cereus reveals a major oxidative response and inactivation-associated radical formation.

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