Unknown,Transcriptomics,Genomics,Proteomics

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Monitoring the ethanol stress response of a sigM deletion strain of B. cereus ATCC 14579.

ABSTRACT: 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.

ORGANISM(S): Bacillus cereus ATCC 14579

SUBMITTER: Menno Voort

PROVIDER: E-GEOD-9856 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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: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.

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Monitoring the ethanol stress response of a sigM deletion strain of B. cereus ATCC 14579.

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