Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Benzoate induced transcriptomic response in Bacillus subtilis

ABSTRACT: Bacillus subtilis strain AG174 was grown in the presence and absence of benzoate (30 mM). Benzoate was used in order to equalize the external and internal pH. The cultures were grown at external pH 7.0, and the addition of benzoate did not change the external pH. Microarray analysis was performed on cDNA synthesized from bacterial RNA. Real-time PCR of highly up-regulated genes confirmed the results of the microarray. These data were compared to previous B.subtilis microarray results, where genes regulated by external pH were identified. Overnight cultures were diluted 1:500 in potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM MOPS at pH 7.0. Bacteria were cultured in baffled flasks (less than 10% volume) with rotation at 220 rpm, incubated at 37 Â°C to an optical density at 600 nm of 0.2. Four cultures were grown in the presence of 30 mM benzoate and four cultures were grown without benzoate. One sample was taken from each of four replicate cultures of 0 mM benzoate and 30 mM benzoate. RNA was isolated using 10% saturated phenol-ethanol solution and the RNeasy Kit. Gene expression profiles were obtained with standard Affymetrix procedures.

ORGANISM(S): Bacillus subtilis

SUBMITTER: Melanie Berkmen

PROVIDER: E-GEOD-12263 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Cytoplasmic acidification and the benzoate transcriptome in Bacillus subtilis.

Kitko Ryan D RD Cleeton Rebecca L RL Armentrout Erin I EI Lee Grace E GE Noguchi Ken K Berkmen Melanie B MB Jones Brian D BD Slonczewski Joan L JL

PloS one 20091214 12

<h4>Background</h4>Bacillus subtilis encounters a wide range of environmental pH. The bacteria maintain cytoplasmic pH within a narrow range. Response to acid stress is a poorly understood function of external pH and of permeant acids that conduct protons into the cytoplasm.<h4>Methods and principal findings</h4>Cytoplasmic acidification and the benzoate transcriptome were observed in Bacillus subtilis. Cytoplasmic pH was measured with 4-s time resolution using GFPmut3b fluorimetry. Rapid extern ...[more]

PMID: 20011599

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Project description:Insight into the mechanisms for the anaerobic metabolism of aromatic compounds by the hyperthermophilic archaeon Ferroglobus placidus is expected to improve understanding of the degradation of aromatics in hot (> 80 °C) environments and to identify enzymes that might have biotechnological applications. Analysis of the F. placidus genome revealed genes predicted to encode enzymes homologous to those previously identified as playing a role in benzoate and phenol metabolism in mesophilic bacteria. Surprisingly, F. placidus lacks genes for an ATP-independent class II benzoyl-CoA reductase found in all strictly anaerobic bacteria, but instead has two sets of genes for ATP-consuming class I benzoyl-CoA reductases, similar to those found in facultative bacteria. The lower portion of the benzoate degradation pathway appears to be more similar to that found in the phototroph Rhodopseudomonas palustris, than the pathway reported for all heterotrophic anaerobic benzoate degraders. Many of the genes predicted to be involved in benzoate metabolism were found in one of two gene clusters. Genes for a phenol carboxylation proceeding through a phenylphosphate intermediate and for conversion of p-hydroxybenzoate to benzoyl-CoA were identified in a single gene cluster. Analysis of transcript abundance with a whole-genome microarray and quantitative PCR demonstrated that most of the genes predicted to be involved in benzoate or phenol metabolism had higher transcript abundance during growth on those substrates versus growth on acetate. These results suggest that the general strategies for benzoate and phenol metabolism may be highly conserved between microorganisms living in moderate and hot environments, and that anaerobic metabolism of aromatic compounds might be analyzed in a wide range of environments with similar molecular targets. A four chip study using total RNA recovered from two separate cultures of Ferroglobus placidus DSM 10642 grown with 1 mM sodium benzoate (experimental condition) and two separate cultures of Ferroglobus placidus DSM 10642 grown on 10 mM acetate (control condition). Each chip measures the expression level of 2613 genes from Ferroglobus placidus DSM 10642 with nine 45-60-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.

Project description:Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown with aeration to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with a significance level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins. Experiment Overall Design: Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Overnight cultures were diluted 1:1000 in potassium-modified Luria-Bertani medium (LBK) buffered with 50 mM HOMOPIPES at pH 5.0, pH 7.0, and pH 8.7. Bacteria were cultured in baffled flasks (less than 10% volume filled) with rotation at 240 rpm, incubated at 37Â°C to an optical density at 600 nm of 0.3. For each of the three pH conditions, RNA was isolated from five independent cultures. Labeled cDNA was hybridized to Affymetrix antisense arrays according to standard procedures. To analyze the expression levels, Dchip software was used to generate model-based expression indices normalized to sample pH 7 replicate 1. ANOVA was used to identify genes with sitnificant expression differences among the three pH classes (p = 0.001). For genes showing significant differences, the Log2 expression ratios were determined for each pair of pH classes, and significance was determined by Tukey's test (p = 0.001).

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Dataset Information

Benzoate induced transcriptomic response in Bacillus subtilis

Publications

Cytoplasmic acidification and the benzoate transcriptome in Bacillus subtilis.

Similar Datasets

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