Unknown,Transcriptomics,Genomics,Proteomics

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Functional Genomic Study of Exogenous n-Butanol Stress in Escherichia coli

ABSTRACT: n-Butanol has been proposed as an alternative biofuel to ethanol, and both Escherichia coli and Saccharomyces cerevisiae have been engineered to produce it. Unfortunately, n-butanol is more toxic than ethanol to these organisms. To understand the basis for its toxicity, cell wide studies were conducted at the transcript, protein and metabolite levels to obtain a global view of the n-butanol stress response. Analysis of the data indicate that n-butanol stress has components common to other stress responses and includes perturbation in respiratory functions (nuo, cyo operons), oxidative stress (sodC, katG, yqhD), heat shock and cell envelope stress (rpoE, clpB, htpG, degP, cpxPR), metabolite transport (malE, opp operon) and biosynthesis. Inducible expression of the yqhD gene was found to improve the hostâs tolerance to exogenous n-butanol and confirms the role of this gene in coping with butanol stress. To survey for other potential candidates that may serve to improve host tolerance, mutant strains in several candidates which show changes at the transcript and protein levels were examined for sensitivity during butanol exposure. Chassis engineering based on these cues may be required in a high production titer, butanol-producing host. This comparison is between E. coli DH1 cells treated with 0.8% n-butanol and untreated cells at 0, 30, 80, and 195 minutes after addition. 3 biological replicates were grown, total RNA was extracted, labeled, and hybridized on 3 slides for each time point. http://www.microbesonline.org/cgi-bin/microarray/viewExp.cgi?expId=1266

ORGANISM(S): Escherichia coli

SUBMITTER: Jay Keasling

PROVIDER: E-GEOD-16973 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Desulfovibrio vulgaris has been studied extensively for its potential in the bioremediation of heavy metals and radionuclides. Hydrocarbons and solvents, as frequent environmental co-contaminants, have been reported to inhibit microbial activities and thereby pose a limitation on bioremediation efficiency. As a part of the Genomes: GTL project to deduce the stress response pathways in metal/radionuclide reducing bacteria, we studied the responses of D. vulgaris to ethanol, which is a solvent and co-contaminant frequently encountered at contaminated DOE sites. Growth experiments in closed vessels at 37 Â°C indicated that D. vulgaris could maintain normal growth with 1%(v/v) ethanol. The growth rates were reduced with increasing ethanol concentrations at 2% and 5%. Growth ceased when ethanol concentration was raised to 5%(v/v). Cell lysis was apparent with decreasing optical density following 10% ethanol addition. To assess the mechanism of ethanol inhibition, genome-wide transcriptional profiles were analyzed from D. vulgaris cultures following ethanol (5% v/v) treatment using whole-genome microarrays. The ethanol treatment altered the expression of a large number of genes in the D. vulgaris genome, of which 354 were up-regulated greater than 2 fold and 217 were down-regulated by over 2 fold. As expected, changes in the transcriptional profile were similar to those of the stress response to acetone, which is also a solvent. Transcripts highly up-regulated included genes encoding the flagella structural subunits, suggesting motility as a mechanism of solvent stress response. Another group of genes highly induced were chaperones, such as dnaJ, groES, and hsp20, indicating the importance of maintaining proper protein folding under ethanol stress. Down-regulated genes included two groups of genes, ribosomal proteins and amino acid transporters, consistent with the growth inhibition by ethanol observed in growth studies. These results were interpreted that D. vulgaris responds to elevated solvent levels by increased motility and maintenance of proper protein functions. Current work is focused on the analysis of regulatory pathways based on temporal transcriptional dynamics. Keywords: Stress response Desulfovibrio vulgaris from our laboratory culture collection was cultured at 37Â°C in Lactate-Sulfate medium to mid-log phase. Subsequently, ethanol (5% v/v) was added into triplicate cultures. Gene expression profiles 30 and 100 minutes following ethanol exposure were compared with those of the control cultures without ethanol treatment. Total RNA was harvested from three replicate control and treated cultures at two time points following ethanol addition. RNA extraction, purification, and labeling were performed independently on each cell sample.Two samples of each total RNA preparation were labeled, one with Cy3-dUTP and another with Cy5-dUTP for microarray hybridization (Dye swap).

Project description:Desulfovibrio vulgaris has been studied extensively for its potential in the bioremediation of heavy metals and radionuclides. Hydrocarbons and solvents, as frequent environmental co-contaminants, have been reported to inhibit microbial activities and thereby pose a limitation on bioremediation efficiency. As a part of the Genomes: GTL project to deduce the stress response pathways in metal/radionuclide reducing bacteria, we studied the responses of D. vulgaris to acetone, which is a ketone solvent frequently observed at contaminated DOE sites. Growth experiments in closed vessels at 37 Â°C indicated that D. vulgaris could maintain normal growth with 3%(v/v) acetone following a 1-h lag phase. When the acetone concentration was raised to 5%(v/v), we observed a 2-h lag phase followed by a growth rate which was only 15% that of normal. At an acetone concentration of 8%(v/v), no active growth was observed following 10 hours of incubation. To assess the mechanism of acetone inhibition, genome-wide transcriptional profiles were analyzed from D. vulgaris cultures following acetone (5% v/v) treatment using whole-genome microarrays. This acetone shock altered the expression of a large number of genes in the D. vulgaris genome, of which 309 were up-regulated greater than 2 fold and 199 were down-regulated by over 2 fold. Transcripts highly up-regulated included genes encoding the flagella structural subunits, flgB (15 fold), fliE (11 fold), and flgH (10 fold). Another group of genes highly induced were chaperones, such as dnaJ (11 fold), groES (8 fold), and hsp20 (8 fold). Down-regulated genes included two groups of genes, ribosomal proteins and amino acid transporters, suggesting a state of growth arrest upon acetone addition. These results were interpreted to mean that D. vulgaris responds to elevated solvent levels by increased motility and maintenance of proper protein functions. Current work is focused on the analysis of regulatory pathways based on temporal transcriptional dynamics. Keywords: Stress response Desulfovibrio vulgaris from our laboratory culture collection was cultured at 37Â°C in Lactate-Sulfate medium to mid-log phase. Subsequently, acetone (5% v/v) was added into triplicate cultures. Gene expression profiles 30 and 100 minutes following acetone exposure were compared with those of the control cultures without acetone treatment. Total RNA was harvested from three replicate control and treated cultures at two time points following acetone addition. RNA extraction, purification, and labeling were performed independently on each cell sample.Two samples of each total RNA preparation were labeled, one with Cy3-dUTP and another with Cy5-dUTP for microarray hybridization (Dye swap).

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Functional Genomic Study of Exogenous n-Butanol Stress in Escherichia coli

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