ABSTRACT: In this study we focus on two Saccharomyces cerevisiae strains with varying production of heterologous α-amylase and we compare the metabolic fluxes and transcriptional regulation at aerobic and anaerobic conditions, in particular with the objective to identify the final electron acceptor for protein folding. We found that anaerobic conditions showed high amount of amylase productions when comparing to aerobic conditions and the genome-scale transcriptional analysis suggested that genes related to the endoplasmic reticulum (ER), lipid synthesis and stress responses were generally up-regulated at anaerobic conditions. Moreover, we proposed a model for the electron transfer from ER to the final electron acceptor, fumarate under anaerobic conditions. Three Saccharomyces cerevisiae strains with varied amylase productions were selected at early glucose phase in batch fermentations for RNA extraction and hybridization on Affymetrix microarrays. Biological triplicates were applied, and strains with empty plasmid (no amylase productions) were used as control strain.
Project description:In this study we focus on two Saccharomyces cerevisiae (CEN. PK series) strains producing either insulin precursor or amylase and we compare the transcriptional regulation at different dilution rates, in particular with the objective to identify the relationship between cell metabolism and recombinant protein production. We found that anaerobic conditions showed high amount of amylase productions when comparing to aerobic conditions and the genome-scale transcriptional analysis suggested that genes related to the endoplasmic reticulum (ER), lipid synthesis and stress responses were generally up-regulated at anaerobic conditions. Moreover, we proposed a model for the electron transfer from ER to the final electron acceptor, fumarate under anaerobic conditions. Two Saccharomyces cerevisiae strains producing either insulin precursor or amylase were selected at different dilution rates in chemostat cultivation for RNA extraction and hybridization on Affymetrix microarrays. Biological triplicates were applied.
Project description:High-resolution tiling analysis of the MR-1 transcriptome under diverse growth conditions The conditions include aerobic growth in Luria-Bertani broth (LB), aerobic growth in defined lactate minimal medium, anaerobic growth in defined lactate minimal medium with 20mM dimethyl sulfoxide as the electron acceptor, anaerobic growth in defined lactate minimal medium with 10mM iron (III) citrate as the electron acceptor, 10 minutes post heat shock at 42oC see GSE39468 for tiling data on lactate minimal media Four slides hybridized to mRNA and one “genomic control” array hybridized to genomic DNA
Project description:Transcriptomic study of A. ferrooxidans was explored either during aerobic growth with sulfur as an electron source and oxygen as final electron acceptor or in anaerobic conditions with ferric iron as the final electron receptor. Differential RNA levels were related to changes in cellular functions that were used to develop a preliminary model for A. ferrooxidans electron transport during dissimilatory ferric iron reduction.
Project description:Responses of Escherichia coli MG1655 as they grow anaerobically in M9 + glucose + fumarate (as electron acceptor) vs Aerobic growth OD 0.4 Keywords: time course Overall design: Escherichia coli cells sampled at several points (OD 0.15, 0.30, 0.63, 0.85, 1.07, 1.29) in anaerobic growth in M9 + glucose vs Aerobic growth OD 0.4
Project description:This study is aimed for the identification of novel small RNAs under different ethanol producing conditions. We have applied transcriptome analysis to facilitate identification and validation of 15 novel sRNAs in Zymomonas mobilis. We furthermore characterize their expression in the context of high and low levels of intracellular ethanol. Here, we report that 3 of the sRNAs (Zms2, Zms4 and Zms6) are differentially expressed under aerobic and anaerobic conditions, when low and high ethanol productions are observed respectively. These data suggests that in this organism regulatory RNAs can be associated with metabolic functions involved in ethanol stress responses. Z. mobilis was grown under aerobic and anaerobic conditions which showed low and high ethanol production, respectively. Each samples were sequenced for identification of small RNA candidates and differentially expressed candidates between two conditions.
Project description:Transcriptional profiling of Shewanella OS185 and OS195 strains grown under aerobic, anaerobic thiosulfate and nitrate respiratory conditions. Overall design: The Shewanella OS185 and OS195 strains were grown under aerobic, anaerobic thiosulfate and nitrate respiratory conditions. Three replicates were included. Therefore, there were a total of 18 samples analyzed. All sample cDNAs were labeled with Cy5. The same reference cDNA pool was labeled Cy3, and were used in all arrays.The reference cDNA pool was constructed using RNA from cells of all four strains OS155, OS185, OS195 and OS223 grown in HEPES medium containing either 10 mM sodium fumarate, 5 mM sodium nitrate or 10 mM sodium thiosulfate under anaerobic condition, or 10 mM sodium chloride under aerobic condition.
Project description:G. sulfurreducens (ATCC #51573) was obtained from the laboratory culture collection of Dr. Derek Lovley. Cells were grown under strict anaerobic conditions at 30 °C in chemostats, (see Esteve-Núñez, A., M. M. Rothermich, M. Sharma, and D. R. Lovley. 2004. Growth of Geobacter sulfurreducens under nutrient-limiting conditions in continuous culture. Environ. Microbiol.:in press. for more information), with acetate (5 mM) as the electron donor and Fe(III) citrate (55 mM) or fumarate (27.5 mM) as the electron acceptor. Under these conditions acetate is the substrate limiting growth. Cultures were maintained at a dilution rate of 0.05 h-1 for 5 culture vessel volumes to ensure that cells were at steady-state prior to harvesting. Cells were harvested by centrifugation at 4 °C and the cell pellet was flash-frozen in liquid nitrogen and then stored at 80 °C prior to RNA extraction. Three cultures of acetate limited growth with fumarate as the electron acceptor and three cultures of acetate limited growth with Fe(III) as the electron acceptor were grown. Each culture vessel was harvested and extracted for total RNA separately to produce three biological replicates for this experiment.
Project description:Comparisson of expression profiling of a etrA deletion mutant strain (experimental sample) with that of the wild type Shewanella oneidensis MR-1 strain to assess global direct/indirect genetic regulation EtrA in Shewanella oneidensis MR-1 shares 73.6% and 50.8% amino acid sequence identity with the oxygen-sensing regulator Fnr in E. coli and Anr in Pseudomonas aeruginosa, respectively; however, its regulatory role of anaerobic metabolism in Shewanella spp. is complex and not well understood. Whole-genome expression profiling using a etrA gene deletion mutant as the experimental sample and the wild type strain as the reference, determine that EtrA fine-tunes the expression of genes involved in various anaerobic metabolic pathways, including nitrate, fumarate and dimethyl sulfoxide reduction. Moreover, genes involved in prophage activation and and genes implicated in aerobic metabolism were also differentially expressed. In contrast to previous studies that attributed a minor regulatory role to EtrA in Shewanella spp., this study demonstrates that EtrA acts as a global transcriptional regulator and cofers physiological advantages to the strain under certain growth conditions. Overall design: Total RNA was extracted from three biological replicate cultures of both S. oneidensis etrA deletion mutant and the wild type strain, grown individually on anaerobic minimal medium supplemented with 2 mM nitrate as the electron acceptor and lactate as the electron donor. Total RNA was collected from mid-log phase cultures. cDNA from each sample was prepared and labeled with Cy-dyes. Three biological replicates per treatment were used for the hybridization of six microarray slides including technical replicates (dye-swap) per experiment.
Project description:The anaerobic metabolism of the opportunistic pathogen Pseudomonas aeruginosa is important for growth and survival during persistent infections. The two Fnr-type transcription factors Anr and Dnr regulate different parts of the underlying network. Both are proposed to bind to a non-distinguishable DNA sequence named Anr box. The aim of this study was the identification of genes induced under anaerobic conditions in the P. aeruginosa wild type and identification of genes under control of the Anr or Dnr regulators. Overall design: We performed three comparisons to identify genes induced under anaerobic denitrifying conditions in the P. aeruginosa wild type strain and genes which are under control of the Anr or Dnr regulators under these anaerobic conditions. Since the anr and dnr mutant strains do not grow under anaerobic denitrifying conditions, we applied anaerobic shift experiments. Pseudomonas aeruginosa was grown in a modified AB minimal medium, containing 25 µM FeSO4, 20 mM glucose and 50 mM NaNO3. The 200 ml aerobic cultures were grown in 1 l Erlenmeyer flasks at 37 oC and 300 rpm. The aerobic culture was grown to an OD578 of 0.3. For the aerobic culture, cells were harvested at this point. For the anaerobic shift experiments 130 ml of the respective aerobic culture were transferred to a 135 ml sealed serum flask. Control experiments verified that oxygen tension decreased within 3 - 5 min below the detection limit of an oxygen electrode. The cells were harvested after incubation for additional 2h under anaerobic conditions. Within these 2h incubation period no growth of the wild type, the anr mutant or the dnr mutant strain was observed. First comparison: Identification of genes induced or repressed under anaerobic conditions in the P. aeruginosa wild type PAO1. Here we compared the transcriptome profile of P. aeruginosa PAO1 grown under aerobic conditions up to an OD578 of 0.3 with the transcriptome profile of the PAO1 strain, which was first grown under aerobic conditions up to an OD578 of 0.3 and than shifted to anaerobic conditions by transfer to a sealed serum flask and further incubated for two hours under anaerobic conditions. Second comparison: Identification of genes regulated differently in the anr mutant strain PAO6261. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa anr mutant strain PAO6261. Both strains were harvested after 2h incubation under anaerobic conditions. Third comparison: Identification of genes regulated differently in the dnr mutant strain RM536. Here we compared the transcriptome profile of the P. aeruginosa wild type PAO1 with the transcriptome profile of the P. aeruginosa dnr mutant strain RM536. Both strains were harvested after 2h incubation under anaerobic conditions.
Project description:Listeria monocytogenes is a gram-positive, facultative anaerobe food-borne pathogen that is the causative agent of listeriosis. Upon ingestion, L. monocytogenes is subjected to a variety of non-specific host defenses such as bile. The main component of bile is bile salts which is known to be bactericidal through inducing DNA damage, oxidative damage, membrane instability, and protein misfolding. Previous studies have focused mainly on aerobic conditions; however the human GI tract is an environment ranging from microaerophilic to anaerobic. The bile salt hydrolase, an enzyme known to reduce toxicity of bile through hydrolysis, has been shown to have an increase in activity under anaerobic conditions. Therefore, the purpose of this study was to determine the effect of oxygen on bile resistance and determine the bile specific proteome that is responsible for this. To do this, we performed survival assays on virulent strains: F2365, 10403S, EGDe and avirulent strain, HCC23. Results showed an increase in viability for all virulent strains when exposed to porcine bile under anaerobic conditions. Interestingly, an increase in resistance was seen in HCC23 only under aerobic conditions. We then used a total proteomic analysis approach to compare the bile specific proteome under both aerobic and anaerobic conditions. Results showed many difference between all strains including an increase in abundance of proteins associated with stress responses, repair, cell morphology, and cell division under anaerobic conditions. Response to bile salt stress showed to be strain dependent. This study not only identified proteins responsible for L. monocytogenes bile resistance but also differerences between aerobic and anaerobic conditions thus suggesting that oxygen availability is not only a stressor but in some way providing assistance to overcoming bile salts.