High succinic acid tolerance E.coli (DST160)
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ABSTRACT:
ORGANISM(S): Escherichia coli str. K-12 substr. W3110 Escherichia coli
PROVIDER: GSE17991 | GEO | 2009/10/30
SECONDARY ACCESSION(S): PRJNA120029
REPOSITORIES: GEO
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Project description:To understand cell responses against high succinate stress, a continuous culture was performed for 9 months using a wild Escherichia coli under gradually increasing succinate stress. The final adapted strain against high succinate stress, named DST160, was able to grow with no lag phase in medium containing 1.35 M of succinate stress while wild W3110 showed more than 38 h lag phase. The maximum growth rate and growth yield of DST160 under the stress condition were 0.20 h-1 and 0.192 g-cell/g-glucose, which were 53.8 % and 12.3 % higher than those of W3110 (0.13 h-1 and 0.171 g-cell/g-glucose, respectively). A single colony of E. coli W3110 was inoculated into a 15-mL tube containing 4 mL of LB medium. After 12 h cultivation in a shaking incubator at 37℃ and 220 rpm, pre-culture (1 mL) was transferred to a 250 mL-fermentor (Mini-chemostat fermentor; Biotron Inc., Bucheon, Korea) containing 100 mL medium consisted of M9-glucose minimal medium (3 g glucose , 0.8 g NH4Cl, 0.5 g NaCl, 7.5 g Na2HPO4•2H2O, 3 g KH2PO4 with separately added trace elements of 0.2 g MgSO4•7H2O, 0.1 g CaCl2, 1 mg thiamine) per liter supplemented with 254 mmole of succinate (30 g succinic acid) per liter. The initial pH of the medium was set to be pH 7.5 by adding 6N NaOH. The fermentor was operated at 37℃, 350 rpm with air flushing (100 mL/min.). After 12 h batch mode growth, feeding and outlet pumps were turned on at flow rate of 0.167 mL/min (Dilution rate=0.1 h-1). The fresh medium reservoir (10 L) contained the same contents and pH with the initial medium. Every 10 turnover time (100 h), the reservoir was changed with the same medium composition except succinate concentration. The succinate concentration of the reservoir was gradually increased by 25.4 ~ 84.7 mM (3 ~ 10 g/L of succinic acid) at a time than previous states depending on the cell concentrations at the outlet. When the cell concentration was reduced below O.D.=0.2, succinate feeding pump was temporarily stopped to go back to the slightly lower succinate concentration. The continuous culture was performed until the steady state when succinate in the reservoir reached 1.35 M (160 g/L in succinic acid form). The final adapted cells in 100 mL culture were harvested by centrifuge and frozen rapidly in a liquid nitrogen tank for further analyses including DNA microarray. To understand the change of up/down regulations in the high-succinate adapted E. coli, total mRNAs of control cells and the adapted E. coli cells were extracted using a RNA extraction kit (RNeasy mini kit; Quiagen, Hilden, Germany), and the transcriptomes were compared using DNA microarray. Hybridization and washing were accomplished by a facility of Digital Genomics Co (Seoul, Korea). DNA microarray were scanned using an Axon GenePix 4000B Scanner (Axon Instruments, Union City, CA, USA), and the scanned images were analyzed with GenePix Pro 3.0 software to obtain gene expression ratios.
2009-10-29 | E-GEOD-17991 | biostudies-arrayexpress
Project description:Pseudomonads, unlike enteric bacteria, prefer to utilize tricarboxylic acid cycle intermediates over glucose. The crc gene has been implicated to impart repression on glucose and other metabolic enzymes in various Pseudomonas species when grown in a mixture of glucose and various organic acids. The crc gene of P. fluorescens strain MB101 has been identified and a crc deletion derivative was constructed. Unlike a crc mutant of P. aeruginosa described earlier, the P. fluorescens deletion mutant crc did not begin to catabolize glucose until most of the succinate has been utilized in a medium containing both carbon sources. The transition phase during diauxic growth in succinate/glucose media was found to be shorter in a strain lacking crc and considerably longer in a strain overproducing Crc as compared to the wild type. DNA microarray analyses were performed with wild-type and crc mutant strains during the transition phase from succinate to glucose. In the wild-type strain, high expression signals were obtained for genes normally induced in cells that approach or are in stationary phase. By contrast, deletion mutant crc cells showed higher expression signals for genes associated with rapid growth than the wild-type strain. Taken together, this data indicates that Crc influences the length of the lag during the transition phase from succinate to glucose by directly or indirectly affecting the mRNA levels of target genes. Keywords: time course strain comparison
2008-09-11 | GSE5829 | GEO
Project description:Pseudomonads, unlike enteric bacteria, prefer to utilize tricarboxylic acid cycle intermediates over glucose. The crc gene has been implicated to impart repression on glucose and other metabolic enzymes in various Pseudomonas species when grown in a mixture of glucose and various organic acids. The crc gene of P. fluorescens strain MB101 has been identified and a crc deletion derivative was constructed. Unlike a crc mutant of P. aeruginosa described earlier, the P. fluorescens ïcrc did not begin to catabolize glucose until most of the succinate has been utilized in a medium containing both carbon sources. The transition phase during diauxic growth in succinate/glucose media was found to be shorter in a strain lacking crc and considerably longer in a strain overproducing Crc as compared to the wild type. DNA microarray analyses were performed with wild-type and crc mutant strains during the transition phase from succinate to glucose. In the wild-type strain, high expression signals were obtained for genes normally induced in cells that approach or are in stationary phase. By contrast, ïcrc cells showed higher expression signals for genes associated with rapid growth than the wild-type strain. Taken together, this data indicates that Crc influences the length of the lag during the transition phase from succinate to glucose by directly or indirectly affecting the mRNA levels of target genes. Keywords: time course strain comparison These are time course experiments to compare crc mutant strain to the wildtype MB101 strain at 5, 6, 7, 8, and 9 ht time points. Dye swaps were included in each time point comparison. There are 10 pairs in total.
2010-06-19 | E-GEOD-5829 | biostudies-arrayexpress
Project description:Purpose: To understand how D654Y mutated RpoB affect gene expression under either normal or osmotic stress conditions in Escherichia coli . Methods: RNA-seq libraries were constructed for four samples, including (I) The parental strain Suc-T110 grown at normal condition (5%w/v glucose); (II)RpoB mutant RpoBD645Y [Suc-T110::rpoB (D654Y), RNA-seq library named NZ-502] grown at normal condition;(III) Suc-T110 grown at osmotic stress condition(12%w/v glucose);(IV) NZ-502 grown at osmotic stress condition. For preparation of RNA samples, Fresh colonies were picked from New NBS mineral salts plates containing 20 g liter-1 glucose, inoculated into 250 ml flasks containing 30 ml NBS mineral salts medium with 20 g liter-1 glucose, and grown at 37°C and 250 rpm for 12 h. Cultures were then transferred to a 500 ml fermentation vessel containing 250 ml NBS mineral salts medium with either 5%w/v or 12%w/v glucose. After 48 h, 1 ml culture was harvested by centrifugation, frozen in liquid nitrogen and sent to Beijing Genomics Institute (BGI, Shenzhen, China). The four 90-nt paired-end RNA-seq libraries were generated commercially at Beijing Genomics Institute by using the HiSeq™ 2000 platform. Sequencing data was handled essentially with Bowtie2, NOIseq. Expression levels are presented as Reads Per Kilobase of transcript per Million mapped reads (RPKM). Results: we first comparative accessed expression levels of Suc-T110 and NZ-502 under normal condition. Among the 4200 predicted genes, 128 genes were found altered their expression in NZ502 compared with Suc-T110; with 38 targets specific up-regulated while 90 targets down-regulated. Genes engaged in biological function of amino acid metabolism (P-value=4.60E-05) were significantly enriched among in induced gene set. Under osmotic stress condition, 244 differential expressed genes were identified in NZ-502 when compared with Suc-T110, with 132 targets specific up-regulated while 112 targets down-regulated, Functional enrichment of 132 up regulated genes showed the most significantly enriched physiological function of NZ-502 upon hyperosmotic stress was associated with carbon source transportation (FunCat term: 20.01.03 C-compound and carbohydrate transport, P-value=9.51E-05), which containing a group of genes encoding diverse sugar transporters. Conclusions: Transcriptome profiling suggested that derepression of sugar transporters might be the primary cause for NZ-502 to resist osmotic stress.
2017-02-04 | GSE84769 | GEO
Project description:Escherichia coli exhibits diauxic growth in sugar mixtures due to CRP-mediated catabolite repression and inducer exclusion related to phosphotransferase system enzyme activity. Replacement of the native crp gene with a catabolite repression mutant (referred to as crp*) alleviates diauxic effects in E. coli and enables co-utilization of glucose and other sugars. While previous studies have examined the effects of expressing CRP* mutants on the expression of specific catabolic genes, little is known about the global transcriptional effects of CRP* expression. In this study, we compare the transcriptome of E. coli W3110 (expressing wild-type CRP) to that of mutant strain PC05 (expressing CRP*) in the presence and absence of glucose. Experiment Overall Design: Four different conditions were tested in this study: W3110 in LB medium (WT), W3110 in LB+glucose medium (WT G), PC05 in LB medium (CRP*), and PC05 in LB+glucose medium (CRP* G).
2009-08-21 | E-GEOD-17584 | biostudies-arrayexpress
Project description:Clostridium acetobutylicum was grown in a batch-culture with minimal medium containing glucose and xylose as substrate. Diauxie growth was observed after glucose was consumed. Following the organism grows on xylose. Transcriptional analysis was done to pursue the cellular processes during the switch from growth on glucose to growth on xylose. We compared DNA-Microarray data from cells grown during the exponential phase on glucose (A), with cells growing during the start of diauxie growth lag (B), during the end of diauxie growth lag (C) and during exponential growth on xylose (D). We used cells grown in a continuous culture with glucose as substrate as common reference for the samples A-D.
2010-09-30 | E-MEXP-2613 | biostudies-arrayexpress
Project description:Gene expression in E coli W3110 strains with either ybaO over-expression (W3110/pcutR) or ybaO deletion (W3110/ΔcutR) were measured with cysteine challenge.
2017-06-13 | GSE90066 | GEO
Project description:In metabolically versatile bacteria, carbon catabolite repression (CCR) facilitates the preferential assimilation of the most efficient carbon sources, improving growth rate and fitness. In Pseudomonas putida, the Crc protein and the CrcZ and CrcY small RNAs (sRNAs), which are believed to antagonise Crc, are key players in CCR. Contrary to what occurs in other bacterial species, succinate or glucose elicit a weak CCR in this bacterium. We combined metabolic, transcriptomics and constraints-based metabolic flux analyses to clarify whether P. putida prefers succinate or glucose, and the role of the Crc/CrcZ-CrcY regulatory system in their metabolization. Succinate was consumed faster than glucose and allowed a higher growth rate. However, both compounds were co-metabolised when provided simultaneously. The levels of CrcZ and CrcY were lower when both substrates were present than when only one of them was provided, suggesting a role for Crc in coordinating metabolism. Metabolic reconstructions suggested that, when both substrates are present, Crc works to organize a metabolism in which carbon compounds flow in two opposite directions: from glucose to pyruvate, and from succinate to pyruvate. Therefore, Crc serves not only to favour the assimilation of preferred compounds, but also to balance the carbon fluxes, optimising metabolism and growth.
2015-03-06 | GSE63987 | GEO
Project description:Obesity induces macrophages to drive inflammation in adipose tissue, a crucial step towards the development of type 2 diabetes. The tricarboxylic acid (TCA) cycle intermediate succinate is released from cells under metabolic stress and has recently emerged as a metabolic signal induced by proinflammatory stimuli. We therefore investigated whether succinate receptor 1 (SUCNR1) could play a role in the development of adipose tissue inflammation and type 2 diabetes. Succinate levels were determined in human plasma samples from individuals with type 2 diabetes and non-diabetic participants. Succinate release from adipose tissue explants was studied. Sucnr1 -/- and wild-type (WT) littermate mice were fed a high-fat diet (HFD) or low-fat diet (LFD) for 16 weeks. Serum metabolic variables, adipose tissue inflammation, macrophage migration and glucose tolerance were determined. We show that hypoxia and hyperglycaemia independently drive the release of succinate from mouse adipose tissue (17-fold and up to 18-fold, respectively) and that plasma levels of succinate were higher in participants with type 2 diabetes compared with non-diabetic individuals (+53%; p < 0.01). Sucnr1 -/- mice had significantly reduced numbers of macrophages (0.56 ± 0.07 vs 0.92 ± 0.15 F4/80 cells/adipocytes, p < 0.05) and crown-like structures (0.06 ± 0.02 vs 0.14 ± 0.02, CLS/adipocytes p < 0.01) in adipose tissue and significantly improved glucose tolerance (p < 0.001) compared with WT mice fed an HFD, despite similarly increased body weights. Consistently, macrophages from Sucnr1 -/- mice showed reduced chemotaxis towards medium collected from apoptotic and hypoxic adipocytes (-59%; p < 0.05). Our results reveal that activation of SUCNR1 in macrophages is important for both infiltration and inflammation of adipose tissue in obesity, and suggest that SUCNR1 is a promising therapeutic target in obesity-induced type 2 diabetes.
2017-04-07 | GSE64104 | GEO
Project description:We measured global expression profiles in SFP1 deletion mutants and isogenic wild type strains during lag and log phase growth. To match the physiology of each species as well as minimize the effect on gene expression from differences in early growth rates, we profiled expression during log growth and lag after glucose repletion as previously described (Thompson et al., 2013).
2020-08-29 | GSE119237 | GEO