Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Custom microarray analysis of small RNAs expression in E.coli with a focus on central carbon metabolism

ABSTRACT: In this experiment we used a custom microarray (accession number A-MTAB-550) to compare the expression of known and predicted sRNAs together with the expression of genes of the standard set of microarray probes of Agilent Technologies in E. coli grown to late exponential phase in minimal media supplemented with glucose and pyruvate respectively as the only carbon source.

INSTRUMENT(S): G2565CA DNA microarray scanner (Agilent) for scanning., Chamber type: SureHyb hybridization chamber (Agilent).

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Olatz Ruiz-Larrabeiti

PROVIDER: E-MTAB-5432 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Dietary immunostimulants are considered as an effective tool for enhancing the immune status of cultured organisms and improving their general welfare. Immunostimulants are chemical substances which activate transiently elements of the immune response and may potentially render animals more resistant to infectious diseases. They also reduce the risk of disease outbreaks, if administrated prior to situations known to result in stress and impaired general performance (e.g. handling, change of temperature and environment, weaning to inert diets) or prior to expected increase in exposure to pathogenic microorganisms and parasites. In this study, we have evaluated the effect of dietary LPS derived from Pantoea agglomerans (LPSp, SOMACY SL100, MACROPHI Inc., Japan) in Anguilla anguilla elvers considering the general condition, growth and immune status of the animals. Elvers (1.7 Â± 0.2 g) obtained from local fishermen (Delta del Ebro, Spain) were fed at different doses of c (0, 20 and 40 Î¼g LPSp kg BW-1 day-1) for a period of 70 days (4 replicates per dietary group). LPSp was added to the diet (52% protein, 24% fat, 9% ash) by top coating the pellets with the lyophilized powder of LPSp dissolved in 2% fish oil (diets manufactured by ALLERQUA, Denmark). The trial was conducted in 100 L tanks (40 L functional volume) connected to a recirculation unit (IRTAmarÂ®) and water quality was as follows: temperature, 21 Â± 0.5 ÂºC; pH, 7.5 Â± 0.1; photoperiod, 12 L:12 D and oxygen at saturation levels. At the end of the trial, fish were measured for growth (BW), size dispersion, histological organization of the intestinal mucosa and the transcriptomic responses of the spleen were evaluated by means of a 60K (15000 gene signatures) custom oligonucleotide microarray (Agilent Technologies Inc.). No significant differences in BW nor survival were observed among groups regardless of the dose of LPSp (BW: 3.9 Â± 0.3 g, survival: 98-99%). Micorarray analysis (LIMMA F<0.05; adj. p value < 0.005) resulted in 81 significantly up-regulated genes and only 3 down-regulated genes, in the highest LPSp dose. The lowest dose of LPSp up-regulated 26 genes and down-regulated 50. Both doses resulted in regulation of immune related genes, such as, sensors for detection of viral and bacterial products, innate antiviral response genes, cytokines, adipocytokines, and their regulatory nuclear factors. The pathway enrichment analysis (FatiGO; p < 0.005) of up-regulated genes (40 Î¼g LPSp kg BW-1 day-1) indicated among the most significantly regulated KEGG pathways: cytokine-cytokine receptor interaction, Jak-STAT signalling, natural killer cell mediated cytotoxicity, Toll-like, RIG-I-like and NOD-like receptor signalling and Wnt, Notch and mTOR signalling. In conclusion, LPSp diet highly regulates spleen transcription levels, mainly enhancing the immune response capacity of the immunostimulated elvers. This study was funded by the CENIT-ACUISOST project (CDTI, Spain), IRTA, Basque Government (S-PC08UN03; IT810-13) And UPV/EHU (UFI 11/37). Technical and human support provided by SGIker (UPV/EHU) is acknowledged. Authors are also in debt to G. MaciÃ (IRTA-SCR) for fish care and maintenance during the trial. 21 samples are analyzed, 7 pools of 3 spleens in each experimental group,Control group; Diet of 20ug/kg and 40ug/kg

Project description:Pelagic aggregates function as hotspots for microbial activity and biological carbon pumps for exporting OM fixed by photoautotrophs to sediments in lakes and oceans. In iron-rich (ferruginous) lakes, photoferrotrophic or chemolithoautotrophic bacteria appear to contribute to CO2 fixation by oxidizing reduced iron which leads to the formation of iron-rich pelagic aggregates called iron-snow. In acidic lakes, iron snow is colonized mainly by acidophilic iron-cycling microbes that can trigger interspecies aggregation mechanisms. However, the significance of iron oxidizers in carbon fixation, their general role in iron snow functioning, and the flow of carbon within iron snow is still unclear. Here, we combined a two-year metatranscriptome analysis with a 13CO2 metabolic labeling approach to determine general metabolic activities. Protein-based stable isotope probing (protein-SIP) was used to trace the 13CO2 incorporation in iron snow microcosms over time under both oxic and anoxic conditions. Analysis of our mRNA-derived metatranscriptome data identified four key players (Leptospirillum, Ferrovum, Acidithrix, Acidiphilium) with relative abundances (59.6%-85.7%) in iron snow encoding a variety of ecologically relevant pathways, including carbon fixation, polysaccharide biosynthesis, and flagellar-based motility. We did not detect transcriptional activity for carbon fixation from archaea or eukaryotes. The largest numbers of expressed genes (3008, 2991, 2936) matched to the genomes of our previously obtained iron snow isolates (Acidithrix sp. C25, Acidiphilium sp. C61, Acidocella sp. C78) separately. 13CO2 incorporation studies identified Leptospirillum and Ferrovum, as the main active chemolithoautotrophs under oxic conditions, and Ferrovum was the main active organism under anoxic conditions as well. Small amounts of labeled 13C (Relative isotope abundance: 1.0%-5.3%) were found in the heterotrophic Acidiphilium and Acidocella. Overall, our data show that iron oxidizers play an important role in the formation of iron minerals and CO2 fixation, but the majority of fixed C apparently did not reach other iron snow microbes. This finding suggests that most of the fixed C will be directly exported to the sediment without feeding heterotrophs in the water column in acidic ferruginous lakes.

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Custom microarray analysis of small RNAs expression in E.coli with a focus on central carbon metabolism

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