Transcriptome profiling of the S. meliloti EcpR1 sRNA overproducing strain and deletion mutant
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ABSTRACT: To obtain further clues to putative target genes of Sinorhizobium meliloti EcpR1 (SmelC291) sRNA, the cellular responses 15 minutes, 1 hour, and 4 hours post-induction of ecpR1 overexpression and of the ecpR1 deletion mutant (2011ecpR1) during stationary growth were characterized by microarray-based transcriptome profiling.
Project description:Analysis of the RNA-seq reads confirmed that transcription of many of the previously identified Pho-regulon genes of Sinorhizobium meliloti was regulated by the availability of inorganic phosphate in the media. The transcriptional start sites of many of the Pho-regulon genes as determined by RNA-seq reads were found to correspond to those determined from primer extension analysis. mRNA from S. meliloti RmP110 grown in MOPS + 2 mM Pi and MOPS + 20 M-BM-5M Pi M-bM-^@M-^S directional RNA-seq via Illumina sequencing
Project description:In this experiment we compare the transcriptome profiles of a SmelC775 overexpression strain to a control strain overexpressing a control RNA (SmelC812) (Schlueter et al. 2010).
Project description:This experiment studied the effect of FPP accumulation on E. coli. E. coli cells transformed with pMBIS (the S. cerevisiae mevalonate pathway enzymes converting mevalonate to FPP) and fed mevalonate produce large amounts of FPP, which causes toxicity when it accumulates. When coupled with an active amorphadiene synthase (pADS) the cells produce amorphadiene, a non-toxic isoprenoid. To accumulate FPP, but maintain similar protein burden, an amorphadiene synthase with 3 mutations to render it inactive was used (pADSmut) to accumulate FPP. E. coli was transformed with pMBIS and pADS or pMBIS and pADSMut and grown in M9+glucose with varying magnesium concentrations and fed 20 mM mevalonate and induced with 0.5 mM IPTG, then sampled at subsequent time points. This comparison is between E. coli DH1 cells accumulating FPP via the heterologous mevalonate pathway (pMBIS/pADSmut) to cells producing amorphadiene via the same pathway (pMBIS/pADS). Samples were collected 6, 10, 14, and 26.5 hr after addition of IPTG and mevalonate. One biological replicate was used. Total RNA was extracted, reverse transcribed, labeled, and hybridized to multiple slides for technical replicates.
Project description:Transcription profiles in BL21, BL21/pOri1 and BL21/pOri2 were analysed using DNA microarray technology. BL21, BL21/pOri1 or BL21/pOri2 strains were cultured at chemostat status and harvested after the cultivation arrived steady status. Experiment Overall Design: BL21, BL21/pOri1 and BL21/pOri2 were cultured at the exponential growth status or at the same growth rate status, respectively. E. coli BL21, BL21/pOri1 and BL21/pOri2 RNAs were reverse-transcripted into cDNAs, The RNAs from the BL21, BL21/pOri1, BL21/pOri2 were labelled with biotin. The labelled cDNAs were mixed and then hybridized on the microarray slides. Experiments were repeated four times. Most of them have no significant difference comparing plasmid-carrying E. coli and plasmid-free E. coli
Project description:Genome-wide gene expression analysis was performed with the cells in exponential and stationary growth phases. Through these two growth status, 89.6% of currently annotated genes were expressed. High-density oligonucleotide tiling arrays consisting of 379,528 50-mer probes spaced 30 bp apart across the whole Klebsiella pneumoniae MGH 78578 genome was used (Roche NimbleGen).
Project description:Assays 1-4: Effects of DnaA activity on S. meliloti chromosome, pSymA and pSymB replicon bias. Sheared DNA extracts of IPTG-induced vs uninduced cultures of DnaA (Assays 1,2) or Hda (Assays 3,4) overexpressors were labeled with Cy3 or Cy5 and hybridized applying dye-swap replicate design. Assay 5: Hybridization of sheared DNA of logarithmic (Cy3-labeled) vs stationary phase (Cy5-labeled) culture.
Project description:Most aging hypotheses revolve around the accumulation of some sort of damage resulting in gradual physiological decline and ultimately death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend lifespan. However, lowering translational efficiency extends rather than shortens lifespan in C. elegans. We studied turnover of individual proteins in the conserved Insulin/Insulin-like Growth Factor (IGF-1) receptor mutant daf-2 by combining Stable Isotope Labeling by Nitrogen-15 in Caenorhabditis elegans and LC-MS/MS. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, while others remained unchanged, signifying that longevity is not supported by high protein turnover. This slow-down of protein turnover was most prominent for components of the translation machinery and mitochondria. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged in daf-2. The slow-down of protein dynamics and decreased abundance of the translational machinery may point at the importance of anabolic attenuation in lifespan extension as suggested by the hyperfunction theory.
Project description:Protein turnover rates severely decline in aging organisms, including C. elegans. However, limited information is available on turnover dynamics at the individual protein level during aging. We followed changes in protein turnover at one-day resolution using a multiple-pulse 15N-labeling and accurate mass spectrometry approach. Forty percent of the proteome shows gradual slowdown in turnover with age, while only few proteins show increased turnover. Decrease in protein turnover was consistent for the minority of functional protein subsets, including tubulins and vitellogenins, while for most functionally related protein pools randomly diverging turnover patterns were observed with age. Our data suggests severe dysregulation of protein turnover of the translation machinery, whereas protein turnover of UPS and antioxidant systems are well-preserved over time. Hence, we presume that maintenance of quality control mechanisms is a protective strategy in aging worms, although the ultimate proteome collapse is inescapable.
Project description:In this project we used the strain E. coli BW25113 ΔfrmA::frt (Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ-, rph-1, Δ(rhaD-rhaB)568, hsdR514, ΔfrmA(::frt)) from the Keio collection (Baba et al. 2006). The plasmid {pSEVA424_mdh-das} (Low-copy-number, lacIq/Ptrc promoter, RK2 ori, SmR) from Silva-Rocha et al. 2013 was integrated in the strain to express mdh (Methanol dehydrogenase) and das (Dihydroxyacetone synthase) respectively from Acinetobacter gerneri and Pichia angusta. In this experiment, we inoculated 50 mL of M9 minimal medium with 15 mM xylose and with or without 0.15 M methanol, in baffled flasks at 30°C, 150 rpm. At D600=1 and D600=2, 12mL of each culture were centrifuged 1’30 at 14000 rpm before discarding the supernatant and freezing the pellets for 15 sec in liquid nitrogen. RNA extraction were carried out using a RNeasy Midi Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s protocol and the transcriptomic experiments were run at the GeT-Biopuces Platform (Toulouse, France). Prior to the experiment, the quality of the RNA extracted was analyzed by Nanodrop and Cell Bio Analyser (Agilent Technologies, California, USA). The labelling with Cy3, hybridation on the Agilent chip and wash were carried out accordingly to the Agilent protocol and kits. We used the scanner NimbleGen MS 200 (Roche, Bâle, Suisse) with the following parameters: autogain, 5micros resolution, 532nm scan. The Agilent Feature Extraction software was used to extract the data.
Project description:Dihydroxyacetone (DHA) is an attractive molecule produced in a wide range of industries . DHA is found among all the kingdoms as an intermediate of various metabolic pathways and can be used as a carbon source by many organisms. The bacterium Escherichia coli is able to grow on DHA as the sole carbon source albeit at a low growth rate (0.1 h-1). If the topology of DHA metabolic network has been characterized, the function and regulation of the metabolic pathways involved in DHA metabolism remain unsolved. Here, we aimed to better understand DHA metabolism in E. coli BW25113 by exploring its transcriptional pattern on DHA versus glucose. We also studied the pattern of three DHA pathway mutants (dhaKLM, ptsA and glpK).