Transcriptomic response of Dickeya dadantii to DNA relaxation by novobiocin
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Dickeya dadantii
SUBMITTER: Sam Meyer
PROVIDER: E-MTAB-7650 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:We analyze the transcriptomic response of the phytopathogenic enterobacterium Dickeya dadantii ihfA mutant to a DNA supercoiling relaxation shock using the gyrase inhibitor novobiocin. The shock was applied to Dickeya cells grown in minimal medium supplemented with sucrose, in exponential or transition to stationary phase, and in minimal medium with sucrose + PGA (a pectin derivative) in transition to stationary phase.
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Project description:We analyze the transcriptomic response of the phytopathogenic enterobacterium Dickeya dadantii to an increase of DNA supercoiling induced by seconeolitsine, a topoisomerase I inhibitor. The shock was applied to Dickeya cells grown in minimal medium supplemented with sucrose, in exponential or transition to stationary phase.
2023-02-23 | E-MTAB-10134 | biostudies-arrayexpress
Project description:In this study transcriptional start sites (TSS) for the phytopathogen Dickeya dadantii 3937 were determined based on differential RNA-seq (dRNA-seq) as previously described (Sharma and Vogel, 2014). RNAs from E-MTAB-541 (Jiang et al. 2015) were pooled into four samples S1 to S4 reflecting a variety of environmental conditions. Each of them resulted in a combination of stress conditions (pH, NaCl, H2O2) and growth conditions: exponential phase with stress (S1), exponential phase without stress (S2), stationary phase with stress (S3), stationary phase without stress (S4). Those samples were then supplied to Vertis Biotechnologie AG for TEX treatment and Illumina sequencing. Briefly, ribosomal RNA molecules were depleted from the total RNA samples using the Ribo-Zero rRNA Removal Kit for bacteria (Epicentre). From the rRNA depleted RNA samples, the small RNA fractions <200 nt were separated from the large fractions using the RNeasy MinElute Cleanup Kit (Qiagen). TSS cDNA libraries were generated from the rRNA depleted large RNA fractions. First, the samples were fragmented using RNase III. Samples were then poly(A)-tailed using poly(A) polymerase, and split into two halves. One half was treated with Terminator exonuclease (+TEX, Epicentre), while the other one was left untreated (-TEX). 5'PPP structures were then converted into 5'P ends using RNA 5' Polyphosphatase (5'PP, Epicentre). Afterwards, an RNA adapter was ligated to the 5'P of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified using a high fidelity DNA polymerase, and purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). The cDNA pools were then sequenced on an Illumina NextSeq 500 system using 60 bp read length and a single-end, strand-specific protocol. Sequencing reads were trimmed, and both polyA and adapters removed.
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Project description:In this study, based on Nanopore direct RNA-seq where native RNAs are sequenced directly as near full-length transcripts in the 3' to 5' direction, transcription units of the phytopathogen Dickeya dadantii 3937 were validated and transcriptional termination sites were determined. Briefly, D. dadantii cultures were grown in M63 medium supplemented with 0.2% glucose and 0.2% PGA, until the early exponential phase (A600nm = 0.2, condition 1), or the early stationary phase (A600nm = 1.8, condition 2). RNAs were extracted using a frozen acid-phenol method, as previously described (Hommais et al. 2008), and treated successively with Roche and Biolabs DNases. Two samples were prepared: 50 µg of RNAs from each condition were pulled into one sample (sample 1), whereas the other one contained 100 µg of RNAs from condition 2 (sample 2). Both samples were then supplied to Vertis Biotechnologie AG for Nanopore native RNA-seq: total RNA preparations were first examined by capillary electrophoresis. For sample 1, ribosomal RNA molecules were depleted using an in-house developed protocol (recovery rate = 84%), whereas no ribodepletion was performed for sample 2. 3' ends of RNA were then poly(A)-tailed using poly(A) polymerase, and the Direct RNA sequencing kit (SQK-RNA002) was used to prepare the library for 1D sequencing on the Oxford Nanopore sequencing device. The direct RNA libraries were sequenced on a MinION device (MIN-101B) using standard settings. Basecalling of the fast5 files was performed using Guppy (version 3.6.1) with the following settings: --flowcell FLO-MIN106 --kit SQK-RNA002 --cpu_threads_per_caller 12--compress_fastq --reverse_sequence true --trim_strategy rna. Reads smaller than 50 nt were removed. 466 393 and 556 850 reads were generated for sample 1 and 2, respectively.
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Project description:Growth in 0.3 M NaCl M63 minimal medium affects the transcriptome of Dickeya dadantii 3937.in comparison to that of cells grown in M63 medium. NimbleGen design name 2005-05-31_Ogg_Erwinia_24mer, NimbleGen design ID 2181 A 1:2 expression design for 4597 genes from Dickeya dadantii 3937 with 20 24-mer probe pairs (PM/MM) per gene. Each probe is replicated 2 times. The design includes random GC and other control probes. Protocol: High-density DNA array prepared with Maskless Array Synthesizer (MAS) technology. See manufacturer's website at http://www.nimblegen.com/.
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Project description:In this study transcriptional start sites (TSS) of E. coli were determined for several growth conditions To detect the complement of transcripts expressed from E. coli, we collected two independent biological replicates (B1 and B2 samples) from MG1655 wild type strain grown to exponential (OD 600 ~0.4) or stationary phase (OD 600 ~2.0) in LB medium (samples LB 0.4 and LB 2.0, respectively) as well as grown to exponential phase (OD 600 ~0.4) in M63 minimal glucose medium (sample M63 0.4). For all six samples, total RNA was extracted and subjected to differential RNA-seq (dRNA-seq) library preparation for primary transcriptome analysis as described previously (Sharma et al., 2010). Specifically, prior to cDNA library construction half of each RNA sample was treated with 5M-bM-^@M-^Y terminator exonuclease (+TEX samples), which degrades RNAs containing a 5M-bM-^@M-^Y-mono-phosphate (5M-bM-^@M-^Y-P) and, thus, enriching enriches for primary transcripts containing 5M-bM-^@M-^Y-tri-phosphates (5M-bM-^@M-^Y-PPP). The other half of each sample was left untreated (-TEX samples) and thus contains both primary transcripts (5M-bM-^@M-^Y-PPP) and processed RNAs (5M-bM-^@M-^Y-P).
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Project description:We have analyzed the transcriptomes of Dickeya dadantii wild type, fis and hns strains in condition of DNA relaxation induced by novobiocin, a DNA gyrase inhibitor. For DNA relaxation novobiocin was added for 15 min to 100µg/ml final concentration to exponentially growing wild type, hns and fis strains (OD600= 0.2) grown in M63 medium supplemented by sucrose 0.2% (w/v). At this concentration, novobiocin has no impact on D. dadantii growth The micro-arrays used in this study were custom designed and produced by Roche NimbleGen, Inc. (Madison, WI) based on the annotated sequence of D. dadantii, available at Genbank accession number n° CP002038, which comprises 4597 CDS. The 4plex expression micro-arrays consist of 60-mer oligonucleotides, triplicated in three blocks on the array (5 oligonucleotides per CDS). For micro-array analyses, cDNA was synthesized, labeled and hybridized by Roche NimbleGen Inc.
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Project description:We have analysed the global gene expression of the plant pathogen Dickeya dadantii 3937 when grown in vitro under different growth and stress conditions. A multi-factorial design with 32 different experimental conditions was constructed, with two biological replicates for each condition. Cells were grown to exponential phase or stationary phases in four different growth media: M63 supplemented with 0.2% sucrose as carbon source, with or without 0.2% (w/v) polygalacturonate (PGA) and with or without 0.1% Saintpaulia leaf extracts (1 g leaves in 1 L M63). Cells grown in these four media were subjected to different stresses: (i) acid stress, by an incubation of 15 min in the presence of 30 mM malic acid; (ii) oxidative stress, by an incubation of 15 min in the presence of 100 _M H2O2 or (iii) osmotic stress, by an incubation of 15 min in the presence of 300 mM NaCl. The micro-arrays used in this study were custom designed and produced by Roche NimbleGen, Inc. (Madison, WI) based on the annotated sequence of D. dadantii, available at Genbank accession number nM-! CP002038, which comprises 4597 CDS. The 4plex expression micro-arrays consist of 60-mer oligonucleotides, triplicated in three blocks on the array (5 oligonucleotides per CDS).
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