High-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of Pseudomonas putida mt-2
Ontology highlight
ABSTRACT:
ORGANISM(S): Pseudomonas putida
SUBMITTER: Juan Oliveros
PROVIDER: E-GEOD-71853 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
High-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of Pseudomonas putida mt-2.
Kim Juhyun J Pérez-Pantoja Danilo D Silva-Rocha Rafael R Oliveros Juan Carlos JC de Lorenzo Víctor V
Environmental microbiology 20151022 10
Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3'-ter ...[more]
Similar Datasets
Project description:Pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the TOL plasmid pWW0 along with other chromosomally-encoded activities. Tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. All xyl sequences were transcribed in response to aromatic substrates and the 3'-termini of both upper and lower mRNA operons extended beyond the ir coding regions, i.e., the 3â-end of the lower operon mRNA penetrated into the convergent xylS regulatory gene. Furthermore, xylR mRNA for the master m-xylene responsive regulator of the system was decreased by aromatic substrates, while the cognate upper operon mRNA was evenly stable throughout its full length. RNA-seq confirmed these data at a single-nucleotide level and refined the formerly misannotated xylL sequence. The chromosomal ortho route for degradation of benzoate (the ben, cat clusters and some pca genes) was activated by this aromatic, but not by the TOL substrates, toluene or m-xylene. We advocate this scenario as a testbed of natural 16 retroactivity between a pre-existing metabolic network and a new biochemical pathway implanted through gene transfer. Genome Analyzer IIx system (1x75bp) was used for sequencing total RNA extracted from P. putida (mt-2) containing the plasmid pWW0, in two different physiological states (treated with m-xylene and in C-runout condition).
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Project description:We used Affymetrix microarrays to monitore the gene expression of P. putida after a cold shock from 30°C to 10°C Culture was grown in minimal medium supplemented with succinate at 30°C. In mid-exp. phase cells were harvested for subsequent RNA extraction (standard condition) and medium was cooled down to 10°C. Two hrs after reaching the temperature, cells were again harvested for RNA extraction.
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Project description:Pseudomonas putida KT2440 is a metabolically versatile soil bacterium useful both as a model biodegradative organism and as a host of catalytic activities of biotechnological interest. In this report, we present the high-resolution transcriptome of P. putida grown in different carbon sources as revealed by deep sequencing of the corresponding RNA pools. Examination of the data from growth on glycolytic (glucose, fructose) and gluconeogenic (succinate or glycerol) substrates revealed that > 20% of the P. putida genome is differentially expressed depending on the ensuing metabolic regime. Changes affected not only metabolic genes but also a suite of global regulators, e.g. the rpoS sigma subunit of RNAP, various cold-shock proteins and the three HU histone-like proteins. Specifically, the genes encoding HU subunit variants hupA, hupB and hupN drastically altered their expression levels (and thus their ability to form heterodimeric combinations) under the different growth conditions. Furthermore, we found that the two small RNAs crcZ and crcY, known to inhibit the Crc protein that mediates catabolite repression in P. putida, were both down-regulated by glucose. cDNA libraries from Pseudomonas supplemented with different carbon sources (glucose, glycerol, fructose, succinate) were sequenced using HiSeq 2000 to yield 91 paired-end reads. Gene expression values were compared.
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Project description:We used cDNA generated from total mRNA for RNA-Seq analysis (Genome Analyzer II at GATC Biotech AG) to monitore the gene expression of P. putida after a cold shock from 30M-BM-0C to 10M-BM-0C Culture was grown in minimal medium supplemented with succinate at 30M-BM-0C. In mid-exp. phase cells were harvested for subsequent RNA extraction (standard condition) and medium was cooled down to 10M-BM-0C. Two hrs after reaching the temperature, cells were again harvested for RNA extraction.
2011-02-01 | E-GEOD-24175 | biostudies-arrayexpress
Project description:We used Progenika oligonucleotide arrays to monitore the gene expression of P. putida after a cold shock from 30°C to 10°C Culture was grown in minimal medium supplemented with succinate at 30°C. In mid-exp. phase cells were harvested for subsequent RNA extraction (standard condition) and medium was cooled down to 10°C. Two hrs after reaching the temperature, cells were again harvested for RNA extraction. Data from four replicate hybridizations (two dye-swaps) were combined into a single ratio value. Individual normalized signal intensity VALUES for each channel of each array are provided in the FullNormalizedMatrix.txt file available on the Series record.
2011-03-24 | E-GEOD-24174 | biostudies-arrayexpress
Project description:Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Here, we explored membrane adaptations of Pseudomonas putida KT2440, in particular outer membrane vesicle (OMV) formation in response to 1-octanol, PQS and prodigiosin, causing chemical membrane stress via RNA-seq of mRNA. Pseudomonas putida wild type KT2440 (Nelson et al. 2002) and the derived strains P. putida pig21 were cultivated in biological triplicates under continuous shaking (130 rpm) at 30 °C in 10 mL LB (lysogeny broth) medium (10 g L-1 tryptone, 5 g L-1 yeast extract, 10 g L-1 sodium chloride; Carl Roth®, Karlsruhe, Germany). Antibiotics were added to the culture medium when appropriate to the following final concentrations: 25 µg mL-1 kanamycin, 25 µg mL-1 irgasan, 25 µg mL-1 gentamicin, 50 µg mL-1 tetracycline. For chemical induction of OMV formation, P. putida KT2440 was exposed to 1 mM 1-octanol or 50 µM PQS (Pseudomonas quinolone signal) after reaching the logarithmic growth phase. For transcriptome analysis, cells were cultivated as described above, the cell pellet was harvested after 7 h, adjusted to an optical density (OD700 nm) of 1 and flash frozen . Total RNA was isolated from 3 biological replicates using Quick-RNA Miniprep Plus kit (Zymo Research). The samples were treated with DNase (Zymo Research) and RNA was again purified with an RNA Clean&Concentrator-5 kit (Zymo Research). Ribosomal rRNA was removed with a riboPOOL for bacteria (siTOOLs Biotech GmbH). The purity of RNA and removal of rRNA was then tested with an Agilent RNA Pico 6000 kit and an Agilent 2100 Bioanalyzer (Agilent Technologies). TruSeq Stranded mRNA Sample Preparation guide (Illumina) was then used to construct the cDNA library. The constructed cDNA library was then sequenced with Illumina NextSeq500 high output mode paired end using a read length of 75 bases.
2023-01-09 | E-MTAB-12470 | biostudies-arrayexpress
Project description:In this study we exploited next-generation Illumina sequencing technology (Wang et al., 2009) to refine the current annotation of the KT2440 genome. Transcriptome sequencing data were queried for yet undescribed small RNAs and ORFs and employed to validate predicted operons and gene coordinates. Expression profiles were measured at 10°C and 30°C to cover the physiologic temperature profile of this mesophilic bacterium. Moreover we compared the sensitivity and specificity of transcriptome data by taking the same RNA preparations for cDNA sequencing and hybridization of Progenika and Affymetrix microarrays. This SuperSeries is composed of the SubSeries listed below. P. putida KT2440 (strain DSM6125) (Bagdasarian et al., 1981) was obtained from the German Resource Centre for Biological Material (DSMZ, (Braunschweig, Germany). Bacterial cultures were inoculated from a frozen stock culture of P. putida wild type, and incubated at 30°C for 8 hours at 250 rpm in LB medium. An aliquot of 0.2 mL was added to 20 mL M9 medium (Na2HPO4 33.9 g/L, KH2PO4 15.0 g/L, NaCl 2.5 g/L, NH4Cl 5.0 g/L, MgSO4 2 mM, CaCl2 0.1 mM, FeSO4 x 7 H2O 0.01 mM, pH 6.8) supplemented with 15 mM succinate as sole carbon source in a 100 mL flask and incubated overnight at 30°C. For RNA extraction, bacteria were grown in a 1.5 L batch culture (M9 + 15 mM succinate) using the BioFlo 110 Fermenter (New Brunswick Scientific Co., Edison, NJ) to ensure constant pH, aeration and agitation. When cultures reached mid-exponential phase (OD600 ~ 0.8) the temperature was decreased from 30°C to 10°C. Three samples, each for parallel RNA extraction, were subsequently taken immediately before temperature downshift (30°C) and two hours after the media had been cooled to 10°C. The gene expression profiles of P. putida KT2440 at 30°C and 10°C were analyzed using three different transcriptome platforms: RNA-Seq (Illumina cDNA sequencing), Affymetrix microarrays and Progenika oligonucleotide microarrays. For a detailed description of sample preparation (RNA, cDNA , labelling, hybridization etc.) refer to individual Series.
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2016-07-19 | E-GEOD-82336 | biostudies-arrayexpress