Project description:A sub-genomic array of structural and regulatory genes of the TOL plasmid pWW0 of Pseudomonas putida mt-2 has been taiolored for inferring the genetic network of m-xylene metabolism through expression profiling of xyl genes. To this end we visualized the response to m-xylene, o-xylene, 3MBA and to a heat shock.
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).
Project description:The study focuses on an extensive biochemical fractionation with in-depth quantitative mass spectrometric profiling in the mitochondrial (mt) extracts of cultured human NTera2 embryonal carcinoma stem cells (i.e. ECSCs or undifferentiated state) and upon exposure to retinoic acid-induced differentiated neurons (DNs) to establish a network of high-quality mt protein-protein interactions. The resulting network showed that most of the native mt protein complexes with predicted subunits are previously unreported and endured extensive changes during neuronal differentiation and influence neuronal function and neurodegenerative disorder attributes.
