Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

High-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of Pseudomonas putida mt-2 [RNA-seq]

ABSTRACT: 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).

ORGANISM(S): Pseudomonas putida

SUBMITTER: Juan Oliveros

PROVIDER: E-GEOD-71852 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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]

PMID: 26373670

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Project description:Strains of Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatics obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimizing the conversion of biomass to fuels and chemicals. The functioning of CCR in a P. putida strain, P. putida M2, capable of growing on both hexose and pentose sugars as well as aromatics, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 was able to co-utilize sugars and aromatics simultaneously; however, during co-cultivation with glucose and phenylpropanoid aromatics (p-coumarate and ferulate), intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis of these co- cultivations revealed that glucose exerted stronger repression compared to xylose on the proteins involved in aromatic catabolism. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during co-cultivation with aromatics implying simultaneous catabolite repression by sugars and aromatics. Downregulation of crc mediated by CRISPRi led to faster growth and uptake of both glucose and p-coumarate in the CRISPRi strains compared to the control while no difference was observed on xylose + p-coumarate. The increased abundance of the Eda protein and amino acids biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that the levels of CrcY and CrcZ homologs in M2, previously identified as sRNAs involved in CCR in P. putida strains, were lower under strong CCR (glucose + p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).

Dataset Information

High-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of Pseudomonas putida mt-2 [RNA-seq]

Publications

High-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of Pseudomonas putida mt-2.

Similar Datasets

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