Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide transcription start sites mapping in Methylorubrum grown using dichloromethane and methanol

ABSTRACT: Dichloromethane (DCM, methylene chloride) is a toxic halogenated volatile organic compound massively used for industrial applications, and consequently often detected in the environment as a major pollutant. DCM biotransformation offers a sustainable decontamination strategy of polluted sites. Among methylotrophic bacteria able to use DCM as sole source of carbon and energy for growth, Methylorubrum extorquens DM4 (formerly named Methyobacterium extorquens) is a longstanding reference Alphaproteobacteria strain. Here, its primary transcriptome was obtained using a differential RNA-seq (dRNA-seq) approach to provide the first transcription start site (TSS) genome-wide landscape of a methylotroph using DCM.

ORGANISM(S): Methylorubrum extorquens

SUBMITTER: Francoise Bringel

PROVIDER: E-MTAB-11726 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Dichloromethane (DCM, methylene chloride) is a toxic chemical with substantial ozone-depleting capacity that is released by anthropogenic activities and natural processes. Specialized anaerobic bacteria metabolize DCM; however, the genetic basis for this process has remained elusive. Comparative genomics of three known anaerobic DCM-degrading bacteria, including the bacterial isolate, Dehalobacterium formicoaceticum strain DMC (Defo), revealed a homologous gene cluster, designated the methylene chloride catabolism (mec) gene cassette, comprising eight to ten genes with predicted 79.6-99.7% amino acid identity. Functional annotation identified genes encoding a corrinoid-dependent methyltransferase system. To support these observations at the functional level, global proteome analysis of Defo cultures growing with DCM as the sole energy source was performed. Briefly, axenic cultures (n = 3 biological replicates) of Defo were grown in 100 mL of anoxic mineral basal salt medium with 0.2 mM sodium sulfide, 0.2 mM L-cysteine and 30 mM bicarbonate (pH 7.3) under a headspace of N2/CO2 (80:20, vol/vol) with 156 umol (10 uL) of DCM. Cultures were initiated with a 5% (vol/vol) inoculum, incubated at 30C in the dark without agitation, and provided one additional feeding of DCM once the initial amendment was consumed. Biomass for proteomic analyses was collected after 2 weeks of incubation when approximately 95% of the second DCM feedings were consumed. After protein extraction and digestion, global proteomics analyses of 2 ug peptide solutions were performed with an Orbitrap Q Exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, US) equipped with a nano-electrospray source (ESI) interfaced with a Proxeon EASY-nLC 1200 system. Tandem mass spectrometry data (MS/MS) were searched against protein databases from the IMG Defo annotated genome (ID. 2757320304) to which common laboratory contaminant proteins were appended. For standard database searching, the peptide MS/MS data was searched using Proteome Discoverer v2.4. The MS/MS data were searched using the SEQUEST HT algorithm. Peptide spectrum match (PSM) confidence was evaluated with Percolator. PSM and peptides were considered identified at a q value of < 0.01. The shotgun proteomics analysis reported here revealed high expression of proteins encoded on the mec gene cluster during Defo anaerobic growth with DCM.

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Genome-wide transcription start sites mapping in Methylorubrum grown using dichloromethane and methanol

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