Unknown,Transcriptomics,Genomics,Proteomics

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Genomic and Transcriptomic Analyses of the Facultative Methanotroph, Methylocystis Strain SB2

ABSTRACT: Here we present the assembled genome of the facultative methanotroph, Methylocystis strain SB2, along with assessment of its transcriptome when grown on methane vs. ethanol. As expected, transcriptomic analyses indicate methane is converted to carbon dioxide via the canonical methane oxidation pathway for energy generation, and that carbon is assimilated at the level of formaldehyde via the serine cycle. When grown on ethanol, it appears this strain converts ethanol to acetyl-CoA and then utilizes the TCA cycle for energy generation and the ethylmalonyl CoA pathway for the production of biomass. All cultures were grown in triplicates for subsequent DNA and RNA extraction as well as for subsequent sequencing using Illumina. Transcriptomic analysis results presented in this Series.

ORGANISM(S): Methylocystis sp. SB2

SUBMITTER: Alexey Vorobev

PROVIDER: E-GEOD-51345 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Our goal is to convert methane efficiently into liquid fuels that may be more readily transported. Since aerobic oxidation of methane is less efficient, we focused on anaerobic processes to capture methane, which are accomplished by anaerobic methanotrophic archaea (ANME) in consortia. However, no pure culture capable of oxidizing and growing on methane anaerobically has been isolated. In this study, Methanosarcina acetivorans, an archaeal methanogen, was metabolically engineered to take up methane, rather than to generate it. To capture methane, we cloned the DNA coding for the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable archaeal organism from a Black Sea mat into M. acetivorans to effectively run methanogenesis in reverse. The engineered strain produces primarily acetate, and our results demonstrate that pure cultures can grow anaerobically on methane. Differential gene analysis of two growth conditions (three biological replicates each) was performed: (i) M. acetivorans/pES1-MATmcr3 grown on methane and (ii) M. acetivorans/pES1-MATmcr3 grown on methanol. All starter cultures (200 mL) were grown on methanol for 5 days, and harvested by centrifugation. Cell pellets were washed three times with HS medium, and resuspended using 5 mL HS medium, 2 Âµg/mL puromycin, and 0.1 mM FeCl3. For condition (i), methane was filled into the headspace of the cultures. For condition (ii), 150 mM methanol was added. All cultures were incubated at 37C for 5 days, followed by rapid centrifugation in the presence of 50 ÂµL RNAlater solution (Ambion, Austin, TX) per mL of culture. Total RNA was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) were then digested with terminator 5â-phosphate-dependent exonuclease (Epicentre, Madison, WI) to partially remove ribosomal RNA. Digested RNA were cleaned up using AgenCourt RNAClean XP beads (AgenCourt Bioscience, Beverly, MA) and used for cDNA library construction using the TruSeq Stranded mRNA Library kit (Illumina). Pooled and barcoded cDNA library was then sequenced on a HiSeq sequencing platform (Illumina). Obtained reads were mapped to the reference genome of M. acetivorans (Genbank accession NC_003552.1) using STAR. The mapped reads were assembled using Cufflink v2.2.1 to identify potential novel transcripts. Assembled, unannotated novel transcripts for all the strains were combined with the list of known genes. Differential expression of genes and potential novel transcripts were determined using Cuffdiff at a significance cutoff at q < 0.07 with a false discovery rate of 0.05. Expression levels of gene transcripts are expressed as fragments per kilobase of transcript per million mapped fragments (FPKM), and expression changes are determined by the ratio of FPKM of culture replicates grown on methane to FPKM of culture replicates grown on methanol.

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Genomic and Transcriptomic Analyses of the Facultative Methanotroph, Methylocystis Strain SB2

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