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Differential expression analysis of a hyperthermophilic archeaon Methanocaldococcus jannaschii upon hydrogen limitation and hydrogen syntrophy with Thermococcus paralvinellae

ABSTRACT: The purpose of this experiment was to compare the transcriptomes of M. jannaschii using RNA-Seq gene expression analyses to understand the physiology of this organism when it is grown under H2-replete, H2-limited and H2-syntrophy conditions. The RNA-seq reads were mapped to both M. jannaschii and T. paralvinellae genomes using BBSplit from BBMap package. BBSplit is an aligner tool that bins sequencing reads by mapping to them multiple references simultaneously and separates the reads that map to multiple references to a special "ambiguous" file for each of them. For further analyses we removed all ambiguously mapped reads to both genomes and worked with only the reads that unambiguously map to M. jannaschii genome. The mapped reads for M. jannaschii were then aligned to the M. jannaschii genome again and sorted using the STAR aligner version 2.5.1b . Aligned sequence reads were assigned to genomic features and quantified using featureCounts read summarization tool. Genes that were differentially expressed were identified using ‘DESeq2’ in the Bioconductor software framework in R. The differential gene expression analyses showed that the enzyme responsible for the reduction of methenyl group to a methylene group during carbon fixation switches from a H2-dependent enzyme to a coenzyme F420-dependent enzyme with decreasing H2 availability and into syntrophy. During syntrophy, the genes for energy generation on the membrane decreased in their expression levels.

ORGANISM(S): Methanocaldococcus jannaschii Thermococcus paralvinellae

PROVIDER: GSE112986 | GEO | 2019/03/11

REPOSITORIES: GEO

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Project description:Purpose: Compare the transcriptomes of T. paralvinellae using RNA-Seq gene expression analyses to understand the physiology of this organism when it is grown on different carbon sources, under optimal and hydrogen stress conditions. Methods: T. paralvinellae was grown in triplicate at with either 0.5% maltose or 0.5% tryptone to investigate its catabolic pathways when grown on a sugar and peptides, respectively. It was also grown on 1% formate to determine the effect of formate on cell growth, metabolite production, and gene expression. It was also grown separately on maltose or tryptone media as before, except with hydrogen in the media to examine the effects of hydrogen inhibition. The cells remaining in the 2-liter bioreactor were concentrated by centrifugation. The resulting cell pellets were resuspended in TRIzol (Invitrogen) and total RNA was extracted using a Direct-zol RNA extraction kit (Zymo). RNA quantity was determined with Qubit fluorometry. RNA integrity was checked by a bioanalyzer and a Nanodrop spectrometer. Genewiz sequencing facilities performed rRNA removal, library construction, multiplexing and sequence the RNA with HiSeq2500, 2×100 Paired-End. We aligned the RNAseq reads to T. paralvinellae genome (STAR) and assigned aligned sequence reads to genomic features (featureCounts). We then quantified transcriptome and reported pairwise significant genes that are differentially expressed by DESeq available under Bioconductor (www.bioconductor.org) on Galaxy platform and in R. Results: Sequencing depths ranged from 29,974,378 to 45,240,342 sequences, with a mean of 38,990,002 and a median of 38,951,352 reads per sample. Of 2,138 genes annotated in the T. paralvinellae genome, 2084 transcripts were detected by RNA-Seq.Of the 2084 genes detected by transcriptomics, hydrogen stress caused only 48 genes to be differentially regulated (log2FC > 1, P < 0.05) with maltose and tryptone carbon sources. These included mostly genes associated with formate-dependent metabolism and transporters. The upregulated genes upon growth under H2 stress conditions indicate intrinsic formate production via formate hydrogen lyase and format secretion.

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Differential expression analysis of a hyperthermophilic archeaon Methanocaldococcus jannaschii upon hydrogen limitation and hydrogen syntrophy with Thermococcus paralvinellae

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