Project description:Differential expression analysis of a hyperthermophilic archeaon Methanocaldococcus jannaschii upon hydrogen limitation and hydrogen syntrophy with Thermococcus paralvinellae
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.
Project description:A hyperthermophilic archaeon Thermococcus kodakarensis can grow in a wide range of temperatures from 60°C to 93°C, optimally at 85°C. To screen the genes that being specifically expressed at lowest growth temperature, 60°C, we investigated the transcriptional profilings of T. kodakarensis grown at 60˚C by comparing with those grown at 85°C.
Project description:Hyperthermophilic archeaon, Thermococcus onnurineus NA1 has known as a strict anaerobe. To date, a few of studies have been reported that strict anaerobe can grow using oxygen. However, the research of the growth enhancement of strict anaerobic archaeon belonging to the order of Thermococcales using the oxygen, in which has never been reported so far. In this study, we showed that the growth of T. onnurineus NA1 strain increased under various oxygen concentrations and we observed that oxygen was decreased in the headspace during the growth of cell. Genome-wide transcriptomic analysis was carried out to evaluate alterations in gene expression induced by O2 and to explain the physiological effects of oxidative stress on the growth of T. onnurineus NA1.
Project description:A hyperthermophilic archaeon Thermococcus kodakarensis can grow in a wide range of temperatures from 60°C to 93°C, optimally at 85°C. To screen the genes that being specifically expressed at lowest growth temperature, 60°C, we investigated the transcriptional profilings of T. kodakarensis grown at 60ËC by comparing with those grown at 85°C. One-condition experiment, KU216 grown at optimal growth temperature, 85°C vs. KU216 grown at lowest growth temperature, 60°C. Technical replicates: 2 KU216 grown at 60°C, independently measured. One replicate per array.
Project description: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.
