Project description:Growth and transcriptional profiles of the barophilic methanarchaeon Methanocaldococcus jannaschii were studied at temperatures up to 98C and pressures up to 500 atm. Application of 500 atm of hyperbaric pressure shifted the optimal growth temperature upwards, and heat shock from 88C to 98C at 500 atm resulted in termination of growth. Pressure shock of M. jannaschii from 7.8 to 500 atm over 15-min, the first pressure upshift reported for a barophile, did not accelerate growth. Transcriptional profiles indicated a similar pressure response under growth and heat shock at 500 atm and pressure shock to 500 atm suggesting that the commonly affected genes are important for high-pressure adaptation. Factorial microarray design allowed de-convolution of the interacting effect of elevated pressure and heat shock on expression profiles, thus suggesting genes that may contribute to the organism’s survival in the turbulent in situ conditions of deep-sea hydrothermal vents. Keywords: stress response, time course, high pressure, heat shock, pressure shock
Project description:Barophilic growth of the hyperthermophilic methanarchaeon Methanocaldococcus jannaschii occurred when gas-substrate availability did not limit growth. In contrast, when growth was limited by gas transfer, no enhancement of growth was evident and a stress response was exhibited at both high and low pressure. A pressure-induced transcriptional response was evident, regardless of whether growth was enhanced by pressure. High-pressure adaptation of a barophilic organism can thus occur at the transcriptional level, even though the cells are stressed by low substrate availability and do not exhibit accelerated growth. Keywords: stress response, gas substrate limitation, bioreactor volume, high pressure
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.
Project description:Transcriptional response to growth with arsenite by the haloalkaliphilic sulfur-oxidizer Thioalkalivibrio thiocyanoxidans ARh2 and Tv. jannaschii by performing RNA-seq analysis
Project description:GMP synthetases are enzymes that catalyze the conversion of XMP to GMP. The two-subunit type GMP synthetases are composed of a glutamine amidotransferase (GATase) subunit that catalyzes the conversion of Gln to Glu and ammonia, and the ATP pyrophosphatase (ATPPase) subunit that catalyzes the formation of AMP-XMP from ATP and XMP. The inactive GATase subunit is allosterically activated by the binding of substrates to the ATPPase subunit. Upon activation, the GATase subunit binds Gln and hydrolyzes it producing ammonia which is tunnelled to the ATPPase subunit. The two subunits form a tight complex to enable domain crosstalk. However, the Methanocaldococcus jannaschii GMP synthetase (MjGMPS) is unique as the GATase (MjGATase) and ATPPase (MjATPPase) subunits interact transiently. Here, we employed enzyme kinetics, X-ray crystallography, cross-linking mass spectrometry (XL-MS) and integrative modelling to understand the mechanistic basis for the various steps in the catalytic cycle of MjGMPS.
