ABSTRACT: Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). Four samples of each strain were grown in batch culture. Differences in transcriptional expression between S40 and S2 were measured using cDNA arrays, with flip dye experiments for each biological replicate. Keywords: mutant, archaea, carbon, hydrogenase, methanogen
Project description:Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). The S40 and S2 strains were grown in 14N and 15N media with acetate separately. A biological replicate was made by switching the media. Mass spectrometry based quantitative proteomics were done on the mixtures to investigate the differences in expression patterns between S40 and S2. Keywords: isotope labeling mass spectrometry, quantitative proteomics
Project description:Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). Four samples of each strain were grown in batch culture. Differences in transcriptional expression between S40 and S2 were measured using cDNA arrays, with flip dye experiments for each biological replicate. 16 replicates were used for the experiment. Four biological replicates of the wild type (S2) and mutant (S40) strains where compared using dye flips. Each slide contained two copies of the array yielding four technical replicates for each biological replicate.
Project description:Methanococcus maripaludis is a methanogenic Archaea that conserves energy from molecular hydrogen to reduce carbon dioxide to methane. Chemostat grown cultures limited for phosphate or leucine were compared to determine the regulatory response to leucine limitation. Keywords: archaea, hydrogen, leucine, phosphate, nutrient limitation, growth rate, methanogen
Project description:Methanococcus maripaludis is a methanogenic Archaea that conserves energy from molecular hydrogen to reduce carbon dioxide to methane. Chemostat grown cultures limited for hydrogen, phosphate, or leucine were compared to determine the regulatory response to hydrogen limitation. This was done by comparing hydrogen limited cultures to both leucine limited and phosphate limited cultures. Slow and rapid growing samples limited for either hydrogen or phosphate were compared to determine the regulatory effects of growth rate. Keywords: archaea, hydrogen, leucine, phosphate, nutrient limitation, growth rate, methanogen
Project description:We hypothesize that FeSaq and FeSMack can serve as sources of S and Fe to support the growth and methanogenesis of methanogens. A model methanogen Methanococcus maripaludis S2 (MmS2) was inoculated in a growth medium with synthetic FeSaq and FeSMack.
Project description:Genome reorganization by large scale indels, gene displacements, and horizontal gene transfers allow an organism to re-organize genes into operons (“operonization”) and explore novel strategies for adapting to its changing environment. We have characterized the process of operonization by mapping and comparing transcriptome structures (TSs) of four phylogenetically diverse exptremophilic archaea: a hydrogenotrophic methanogen (Methanococcus maripaludis S2), an anaerobic thermophile (Pyrococcus furiosis DSM 3638), an acidophilic and aerobic thermophile (Sulfolobus solfataricus P2), and a photoheterotrophic halophile (Halobacterium salinarum NRC-1). We demonstrate how the evolution of new transcriptional elements (promoters and terminators) is utilized as a mechanism to incorporate translocated, inverted, and newly acquired genes into existing gene regulatory programs. This SuperSeries is composed of the following subset Series: GSE26777: Methanococcus maripaludis S2 growth curve, tiling arrays GSE26778: Pyrococcus furiosus DSM 3638 growth curve, tiling arrays GSE26779: Sulfolobus solfataricus P2 growth curve, tiling arrays Refer to individual Series
Project description:Experimentally mapped transcriptome structure of Methanococcus maripaludis S2 by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 14 nt).
Project description:Richards2016 - Genome-scale metabolic
reconstruction of Methanococcus maripaludis (iMR539)
This model is described in the article:
Exploring Hydrogenotrophic
Methanogenesis: a Genome Scale Metabolic Reconstruction of
Methanococcus maripaludis.
Richards MA, Lie TJ, Zhang J,
Ragsdale SW, Leigh JA, Price ND.
J. Bacteriol. 2016 Dec; 198(24):
3379-3390
Abstract:
Hydrogenotrophic methanogenesis occurs in multiple
environments, ranging from the intestinal tracts of animals to
anaerobic sediments and hot springs. Energy conservation in
hydrogenotrophic methanogens was long a mystery; only within
the last decade was it reported that net energy conservation
for growth depends on electron bifurcation. In this work, we
focus on Methanococcus maripaludis, a well-studied
hydrogenotrophic marine methanogen. To better understand
hydrogenotrophic methanogenesis and compare it with
methylotrophic methanogenesis that utilizes oxidative
phosphorylation rather than electron bifurcation, we have built
iMR539, a genome scale metabolic reconstruction that accounts
for 539 of the 1,722 protein-coding genes of M. maripaludis
strain S2. Our reconstructed metabolic network uses recent
literature to not only represent the central electron
bifurcation reaction but also incorporate vital biosynthesis
and assimilation pathways, including unique cofactor and
coenzyme syntheses. We show that our model accurately predicts
experimental growth and gene knockout data, with 93% accuracy
and a Matthews correlation coefficient of 0.78. Furthermore, we
use our metabolic network reconstruction to probe the
implications of electron bifurcation by showing its
essentiality, as well as investigating the infeasibility of
aceticlastic methanogenesis in the network. Additionally, we
demonstrate a method of applying thermodynamic constraints to a
metabolic model to quickly estimate overall free-energy changes
between what comes in and out of the cell. Finally, we describe
a novel reconstruction-specific computational toolbox we
created to improve usability. Together, our results provide a
computational network for exploring hydrogenotrophic
methanogenesis and confirm the importance of electron
bifurcation in this process.Understanding and applying
hydrogenotrophic methanogenesis is a promising avenue for
developing new bioenergy technologies around methane gas.
Although a significant portion of biological methane is
generated through this environmentally ubiquitous pathway,
existing methanogen models portray the more traditional energy
conservation mechanisms that are found in other methanogens. We
have constructed a genome scale metabolic network of
Methanococcus maripaludis that explicitly accounts for all
major reactions involved in hydrogenotrophic methanogenesis.
Our reconstruction demonstrates the importance of electron
bifurcation in central metabolism, providing both a window into
hydrogenotrophic methanogenesis and a hypothesis-generating
platform to fuel metabolic engineering efforts.
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