Towards a Computational Model of a Methane Producing Archaeum

Abstract

Progress towards a complete model of the methanogenic archaeum Methanosarcina acetivorans is reported. We characterized size distribution of the cells using differential interference contrast microscopy, finding them to be ellipsoidal with mean length and width of 2.9 μm and 2.3 μm, respectively, when grown on methanol and 30% smaller when grown on acetate. We used the single molecule pull down (SiMPull) technique to measure average copy number of the Mcr complex and ribosomes. A kinetic model for the methanogenesis pathways based on biochemical studies and recent metabolic reconstructions for several related methanogens is presented. In this model, 26 reactions in the methanogenesis pathways are coupled to a cell mass production reaction that updates enzyme concentrations. RNA expression data (RNA-seq) measured for cell cultures grown on acetate and methanol is used to estimate relative protein production per mole of ATP consumed. The model captures the experimentally observed methane production rates for cells growing on methanol and is most sensitive to the number of methyl-coenzyme-M reductase (Mcr) and methyl-tetrahydromethanopterin:coenzyme-M methyltransferase (Mtr) proteins. A draft transcriptional regulation network based on known interactions is proposed which we intend to integrate with the kinetic model to allow dynamic regulation.

Single‐molecule pull‐down (SiMPull) for new‐age biochemistry

“…SiMPull is a sensitive way of measuring the concentrations of proteins in cells because it can detect changes in picomolar concentrations in the cell lysate. The copy number per cell can be estimated based on number of cells used, and the sample volume . SiMPull is also a very reproducible and robust way of estimating concentrations because we found that similar numbers of proteins are pulled‐down at a specific concentration each time the experiment is repeated (within about 20% error) .…”

“…In this review, we will discuss the detailed methodology of the assay. We will highlight how SiMPull can be extended to diverse biological macromolecules, not just proteins , and serve as a valuable tool for characterizing various biochemical properties and processes . We will conclude by extending the applications of SiMPull to the realm of biosensors, and draw comparisons with other major biosensing techniques presently available.…”

Genome-wide gene expression and RNA half-life measurements allow predictions of regulation and metabolic behavior in Methanosarcina acetivorans

“…RNA was isolated as previously reported [85]. Briefly, M. acetivorans C2A wild type was adapted to grown, TMA or acetate for 33 generations.…”

Single-Molecule Pull-Down FRET to Dissect the Mechanisms of Biomolecular Machines

“…Selective pull-down is achieved through capture of a bait protein using a high-affinity antibody either specific for the bait protein itself or to a purification tag appended to the protein (TAP, Flag, etc). The immobilized bait is then typically used to capture one or more, fluorescently-labeled prey protein (as well as any interacting partners that might form a complex) in order to study stoichiometry of the bait-prey complex (Bharill, Fu, Palty & Isacoff, 2014; Jain et al, 2011; Panter, Jain, Leonhardt, Ha & Cresswell, 2012; Peterson, Labhsetwar, Ellermeier, Kohler, Jain, Ha, et al, 2014; Shen, Chakraborty, Jain, Giri, Ha, Prasanth, et al, 2012) or conformational dynamics of a protein or protein complex (Zhou, Kunzelmann, Webb & Ha, 2011; Zhou, Zhang, Bochman, Zakian & Ha, 2014). …”