Recombinant Methanosarcina barkeri Tetrahydromethanopterin S-methyltransferase subunit G (mtrG)

What is the functional role of mtrG in the methanogenesis pathway of M. barkeri?

The mtrG protein functions as a crucial subunit of the membrane-bound N5-methyl-tetrahydrosarcinapterin (CH3-H4SPT):coenzyme M (CoM) methyltransferase complex encoded by the mtrECDBAFGH operon in M. barkeri. This complex catalyzes the energy-conserving (sodium-pumping) methyl transfer from CH3-H4SPT to CoM during growth on H2/CO2 or acetate . In the reverse direction, it catalyzes the endergonic transfer from methyl-CoM to H4SPT during growth on methylotrophic substrates like methanol, which is driven by sodium uptake .

To study this function experimentally:

• Measure methyltransferase activity using spectrophotometric assays that track the decrease in absorbance at 415 nm when methyl groups are transferred from methyl-H4SPT to CoM

• Verify specific activity in cell extracts before and after genetic manipulation

• Compare wild-type activity (approximately 27.7 ± 0.1 nmol/min/mg extract) with that of mutant strains

What methods can be used to express and purify recombinant mtrG for biochemical studies?

Methodology for expression and purification of recombinant mtrG:

• Cloning Approach:

  • Amplify the mtrG gene from M. barkeri genomic DNA using PCR with gene-specific primers

  • Clone into an expression vector with a suitable tag (His-tag recommended for archaeal proteins)

  • Transform into an E. coli expression strain optimized for archaeal proteins (e.g., Rosetta or BL21-CodonPlus)

• Expression Optimization:

  • Test expression at lower temperatures (18-25°C) to enhance proper folding

  • Consider co-expression with archaeal chaperones if inclusion body formation occurs

  • Monitor expression using SDS-PAGE and Western blotting with anti-His antibodies

• Purification Protocol:

  • Use immobilized metal affinity chromatography (IMAC) for initial purification

  • Apply size exclusion chromatography to achieve higher purity

  • Verify protein identity using mass spectrometry

• Activity Verification:

  • Develop reconstitution assays with other Mtr subunits to test functionality

  • Compare activity with native protein complex isolated from M. barkeri

How can the involvement of mtrG in methanogenesis be verified through gene deletion studies?

To verify mtrG involvement through gene deletion:

• Genetic System Setup:

  • Utilize homologous recombination-mediated gene replacement techniques established for M. barkeri

  • Construct a deletion cassette containing a selectable marker (e.g., puromycin resistance cassette pac-ori-aph) flanked by upstream and downstream regions of the target gene

  • Create either a specific mtrG deletion or a complete mtr operon deletion for comparative studies

• Transformation Protocol:

  • Linearize the plasmid containing the deletion cassette

  • Introduce into M. barkeri using liposome-mediated transformation

  • Select transformants on media containing the appropriate antibiotic (e.g., puromycin)

• Verification of Deletion Mutants:

  • Confirm successful deletion using:

    • Southern blot analysis to verify the replacement event

    • PCR verification of the deletion junction

    • RT-PCR to confirm absence of target gene expression

• Phenotypic Characterization:

  • Test growth on different substrates (methanol, H2/CO2, acetate, methanol+acetate)

  • Compare growth rates and final cell densities with wild-type

  • Document substrate consumption rates and product formation

What alternative methanogenesis pathways are activated when mtrG function is disrupted?

When mtrG function is disrupted (as part of the mtr operon deletion), M. barkeri activates alternative methanogenic pathways:

How do mutations in the mtrG gene impact the sodium-pumping function of the Mtr complex?

The impact of mtrG mutations on sodium pumping can be investigated through:

How can contradictory experimental results involving mtrG function be reconciled through systematic analysis?

When facing contradictory experimental results regarding mtrG function, employ the following systematic analysis approach:

• Experimental Design Framework:

  • Implement single-case experimental designs (SCEDs) to analyze individual variations

  • Use reversal designs (A-B-A) where possible to establish causality

  • Consider multiple baseline designs to control for confounding variables

  • Employ combined reversal and multiple baseline designs for robust validation

• Data Contradiction Analysis Matrix:

  Contradiction Type	Analysis Method	Resolution Strategy
  Growth phenotype discrepancies	Standardize media composition and growth conditions	Test across multiple strain backgrounds
  Enzymatic activity variations	Use multiple activity assay methods	Perform enzyme kinetics under various conditions
  Expression level differences	Normalize to multiple housekeeping genes	Verify with both RNA and protein level analysis
  Genetic complementation failures	Check expression of complemented gene	Test alternative promoters and regulatory elements

• Contradiction Detection Model:

  • Develop a contradiction detection framework similar to dialogue contradiction detection systems

  • Train the model on existing contradictory data in scientific literature

  • Apply to experimental datasets to identify potential contradictions early

• Integration with Genome-Scale Models:

  • Incorporate experimental data into genome-scale metabolic models

  • Use constraint-based methods to simulate metabolic fluxes under different conditions

  • Identify network-level effects that might explain seemingly contradictory local observations

What is the optimal protocol for incorporating recombinant mtrG into liposomes for in vitro methyltransferase activity assays?

For reconstituting recombinant mtrG into liposomes:

• Liposome Preparation:

  • Use archaeal lipid extracts or synthetic lipid mixtures that mimic archaeal membranes

  • Prepare small unilamellar vesicles (SUVs) by sonication or extrusion

  • Form a lipid film, hydrate, and extrude through polycarbonate membranes (100 nm pore size)

• Protein Incorporation:

  • Method 1: Direct incorporation during liposome formation

    • Mix purified mtrG with lipids in detergent

    • Remove detergent using Bio-Beads or dialysis

  • Method 2: Liposome-mediated transformation approach as used for M. barkeri

    • Form proteoliposomes using gentle detergent removal

    • Verify protein orientation using protease accessibility assays

• Reconstitution of the Complete Mtr Complex:

  • Co-reconstitute all individually purified Mtr subunits (mtrECDBAFGH)

  • Alternatively, purify the intact complex from M. barkeri and reconstitute it as a control

  • Create a series of proteoliposomes with different subunit compositions to determine the minimal functional unit

• Activity Measurement Protocol:

  • Establish a sodium gradient across the liposome membrane

  • Add substrates (CH3-H4SPT and CoM) to initiate the reaction

  • Monitor methyl transfer using:

    • Spectrophotometric assays (absorbance at 415 nm)

    • Radiolabeled substrates to track methyl group transfer

    • Sodium ion movement using sodium-selective electrodes

• Data Analysis:

  • Calculate specific activity (nmol/min/mg protein)

  • Determine the stoichiometry of sodium ions translocated per methyl group transferred

  • Compare with native enzyme complex activities from wild-type M. barkeri extracts (27.7 ± 0.1 nmol/min/mg)