Methylobacterium extorquens

Methylobacterium extorquens is a gram-negative, plant-associated bacterium capable of growth on methanol and other C₁ compounds (methylotrophy). It is a member of the α-proteobacteria and produces Hopanoids as a substantial component of its membrane lipidome.

Role in Our Research

M. extorquens became the central model organism for understanding hopanoid function in living bacteria — the in vivo counterpart to the model membrane work with Diplopterol.

We showed that hopanoids are localized to the outer membrane (OM) of M. extorquens, where they interact with lipid A to produce a highly ordered bilayer, analogous to sterol-sphingolipid ordering in eukaryotic plasma membranes. A squalene-hopene cyclase deletion mutant (ΔSHC) exhibits reduced OM order, impaired energy-dependent multidrug efflux, and >1,000-fold increased sensitivity to the detergent Triton X-100 (Sáenz et al., 2015, PNAS).

Lipid Composition

• Major hopanoids: Diplopterol and 2-methyl-diplopterol, comprising ~19 mol% of total lipids in the OM.
• Polar hopanoids: BHT-CE (bacteriohopanetetrol cyclitol ether) and BHT-GCE (guanidine-modified BHT-CE), also enriched in the OM.
• Phospholipids: virtually all unsaturated (~90% with double bonds in both acyl chains), primarily in the inner membrane. This all-unsaturated phospholipid composition explains why diplopterol interacts preferentially with lipid A (saturated) rather than phospholipids in this organism.
• LPS/Lipid A: present in the outer leaflet of the OM, with conserved saturated acyl chains and structural features analogous to sphingolipids (amide-linked backbone, hydroxylations).

Lipidome Adaptation

Using shotgun lipidomics, we showed that M. extorquens has a simple yet adaptive lipidome of ~27 species. Only 11 lipids account for 90% of total variability across environmental perturbations, with temperature as the dominant driver of remodeling. Acyl chain changes are distributed unevenly across headgroup classes, enabling fine-tuned control of membrane properties (Chwastek et al., 2020, Cell Reports). The primary unsaturation position is Δ11 (ω7), which optimizes interactions with Diplopterol (Nguyen et al., 2024, Biophysical Journal).

Isoprenoid Lipid Diversity

M. extorquens produces both hopanoids and C₃₀ carotenoids (squalene-derived, not the expected C₄₀ phytoene pathway). Disruption of hopanoid synthesis impairs growth at elevated temperatures, increases membrane permeability, and reduces tolerance to low divalent cations. Carotenoid disruption has minimal membrane phenotypes but increases oxidative stress sensitivity (Rizk et al., 2021, Molecular Microbiology).

Additional Studies

We characterized the LPS structure of M. extorquens and its role in methanol tolerance, revealing that LPS modifications contribute to the organism’s resistance to its primary carbon source (Gnädig et al., 2023, Front. Microbiol.). Collaborative work showed that extracellular polysaccharides (EPS) produced by M. extorquens play a role in plant abiotic stress tolerance (Ramírez-Villacis et al., 2022, Front. Microbiol.).

Key Experimental Tools

• ΔSHC mutant: squalene-hopene cyclase knockout, unable to synthesize hopanoids. Non-lethal but exhibits clear phenotypes in membrane order, detergent sensitivity, and efflux capacity.
• ΔhpnE mutant: hydroxysqualene oxidoreductase knockout, disrupts both hopanoid and carotenoid (C₃₀) biosynthesis.
• Methyl-β-cyclodextrin loading: used to deplete or load hopanoids/cholesterol into purified OM fractions, demonstrating reversibility and functional interchangeability.
• Membrane fractionation: gradient centrifugation separates OM from inner membrane fractions for independent biophysical analysis.