Functional diversity of isoprenoid lipids in Methylobacterium extorquens PA1

Rizk, S.; Henke, P.; Santana-Molina, C.; Martens, G.; Gnädig, M.; Nguyen, N. A.; Devos, D. P.; Neumann-Schaal, M.; Sáenz, J. P. (2021) Molecular Microbiology, 116, 1064–1078. DOI: 10.1111/mmi.14794

Summary

Hopanoids and carotenoids are two major isoprenoid-derived lipid classes in prokaryotes, both proposed as sterol surrogates. Using [[methylobacterium-extorquens|Methylobacterium extorquens]] as a model — which produces both in its outer membrane — we genetically disrupted each pathway to compare their contributions to membrane function and cellular fitness. We made the surprising discovery that carotenoid biosynthesis in M. extorquens uses squalene (a C₃₀ backbone) rather than the canonical C₄₀ phytoene-derived pathway, likely acquired through lateral gene transfer from Planctomycetes. Hopanoids, but not carotenoids, proved essential for growth at higher temperatures, maintaining low membrane permeability, and tolerating low divalent cation concentrations.

Key Findings

• Biosynthetic pathway surprise: Deleting phytoene synthase (crtB) did not eliminate pigmentation; deleting hydroxysqualene oxidoreductase (hpnE) did. This revealed that M. extorquens carotenoids have a C₃₀ squalene-derived backbone, not the expected C₄₀ phytoene-derived structure.
• Lateral gene transfer: Phylogenetic analysis suggested the C₃₀ carotenoid pathway was acquired from Planctomycetes via horizontal gene exchange.
• Membrane phenotypes of ΔhpnE: The ΔhpnE mutant (lacking both hopanoids and carotenoids, since both depend on squalene) showed impaired growth at 35 °C, increased membrane permeability, and sensitivity to low divalent cation concentrations — phenotypes consistent with loss of hopanoid-mediated lipid ordering, though carotenoid loss cannot be fully excluded as a contributing factor. The ΔcrtB mutant (which retains both hopanoids and carotenoids via the squalene pathway) showed no major growth or membrane phenotypes and is therefore not informative for separating the two pathways.
• Oxidative stress sensitivity: Carotenoid-deficient cells (ΔhpnE) showed slightly increased sensitivity to oxidative stress, consistent with a photoprotective role for carotenoids.
• Diplopterol content in WT: ~19 arbitrary units (LC-MS), absent in ΔhpnE; squalene accumulates in Δshc (318.4 ± 2.8 units); hydroxysqualene accumulates in ΔhpnE (84.8 ± 10.1 units).

Methods

• Genetic knockouts: Deletion of hpnE (squalene biosynthesis) and crtB (phytoene biosynthesis) in M. extorquens PA1.
• TLC and LC-MS: to verify hopanoid and carotenoid content in mutant strains.
• Absorbance spectrometry: to assess carotenoid pigmentation.
• Growth assays: across temperatures (22–38 °C), divalent cation concentrations, and oxidative stress (H₂O₂).
• Phylogenetic analysis: of C₃₀ carotenoid biosynthetic genes to determine origin.

Significance

This paper dissected the distinct functional roles of two bacterial isoprenoid lipid classes. While both have been proposed as sterol analogues, hopanoids emerged as the critical player for membrane robustness and stress tolerance, with carotenoids playing a more specialized role in oxidative stress protection. The biosynthetic surprise — C₃₀ carotenoids from squalene — highlighted unexpected metabolic flexibility in isoprenoid biosynthesis and provided a cautionary tale about assuming canonical pathways.