Hopanoid enrichment in a detergent resistant membrane fraction of Crocosphaera watsonii

Sáenz, J. P. (2010) Organic Geochemistry, 41(8), 853–856. DOI: 10.1016/j.orggeochem.2010.05.005

Summary

Hopanoids have long been proposed as bacterial “sterol surrogates,” but their association with membrane lateral organization had not been tested. We extracted [[crocosphaera-watsonii|Crocosphaera watsonii]] membranes with the non-ionic detergent Triton X-100 and showed that bacteriohopanetetrol cyclitol ether (BHT-CE), the principal hopanoid in this organism, is enriched 10-fold in the detergent-resistant membrane (DRM) fraction. Detergent resistance is the established biochemical criterion for association with lipid raft-like domains in eukaryotic cells. This was the first evidence that hopanoids may be associated with putative lipid rafts in bacterial membranes.

Key Findings

• BHT-CE is enriched ~10-fold in the DRM fraction of C. watsonii relative to the detergent-soluble fraction, based on peak area normalized to sucrose buffer volume.
• Bacteriohopanetetrol (BHT), present in trace amounts (~0.1% of BHT-CE abundance), was detected only in the detergent-soluble fraction — not in the DRM — suggesting that hopanoid side-chain structure determines intracellular associations and detergent resistance.
• The differential partitioning of BHT-CE and BHT between DRM and soluble fractions confirms that the TX-100 extraction method separates compositionally distinct membrane components.
• These results are consistent with the hypothesis that hopanoids, like sterols in eukaryotes, are associated with liquid-ordered membrane domains.

Methods

• C. watsonii (WH8501) grown in SN media at 28 °C (14:10 light:dark cycle), harvested by centrifugation and homogenized by French pressure cell.
• Triton X-100 extraction following Lingwood and Simons (2007), with DRM/soluble fractions separated by floatation ultracentrifugation on a sucrose gradient.
• UV absorbance at 275 nm to confirm DRM vs. soluble fraction identity.
• Hopanoid analysis by reversed-phase HPLC-APCI/MSⁿ (Talbot et al., 2003).

Significance

This short paper planted the seed for the entire hopanoid-lipid ordering research program. By demonstrating that a specific hopanoid (BHT-CE) partitions into detergent-resistant domains, it provided the first direct evidence connecting hopanoids to membrane lateral organization — the functional property of sterols that had been hypothesized but never tested for hopanoids. The work was performed during the PhD at MIT-WHOI and set the stage for the biophysical characterization of hopanoid ordering that followed at MPI-CBG (Sáenz et al., 2012, PNAS).