Ladderane Phospholipids Form Dense Membranes with Low Proton Permeability

Abstract

Anaerobic ammonium oxidizing (anammox) bacteria form a critical part of the nitrogen cycle by converting ammonium and nitrite to nitrogen gas. Anammox bacteria carry out their metabolism in the anammoxosome, a membrane-bound organelle that is comprised of the unique ladderane lipids. Ladderane lipids, named for the ladder-like structure of fused cyclobutane rings in their fatty acid tails, have not been found anywhere else in nature, suggesting that they play a critical role in the anammox metabolism. It has been hypothesized that ladderane lipids prevent the diffusion of hydrazine (an anammox intermediate), protons, or other species across the anammoxosome membrane. As anammox bacteria have not been grown in axenic culture and grow extremely slowly in enrichment cultures, researchers have not been able to isolate sufficient quantities of pure ladderane lipids to determine the biophysical properties of ladderane membranes. Without knowledge of the physical properties of ladderane lipids or genetic tools for studying the lipids in vivo, their biological function remains unknown. We have developed efficient synthetic routes to naturally occurring ladderane phospholipids and unnatural analogs. We show that ladderane lipids have physical properties that are distinct from conventional straight-chain lipids. Ladderane lipids form dense bilayers with slow lateral diffusion and dense monolayers with low compressibility. By varying the identities of the fatty acid tails, we establish structure-function relationships for the different ladderane structures. These physical properties result in membranes with much slower rates of transbilayer diffusion of protons, which are pumped across the anammoxosome membrane and used to produce ATP. These results suggest that ladderane lipids in the anammoxosome may prevent the dissipation of the proton gradient during the slow anammox metabolism. This role for ladderane lipids in the anammoxosome may partially explain why anammox bacteria evolved such unique lipids.