The functionality of cellular membranes relies on the molecular order imparted by lipids. In eukaryotes, sterols such as cholesterol modulate membrane order, yet they are not typically found in prokaryotes. The structurally similar bacterial hopanoids exhibit similar ordering properties as sterols in vitro, but their exact physiological role in living bacteria is relatively uncharted. We present evidence that hopanoids interact with glycolipids in bacterial outer membranes to form a highly ordered bilayer in a manner analogous to the interaction of sterols with sphingolipids in eukaryotic plasma membranes. Furthermore, multidrug transport is impaired in a hopanoid-deficient mutant of the gram-negative Methylobacterium extorquens, which introduces a link between membrane order and an energy-dependent, membrane-associated function in prokaryotes. Thus, we reveal a convergence in the architecture of bacterial and eukaryotic membranes and implicate the biosynthetic pathways of hopanoids and other order-modulating lipids as potential targets to fight pathogenic multidrug resistance.

Liquid-ordered phases are one of two biochemically active membrane states, which until now were thought to be a unique consequence of the interactions between eukaryotic membrane lipids. The formation of a liquid-ordered phase depends crucially on the ordering properties of sterols. However, it is not known whether this capacity exists in organisms that lack sterols, such as bacteria. We show that diplopterol, the simplest bacterial hopanoid, has similar properties and that hopanoids are bacterial "sterol surrogates" with the ability to order saturated lipids and to form a liquid-ordered phase in model membranes. These observations suggest that the evolution of an ordered biochemically active liquid membrane could have evolved before the oxygenation of Earth's surface and the emergence of sterols.lipid order | polycyclic isoprenoids | bacterial membrane organization | membrane phase evolution | organic geochemistry T he capacity for sterols to modulate the ordering of lipids forms the basis for a membrane organizing principle in eukaryotes (1). The emergence of sterol-like ordering was likely a critical step in the evolution of biological membranes, allowing cells to control fluidity without compromising membrane integrity and providing a means to compartmentalize membranes into functional domains (2-4). It is not known, however, to what extent such membrane-ordering properties span the domains of life. Prokaryotes generally lack sterols; however, some bacteria produce hopanoids (5, 6), which are structurally similar (Fig. 1A) (7), and their cyclization is catalyzed by related enzymes (8). These similarities inspired the hypothesis that hopanoids are bacterial sterol surrogates (9) and led us to examine whether hopanoids might share the properties of sterols in membranes. Results and DiscussionEffects of Cholesterol and Diplopterol on the Phase Behavior and Ordering of Sphingomyelin in Model Membranes. Sterols and sphingolipids are closely associated in eukaryotic membranes, and the nature of their interactions has been extensively characterized. Therefore, we chose to test whether diplopterol behaves similarly to cholesterol in this well-defined system. Sterols interact with sphingolipids in vitro to form a liquid-ordered (L o ) phase that represents a thermodynamic intermediate between liquid-disordered (L d ) and crystalline gel phases (Fig. 1B) (10). The interactions leading to the formation of a L o phase derive from the ability of sterols to simultaneously inhibit the formation of the gel phase (by intercalating between sphingolipids and preventing their crystallization) and to order saturated acyl chains. To test whether these properties are also exhibited by hopanoids, we examined the effect of diplopterol on N-stearoyl-D-erythro-sphingosylphosphorylcholine (SM; Fig. S1), a synthetic sphingolipid.Monolayer experiments provide an approach to study the gelliquid phase transition of SM. Lipids are spread out over an airwater interface to form a monolayer and lateral pressure (measured as surface tension) is...

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Highlights d 3D spatial resolution of a fully dynamical whole-cell kinetic model d Detailed single-reaction, single-cell accounting of timedependent ATP costs d Genome-wide mRNA half-lives emerge from lengthdependent kinetics and diffusion d Connections among metabolism, genetic information, and cell growth are revealed

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Highlights d We examine the lipidome of the Gram-negative bacterium Methylobacterium extorquens d At least 11 out of 27 total lipid species contribute to adaptation to varying conditions d Remodeling of acyl chains is unevenly distributed across all lipid classes d Headgroup-specific acyl chain remodeling is implicated as an adaptive mechanism