Fluidity is the way to life: lipid phase separation in bacterial membranes

A hallmark of biological membranes is the dynamic localization of lipids and proteins. Lipids respond to temperature reduction below a critical point with phase separation, and poikilothermic animals and also bacteria adapt their lipid content to prevent gel phase formation in membranes. In a new study, Gohrbandt et al (2022) show that reduced membrane fluidity in bacterial cells causes reversible phase separation without membrane rupture in vivo, highlighting the physical robustness of biological membranes.

A new study provides a first example of a co‐existing gel/fluid phase separation in a bacterial cell.

Membranes are complex assemblies of diverse lipid species and proteins, which regulate the exchange of information and content between the cell and the environment. A functional composition of the plasma membrane is essential for cell survival and the regulation of its lipid composition has profound effects on membrane protein function and localization. The molecular mechanisms underlying this regulation are subject of intense research and require insights into the physicochemical properties of lipids. Lipids are amphipathic molecules with hydrophobic fatty acid acyl chains and a polar head group. The acyl chains can be saturated (SFA), unsaturated (UFA), or branched (BCFA) and this diversity has direct effects on their physical properties. Phospholipids with saturated acyl chains are densely packed and form non‐fluid gel phases, characterized by restricted lipid mobility (S_o domains). Lipids with unsaturated or branched chain fatty acids form liquid fluid membranes.

All cells adapt their membrane lipid composition to changing temperatures (or other stressors) by modulating the degree of fatty acid saturation/unsaturation in a head‐group‐specific process termed homeoviscous adaption (Ernst et al, 2016; Chwastek et al, 2020). Usually, biological membranes exist in liquid phases, where lipids are highly dynamic. If the surrounding temperature falls below a critical value, the membrane rigidifies into a gel state with drastically reduced lipid dynamics. It is assumed that gel states do not support membrane protein function and therefore, are not found in living cells. However, a liquid–liquid demixing of membranes in living cells is observed. This demixing can occur when saturated and unsaturated acyl chains, as well as sterols/hopanoids, are present in the membrane. Sterols and their bacterial surrogates, hopanoids, allow demixing below a miscibility transition temperature into liquid ordered (L_o) domains, which exhibit reduced lipid mobility, but retain lateral fluidity of the membrane. Lipids with unsaturated, straight acyl chains sort into these L_o regions, while lipids with unsaturated, branched, or shorter fatty acids cluster in liquid disordered (L_d) domains of the membrane. This phase separation is observed in model membranes, but has recently also been shown in vivo, for example, in the vacuolar membrane of yeast (Leveille et al, 2022).

L_o domain formation has also been associated with lipid raft formation. Lipid rafts have been controversially discussed and proving their existence in vivo has been particularly challenging, as their size is below the diffraction limit of light and hence, difficult to observe microscopically. However, in recent years, evidence for lateral demixing of membranes into nanoscale domains has, indeed, been found in Bacillus subtilis (Nickels et al, 2017). A functional relevance for lipid heterogeneity and L_o domain formation is likely a stabilizing effect in response to temperature changes and other membrane destabilizing environmental conditions (Nickels et al, 2019).

The high complexity of membrane lipid composition has been a major obstacle to analyze membrane viscosity, lipid packing and phase behavior in vivo. So far, phase separation has been tested mainly by using in vitro settings with synthetic membranes or membrane vesicles derived from cells. In a new publication, the laboratories of Gabriele Deckers‐Hebestreit and Henrik Strahl provide the experimental framework to study membrane phase separation in two bacterial model organisms (Gohrbandt et al, 2022). They use genetically modified strains of gram‐positive B. subtilis and the gram‐negative E. coli to meticulously manipulate the lipid composition. The authors generated a B. subtilis strain, in which the enzymes for branched chain fatty acid synthesis have been deleted (Δbkd). Synthesis of BCFAs can then be fine‐tuned by external addition of precursor molecules. Furthermore, the desaturase gene (des) was deleted to prevent fluidity adaption. For experiments in E. coli, they used a temperature‐sensitive FabA mutant. FabA is critically involved in the generation of unsaturated lipid acyl chains. Growth of these bacteria under conditions in which lipid adaption cannot take place leads to changes in membrane lipid composition and fluidity.

In the current study, synthetic reduction of membrane fluidity resulted in a remarkable phase separation into gel phase and fluid lipid domains. These data are the first example of a co‐existing gel/fluid phase separation in a bacterial cell. Importantly, this phase transition is reversible, supporting the notion that gel phase separation does not result in membrane rupture and hence does not lead to immediate cell death. The authors show that the permeability barrier function of the membrane is retained despite this drastic lipid demixing. However, membrane proteins are excluded from the gel phase areas and instead segregated into the liquid membrane regions. The crowding of membrane proteins in these domains impairs their function, such that crucial cellular processes, including cell wall synthesis, cell division, chromosome segregation, and energy production, are disturbed. Overall, a significant drop in membrane potential was observed under conditions of low membrane fluidity, which could be explained by reduced diffusion of the membrane integral electron carrier ubiquinone or non‐functional respiratory chain proteins. Thus, depolarization of the membrane at low fluidity was not triggered by increased ion permeability across the membrane, but rather changes in membrane protein function. It is known that proteins binding with amphipathic helices to the membrane prefer liquid fluid (L_d) regions that facilitate insertion of the helix into the lipid head groups. Many proteins involved in cell elongation, such as MreB, or in cytokinesis, such as MinD, or FtsA are localized via such a membrane interaction (Strahl et al, 2014). Altered phase separation has, therefore, immediate effects on cell wall synthesis and cell division.

Despite the drastic phenotypes induced by lowering the fluidity of bacterial membranes, these experiments also provide stunning evidence for the robustness of biological membranes and the resilience of bacteria against environmental changes. A fascinating question is, therefore, why membrane fluidity is so tightly regulated and poised at a critical point. One answer may be that systems close to critical points are able to react fast and efficiently to any changes. Interestingly, membrane fluidity may also be regulated by specialized proteins. In vivo and in vitro analyses, including solid state NMR experiments, have recently revealed that bacterial flotillins increase the dynamics of the lipid acyl chains within the membrane bilayer, thereby fluidizing the membrane (Bach & Bramkamp, 2013; Zielinska et al, 2020). Apparently, bacteria have evolved a multilayered tool‐set that maintains membrane fluidity by modulating lipid composition and also protein–lipid interactions. The fact that not all of these factors are triggered by temperature changes suggests that membrane fluidity is not exclusively required for temperature adaption, but also to maintain diverse properties under varying nutrient and environmental conditions.

The bacterial strains constructed by Gohrbandt and colleagues in their landmark study will be a useful resource for many further experiments aiming at deciphering the role of membrane fluidity, lipid diversity, and phase separation. Membrane and lipid research still has many unanswered questions, yet insights into cellular self‐organization are essential for the eventual construction of synthetic cells (Fig 1). Furthermore, the membrane offers an ideal target for antimicrobial substances and precise knowledge about the physicochemical characteristics of bacterial membranes is therefore a prerequisite to understanding the mode of action of novel membrane‐targeting antibiotics (Müller et al, 2016).

Figure 1. Reduction of membrane fluidity triggers phase separation in vivo .

Membranes in bacteria are maintained in a fluid state. Lipid bilayers are mainly in liquid disordered phase (L_d, blue) and may phase separate into nanoscale, hopanoid containing liquid‐ordered phases (green, L_o). Reduction of fluidity by preventing acyl chain modification leads to massive phase separation into gel phase (S_o) phases that cover large parts of the plasma membrane. Gel phase formation pushes membrane proteins such as the ATP‐synthase complex into the small remaining fluid phases, thereby restricting their mobility (arrows). Membrane polarization (proton motive force, pmf) is dissipated under these conditions, but the semi‐permeability and integrity of the membrane remains functional. The process of phase separation is reversible when fluidity is restored. Thus, in vivo gel phase formation is not per se lethal.

The EMBO Journal (2022) 41: e110737.