Environmentally induced lipidome adaptation in the bacterial model organism M. extorquens

Cells, from microbes to man, adapt their membranes in response to the environment to maintain their properties and functions. To adapt, lipid composition is homeostatically regulated to conserve optimal membrane properties. Global patterns of lipidome remodelling are poorly understood, particularly in model organisms with simple lipid compositions that can provide insight into fundamental principles underlying membrane adaptation. Using shotgun lipidomics, we examined the simple yet adaptive lipidome of the plant-associated Gram-negative bacterium Methylobacterium extorquens over varying temperature, hyperosmotic and detergent stress, carbon sources, and cell density. We observed that as few as ten lipids account for 90% of the total changes, thus constraining the upper limit of variable lipids required for an adaptive living membrane. Across all conditions, the highest degree of lipidomic variability was observed for changing growth temperature. We also revealed that variations in lipid structural features are not monotonic over a given range of conditions and are heterogeneous across lipid classes. Interestingly, phosphotidylcholine showed the most extreme acyl chain remodeling among all lipid classes, suggesting a new link to its importance in bacterial-host interactions and pathogenicity. These patterns in lipidomic remodeling suggest a highly adaptive mechanism with many degrees of freedom and constrain the lipidomic requirements for an adaptive membrane.


Introduction 35
All organisms have at least one membrane that is crucial for compartmentalizing and coordinating 36 biochemical processes within the cell. And yet, nearly a century since the discovery that membranes 37 are made of a lipid bilayer (Gorter and Grendel, 1925) and half a century since integral membrane 38 proteins were proposed in the Fluid Mosaic model (Singer and Nicolson, 1972) we still lack 39 varies relative to its abundance, such that even a very low abundance lipid that has a small change in 134 relation to a total lipidome could have a high variance over mean. However, in this study we aimed 135 to characterize variations that would have an impact on the bulk adaptive properties of the membrane. 136 Since small changes in abundance do not have a considerable effect on the bulk adaptive properties 137 of the membrane, we focused on lipids with high absolute changes in abundance. Therefore, we 138 calculated lipid variability as the standard deviation of abundances over a range of conditions. The 139 total lipidomic variability was then calculated as the aggregate standard deviation of relative 140 abundance for all individual lipid species over the range of the various conditions (e.g. temperature, 141 growth stage, etc.) (Figure 3a). This analysis revealed that temperature has by far the largest effect 142 on lipidomic remodeling, with more than 2-fold more variability than any other condition. It is surprising that a membrane destabilizing detergent (Helenius, 1975)

did not result in lipidomic 159
remodeling. This could possibly be attributed to the ability of Gram-negative bacteria to resist 160 chemicals through the robustness imparted by barrier function of lipopolysaccharide at the cell 161 surface and active removal of toxins by multidrug transporters (Nikaido, 1996;2003;Nikaido and 162 Vaara, 1985;Piddock, 2006). The insensitivity of the lipidome to methanol was also unexpected. We then asked how many and which lipid species are highly variable for each set of conditions. We 183 defined the highly variable lipid species as those that account for 90% of the total lipidomic variability 184 within any given condition. By this measure we observed that 10-13 lipids are highly variable over 185 any given condition (Figure 4c), meaning that only around 30% of the lipidome is involved in 186 remodeling during adaptation. Of these 10-13 highly variable lipids, 8 are highly variable over all of 187 the conditions tested. These results revealed that only a fraction of the lipidome is involved in adaptive 188 remodeling and point towards the minimum complexity of lipid species required for membrane 189 adaptation. In principle, it is possible that as few as 8 lipid species could support adaptation over the 190 range of experimental conditions examined in this study. On the other hand, constraining total unsaturation while internally shifting double bonds from PC to 291 CL might be a means of varying intrinsic curvature while maintaining total membrane viscosity. 292 293

Summary and Outlook 294
In this study, we systematically characterized the lipidome of M. extorquens over a range of growth 295 conditions to gain insights into membrane homeostatic adaptation. Countless studies have addressed 296 phospholipid acyl chain composition (e.g. as measured by GC or GC-MS) and have revealed an 297 important role of membrane lipid composition to various phenotypic aspects. However, phospholipid 298 headgroups and acyl chains jointly determine membrane properties. Using shotgun lipidomics, we 299 provide a near-complete lipidome down to the species level. This allowed us to map how structural 300 features of lipids (e.g. head group, saturation, chain length) are recombined to achieve adaptation. 301 We demonstrated that membranes composed of only a few lipid species are capable of adapting 302 specifically to a broad range of conditions, in particular temperature. Additionally, by assessing how lipid structural features are recombined through adaptation, we reveal that even a simple lipidome 304 exhibits complex patterns of remodelling that were previously undefined. We propose that it is 305 essential to assess lipidomic data in terms of the composite structural features conferred by binned 306 combinations of lipids, since the combination of these structural features ultimately dictates the 307 physical state and functionality of membranes. 308 for growth phases is a conceptual drawing. 523 Figure 6 -Supplement 1 Overview of compositional changes across exemplary experimental 524 conditions reveals the lipidome is robust in most growth conditions except in drastic temperature 525 changes when the lipidome is markedly different. 526 Figure 6 -Supplement 2.
Step-wise adaptation of lipids by feature sampled at varying growth 527 temperatures. 528 Figure 6 -Supplement 3.
Step-wise adaptation of lipids by feature sampled at early, late, mid and 529 stationary growth phase. 530 Figure 6 -Supplement 4.
Step-wise adaptation of lipids by feature sampled at varying salt 531 concentrations (NaCl         Step-wise adaptation of diplopterols when changing temperature, growth phase, and salt condition.  Step-wise adaptation of lipids by feature sampled at varying growth temperatures.  Step-wise adaptation of lipids by feature sampled at early, late, mid and stationary growth phase. Step-wise adaptation of lipids by feature sampled at varying salt concentrations (NaCl).