Chwastek, G. | bioRxiv Science

bioRxiv2019

New method for high-throughput measurements of viscosity in submicrometer-sized membrane systems

In order to unravel the underlying principles of membrane adaptation in small systems like bacterial cells, robust approaches to characterize membrane fluidity are needed. Currently available relevant methods require advanced instrumentation and are not suitable for high throughput settings needed to elucidate the biochemical pathways involved in adaptation. We developed a fast, robust, and financially accessible quantitative method to measure microviscosity of lipid membranes in bulk suspension using a commercially available plate reader. Our approach, which is suitable for high-throughput screening, is based on the simultaneous measurements of absorbance and fluorescence emission of a viscosity-sensitive fluorescent dye DCVJ incorporated into a lipid membrane. We validated our method using artificial membranes with various lipid compositions over a range of temperatures and observed values that were in good agreement with previously published results. Using our approach, we were able to detect a lipid phase transition in the ruminant pathogen Mycoplasma mycoides.

biophysics↗

bioRxiv2019

Minimal requirements for membrane adaptation of 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.

biochemistry↗