Lipid phase separation impairs membrane thickness sensing by the sensor kinase DesK.

Unlabelled: Membrane fluidity and thickness have emerged as crucial factors for the activity of and resistance to several antimicrobials. However, the lack of tools to study membrane fluidity and, in particular, thickness in living bacteria limits our understanding of this interplay. The histidine kinase/phosphatase DesK is a molecular sensor that directly detects membrane thickness. It controls activity of DesR, which regulates expression of the lipid desaturase Des, known for its role in cold adaptation and daptomycin susceptibility. We hypothesized that this property could be exploited to develop biosensors and reporters for antibiotic-induced changes in membrane fluidity and thickness. To test this, we designed three assays based on the system: activation of the promoter as reporter for membrane thickening, localization of DesK-GFP(green-fluorescent protein) as proxy for rigidified membrane domains, and antibiotic sensitivity of , , and deletion mutants as readout for the importance of membrane rigidification/thickening under the tested condition. While we could not confirm the suitability of the system as reporter for antibiotic-induced changes in membrane thickness, we did observe that expression is only activated by mild temperature shocks, likely due to partitioning of the sensor DesK into fluid membrane domains upon phase separation, precluding effective thickness sensing under harsh cold shock and antibiotic stress conditions. Similarly, we did not observe any sensitivity of the deletion mutants to either temperature or antibiotic stress, raising the question to what extent the system contributes to fluidity adaptation under these conditions.

Importance: The des system is a prime model for direct molecular membrane thickness sensor and, as such, has been well studied . Our study shows that our understanding of its function and its importance under temperature and antibiotic stress is still very limited. Specifically, our results suggest that (i) the system senses very subtle membrane fluidity changes that escape detection by established fluidity reporters like laurdan; (ii) membrane thickness sensing by DesK is impaired by phase separation due to partitioning of the protein into the fluid phase; and (iii) fluidity adaptations by Des are too subtle to elicit growth defects under rigidifying conditions, raising the question of how much the system contributes to adaptation of overall membrane fluidity.