Low membrane fluidity triggers lipid phase separation and protein segregation in living bacteria

All living organisms adapt their membrane lipid composition in response to changes in their environment or diet. These conserved membrane‐adaptive processes have been studied extensively. However, key concepts of membrane biology linked to regulation of lipid composition including homeoviscous adaptation maintaining stable levels of membrane fluidity, and gel‐fluid phase separation resulting in domain formation, heavily rely upon in vitro studies with model membranes or lipid extracts. Using the bacterial model organisms Escherichia coli and Bacillus subtilis , we now show that inadequate in vivo membrane fluidity interferes with essential complex cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Furthermore, we demonstrate that very low membrane fluidity is indeed capable of triggering large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living cells; a process that coincides with the minimal level of fluidity capable of supporting growth. Importantly, the in vivo lipid phase separation is not associated with a breakdown of the membrane diffusion barrier function, thus explaining why the phase separation process induced by low fluidity is biologically reversible. SYNOPSIS Key concepts of membrane biology linked to regulation of lipid composition have been predominantly assessed in vitro via model membranes or lipid extracts. Here, living bacteria are found to be surprisingly tolerant towards changes in membrane fluidity, thus questioning the dogma that careful regulation of membrane fluidity is critical for supporting general activities of membrane‐associated processes. Low membrane fluidity triggers reversible, large‐scale lipid phase separation and protein segregation in intact, protein‐crowded membranes of living bacteria. In vivo gel‐fluid phase separation determines the minimal level of fluidity capable of supporting growth, but is not associated with a breakdown of the membrane diffusion barrier function. Lipid phase separation drives segregation of membrane proteins into the fluid phase and severely limits and confines lateral diffusion of membrane proteins. Very low levels of membrane fluidity interfere with essential cellular processes including cytokinesis, envelope expansion, chromosome replication/segregation and maintenance of membrane potential. Graphical Abstract Essential cellular processes in bacteria, includ