How dehydration with glycerol protects bacteria cell membranes against deleterious effects of supercooling

Abstract

Water crystallization and thawing has long been incriminated in cell death in cryopreservation protocols. We recently studied the role of cold-induced injuries in the occurrence of cell death during freezing of Escherichia coli K12TG1. We showed that extreme cold stress, arising under the supercooling step, is an important cause of cell death in cryopreservation protocols, involving strong cell membrane damages. When glycerol was added in the supercooled medium, cells were protected against cold damages and remained viable. In the present work we examine the mechanisms involved in membrane protection by glycerol. For this purpose, we evaluated the membrane organization of E. coli under cooling at subzero temperatures with or without glycerol. We measured generalized polarization (GP) and lifetime of Laurdan, a membrane marker used to investigate membrane fluidity. Glycerol had no effect on membrane main phase transition under cooling. However, GP values were significantly higher when glycerol was present in the cell suspension at low temperature. Furthermore, lifetime measurements revealed the coexistence of two domains of different orders in cell membranes. Glycerol acted as a buffer on lifetime of the ordered phase, which could explain the stabilizing effect of glycerol on cell membrane upon supercooling.