Membrane physical state as key parameter for the resistance of the gram-negative <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> to hyperosmotic treatments

The survival of Bradyrhizobium japonicum under hyperosmotic treatments achieved at various temperatures was investigated. The bacterial viability was measured at a combination of different levels of osmotic pressure (1.4–49.2 MPa) in glycerol solutions and temperature (4–28°C). Viability was dependent on these two variables, with low temperatures (10 and 4°C) exhibiting a protective effect against exposure to high levels of osmotic pressure. To understand these results, the relation between membrane physical state and structure of whole cells and osmotic shock tolerance of B. japonicum was studied. Membrane physical changes were evaluated by using 1,3-diphenyl-1,3,5-hexatriene (DPH) and Laurdan (6-dodecanoil-2-dimethylaminonaphtelene) as probes. The results showed that the membrane of B. japonicum was subjected to a progressive phase transition from the liquid-crystalline to the gel phase during cooling between 28 and 4°C. Accordingly, under isotonic conditions, the Laurdan GP spectra showed that, in the range 12–28°C, membrane lipids were in the liquid-crystalline phase, and in a gel phase at 4°C. The study of the variation in anisotropy of DPH revealed that cooling cells before the hyperosmotic treatment could induce opposite effects to the fluidizing effect of the hyperosmotic shock. Cell resistance was finally related to modifications of the membrane structure depending on combined effects of cooling and dehydration.