Energy metabolism response to low-temperature and frozen conditions in Psychrobacter cryohalolentis

Abstract

Studies of cold-active enzymes have provided basic information on the molecular and biochemical properties of psychrophiles; however, the physiological strategies that compensate for low-temperature metabolism remain poorly understood. We investigated the cellular pools of ATP and ADP in Psychrobacter cryohalolentis K5 incubated at eight temperatures between 22 degrees C and -80 degrees C. Cellular ATP and ADP concentrations increased with decreasing temperature, and the most significant increases were observed in cells that were incubated as frozen suspensions (<-5 degrees C). Respiratory uncoupling significantly decreased this temperature-dependent response, indicating that the proton motive force was required for energy adaptation to frozen conditions. Since ATP and ADP are key substrates in metabolic and energy conservation reactions, increasing their concentrations may provide a strategy for offsetting the kinetic temperature effect, thereby maintaining reaction rates at low temperature. The adenylate levels increased significantly <1 h after freezing and also when the cells were osmotically shocked to simulate the elevated solute concentrations encountered in the liquid fraction of the ice. Together, these data demonstrate that a substantial change in cellular energy metabolism is required for the cell to adapt to the low temperature and water activity conditions encountered during freezing. This physiological response may represent a critical biochemical compensation mechanism at low temperature, have relevance to cellular survival during freezing, and be important for the persistence of microorganisms in icy environments.

FIG. 1.

Cellular concentrations of ATP and ADP in P. cryohalolentis K5 at temperatures of −80°C to 22°C after 88 h of incubation. Samples at temperatures of ≥−5°C remained liquid for the duration of the experiment. Errors bars denote the standard deviation from the mean (n = 3). An asterisk indicates a significant difference (P < 0.05) from the value measured at the next warmer (right side of error bar) or colder (left side of error bar) temperature (for ATP, P_{−5°C/−15°C} = 0.01 and P_{−15°C/−20°C} < 0.01; for ADP, P_22°C/15°C = 0.05, P_{−5°C/−15°C} <0.01, and P_{−15°C/−20°C} < 0.01).

FIG. 2.

Time-dependent changes in ATP and ADP concentrations and the ATP/ADP ratio at temperatures between −80°C and 22°C. The cell suspensions in panels A, C, and E were unamended, and those in panels B, D, and F were incubated in the presence of an uncoupler (CCCP). The dashed horizontal line indicates adenylate concentrations and ratios at t = 0, open triangles are measurements after 2.5 h, closed triangles are measurements after 21 h, and circles are measurements after 45 h. As indicated by the vertical dashed line, samples remained liquid at temperatures of −5°C and above. The values plotted are averages of triplicate experiments. Errors are not shown for visual clarity, and the coefficients of variation averaged 18 and 26% for ATP and ADP, respectively.

FIG. 3.

Concentrations of ATP (circles) and ADP (triangles) in cells cultured at 15°C and subsequently transferred to −20°C (representing t = 0). After 78 min of incubation, a final concentration of 1 M NaCl was added to half of the cell suspensions (open symbols) and freezing was induced in the remaining samples (closed symbols after 78 min). Errors bars denote standard deviations from the means (n = 3).

FIG. 4.

Linear relationship (r² = 0.79) between temperature and the inferred rate of cellular ATP accumulation by respiration. Calculations are based on the differences between ATP concentrations in respiring cells and those in cells treated with the uncoupler CCCP between 2.5 and 21 h of incubation.