ACQUIRED THERMOTOLERANCE AND STRESSED-PHASE GROWTH OF THE EXTREMELY THERMOACIDOPHILIC ARCHAEON METALLOSPHAERA-SEDULA IN CONTINUOUS-CULTURE

The response of an extremely thermoacidophilic archaeon, Metallosphaer a sedula (growth temperature range, 50 to 79 degrees C; optimum temper ature, 74 degrees C; optimum pH, 2.0), to thermal stress was investiga ted by using a 10-liter continuous cultivation system. M. sedula, grow ing at 74 degrees C, pH 2.0, and a dilution rate of 0.04 hr(-1), was s ubjected to both abrupt and gradual temperature shifts in continuous c ulture to determine the responses of cell density levels and protein s ynthesis patterns. An abrupt temperature shift from 74 to 79 degrees C resulted in little, if any, changes in cell density and a small incre ase in total protein per cell. When the culture temperature was shifte d further to 80.5 degrees C, cell density dropped to below 5 x 10(6) c ells/ml from 10(8) cells/ml, leading to washout of the culture. Operat ion at this temperature and slightly higher temperatures, however, cou ld be achieved by exposing the culture to thermal stress more graduall y (0.5 degrees C increments). As a result, stable operation could be m aintained at temperatures of up to 81 degrees C, and the washout tempe rature could be increased to 82.5 degrees C. Continuous culture operat ion at 81 degrees C for 100 h (stressed phase) led to an approximately sevenfold lower steady-state cell density than that observed for oper ation at or below 79 degrees C. However, sodium dodecyl sulfate-polyac rylamide gel electrophoresis analysis (both one and two dimensional) r evealed significantly higher levels (sixfoId increase) of a 66-kDa str ess response protein (MseHSP60), immunologically related to Thermophil ic Factor 55 from Sulfolobus shibatae (J. D. Trent, J. Osipiuk, and T. Pinkau, J. Bacteriol. 172:1478-1484, 1990). If the acclimated culture was returned to a lower temperature (i.e., 74 degrees C), the amount of MseHSP60 returned to levels observed prior to thermal acclimation. Furthermore, when the previously acclimated culture (at 81 degrees C) was shifted back from 74 to 81 degrees C, without going through gradua l acclimation steps, the result was the immediate onset of washout, su ggesting no residual thermotolerance. This study shows that gradual th ermal acclimation of M. sedula could only extend the temperature range of stable growth for this organism by 2 degrees C above its maximal g rowth temperature, albeit at reduced cell densities. Also, this invest igation illustrates the utility of continuous culture for characterizi ng heat shock response and assessing maximum growth temperatures for e xtremely thermophilic microorganisms.