We have studied the stress response of a rifampin-resistant mutant of Francisella tularensis LVS. This mutant, Rif 7, was avirulent with an intraperitoneally administered 50% lethal dose greater than 10(7) CFU in a murine model of infection. Exposure of Rif 7 to heat stress for 5 h in vitro resulted in a 2-log decrease in its 50% lethal dose (P < 0.02). The increase in virulence was dependent on the time of exposure to high temperature and was maximal at 5 h. Envelope preparations from heat-stressed cells showed increased levels of several proteins. Notable among these were polypeptides with approximate molecular masses of 16, 60, and 75 kDa. Increases in both virulence and envelope protein levels were reversed when heat-treated cells were subsequently grown at 37 degrees C. Inhibition of protein synthesis by actinomycin D during heat stress blocked the increase in virulence of Rif 7. Cell-free media from the heat-stressed Rif 7 reacted with the whole spectrum of bacterial proteins were not toxic to mice. Hyperimmune serum against Rif 7 reacted with the whole spectrum of bacterial proteins in Western blots (immunoblots), although its reaction with 34- and 45-kDa proteins and two 60- and 75-kDa proteins upregulated during heat stress was weak. Other stress conditions, low iron and low pH, caused similar increases in the virulence of Rif 7. However, examination of the protein profile did not reveal any major common polypeptides induced by different stresses. Heat-treated Rif 7 bacteria were fully able to replicate in macrophages in vitro and in the host tissues, even though heat treatment only partially restored virulence.