STONIER, TOM (Manhattan College, Bronx, N.Y.), ROBERT E. BEARDSLEY, LOWELL PARSONS, AND JAMES MCSHARRY. Agrobacterium tumefaciens Conn. III. Effect of thermal shock on bacteria in relation to tumor-inducing ability. J. Bacteriol. 91: 266-269. 1966.-Bacteria heated to 42 C for 30 min exhibit a decrease in tumorinitiating ability without a detectable loss in viability. The thermal shock inhibits subsequent bacterial growth for up to 1.5 hr. As bacterial growth recovers, so does tumor-initiating ability. Respiration of the culture is somewhat increased by the heat treatment. The data suggest that living, actively respiring bacteria do not induce tumors unless they are also growing. The results also point to the necessity for excluding bacterial growth inhibition when interpreting data on the effect of various agents on tumor initiation. The manner in which the crown gall organism, Agrobacterium tumefaciens Conn, converts normal plant cells into tumor cells has remained a mystery since the demonstration by Braun and White that the tumor cells retain their morphogenetic autonomy even when the bacteria are no longer present (6, 14). The nature of the hypothetical tumor-inducing principle released by the bacteria has remained a matter for speculation (for reviews, see 3, 5, 8). The concept that the tumor-inducing principle is heat-labile was suggested by Braun and Mandle (4), who stated that "inactivation of the tumorinducing principle itself occurs at 32 C." In a subsequent publication, Braun (1) reported that between 27.5 and 28.8 C there was a progressive decrease in the size and weight of the resulting tumors and a delay in the inception period. Tumors were not observed above 29 C. Calculations of the activation energy implied values of the order of 80 kcal/mole. Braun interpreted the results to suggest that "either the tumor-inducing principle itself or something intimately associated with its inactivation is a factor of complex structure." This interpretation of the data was recently questioned by Lipetz (9), who provided evidence to indicate that the response of infected plants to temperature, in the range studied by Braun and Mandle, may reflect effects on the host rather than inactivation of a specific tumorinducing principle. More recently, Lippincott and Lippincott (11) demonstrated that bacteria exposed to heat treatment lose, in part, their ability to produce tumors, without a loss in viability. This finding is of considerable interest, especially in view of the fact that the "results suggest that a first-order, unimolecular event is responsible for the loss of TIA [tumor-initiating ability]." These authors calculated a heat of inactivation of 56.7 kcal/ mole and suggested further that a "particular protein or nucleoprotein active in the process of infection may be inactivated by the treatment." The results presented here confirm the observation of the Lippincotts that bacteria exposed to temperatures in the range 39 to 45 C, for periods of less than 1 hr, exhibit a decrease in tumorinitiating ability without a detectable loss in viability. Our results extend this finding to indicate that the effect of 42 C on the tumor-initiating ability of the bacteria is transient and nonspecific in respect to the tumor-initating process, insofar as the thermal shock temporarily inhibits bacterial growth. MATERIALS AND METHODS All experiments were conducted with strain B6 of A. tumefaciens growing, and treated, in media described by Lippincott and Heberlein (10). Viability counts were obtained in the standard manner by dilution and plating on agar plates (7). Samples and dilution tubes were agitated with a Vortex mixer to reduce 266 on O cber 6, 2017 by gest http/jb.asm .rg/ D ow nladed fom A. TUMEFACIENS THERMAL SHOCK clumping. The tumor-initiating ability of the bacteria was assayed against pinto bean leaves according to the technique described by Lippincott and Heberlein (10). Respiration was measured by following oxygen uptake in a Warburg vessel (12), and by following the reduction of methylene blue in an atmosphere of nitrogen (13). In both instances, the bacterial cultures were supplemented with 0.02 M sucrose in addition to the 0.5% sucrose initially present in the growth medium. All phases of the experiments except the thermal treatments were conducted at 27 C. Temperature treatments consisted of immersion of shaking bottles (containing 30 ml of bacterial cultures) for various periods of time in a hot-water bath kept at 42 C. Control cultures were immersed in a bath kept at 27 C. A 2-mi amount of sample was inoculated into 18 ml of fresh medium immediately after thermal treatment, and growth was measured by following changes in turbidity with a Coleman Jr. spectrophotometer at 600 m,.