Cells of Escherichia coli mounted on a hydrophobic filter membrane were dried under various vapor pressures. A mutant defective in deoxyribonucleic acid repair (uvrA recA) was more sensitive to drying at a water activity of 0.53 or below than the parent strain but not at a water activity of 0.75 and above. Sucrose gradient studies showed that single-and double-strand breaks of deoxyribonucleic acid occurred at a water activity of 0.53 or below, but no breaks could be observed at a water activity of 0.75 or above. These results were observed in all cells rehydrated with 0.03 M tris(hydroxymethyl)aminomethane-hydrochloride buffer solution at 0 or 37°C, in the presence or absence of oxygen, with saturated water vapor or with a hypertonic solution followed by a gradual dilution. Freezable water was detected in the cells only at a water activity above 0.75 by differential scanning calorimetry. Removal of unfreezable water of cells in the drying, therefore, might induce deoxyribonucleic acid strand breaks.

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Drying of Escherichia coli to a required cellular water level was conducted on a hydrophobic membrane at the corresponding relative humidity. Mutation from an arginine auxotroph to the prototroph was induced by drying to a water activity (au,) of 0.53 and below, but not to an a,, of 0.75 and above. The critical a,,. below which mutation occurred in the course of drying was similar to that for induction of deoxyribonucleic acid (DNA) strand breakage in the bacteria. Some ultraviolet or-y-irradiation-sensitive strains, e.g., strains carrying recA, recB, and uvrA recA were more sensitive to drying than the wild-type strains or strains carrying uvrA and polA. The DNA strand breakage of every strain was observed to be to a similar extent after drying to an a,,. of <0.53. The drying-resistant strains repaired the damaged DNA partially during postdrying incubation in a growth medium but not in phosphate buffer solution, while the drying-sensitive strains could not at all. Significant mutation on drying occurred in the wild-type strains, strains carrying uvrA andpolA, but not in strains carrying recA. It is, therefore, concluded that the mutation is caused by errors in rec-dependent repair of the dryinginduced breakage in DNA. Induction of mutation by several methods of drying has been found in various bacteria and yeasts (2, 5, 7, 12, 14, 18), but no detailed work has been done on the cause of mutation except for some on freeze-drying (2, 9, 14). We devised a new method of drying, in which bacteria on a hydrophobic membrane were equilibrated quickly at a given relative humidity (RH) being dried to the corresponding water activity (a,,). Using this method we found in a previous work (1) that single-strand breaks of deoxyribonucleic acid (DNA) were induced at an a, of 0.53 and below during the drying of Escherichia coli, depending on the final cellular water level, aw. Mutagenesis in bacteria is generally thought to occur as a result of errors in the repair of damaged DNA (20, 21). Therefore, such DNA breaks induced by drying might be the cause of induction of mutation if the breakage was the only damage to DNA. To obtain positive proof for the hypothesis, we investigated, in this work, the a,, dependency of induction of mutation and the repair mechanism of strand breaks of DNA. MATERIALS AND METHODS Bacterial strains. E. coli F3294 (wild type for repair, argF), F3295 (uvrA hcr argF), F3296 (polA argF), and F3297 (recA phr argF), were used in the mutation experiments.

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