Anhydrobiotic engineering aims to increase the level of desiccation tolerance in sensitive organisms to that observed in true anhydrobiotes. In addition to a suitable extracellular drying excipient, a key factor for anhydrobiotic engineering of gram-negative enterobacteria seems to be the generation of high intracellular concentrations of the nonreducing disaccharide trehalose, which can be achieved by osmotic induction. In the soil bacterium Pseudomonas putida KT2440, however, only limited amounts of trehalose are naturally accumulated in defined high-osmolarity medium, correlating with relatively poor survival of desiccated cultures. Based on the enterobacterial model, it was proposed that increasing intracellular trehalose concentration in P. putida KT2440 should improve survival. Using genetic engineering techniques, intracellular trehalose concentrations were obtained which were similar to or greater than those in enterobacteria, but this did not translate into improved desiccation tolerance. Therefore, at least for some populations of microorganisms, trehalose does not appear to provide full protection against desiccation damage, even when present at high concentrations both inside and outside the cell. For P. putida KT2440, it was shown that this was not due to a natural limit in desiccation tolerance since successful anhydrobiotic engineering was achieved by use of a different drying excipient, hydroxyectoine, with osmotically preconditioned bacteria for which 40 to 60% viability was maintained over extended periods (up to 42 days) in the dry state. Hydroxyectoine therefore has considerable potential for the improvement of desiccation tolerance in sensitive microorganisms, particularly for those recalcitrant to trehalose.Disaccharides and other polyols have been shown to be highly effective stabilizers of dried biological molecules and membranes in vitro, and the protection conferred by trehalose, in particular, has attracted considerable attention (7, 9, 10). Trehalose is also thought to be crucial for the survival of many anhydrobiotic organisms, which are able to maintain viability throughout long periods in a dried state (reviewed in reference 8). It is therefore reasonable to suppose that trehalose (or related molecules, such as sucrose) could be used to improve the desiccation tolerance of otherwise sensitive organisms, and several groups have demonstrated this for microorganisms (2,17,21,23,37).However, the stability of the dried bacteria in these experiments falls far short of that observed in anhydrobiotic organisms. For example, Louis et al. (23) showed that Escherichia coli loses between 1 and 4 logs of viability 6 weeks after being dried in 0.5 M trehalose and stored at 4°C. Welsh and Herbert (37) reported maximal survival rates of 4.2 and 6.5% of initial CFU after 50 days at ambient temperature when E. coli was dried in the presence of 0.25 M extracellular trehalose or with a similar concentration of intracellular trehalose, respectively. Billi et al. (2) genetically engineered E. coli to pr...

Anhydrobiotic engineering aims to improve desiccation tolerance in living organisms by adopting the strategies of anhydrobiosis. This was achieved for Escherichia coli and Pseudomonas putida by osmotic induction of intracellular trehalose synthesis and by drying from trehalose solutions, resulting in long-term viability in the dried state.Organisms able to undergo anhydrobiosis survive the loss of essentially all their water, assuming metabolic dormancy in the dried state and resuming normal functions on rehydration. When dry, such organisms are highly resistant to environmental challenge (2, 7). Although bacteria exhibit variable degrees of desiccation tolerance (9), relatively few genera are recognized as anhydrobiotic, the major exceptions being among the cyanobacteria (10). The ability to confer similar desiccation tolerance on otherwise desiccation-sensitive microorganisms, termed anhydrobiotic engineering (6), has numerous potential biotechnological applications. Studies of anhydrobiosis in baker's yeast suggest that both synthesis and export of the disaccharide trehalose are of crucial importance (2, 5). Since many bacteria accumulate trehalose under certain hyperosmotic culture conditions (11), they are ideally suited for anhydrobiotic engineering. One recent study has shown that osmotically induced trehalose synthesis can increase the rate of survival of desiccation for Escherichia coli (13). In this paper, we demonstrate that when trehalose is present both inside and outside the cell, desiccation tolerance and long-term stability of both E. coli and Pseudomonas putida can be comparable to those of anhydrobiotic organisms.E. coli MC4100 was grown in M9 medium with 1% glucose and trace elements (0.015 mM FeSO 4 , 0.015 mM ZnSO 4 , and 0.015 mM MnSO 4 ), with or without osmotic stress (0.6 M NaCl), and harvested in growth and stationary phases. Intracellular trehalose was measured by gas chromatography, essentially as described previously (12); concentrations were calculated with reference to CFU. Stressed E. coli in growth phase contained the highest level of trehalose (230 g/10 9 CFU in a typical experiment), while stressed cells from stationary phase contained somewhat less (150 g/10 9 CFU). Unstressed cells from either growth phase or stationary phase did not contain detectable amounts of trehalose (Ͻ0.5 g/10 9 CFU).P. putida has been reported to accumulate mannitol as the main compatible solute (8). A study of the compatible solute profile of P. putida KT2440 grown in high-salt medium identical to that used for E. coli, except that 0.4 M NaCl was used, has demonstrated high concentrations of mannitol in early growth phase, which decrease rapidly thereafter. Trehalose is produced in high-salt cultures of P. putida, although not until

Trehalose has been shown to play a role in osmotolerance or desiccation tolerance in some microorganisms, anhydrobiotic invertebrates and resurrection plants. To test whether trehalose could improve stress responses of higher eukaryotes, a mouse cell line was genetically engineered to express bacterial trehalose synthase genes. We report that the resulting levels of intracellular trehalose (V V80 mM) are able to confer increased resistance to the partial dehydration resulting from hypertonic stress, but do not enable survival of complete desiccation due to air drying. ß

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