Anhydrobiotic Engineering of Gram-Negative Bacteria

Abstract

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

New isolation method of desiccation-tolerant microorganisms for the bioremediation of arid and semiarid soils

“…Bacteria were grown in M9 minimal medium with PAHs as the sole carbon source at 30°C [21]. When PAHs were used, chips of naphthalene, anthracene, phenanthrene or pyrene were placed on the lid to avoid direct contact with cells; therefore, they were provided in vapour form.…”

Hydroxyectoine Is Superior to Trehalose for Anhydrobiotic Engineering of Pseudomonas putida KT2440

“…Billi et al (2) genetically engineered E. coli to produce up to 25 mM sucrose, and after freeze drying and immediate rehydration, 2.5% of the bacteria were viable. While these survival rates are clearly higher than those of controls, they do not match those of anhydrobiotic microorganisms such as Deinococcus radiodurans, for which in excess of 60% survival is observed after a 6-week storage period at ambient temperature (27).Anhydrobiotic engineering aims to achieve levels of desiccation tolerance in sensitive organisms which are comparable to those of anhydrobiotes (15,16). This has been demonstrated for the gram-negative enterobacteria E. coli and Salmonella enterica serovar Typhimurium, for which between 50 and 80% survival after vacuum drying has been maintained over storage periods of up to 6 weeks at above-ambient temperatures (4, 15; A. G. Tunnacliffe, D. T. Welsh, B. J. Roser, K. S. Dhaliwal, and C. Colaço, 1996, PCT patent application WO9824882A1).…”

“…A crucial element of this success is thought to be the presence of high concentrations of trehalose both inside and outside the cell prior to drying (35). Growth in defined medium of high osmolarity can result in intracellular concentrations of trehalose between 190 and 400 mM in E. coli and S. enterica serovar Typhimurium (4,13,16,20,36), in which the disaccharide is used as a compatible solute (reviewed in reference 33). This has been exploited for the anhydrobiotic engineering of these enterobacterial species, which are first osmotically preconditioned by growth in an appropriate medium and then dried in a trehalose solution.…”

“…Anhydrobiotic engineering aims to achieve levels of desiccation tolerance in sensitive organisms which are comparable to those of anhydrobiotes (15,16). This has been demonstrated for the gram-negative enterobacteria E. coli and Salmonella enterica serovar Typhimurium, for which between 50 and 80% survival after vacuum drying has been maintained over storage periods of up to 6 weeks at above-ambient temperatures (4, 15; A. G. Tunnacliffe, D. T. Welsh, B. J. Roser, K. S. Dhaliwal, and C. Colaço, 1996, PCT patent application WO9824882A1).…”

“…A similar approach with the gram-negative soil bacterium Pseudomonas putida KT2440 was, however, less successful, since long-term viability after drying was Յ20% that of nondried controls (16). This was attributed to the fact that although trehalose is synthesized by P. putida during growth in high-osmolarity defined medium, the intracellular concentration never naturally exceeds 50 mM.…”