Anhydrobiotic engineering

Castro, Arcadio García de; Lapinski, Jens; Tunnacliffe, Alan

doi:10.1038/75237

Cited by 39 publications

(23 citation statements)

References 4 publications

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“…Further contributions to the debate on the role of disaccharides come from attempts to create anhydrobiotic cell types from desiccation-sensitive cells, an approach we have called 'anhydrobiotic engineering' (Garcǵa de Castro et al 2000b). Current excitement surrounds work on mammalian cells, which are normally sensitive to dehydration: loss of up to 65% of cellular water from cell lines (e.g.…”

Section: Reappraisal Of the Role Of Disaccharidesmentioning

confidence: 99%

Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis

Tunnacliffe

Lapinski

2003

Phil. Trans. R. Soc. Lond. B

184

136

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In 1702, Van Leeuwenhoek was the first to describe the phenomenon of anhydrobiosis in a species of bdelloid rotifer, Philodina roseola. It is the purpose of this review to examine what has been learned since then about the extreme desiccation tolerance in rotifers and how this compares with our understanding of anhydrobiosis in other organisms. Remarkably, much of what is known today about the requirements for successful anhydrobiosis, and the degree of biostability conferred by the dry state, was already determined in principle by the time of Spallanzani in the late 18th century. Most modern research on anhydrobiosis has emphasized the importance of the non-reducing disaccharides trehalose and sucrose, one or other sugar being present at high concentrations during desiccation of anhydrobiotic nematodes, brine shrimp cysts, bakers' yeast, resurrection plants and plant seeds. These sugars are proposed to act as water replacement molecules, and as thermodynamic and kinetic stabilizers of biomolecules and membranes. In apparent contradiction of the prevailing models, recent experiments from our laboratory show that bdelloid rotifers undergo anhydrobiosis without producing trehalose or any analogous molecule. This has prompted us to critically re-examine the association of disaccharides with anhydrobiosis in the literature. Surprisingly, current hypotheses are based almost entirely on in vitro data: there is very limited information which is more than simply correlative in the literature on living systems. In many species, disaccharide accumulation occurs at approximately the same time as desiccation tolerance is acquired. However, several studies indicate that these sugars are not sufficient for anhydrobiosis; furthermore, there is no conclusive evidence, through mutagenesis or functional knockout experiments, for example, that sugars are necessary for anhydrobiosis. Indeed, some plant seeds and micro-organisms, like the rotifer, exhibit excellent desiccation tolerance in the absence of high intracellular sugar concentrations. Accordingly, it seems appropriate to call for a re-evaluation of our understanding of anhydrobiosis and to embark on new experimental programmes to determine the key molecular mechanisms involved.

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Section: Reappraisal Of the Role Of Disaccharidesmentioning

confidence: 99%

Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis

Tunnacliffe

Lapinski

2003

Phil. Trans. R. Soc. Lond. B

184

136

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“…This has been termed anhydrobiotic engineering (9), in reference to anhydrobiotic organisms which naturally exhibit extreme desiccation tolerance (4,6,14). In this report, we describe a new approach to preserving bacteria by drying and then encapsulating the bacteria in plastic, and we demonstrate a potential application as a seed coating.…”

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confidence: 99%

Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida

Manzanera

Vı́lchez

Tunnacliffe

2004

Appl Environ Microbiol

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Escherichia coli and Pseudomonas putida dried in hydroxyectoine or trehalose are shown to be highly resistant to the organic solvents chloroform and acetone, and consequently, they can be encapsulated in a viable form in solid plastic materials. Bacteria are recovered by rehydration after physical disruption of the plastic. P. putida incorporated into a plastic coating of maize seeds was shown to colonize roots efficiently after germination.Previously, we have shown how osmotic preconditioning of bacteria, followed by drying in the presence of glass-forming protectant molecules, such as trehalose or hydroxyectoine, results in a high level of desiccation tolerance, where viability is maintained throughout extended storage periods at above-ambient temperatures (8,12). This has been termed anhydrobiotic engineering (9), in reference to anhydrobiotic organisms which naturally exhibit extreme desiccation tolerance (4,6,14). In this report, we describe a new approach to preserving bacteria by drying and then encapsulating the bacteria in plastic, and we demonstrate a potential application as a seed coating.Glasses, including those derived from organic materials, are high-viscosity liquids that slow molecular diffusion and the rates of chemical reactions, including degradative processes, dramatically (7). Consequently, biological molecules embedded in some organic glasses exhibit a high degree of stability (2, 6). For example, proteins dried in a trehalose glass are protected from denaturation by organic solvents (5, 10). Whether anhydrobiotically engineered bacteria are similarly tolerant of chemical stress, as is observed for some bacterial spores (1, 13), is not known, however. We therefore tested the resistance of dried samples of Escherichia coli and Pseudomonas putida to different pure organic solvents. Growth of E. coli MC4100 and P. putida KT2440 (strains cited in reference 12) in hypersaline minimal medium (HMM), harvesting, and vacuum drying in 1 M trehalose plus 1.5% (wt/vol) polyvinylpyrrolidone (PVP; viscosity enhancer), or 1 M hydroxyectoine plus 1.5% (wt/vol) PVP, were performed as previously described (8, 12). Dried samples of E. coli and P. putida containing approximately 10 8 cells were mixed with 200 l of pure acetone, chloroform, or ethanol and incubated for 5 min. Solvents were then removed under vacuum for 25 to 135 min, depending on the solvent. Control experiments were done using fresh E. coli and P. putida, and survival of solvent-treated bacteria was compared to that of untreated cells, measured by plating a dilution series and colony counting. As expected, survival of nondried E. coli and P. putida was below detection levels. Remarkably, dried bacteria tolerated acetone and chloroform treatment to a high degree, with survival rates above 90% in some instances ( Table 1). The experiment was repeated three times, giving similar rates of survival. However, ethanol treatment of dried cells of both species resulted in very low or undetectable survival. This is consistent with the partial solubili...

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“…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).…”

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Anhydrobiotic Engineering of Gram-Negative Bacteria

Castro

Bredholt

Strøm

et al. 2000

Appl Environ Microbiol

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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

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Anhydrobiotic engineering

Cited by 39 publications

References 4 publications

Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis

Resurrecting Van Leeuwenhoek's rotifers: a reappraisal of the role of disaccharides in anhydrobiosis

Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida

Anhydrobiotic Engineering of Gram-Negative Bacteria

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