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

Abstract

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...

The ectD Gene, Which Is Involved in the Synthesis of the Compatible Solute Hydroxyectoine, Is Essential for Thermoprotection of the Halophilic Bacterium Chromohalobacter salexigens

“…In vitro studies have suggested that the protective effect of hydroxyectoine against different types of stress is superior to that of ectoine or other compatible solutes. Thus, hydroxyectoine conferred protection to E. coli against osmotic and heat stress (35), and its capacity to provide desiccation tolerance to E. coli and Pseudomonas putida was comparable to that of trehalose (36,37). On the other hand, Halomonas elongata and Streptomyces griseus accumulated hydroxyectoine in response to a temperature upshift, providing the first evidence that hydroxyectoine might function as a thermoprotectant in vivo (35,57).…”

Osmotically Induced Synthesis of the Compatible Solute Hydroxyectoine Is Mediated by an Evolutionarily Conserved Ectoine Hydroxylase

“…Like ectoine, 5-hydroxyectoine serves as a compatible solute in vivo, functions as an osmoprotectant, and exhibits protein stabilizing properties in vitro (22)(23)(24)(25). The ability of a given microorganism to synthesize 5-hydroxyectoine invariably depends on its ability to produce ectoine, suggesting that 5-hydroxyectoine formation occurs either directly from ectoine (26,27) or from one of its biosynthetic intermediates (17).…”

Plastic Encapsulation of Stabilized Escherichia coli and Pseudomonas putida

“…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).…”

“…One factor which can have a detrimental effect on dried microorganisms over the long term is humidity in the environment; increasing moisture content of the dried sample compromises viability. Storage under vacuum or in an inert atmosphere can prevent this (8,12) but is costly and unwieldy. Long-term storage of culture collections and libraries should be facilitated by the plastic encapsulation procedure we describe, since the dried bacteria are isolated from atmospheric conditions but can be recovered easily.…”

“…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.…”