Hydroxyectoine is superior to trehalose for anhydrobiotic engineering of Pseudomonas putida KT2440.

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.

Assessing chemical toxicity with the bioluminescent photobacterium (Vibrio fischeri): a comparison of three commercial systems.

The inhibition of light emitted by the bioluminescent bacterium, Vibrio fischeri, is the basis for several toxicity bioassays. The inhibitory effects of 81 chemicals, after 5 min contact time, were studied at eight concentrations using reagents from three commercial assay systems (ToxAlert 10, Microtox and LUMIStox). Solubility in water was the limiting factor in determining the selection of chemicals for study. The effective nominal concentrations (EC) resulting in 20, 50 and 80% inhibition were determined using Ln dose/Ln gamma plots and the results obtained for each system were compared by linear regression. The chemical concentrations producing 10-90% inhibition extended over 9 orders of magnitude and ranged from a minimum of 0.001 ppm to a maximum of 1,000,000 ppm. The toxicity of many chemicals was apparently related to their pH in solution and at high chemical concentrations, to osmotic imbalance. The fact that the same operator tested the same solutions simultaneously on three different systems reduced sources of error and variability and improved the consistency and reliability of the results. Only five compounds gave EC 50s that varied more than three-fold between assays. These data provide comparisons of toxicity that have not been previously available and demonstrate that, when used under standardised conditions, these bioluminescence-based toxicity assays produce very similar results.