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Applied and Environmental Microbiology, September 2002, p. 4328-4333, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4328-4333.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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

M. Manzanera,1,{dagger} A. García de Castro,1 A. Tøndervik,2 M. Rayner-Brandes,3 A. R. Strøm,2 and A. Tunnacliffe1*

Institute of Biotechnology, University of Cambridge, Cambridge CB2 1QT,1 Merck Chemicals Ltd., Newport NP10 8UL, United Kingdom,3 Department of Biotechnology, Norwegian University of Science and Technology, N-7491 Trondheim, Norway2

Received 27 February 2002/ Accepted 7 June 2002

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.

* Corresponding author. Mailing address: Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, United Kingdom. Phone: 44-1223-766549. Fax: 44-1223-334162. E-mail: at10004{at}biotech.cam.ac.uk.

{dagger} Present address: Department of Genetics, University of Cambridge, Cambridge CB2 3EH, United Kingdom.

Applied and Environmental Microbiology, September 2002, p. 4328-4333, Vol. 68, No. 9
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.9.4328-4333.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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