JB Accepts, published online ahead of print on 30 October 2009

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J. Bacteriol. doi:10.1128/JB.01115-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Thermococcus kodakaraensis mutants deficient in di-myo-inositol phosphate use aspartate to cope with heat stress

Nuno Borges, Rie Matsumi, Tadayuki Imanaka, Haruyuki Atomi, and Helena Santos^*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Rua da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal; Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, 615-8510, Japan

^* To whom correspondence should be addressed. Email: santos{at}itqb.unl.pt.

Many of the marine microorganisms which are adapted to grow at temperatures above 80°C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakaraensis, manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and -glutamate. Under heat stress the level of DIP increased 20-fold as compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, over-expression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85°C) and supraoptimal temperatures (93.7°C) for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakaraensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.