Thermococcus kodakar ensis Mutants Deficient in Di- myo -Inositol Phosphate Use Aspartate To Cope with Heat Stress

Abstract

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 kodakarensis (formerly 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 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, overexpression 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 (93.7°C) temperatures 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. kodakarensis 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.Hyperthermophilic bacteria and archaea isolated from saline environments accumulate unusual organic solutes in response to osmotic as well as heat stress. Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyldi-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27,31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions. The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11,16,17,27,31).

Insights into thermoadaptation and the evolution of mesophily from the bacterial phylum Thermotogae

“…Many temperature studies in the Thermotogae have focused on the accumulation of these organic compounds and polyamines and the elucidation of their biosynthetic pathways in T. maritima and the more moderate thermophile Petrotoga miotherma (Zellner and Kneifel 1993;Jorge et al 2007;Rodrigues et al 2009;Oshima et al 2011;Rodionova et al 2013). Several compatible solutes have so far been found only in thermophiles, including di-myoinositol phosphate, mannosyl-di-myo-inositol phosphate, mannosylglyceramide, and diglycerol phosphate (Borges et al 2010;Gonçalves et al 2012), and novel thermophilic solutes continue to be identified (Jorge et al 2007;Rodrigues et al 2009). However, while these compounds are thermophile-specific and may represent thermophilespecific adaptations, they are not the only compatible solutes used to deal with heat stress.…”

“…Similarly, the compatible solute di-myo-inositol phosphate is thought to be important for heat tolerance in thermophiles and hyperthermophiles (Borges et al 2010). Two key genes involved in the synthesis of this compound (inositol-1-phosphate cytidylyltransferase and di-myo-inositol phosphate phosphate synthase) are suggested to have been laterally transferred from an archaeal lineage to hyperthermophilic marine Thermotoga species, while in other lineages the 2 genes are predicted to have fused before being exchanged among several bacterial and archaeal lineages (Gonçalves et al 2012).…”

“…However, while these compounds are thermophile-specific and may represent thermophilespecific adaptations, they are not the only compatible solutes used to deal with heat stress. When the ability to synthesize di-myo-inositol phosphate was removed from Thermococcus kodakarensis by deleting a key synthesis gene, the growth of this archaeon was unaffected, and aspartate accumulated as an alternative compatible solute (Borges et al 2010). In the Thermotogae, multiple solutes accumulate under stress conditions (Jorge et al 2007;Rodrigues et al 2009).…”

Mannosylglycerate and Di- myo -Inositol Phosphate Have Interchangeable Roles during Adaptation of Pyrococcus furiosus to Heat Stress

“…furiosus is an ideal target organism for investigating the physiological role of MG and DIP because these are the major components of its solute pool, they are produced by de novo synthesis, and, importantly, the organism does not accumulate amino acids or other biomolecules from the external medium to cope with osmotic and heat stress (8). The preferential import of alternative solutes could complicate the interpretation of results, as reported for a DIP-deficient mutant of Thermococcus kodakarensis (15). The biosynthetic pathways of DIP and MG are depicted in Fig.…”

Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis

“…One interesting example involved the in vivo disruption and analysis of a gene encoding reverse gyrase on the T. kodakaraensis genome, which revealed that the gene product functions in adaptation to high temperature, because reverse gyrase is highly conserved only in hyperthermophiles (Atomi et al, 2004). These efficient genetic tools have already been used to investigate several biological systems, such as the glycolysis pathway (Yoshida et al, 2006;Matsumi et al, 2007), natural product biosynthesis (Yokooji et al, 2009;Borges et al, 2010), transcription factors (Kanai et al, 2007(Kanai et al, , 2010, and operon transcription and translation machinery Hirata et al, 2008). However, these useful and powerful techniques have not yet been applied to the analysis of DNA repair.…”