Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (L-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using L-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.
The halotolerant green alga Dunaliella salina accumulates large amounts of beta-carotene when exposed to various stress conditions. Although several studies concerning accumulation and biotechnological production of beta-carotene have been published, the molecular basis and regulation of the genes involved in carotenoid biosynthesis in D. salina are still poorly known. In this paper, we report the isolation and regulation of the lycopene beta-cyclase (Lcy-beta) gene by abiotic stress. The function of this gene was determined by heterologous genetic complementation in E. coli. Gene expression and physiological analyses revealed that D. salina Lcy-beta steady-state transcript and carotenoid levels were up-regulated in response to all stress conditions tested (salt, light and nutrient depletion). The results presented here suggest that nutrient availability is a key factor influencing carotenogenesis as well as carotenoid biosynthesis-related gene expression in D. salina.
The accumulation of compatible solutes was studied in the hyperthermophilic bacterium Aquifex pyrophilus as a function of the temperature and the NaCl concentration of the growth medium. Nuclear magnetic resonance analysis of cell extracts revealed the presence of ␣-and -glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-inositol phosphate, and an additional compound here identified as 1-glyceryl-1-myo-inosityl phosphate. All solutes accumulated by A. pyrophilus are negatively charged at physiological pH. The intracellular levels of di-myo-inositol phosphate increased in response to supraoptimal growth temperature, while ␣-and -glutamate accumulated in response to osmotic stress, especially at growth temperatures below the optimum. The newly discovered compound, 1-glyceryl-1-myo-inosityl phosphate, appears to play a double role in osmo-and thermoprotection, since its intracellular pool increased primarily in response to a combination of osmotic and heat stresses. This work also uncovered the nature of the unknown compound, previously detected in Archaeoglobus fulgidus (L. O. Martins et al., Appl. Environ. Microbiol. 63:896-902, 1997). The curious structural relationship between diglycerol phosphate (found only in Archaeoglobus species), di-myoinositol phosphate (a canonical solute of hyperthermophiles), and the newly identified solute is highlighted. This is the first report on the occurrence of 1-glyceryl-1-myo-inosityl phosphate in living systems.The accumulation of low-molecular-mass organic compounds (compatible solutes) is a common strategy among organisms isolated from saline environments to cope with osmotic stress (4). Hyperthermophiles, organisms adapted to grow at high temperature (near 100°C), are unable to cope with high salinity (more than ϳ7% NaCl). Many, however, thrive in niches of hot seawater and often also use organic solutes for osmoregulation. Compatible solutes of hyperthermophiles belong broadly to the same classes of compounds used by mesophiles, i.e., sugars, amino acids, and polyols, but hyperthermophiles tend to accumulate solutes that are rarely or never encountered in mesophiles, such as di-myo-inositol phosphate, diglycerol phosphate, mannosylglycerate, and derivatives of these three compounds (8,20,21). Interestingly, solutes of hyperthermophiles are generally negatively charged, while mesophiles accumulate primarily neutral or zwitterionic molecules. These trends indicate that compatible solutes in hyperthermophiles may play more complex roles than in mesophiles and are probably part of the strategies used by these organisms to protect cellular structures against heat damage.Recent research efforts, especially in the last decade, have noticeably expanded our knowledge about the nature and accumulation profiles of compatible solutes in hyperthermophiles (21). However, the number of hyperthermophilic organisms examined thus far is relatively low and insufficient to provide a clear picture of the chemical diversity and physiological roles of compatible solutes in organisms ad...
The nutritionally versatile soil bacterium Acinetobacter baylyi ADP1 copes with salt stress by the accumulation of compatible solutes, a strategy that is widespread in nature. This bacterium synthesizes the sugar alcohol mannitol de novo in response to osmotic stress. In a previous study, we identified MtlD, a mannitol-1-phosphate dehydrogenase, which is essential for mannitol biosynthesis and which catalyses the first step in mannitol biosynthesis, the reduction of fructose-6-phosphate (F-6-P) to the intermediate mannitol-1-phosphate (Mtl-1-P). Until now, the identity of the second enzyme, the phosphatase that catalyses the dephosphorylation of Mtl-1-P to mannitol, was elusive. Here we show that MtlD has a unique sequence among known mannitol-1-phosphate dehydrogenases with a haloacid dehalogenase (HAD)-like phosphatase domain at the N-terminus. This domain is indeed shown to have a phosphatase activity. Phosphatase activity is strictly Mg(2+) dependent. Nuclear magnetic resonance analysis revealed that purified MtlD catalyses not only reduction of F-6-P but also dephosphorylation of Mtl-1-P. MtlD of A. baylyi is the first bifunctional enzyme of mannitol biosynthesis that combines Mtl-1-P dehydrogenase and phosphatase activities in a single polypeptide chain. Bioinformatic analysis revealed that the bifunctional enzyme is widespread among Acinetobacter strains but only rarely present in other phylogenetic tribes.
Archaeoglobus fulgidus accumulates di-myo-inositol phosphate (DIP) and diglycerol phosphate (DGP) in response to heat and osmotic stresses, respectively, and the level of glycero-phospho-myo-inositol (GPI) increases primarily when the two stresses are combined. In this work, the pathways for the biosynthesis of these three compatible solutes were established based on the detection of the relevant enzymatic activities and characterization of the intermediate metabolites by nuclear magnetic resonance analysis. The synthesis of DIP proceeds from glucose-6-phosphate via four steps: (i) glucose-6-phosphate was converted into L-myo-inositol 1-phosphate by L-myo-inositol 1-phosphate synthase; (ii) L-myo-inositol 1-phosphate was activated to CDPinositol at the expense of CTP; this is the first demonstration of CDP-inositol synthesis in a biological system; (iii) CDP-inositol was coupled with L-myo-inositol 1-phosphate to yield a phosphorylated intermediate, 1,1-di-myo-inosityl phosphate 3-phosphate (DIPP); (iv) finally, DIPP was dephosphorylated into DIP by the action of a phosphatase. The synthesis of the two other polyol-phosphodiesters, DGP and GPI, proceeds via the condensation of CDP-glycerol with the respective phosphorylated polyol, glycerol 3-phosphate for DGP and L-myo-inositol 1-phosphate for GPI, yielding the respective phosphorylated intermediates, 1X,1X-diglyceryl phosphate 3-phosphate (DGPP) and 1-(1X-glyceryl) myo-inosityl phosphate 3-phosphate (GPIP), which are subsequently dephosphorylated to form the final products. The results disclosed here represent an important step toward the elucidation of the regulatory mechanisms underlying the differential accumulation of these compounds in response to heat and osmotic stresses.Archaeoglobus fulgidus is a hyperthermophilic archaeon first isolated from marine hydrothermal vents (1). In addition to this type strain, designated VC-16, a few others belonging to the same species were isolated from hot marine sediments (strain Z) (34) and oil field water (strain 7324) (2). The type strain is able to grow between 60°C and 90°C, with an optimum around 83°C. Like other marine hyperthermophiles, A. fulgidus is slightly halophilic, displaying optimal growth in medium containing 1.9% NaCl (wt/vol) and unable to grow in medium containing more than 5.5% NaCl. Osmoregulation involves accumulation of organic solutes, some of them very unusual (8,14). The solute pool comprises DGP, DIP, minor amounts of glutamate, and the newly discovered GPI (14, 16). The most striking feature is the occurrence of polyol-phosphodiesters, a class of compatible solutes encountered in organisms thriving in hot environments but totally absent in mesophiles (25, 26). Accordingly, a putative thermo-protective function of cell components in vivo was ascribed to them, and their action as protein stabilizers in vitro was confirmed at least for DIP and DGP (13,22,28).DIP was the first of these compounds to be discovered in members of the genus Pyrococcus (28). Since then, DIP accumulation has been repo...
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