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The compatible solute mannosylglucosylglycerate (MGG), recently identified in Petrotoga miotherma, also accumulates in Petrotoga mobilis in response to hyperosmotic conditions and supraoptimal growth temperatures. Two functionally connected genes encoding a glucosyl-3-phosphoglycerate synthase (GpgS) and an unknown glycosyltransferase (gene Pmob_1143), which we functionally characterized as a mannosylglucosyl-3- phosphoglycerate synthase and designated MggA, were identified in the genome of Ptg. mobilis. This enzyme used the product of GpgS, glucosyl-3-phosphoglycerate (GPG), as well as GDP-mannose to produce mannosylglucosyl-3- phosphoglycerate (MGPG), the phosphorylated precursor of MGG. The MGPG dephosphorylation was determined in cell extracts, and the native enzyme was partially purified and characterized. Surprisingly, a gene encoding a putative glucosylglycerate synthase (Ggs) was also identified in the genome of Ptg. mobilis, and an active Ggs capable of producing glucosylglycerate (GG) from ADP-glucose and D-glycerate was detected in cell extracts and the recombinant enzyme was characterized, as well. Since GG has never been identified in this organism nor was it a substrate for the MggA, we anticipated the existence of a nonphosphorylating pathway for MGG synthesis. We putatively identified the corresponding gene, whose product had some sequence homology with MggA, but it was not possible to recombinantly express a functional enzyme from Ptg. mobilis, which we named mannosylglucosyiglycerate synthase (MggS). In turn, a homologous gene from Thermotoga maritima was successfully expressed, and the synthesis of MGG was confirmed from GDP-mannose and GG. Based on the measurements of the relevant enzyme activities in cell extracts and on the functional characterization of the key enzymes, we propose two alternative pathways for the synthesis of the rare compatible solute MGG in Ptg. mobilis. doi: 10.1128/JB.01424-09

In addition to di-myo-inositol-1,3'-phosphate (DIP), a compatible solute widespread in hyperthermophiles, the organic solute pool of Thermotoga maritima comprises 2.(O.[beta]-D-mannosyl)-di-myo-inositol-1,3'-phosphate (MDIP) and 2.(O-[beta]-D-mannosyl-1,2.0.[beta]-D-mannosyl)-di-myo-inositol-1,3'-phosphate (MMDIP), two newly identified [beta]-l,2-mannosides. In cells grown under heat stress, MDIP was the major solute, accounting for 43% of the total pool; MMDIP and DIP accumulated to similar levels, each corresponding to 11.5% of the total pool. The synthesis of MDIP involved the transfer of the mannosyl group from GDP-mannose to DIP in a single-step reaction catalyzed by MDIP synthase. This enzyme used MDIP as an acceptor of a second mannose residue, yielding the di-mannosylated compound. Minor amounts of the tri-mannosylated form were also detected. With a genomic approach, putative genes for MDIP synthase were identified in the genome of T. maritima, and the assignment was confirmed by functional expression in Escherichia coli. Genes with significant sequence identity were found only in the genomes of Thermotoga spp., Aquifex aeolicus, and Archaeoglobus profundus. MDIP synthase of T. maritima had maximal activity at 95[degrees]C and apparent [K.sub.m] values of 16 mM and 0.7 mM for DIP and GDP-mannose, respectively. The stereochemistry of MDIP was characterized by isotopic labeling and nuclear magnetic resonance (NMR): DIP selectively labeled with carbon 13 at position C1 of the L-inositol moiety was synthesized and used as a substrate for MDIP synthase. This [beta]-1,2-mannosyltransferase is unrelated to known glycosyltransferases, and within the domain Bacteria, it is restricted to members of the two deepest lineages, i.e., the Thermotogales and the Aquificales. To our knowledge, this is the first [beta]-1,2- mannosyltransferase characterized thus far. doi: 10.1128/JB.00598-09

The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Goncalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:L-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and L-myo-inositol-1-phosphate into CDP-L-myo-inositol, as well as the synthesis of DIPP from CDP-L-myo-inositol and L-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and L-myo-inositol-1-P; the DIPP synthase domain used only L-myoinositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-L-myo-inositol and CDP-D-myoinositol, were recognized as alcohol donors. 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 cytidylyitransferase 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.

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 l-phosphate by L-myo-inositol 1-phosphate synthase; (ii) L-myo-inositol 1-phosphate was activated to CDP-inositol 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, 1'X-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.

The accumulation of compatible solutes is studied in the hyperthermophilic bacterium Aquifex pyrophilus as a function of the temperature and the NaCl concentration of the growth medium. The results reveal the presence of alpha- and beta-glutamate, di-mannosyl-di-myo-inositol phosphate, di-myo-imositol phosphate, and a compound identified as 1-glyceryl-1-myo-insoityl phosphate in living systems.

The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and D-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and 'Dehalococcoides ethenogenes.' GpgS had maximal activity at 50[degrees]C. The maximal activity of GpgP was at 50[degrees]C with GPG as the substrate and at 60[degrees]C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.

The environment-dependent multiexponential behavior of N-acetyltyrosinamide (NAYA) and other tyrosine derivatives are revisited. It is demonstrated that observations of double exponential-decays with NAYA is exceptional and restricted to narrow ranges of solvent polarity and viscosity which in turn determine the electron-transfer and the interconversion rate constants, respectively.

A new compatible solute from the hyperthermophile Archaeoglobus fulgidus is discussed. Thermostabilization of proteins by 1,1'- diglycerol phosphate (DGP) has been studied. It was found to be the major solute accumulating mainly in response to salt stress in the archaeon A. fulgidus. Chemical synthesis was reported on because the poor growth yields of A. fulgidus and large amounts of solute required made the organisms not a good source. Indirect evidence for the stereochemistry of the natural solute was found. Rubredoxins and their known structural features are related to this study. DGP is potentially useful as a protein stabilizer in biotech applications.

Nuclear magnetic resonance spectroscopy was used to investigate the effects of salinity and growth temperature on the accumulation of intracellular organic solutes in Thermococcus litoralis, T. celer, T. stetteri and T. zilligii. It was shown that T. celer, T. stetteri and T. litoralis accumulate different solutes in response to two different types of stress. The levels of aspartate, mannosylglycerate and beta-galactopyranosyl-5- hydroxyllysine increased in response to salt stress while the levels of di-myo-inositol-1,1'(3,3')-phosphate responded to temperature stress.