, Volume 17, Issue 1, pp 137–146 | Cite as

Organic solutes in the deepest phylogenetic branches of the Bacteria: identification of α(1–6)glucosyl-α(1–2)glucosylglycerate in Persephonella marina

  • Pedro Lamosa
  • Marta V. Rodrigues
  • Luís G. Gonçalves
  • Jean Carr
  • Rita Ventura
  • Christopher Maycock
  • Neil D. Raven
  • Helena SantosEmail author
Original Paper


The accumulation of organic solutes was investigated in the thermophilic bacteria Persephonella marina and Marinitoga piezophila, two representatives of the deepest lineages in the domain Bacteria. These organisms grow optimally at around 70 °C in medium containing 3 % NaCl. A new disaccharide, accumulating in Persephonella marina, was identified as α(1–6)glucosyl-α(1–2)glucosylglycerate (GGG), by nuclear magnetic resonance. This identification was validated by comparison with the spectra of the compound obtained by chemical synthesis. Besides GGG, the solute pool of Persephonella marina comprised β-glutamate, di-myo-inositol-1,3′-phosphate and 2-O-α-glucosylglycerate. In contrast, amino acids such as α-glutamate, proline and alanine were the dominant components of the solute pool of Marinitoga piezophila and sugar derivatives were absent. The ability of GGG to protect protein structure against heat denaturation was assessed using model proteins. A genomic search for the biosynthetic pathways of known ionic solutes in Aquificales and Thermotogales shows the inability of this analysis to predict the nature of compatible solutes and underlines the need for efficient cultivation techniques.


Compatible solutes Persephonellamarina Thermophiles Glucosylglycerate Glucosylglucosylglycerate 



This work was supported by the European Commission, 6th Framework Programme contract COOP-CT-2003-508644, PRODEP and POCI, Portugal (PTDC/BIO/70806/2006) (POCI/V.5/A0004/2005). Technical assistance by Ana I Mingote is acknowledged. The NMR spectrometers are part of The National NMR Network (REDE/1517/RMN/2005), supported by “Programa Operacional Ciência e Inovação (POCTI) 2010” and Fundação para a Ciência e a Tecnologia (FCT). M.V.R. and L.G.G. received fellowships from FCT (SFRH/BPD/80219/2011 and SFRH/BPD/26905/2006). Data for the amino acid analysis was obtained by the Analytical Laboratory, Analytical Services Unit, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa.

Supplementary material

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Supplementary material 1 (PDF 225 kb)


  1. Alain K, Marteinsson VT, Miroshnichenko ML, Bonch-Osmolovskaya EA, Prieur D, Birrien JL (2002) Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent. Int J Syst Evol Microbiol 52:1331–1339PubMedCrossRefGoogle Scholar
  2. Behrends V, Williams KJ, Jenkins VA, Robertson BD, Bundy JG (2012) Free glucosylglycerate is a novel marker of nitrogen stress in Mycobacterium smegmatis. J Proteome Res 11:3888–3896PubMedCrossRefGoogle Scholar
  3. Borges N, Ramos A, Raven NDH, Sharp RJ, Santos H (2002) Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes. Extremophiles 6:209–216PubMedCrossRefGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  5. Brill J, Hoffmann T, Bleisteiner M, Bremer E (2011) Osmotically controlled synthesis of the compatible solute proline is critical for cellular defense of Bacillus subtilis against high osmolarity. J Bacteriol 193:5335–5346PubMedCrossRefGoogle Scholar
  6. Cánovas D, Borges N, Vargas C, Ventosa A, Nieto JJ, Santos H (1999) Role of Nγ-acetyldiaminobutyrate as an enzyme stabiliser and an intermediate in the biosynthesis of hydroxyectoine. Appl Environ Microbiol 65:3774–3779PubMedGoogle Scholar
  7. Cario A, Jebbar M, Kervarec N, Oger P (2010) Influence of high hydrostatic pressure on the salt and heat stress response in the piezophilic archaeon Thermococcus barophilus. Book of Abstracts of Extremophiles P7:108Google Scholar
  8. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356CrossRefGoogle Scholar
  9. Empadinhas N, Mendes V, Simoes C, Santos MS, Mingote A, Lamosa P, Santos H, da Costa MS (2007) Organic solutes in Rubrobacter xylanophilus: the first example of di-myo-inositol phosphate in a thermophile. Extremophiles 11:667–673PubMedCrossRefGoogle Scholar
  10. Faria TQ, Lima JC, Bastos M, Macanita AL, Santos H (2004) Protein stabilization by osmolytes from hyperthermophiles: effect of mannosylglycerate on the thermal unfolding of recombinant nuclease a from Staphylococcus aureus studied by picosecond time-resolved fluorescence and calorimetry. J Biol Chem 279:48680–48691PubMedCrossRefGoogle Scholar
  11. Faria TQ, Mingote A, Siopa F, Ventura R, Maycock C, Santos H (2008) Design of new enzyme stabilizers inspired by glycosides of hyperthermophilic microorganisms. Carbohydr Res 343:3025–3033PubMedCrossRefGoogle Scholar
  12. Fernandes C, Empadinhas N, da Costa MS (2007) Single-step pathway for synthesis of glucosylglycerate in Persephonella marina. J Bacteriol 189:4014–4019PubMedCrossRefGoogle Scholar
  13. Fernandes C, Mendes V, Costa J, Empadinhas N, Jorge C, Lamosa P, Santos H, da Costa MS (2010) Two alternative pathways for the synthesis of the rare compatible solute mannosylglucosylglycerate in Petrotoga mobilis. J Bacteriol 192:1624–1633PubMedCrossRefGoogle Scholar
  14. Gonçalves LG, Borges N, Serra F, Fernandes PL, Dopazo H, Santos H (2012) Evolution of the biosynthesis of di-myo-inositol phosphate, a marker of adaptation to hot marine environments. Environ Microbiol 14:691–701PubMedCrossRefGoogle Scholar
  15. Götz D, Banta A, Beveridge TJ, Rushdi AI, Simoneit BR, Reysenbach AL (2002) Persephonella marina gen. nov., sp. nov. and Persephonella guaymasensis sp. nov., two novel, thermophilic, hydrogen-oxidizing microaerophiles from deep-sea hydrothermal vents. Int J Syst Evol Microbiol 52:1349–1359PubMedCrossRefGoogle Scholar
  16. Goude R, Renaud S, Bonnassie S, Bernard T, Blanco C (2004) Glutamine, glutamate, and α-glucosylglycerate are the major osmotic solutes accumulated by Erwinia chrysanthemi starin 3937. Appl Environ Microbiol 70:6535–6541PubMedCrossRefGoogle Scholar
  17. Hua SS, Tsai VY, Lichens GM, Noma AT (1982) Accumulation of amino acids in Rhizobium sp. Strain WR1001 in response to sodium chloride salinity. Appl Environ Microbiol 44:135–140PubMedGoogle Scholar
  18. Jorge C, Lamosa P, Santos H (2007) α-d-Mannopyranosyl-(1,2)-α-d-glucopyranosyl-(1,2)glycerate in the thermophilic bacterium Petrotoga miotherma: structure, cellular content and function. FEBS J 274:3120–3127PubMedCrossRefGoogle Scholar
  19. Klähn S, Steglich C, Hess WR, Hagemann M (2010) Glucosylglycerate: a secondary compatible solute common to marine cyanobacteria from nitrogen-poor environments. Environ Microbiol 12:83–94PubMedCrossRefGoogle Scholar
  20. Kollman VH, Hanner JL, London RE, Adame EG, Walker TE (1979) Photosynthetic preparation and characterization of 13C-labeled carbohydrates in Agmenellum quadruplicatum. Carbohydr Res 73:193–202CrossRefGoogle Scholar
  21. Lamosa P, Burke A, Peist R, Huber R, Liu MY, Silva G, Rodrigues-Pousada C, LeGall J, Maycock C, Santos H (2000) Thermostabilization of proteins by diglycerol phosphate, a new compatible solute from the hyperthermophile Archaeoglobus fulgidus. Appl Environ Microbiol 66:1974–1979PubMedCrossRefGoogle Scholar
  22. Lamosa P, Gonçalves LG, Rodrigues M, Martins LO, Raven N, Santos H (2006) Occurrence of 1-glyceryl-1-myo-inosityl-phosphate in hyperthermophiles. Appl Environ Microbiol 72:6169–6173PubMedCrossRefGoogle Scholar
  23. Lourenço EC, Maycock CD, Ventura MR (2009) Synthesis of potassium (2R)-2-O-α-d-glucopyranosyl-(1–>6)-alpha-d-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute. Carbohydr Res 344:2073–2078PubMedCrossRefGoogle Scholar
  24. Martin DD, Ciulla RA, Roberts MF (1999) Osmoadaptation in archaea. Appl Environ Microbiol 65:1815–1825PubMedGoogle Scholar
  25. Martin DD, Bartlett DH, Roberts MF (2002) Solute accumulation in the deep-sea bacterium Photobacterium profundum. Extremophiles 6:507–514PubMedCrossRefGoogle Scholar
  26. Martins LO, Carreto LS, da Costa MS, Santos H (1996) New compatible solutes related to di-myo-inositol-phosphate in members of the order Thermotogales. J Bacteriol 178:5644–5651PubMedGoogle Scholar
  27. Martins LO, Huber R, Huber H, Stetter KO, da Costa MS, Santos H (1997) Organic solutes in hyperthermophilic Archaea. Appl Environ Microbiol 63:896–902PubMedGoogle Scholar
  28. Müller V, Spanheimer R, Santos H (2005) Stress response by solute accumulation in archaea. Curr Opin Microbiol 8:729–736PubMedCrossRefGoogle Scholar
  29. Neves C, da Costa MS, Santos H (2005) Compatible solutes of the hyperthermophile Palaeococcus ferrophilus: osmoadaptation and thermoadaptation in the order thermococcales. Appl Environ Microbiol 71:8091–8098PubMedCrossRefGoogle Scholar
  30. Nunes OC, Manaia CM, da Costa MS, Santos H (1995) Compatible solutes in thermophilic bacteria Rhodothermus marinus and Thermus thermophilus. Appl Environ Microbiol 61:2351–2357PubMedGoogle Scholar
  31. Pospísl S, Halada P, Petrícek M, Sedmera P (2007) Glucosylglycerate is an osmotic solute and an extracellular metabolite produced by Streptomyces caelestis. Folia Microbiol (Praha) 52:451–456CrossRefGoogle Scholar
  32. Ramos A, Raven NDH, Sharp RJ, Bartolucci S, Rossi M, Cannio R, Lebbink J, van der Oost J, de Vos WM, Santos H (1997) Stabilization of enzymes against thermal stress and freeze-drying by mannosylglycerate. Appl Environ Microbiol 63:4020–4025PubMedGoogle Scholar
  33. Roberts MF (2005) Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Systems 1:5PubMedCrossRefGoogle Scholar
  34. Robertson DE, Roberts MF, Belay N, Stetter KO, Boone DR (1990) Occurrence of β-glutamate, a novel osmolyte, in marine methanogenic bacteria. Appl Environ Microbiol 56:1504–1508PubMedGoogle Scholar
  35. Robertson DE, Noll D, Roberts MF (1992) Free amino acid dynamics in marine methanogens. J Biol Chem 267:14893–14901PubMedGoogle Scholar
  36. Rodrigues MV, Borges N, Henriques M, Lamosa P, Ventura R, Fernandes C, Empadinhas N, Maycock C, da Costa MS, Santos H (2007) Bifunctional CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, the key enzyme for di-myo-inositol-phosphate synthesis in several (hyper)thermophiles. J Bacteriol 189:5405–5412PubMedCrossRefGoogle Scholar
  37. Rodrigues MV, Borges N, Almeida CP, Lamosa P, Santos H (2009) A unique beta-1,2-mannosyltransferase of Thermotoga maritima that uses di-myo-inositol phosphate as the mannosyl acceptor. J Bacteriol 191:6105–6115PubMedCrossRefGoogle Scholar
  38. Santos H, da Costa MS (2002) Compatible solutes of organisms that live in hot saline environments. Environ Microbiol 4:501–509PubMedCrossRefGoogle Scholar
  39. Santos H, Lamosa P, Borges N (2006) Characterization and quantification of compatible solutes in (hyper)thermophilic microorganisms. Methods Microbiol 35:173–199CrossRefGoogle Scholar
  40. Santos H, Lamosa P, Faria TQ, Borges N, Neves C (2007) The physiological role, biosynthesis and mode of action of compatible solutes from (hyper)thermophiles. In: Gerday C, Glandorff N (eds) Physiology and biochemistry of extremophiles. ASM Publishers, Washington, DC, pp 86–103Google Scholar
  41. Santos H, Lamosa P, Borges N, Gonçalves LG, Pais T, Rodrigues MV (2011) Organic compatible solutes of prokaryotes that thrive in hot environments: the importance of ionic compounds for thermostabilization. In: Horikoshi K (ed) Extremophiles handbook. Springer, Tokyo, pp 497–520CrossRefGoogle Scholar
  42. Saum SH, Müller V (2007) Salinity-dependent switching of osmolyte strategies in a moderately halophilic bacterium: glutamate induces proline biosynthesis in Halobacillus halophilus. J Bacteriol 189:6968–6975PubMedCrossRefGoogle Scholar
  43. Saum R, Mingote A, Santos H, Müller V (2009) A novel limb in the osmoregulatory network of Methanosarcina mazei Gö1: N(epsilon)-acetyl-beta-lysine can be substituted by glutamate and alanine. Environ Microbiol 11:1056–1065PubMedCrossRefGoogle Scholar
  44. Scholz S, Sonnenbichler J, Schäfer W, Hensel R (1992) Di-myo-inositol-1,1′-phosphate: a new inositol phosphate isolated from Pyrococcus woesei. FEBS Lett 306:239–242PubMedCrossRefGoogle Scholar
  45. Silva Z, Borges N, Martins LO, Wait R, da Costa MS, Santos H (1999) Combined effect of the growth temperature and salinity of the medium on the accumulation of compatible solutes by Rhodothermus marinus and Rhodothermus obamensis. Extremophiles 3:163–172PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2012

Authors and Affiliations

  • Pedro Lamosa
    • 1
  • Marta V. Rodrigues
    • 1
  • Luís G. Gonçalves
    • 1
  • Jean Carr
    • 3
  • Rita Ventura
    • 2
  • Christopher Maycock
    • 2
  • Neil D. Raven
    • 3
  • Helena Santos
    • 1
    Email author
  1. 1.Biology Division, Instituto de Tecnologia Química e BiológicaUniversidade Nova de LisboaOeirasPortugal
  2. 2.Chemistry Division, Instituto de Tecnologia Química e BiológicaUniversidade Nova de LisboaOeirasPortugal
  3. 3.Health Protection AgencySalisbury, WiltshireUK

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