, Volume 19, Issue 1, pp 87–99 | Cite as

The hydroxyectoine gene cluster of the non-halophilic acidophile Acidiphilium cryptum

  • Katharina D. Moritz
  • Birgit Amendt
  • Elisabeth M. H. J. Witt
  • Erwin A. Galinski
Original Paper


Acidiphilium cryptum is an acidophilic, heterotrophic α-Proteobacterium which thrives in acidic, metal-rich environments (e.g. acid mine drainage). Recently, an ectABCDask gene cluster for biosynthesis of the compatible solutes ectoine and hydroxyectoine was detected in the genome sequence of A. cryptum JF-5. We were able to demonstrate that the type strain A. cryptum DSM 2389T is capable of synthesizing the compatible solute hydroxyectoine in response to moderate osmotic stress caused by sodium chloride and aluminium sulphate, respectively. Furthermore, we used the A. cryptum JF-5 sequence to amplify the ectABCDask gene cluster from strain DSM 2389T and achieved heterologous expression of the gene cluster in Escherichia coli. Hence, we could for the first time prove metabolic functionality of the genes responsible for hydroxyectoine biosynthesis in the acidophile A. cryptum. In addition, we present information on specific enzyme activity of A. cryptum DSM 2389T ectoine synthase (EctC) in vitro. In contrast to EctCs from halophilic microorganisms, the A. cryptum enzyme exhibits a higher isoelectric point, thus a lower acidity, and has maximum specific activity in the absence of sodium chloride.


Acidiphilium cryptum Acidophile Compatible solute Ectoine synthase Hydroxyectoine 



The authors would like to thank Marlene Hecker for skilful technical assistance with the osmotic pressure measurements.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The authors declare that the experiments comply with the current laws of the country in which they were performed.

Supplementary material

792_2014_687_MOESM1_ESM.pdf (190 kb)
Supplementary material 1 (PDF 190 kb)


  1. Becker J, Schäfer R, Kohlstedt M, Harder BJ, Borchert NS, Stöveken N, Bremer E, Wittmann C (2013) Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine. Microb Cell Fact 12:110. doi: 10.1186/1475-2859-12-110 PubMedCentralPubMedCrossRefGoogle Scholar
  2. Bertani G (1951) Studies on lysogenesis. I.: The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62:293–300PubMedCentralPubMedGoogle Scholar
  3. Bestvater T, Louis P, Galinski EA (2008) Heterologous ectoine production in Escherichia coli: by-passing the metabolic bottle-neck. Saline Syst 4:12. doi: 10.1186/1746-1448-4-12 PubMedCentralPubMedCrossRefGoogle Scholar
  4. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. doi: 10.1139/o59-099 PubMedCrossRefGoogle Scholar
  5. Borges N, Ramos A, Raven ND, Sharp RJ, Santos H (2002) Comparative study of the thermostabilizing properties of mannosylglycerate and other compatible solutes on model enzymes. Extremophiles 6:209–216. doi: 10.1007/s007920100236 PubMedCrossRefGoogle Scholar
  6. Brown AD (1976) Microbial water stress. Bacteriol Rev 40:803–846PubMedCentralPubMedGoogle Scholar
  7. Bursy J, Pierik AJ, Pica N, Bremer E (2007) Osmotically induced synthesis of the compatible solute hydroxyectoine is mediated by an evolutionarily conserved ectoine hydroxylase. J Biol Chem 282:31147–31155. doi: 10.1074/jbc.M704023200 PubMedCrossRefGoogle Scholar
  8. Cummings DE, Fendorf S, Singh N, Sani RK, Peyton BM, Magnuson TS (2007) Reduction of Cr(VI) under acidic conditions by the facultative Fe(lll)-reducing bacterium Acidiphilium cryptum. Environ Sci Technol 41:146–152. doi: 10.1021/es061333k PubMedCrossRefGoogle Scholar
  9. Dinnbier U, Limpinsel E, Schmid R, Bakker EP (1988) Transient accumulation of potassium glutamate and its replacement by trehalose during adaptation of growing cells of Escherichia coli K-12 to elevated sodium chloride concentrations. Arch Microbiol 150:348–357. doi: 10.1007/BF00408306 PubMedCrossRefGoogle Scholar
  10. Dötsch A, Severin J, Alt W, Galinski EA, Kreft JU (2008) A mathematical model for growth and osmoregulation in halophilic bacteria. Microbiology 154:2956–2969. doi: 10.1099/mic.0.2007/012237-0 PubMedCrossRefGoogle Scholar
  11. Eilert E, Kranz A, Hollenberg CP, Piontek M, Suckow M (2013) Synthesis and release of the bacterial compatible solute 5-hydroxyectoine in Hansenula polymorpha. J Biotechnol 167:85–93. doi: 10.1016/j.jbiotec.2013.02.005 PubMedCrossRefGoogle Scholar
  12. Elevi Bardavid R, Oren A (2012) The amino acid composition of proteins from anaerobic halophilic bacteria of the order Halanaerobiales. Extremophiles 16:567–572. doi: 10.1007/s00792-012-0455-y PubMedCrossRefGoogle Scholar
  13. Fischer J, Quentmeier A, Gansel S, Sabados V, Friedrich CG (2002) Inducible aluminum resistance of Acidiphilium cryptum and aluminum tolerance of other acidophilic bacteria. Arch Microbiol 178:554–558. doi: 10.1007/s00203-002-0482-7 PubMedCrossRefGoogle Scholar
  14. Follmann M, Becker M, Ochrombel I, Ott V, Krämer R, Marin K (2009) Potassium transport in Corynebacterium glutamicum is facilitated by the putative channel protein CglK, which is essential for pH homeostasis and growth at acidic pH. J Bacteriol 191:2944–2952. doi: 10.1128/JB.00074-09 PubMedCentralPubMedCrossRefGoogle Scholar
  15. Fukuchi S, Yoshimune K, Wakayama M, Moriguchi M, Nishikawa K (2003) Unique amino acid composition of proteins in halophilic bacteria. J Mol Biol 327:347–357. doi: 10.1016/S0022-2836(03)00150-5 PubMedCrossRefGoogle Scholar
  16. Galinski EA, Oren A (1991) Isolation and structure determination of a novel compatible solute from the moderately halophilic purple sulfur bacterium Ectothiorhodospira marismortui. Eur J Biochem 198:593–598. doi: 10.1111/j.1432-1033.1991.tb16055.x PubMedCrossRefGoogle Scholar
  17. Galinski EA, Pfeiffer HP, Trüper HG (1985) 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid. A novel cyclic amino acid from halophilic phototrophic bacteria of the genus Ectothiorhodospira. Eur J Biochem 149:135–139. doi: 10.1111/j.1432-1033.1985.tb08903.x PubMedCrossRefGoogle Scholar
  18. Gandbhir M, Rasched I, Marlière P, Mutzel R (1995) Convergent evolution of amino acid usage in archaebacterial and eubacterial lineages adapted to high salt. Res Microbiol 146:113–120. doi: 10.1016/0923-2508(96)80889-8 PubMedCrossRefGoogle Scholar
  19. Göller K, Galinski EA (1999) Protection of a model enzyme (lactate dehydrogenase) against heat, urea and freeze-thaw treatment by compatible solute additives. J Mol Catal B Enzym 7:37–45. doi: 10.1016/S1381-1177(99)00043-0 CrossRefGoogle Scholar
  20. Goltsman DS, Denef VJ, Singer SW, VerBerkmoes NC, Lefsrud M, Mueller RS, Dick GJ, Sun CL, Wheeler KE, Zemla A, Baker BJ, Hauser L, Land M, Shah MB, Thelen MP, Hettich RL, Banfield JF (2009) Community genomic and proteomic analyses of chemoautotrophic iron-oxidizing “Leptospirillum rubarum” (Group II) and “Leptospirillum ferrodiazotrophum” (Group III) bacteria in acid mine drainage biofilms. Appl Environ Microbiol 75:4599–4615. doi: 10.1128/AEM.02943-08 PubMedCentralPubMedCrossRefGoogle Scholar
  21. Guida L, Saidi Z, Hughes MN, Poole RK (1991) Aluminium toxicity and binding to Escherichia coli. Arch Microbiol 156:507–512. doi: 10.1007/BF00245400 PubMedGoogle Scholar
  22. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580. doi: 10.1016/S0022-2836(83)80284-8 PubMedCrossRefGoogle Scholar
  23. Harrison AP (1981) Acidiphilium cryptum gen. nov., sp. nov., heterotrophic bacterium from acidic mineral environments. Int J Syst Bacteriol 31:327–332. doi: 10.1099/00207713-31-3-327 CrossRefGoogle Scholar
  24. Harrison AP, Jarvis BW, Johnson JL (1980) Heterotrophic bacteria from cultures of autotrophic Thiobacillus ferrooxidans: relationships as studied by means of deoxyribonucleic acid homology. J Bacteriol 143:448–454PubMedCentralPubMedGoogle Scholar
  25. Imhoff JF, Trüper HG (1977) Ectothiorhodospira halochloris sp. nov., a new extremely halophilic phototropic bacterium containing bacteriochlorophyll b. Arch Microbiol 114:115–121. doi: 10.1007/BF00410772 CrossRefGoogle Scholar
  26. Inbar L, Lapidot A (1988) The structure and biosynthesis of new tetrahydropyrimidine derivatives in actinomycin D producer Streptomyces parvulus. Use of 13C- and 15N-labeled L-glutamate and 13C and 15N NMR spectroscopy. J Biol Chem 263:16014–16022PubMedGoogle Scholar
  27. Johnson DB, McGinness S (1991) Ferric iron reduction by acidophilic heterotrophic bacteria. Appl Environ Microbiol 57:207–211PubMedCentralPubMedGoogle Scholar
  28. Kishimoto N, Tano T (1987) Acidophilic heterotrophic bacteria isolated from acidic mine drainage, sewage, and soils. J Gen Appl Microbiol 33:11–25. doi: 10.2323/jgam.33.11 CrossRefGoogle Scholar
  29. Koichi M, Mitsuhiko M, Tatsuo N, Yoshio S; Takeda Chem Ind Ltd (Take), assignee (1991) Production of tetrahydropyrimidine derivatives. Jp Patent JP3031265Google Scholar
  30. Kraegeloh A, Kunte HJ (2002) Novel insights into the role of potassium for osmoregulation in Halomonas elongata. Extremophiles 6:453–462. doi: 10.1007/s00792-002-0277-4 PubMedCrossRefGoogle Scholar
  31. Krämer R, Lambert C, Hoischen C, Ebbighausen H (1990) Uptake of glutamate in Corynebacterium glutamicum. 1. Kinetic properties and regulation by internal pH and potassium. Eur J Biochem 194:929–935. doi: 10.1111/j.1432-1033.1990.tb19488.x PubMedCrossRefGoogle Scholar
  32. Kunte HJ, Galinski EA, Trüper HG (1993) A modified FMOC-method for the detection of amino acid-type osmolytes and tetrahydropyrimidines (ectoines). J Microbiol Methods 17:129–136. doi: 10.1016/0167-7012(93)90006-4 CrossRefGoogle Scholar
  33. Kunte HJ, Lentzen G, Galinski EA (2014) Industrial production of the cell protectant ectoine: protection mechanisms, processes, and products. Curr Biotechnol 3:10–25. doi: 10.2174/22115501113026660037 CrossRefGoogle Scholar
  34. Küsel K, Dorsch T, Acker G, Stackebrandt E (1999) Microbial reduction of Fe(III) in acidic sediments: isolation of Acidiphilium cryptum JF-5 capable of coupling the reduction of Fe(III) to the oxidation of glucose. Appl Environ Microbiol 65:3633–3640PubMedCentralPubMedGoogle Scholar
  35. Lang YJ, Bai L, Ren YN, Zhang LH, Nagata S (2011) Production of ectoine through a combined process that uses both growing and resting cells of Halomonas salina DSM 5928T. Extremophiles 15:303–310. doi: 10.1007/s00792-011-0360-9 PubMedCrossRefGoogle Scholar
  36. Larsen PI, Sydnes LK, Landfald B, Strom AR (1987) Osmoregulation in Escherichia coli by accumulation of organic osmolytes: betaines, glutamic acid, and trehalose. Arch Microbiol 147:1–7. doi: 10.1007/BF00492896 PubMedCrossRefGoogle Scholar
  37. Lentzen G, Schwarz T (2006) Extremolytes: natural compounds from extremophiles for versatile applications. Appl Microbiol Biotechnol 72:623–634. doi: 10.1007/s00253-006-0553-9 PubMedCrossRefGoogle Scholar
  38. Lippert K, Galinski EA (1992) Enzyme stabilization by ectoine-type compatible solutes: protection against heating, freezing and drying. Appl Microbiol Biotechnol 37:61–65. doi: 10.1007/BF00174204 CrossRefGoogle Scholar
  39. Lo CC, Bonner CA, Xie G, D’Souza M, Jensen RA (2009) Cohesion group approach for evolutionary analysis of aspartokinase, an enzyme that feeds a branched network of many biochemical pathways. Microbiol Mol Biol Rev 73:594–651. doi: 10.1128/mmbr.00024-09 PubMedCentralPubMedCrossRefGoogle Scholar
  40. Louis P, Trüper HG, Galinski EA (1994) Survival of Escherichia coli during drying and storage in the presence of compatible solutes. Appl Microbiol Biotechnol 41:684–688. doi: 10.1007/BF00167285 CrossRefGoogle Scholar
  41. Magnuson TS, Swenson MW, Paszczynski AJ, Deobald LA, Kerk D, Cummings DE (2010) Proteogenomic and functional analysis of chromate reduction in Acidiphilium cryptum JF-5, an Fe(III)-respiring acidophile. Biometals 23:1129–1138. doi: 10.1007/s10534-010-9360-y PubMedCrossRefGoogle Scholar
  42. Manzanera M, Vilchez S, Tunnacliffe A (2004) High survival and stability rates of Escherichia coli dried in hydroxyectoine. FEMS Microbiol Lett 233:347–352. doi: 10.1016/j.femsle.2004.03.005 PubMedCrossRefGoogle Scholar
  43. Mosier AC, Justice NB, Bowen BP, Baran R, Thomas BC, Northen TR, Banfield JF (2013) Metabolites associated with adaptation of microorganisms to an acidophilic, metal-rich environment identified by stable-isotope-enabled metabolomics. M Bio 4:e00484-12. doi: 10.1128/mBio.00484-12
  44. Mustakhimov II, Reshetnikov AS, Khmelenina VN, Trotsenko YA (2009) EctR—a novel transcriptional regulator of ectoine biosynthesis genes in the haloalcaliphilic methylotrophic bacterium Methylophaga alcalica. Dokl Biochem Biophys 429:305–308. doi: 10.1134/S1607672909060052 PubMedCrossRefGoogle Scholar
  45. Mustakhimov II, Reshetnikov AS, Glukhov AS, Khmelenina VN, Kalyuzhnaya MG, Trotsenko YA (2010) Identification and characterization of EctR1, a new transcriptional regulator of the ectoine biosynthesis genes in the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. J Bacteriol 192:410–417. doi: 10.1128/jb.00553-09 PubMedCentralPubMedCrossRefGoogle Scholar
  46. Ono H, Sawada K, Khunajakr N, Tao T, Yamamoto M, Hiramoto M, Shinmyo A, Takano M, Murooka Y (1999) Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. J Bacteriol 181:91–99PubMedCentralPubMedGoogle Scholar
  47. Oren A, Larimer F, Richardson P, Lapidus A, Csonka LN (2005) How to be moderately halophilic with broad salt tolerance: clues from the genome of Chromohalobacter salexigens. Extremophiles 9:275–279. doi: 10.1007/s00792-005-0442-7 PubMedCrossRefGoogle Scholar
  48. Peters P, Galinski EA, Trüper HG (1990) The biosynthesis of ectoine. FEMS Microbiol Lett 71:157–162. doi: 10.1016/0378-1097(90)90049-V CrossRefGoogle Scholar
  49. Reed CJ, Lewis H, Trejo E, Winston V, Evilia C (2013) Protein adaptations in archaeal extremophiles. Archaea 2013:373275. doi: 10.1155/2013/373275 PubMedCentralPubMedCrossRefGoogle Scholar
  50. Rodríguez-Moya J, Argandoña M, Iglesias-Guerra F, Nieto JJ, Vargas C (2013) Temperature- and salinity-decoupled overproduction of hydroxyectoine by Chromohalobacter salexigens. Appl Environ Microbiol 79:1018–1023. doi: 10.1128/AEM.02774-12 PubMedCentralPubMedCrossRefGoogle Scholar
  51. Salis HM (2011) The ribosome binding site calculator. Meth Enzymol 498:19–42. doi: 10.1016/B978-0-12-385120-8.00002-4 PubMedCrossRefGoogle Scholar
  52. Salis HM, Mirsky EA, Voigt CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol 27:946–950. doi: 10.1038/nbt.1568 PubMedCentralPubMedCrossRefGoogle Scholar
  53. Sauer T (1995) Untersuchungen zur Nutzung von Halomonas elongata für die Gewinnung kompatibler Solute. Dissertation, Rheinische Friedrich-Wilhelms-Universität BonnGoogle Scholar
  54. Sauer T, Galinski EA (1998) Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnol Bioeng 57:306–313. doi: 10.1002/(SICI)1097-0290(19980205)57:3<306::AID-BIT7>3.0.CO;2-L PubMedCrossRefGoogle Scholar
  55. Schubert T, Maskow T, Benndorf D, Harms H, Breuer U (2007) Continuous synthesis and excretion of the compatible solute ectoine by a transgenic, nonhalophilic bacterium. Appl Environ Microbiol 73:3343–3347. doi: 10.1128/AEM.02482-06 PubMedCentralPubMedCrossRefGoogle Scholar
  56. Seip B, Galinski EA, Kurz M (2011) Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster. Appl Environ Microbiol 77:1368–1374. doi: 10.1128/AEM.02124-10 PubMedCentralPubMedCrossRefGoogle Scholar
  57. Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130. doi: 10.1016/0022-2836(86)90385-2 PubMedCrossRefGoogle Scholar
  58. Tanne C, Golovina EA, Hoekstra FA, Meffert A, Galinski EA (2014) Glass-forming property of hydroxyectoine is the cause of its superior function as a desiccation protectant. Front Microbiol 5:150. doi: 10.3389/fmicb.2014.00150 PubMedCentralPubMedCrossRefGoogle Scholar
  59. Voß P (2002) Synthese von kompatiblen Soluten mit ectoinanaloger Struktur und Charakterisierung des protektiven Effektes auf biochemische Modellsysteme und Escherichia coli. Dissertation, Westfälische Wilhelms-Universität Münster,
  60. Widderich N, Höppner A, Pittelkow M, Heider J, Smits SHJ, Bremer E (2014) Biochemical properties of ectoine hydroxylases from extremophiles and their wider taxonomic distribution among microorganisms. PLoS ONE 9:e93809. doi: 10.1371/journal.pone.0093809 PubMedCentralPubMedCrossRefGoogle Scholar
  61. Wilkinson SP, Grove A (2006) Ligand-responsive transcriptional regulation by members of the MarR family of winged helix proteins. Curr Issues Mol Biol 8:51–62PubMedGoogle Scholar
  62. Witt E (2011) Nebenreaktionen der Ectoin-Synthase aus Halomonas elongata DSM 2581T und Entwicklung eines salzinduzierten Expressionssystems. Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
  63. Witt EMHJ, Davies NW, Galinski EA (2011) Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC. Appl Microbiol Biotechnol 91:113–122. doi: 10.1007/s00253-011-3211-9 PubMedCrossRefGoogle Scholar
  64. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119. doi: 10.1016/0378-1119(85)90120-9 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2014

Authors and Affiliations

  • Katharina D. Moritz
    • 1
  • Birgit Amendt
    • 1
  • Elisabeth M. H. J. Witt
    • 1
  • Erwin A. Galinski
    • 1
  1. 1.Institut für Mikrobiologie und BiotechnologieRheinische Friedrich-Wilhelms-Universität BonnBonnGermany

Personalised recommendations