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. 1997 Dec;179(24):7803–7811. doi: 10.1128/jb.179.24.7803-7811.1997

Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum.

C Schleper 1, R V Swanson 1, E J Mathur 1, E F DeLong 1
PMCID: PMC179745  PMID: 9401041

Abstract

Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (alpha-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (alpha-)-type DNA polymerases and 3'-5' exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand- and double-strand-dependent 3'-5' exonuclease activity was detected, as was a marginal 5'-3' exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40 degrees C, with a half-life of 10 min at 46 degrees C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.

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Selected References

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  1. Bintrim S. B., Donohue T. J., Handelsman J., Roberts G. P., Goodman R. M. Molecular phylogeny of Archaea from soil. Proc Natl Acad Sci U S A. 1997 Jan 7;94(1):277–282. doi: 10.1073/pnas.94.1.277. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blanco L., Bernad A., Blasco M. A., Salas M. A general structure for DNA-dependent DNA polymerases. Gene. 1991 Apr;100:27–38. doi: 10.1016/0378-1119(91)90346-d. [DOI] [PubMed] [Google Scholar]
  3. Blöchl E., Rachel R., Burggraf S., Hafenbradl D., Jannasch H. W., Stetter K. O. Pyrolobus fumarii, gen. and sp. nov., represents a novel group of archaea, extending the upper temperature limit for life to 113 degrees C. Extremophiles. 1997 Feb;1(1):14–21. doi: 10.1007/s007920050010. [DOI] [PubMed] [Google Scholar]
  4. Braithwaite D. K., Ito J. Compilation, alignment, and phylogenetic relationships of DNA polymerases. Nucleic Acids Res. 1993 Feb 25;21(4):787–802. doi: 10.1093/nar/21.4.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chong S., Shao Y., Paulus H., Benner J., Perler F. B., Xu M. Q. Protein splicing involving the Saccharomyces cerevisiae VMA intein. The steps in the splicing pathway, side reactions leading to protein cleavage, and establishment of an in vitro splicing system. J Biol Chem. 1996 Sep 6;271(36):22159–22168. doi: 10.1074/jbc.271.36.22159. [DOI] [PubMed] [Google Scholar]
  6. Datukishvili N., Pokholok D., Lottspeich F., Prangishvili D., Rechinsky V. The DNA polymerase-encoding gene from a thermoacidophilic archaeon Sulfolobus acidocaldarius. Gene. 1996 Oct 24;177(1-2):271–273. doi: 10.1016/0378-1119(96)00298-3. [DOI] [PubMed] [Google Scholar]
  7. Davail S., Feller G., Narinx E., Gerday C. Cold adaptation of proteins. Purification, characterization, and sequence of the heat-labile subtilisin from the antarctic psychrophile Bacillus TA41. J Biol Chem. 1994 Jul 1;269(26):17448–17453. [PubMed] [Google Scholar]
  8. DeLong E. F. Archaea in coastal marine environments. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5685–5689. doi: 10.1073/pnas.89.12.5685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. DeLong E. F., Wu K. Y., Prézelin B. B., Jovine R. V. High abundance of Archaea in Antarctic marine picoplankton. Nature. 1994 Oct 20;371(6499):695–697. doi: 10.1038/371695a0. [DOI] [PubMed] [Google Scholar]
  10. Edgell D. R., Klenk H. P., Doolittle W. F. Gene duplications in evolution of archaeal family B DNA polymerases. J Bacteriol. 1997 Apr;179(8):2632–2640. doi: 10.1128/jb.179.8.2632-2640.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Elie C., De Recondo A. M., Forterre P. Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Purification, characterization and immunological properties. Eur J Biochem. 1989 Jan 2;178(3):619–626. doi: 10.1111/j.1432-1033.1989.tb14490.x. [DOI] [PubMed] [Google Scholar]
  12. Feller G., Lonhienne T., Deroanne C., Libioulle C., Van Beeumen J., Gerday C. Purification, characterization, and nucleotide sequence of the thermolabile alpha-amylase from the antarctic psychrotroph Alteromonas haloplanctis A23. J Biol Chem. 1992 Mar 15;267(8):5217–5221. [PubMed] [Google Scholar]
  13. Fuhrman J. A., McCallum K., Davis A. A. Novel major archaebacterial group from marine plankton. Nature. 1992 Mar 12;356(6365):148–149. doi: 10.1038/356148a0. [DOI] [PubMed] [Google Scholar]
  14. Hershberger K. L., Barns S. M., Reysenbach A. L., Dawson S. C., Pace N. R. Wide diversity of Crenarchaeota. Nature. 1996 Dec 5;384(6608):420–420. doi: 10.1038/384420a0. [DOI] [PubMed] [Google Scholar]
  15. Jurgens G., Lindström K., Saano A. Novel group within the kingdom Crenarchaeota from boreal forest soil. Appl Environ Microbiol. 1997 Feb;63(2):803–805. doi: 10.1128/aem.63.2.803-805.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kim U. J., Shizuya H., de Jong P. J., Birren B., Simon M. I. Stable propagation of cosmid sized human DNA inserts in an F factor based vector. Nucleic Acids Res. 1992 Mar 11;20(5):1083–1085. doi: 10.1093/nar/20.5.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Klimczak L. J., Grummt F., Burger K. J. Purification and characterization of DNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Nucleic Acids Res. 1985 Jul 25;13(14):5269–5282. doi: 10.1093/nar/13.14.5269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. MacGregor B. J., Moser D. P., Alm E. W., Nealson K. H., Stahl D. A. Crenarchaeota in Lake Michigan sediment. Appl Environ Microbiol. 1997 Mar;63(3):1178–1181. doi: 10.1128/aem.63.3.1178-1181.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Massana R., Murray A. E., Preston C. M., DeLong E. F. Vertical distribution and phylogenetic characterization of marine planktonic Archaea in the Santa Barbara Channel. Appl Environ Microbiol. 1997 Jan;63(1):50–56. doi: 10.1128/aem.63.1.50-56.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. McInerney J. O., Wilkinson M., Patching J. W., Embley T. M., Powell R. Recovery and phylogenetic analysis of novel archaeal rRNA sequences from a deep-sea deposit feeder. Appl Environ Microbiol. 1995 Apr;61(4):1646–1648. doi: 10.1128/aem.61.4.1646-1648.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Olsen G. J., Woese C. R. Archaeal genomics: an overview. Cell. 1997 Jun 27;89(7):991–994. doi: 10.1016/s0092-8674(00)80284-6. [DOI] [PubMed] [Google Scholar]
  22. Pace N. R. A molecular view of microbial diversity and the biosphere. Science. 1997 May 2;276(5313):734–740. doi: 10.1126/science.276.5313.734. [DOI] [PubMed] [Google Scholar]
  23. Perler F. B., Kumar S., Kong H. Thermostable DNA polymerases. Adv Protein Chem. 1996;48:377–435. doi: 10.1016/s0065-3233(08)60367-8. [DOI] [PubMed] [Google Scholar]
  24. Pisani F. M., De Martino C., Rossi M. A DNA polymerase from the archaeon Sulfolobus solfataricus shows sequence similarity to family B DNA polymerases. Nucleic Acids Res. 1992 Jun 11;20(11):2711–2716. doi: 10.1093/nar/20.11.2711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pisani F. M., Rossi M. Evidence that an archaeal alpha-like DNA polymerase has a modular organization of its associated catalytic activities. J Biol Chem. 1994 Mar 18;269(11):7887–7892. [PubMed] [Google Scholar]
  26. Preston C. M., Wu K. Y., Molinski T. F., DeLong E. F. A psychrophilic crenarchaeon inhabits a marine sponge: Cenarchaeum symbiosum gen. nov., sp. nov. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6241–6246. doi: 10.1073/pnas.93.13.6241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Schleper C., Holben W., Klenk H. P. Recovery of crenarchaeotal ribosomal DNA sequences from freshwater-lake sediments. Appl Environ Microbiol. 1997 Jan;63(1):321–323. doi: 10.1128/aem.63.1.321-323.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Southworth M. W., Kong H., Kucera R. B., Ware J., Jannasch H. W., Perler F. B. Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity. Proc Natl Acad Sci U S A. 1996 May 28;93(11):5281–5285. doi: 10.1073/pnas.93.11.5281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Stein J. L., Marsh T. L., Wu K. Y., Shizuya H., DeLong E. F. Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. J Bacteriol. 1996 Feb;178(3):591–599. doi: 10.1128/jb.178.3.591-599.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Uemori T., Ishino Y., Doi H., Kato I. The hyperthermophilic archaeon Pyrodictium occultum has two alpha-like DNA polymerases. J Bacteriol. 1995 Apr;177(8):2164–2177. doi: 10.1128/jb.177.8.2164-2177.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Wong S. W., Wahl A. F., Yuan P. M., Arai N., Pearson B. E., Arai K., Korn D., Hunkapiller M. W., Wang T. S. Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 1988 Jan;7(1):37–47. doi: 10.1002/j.1460-2075.1988.tb02781.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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