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Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins


Features of the physical environment surrounding an ancestral organism can be inferred by reconstructing sequences1,2,3,4,5,6,7,8,9 of ancient proteins made by those organisms, resurrecting these proteins in the laboratory, and measuring their properties. Here, we resurrect candidate sequences for elongation factors of the Tu family (EF-Tu) found at ancient nodes in the bacterial evolutionary tree, and measure their activities as a function of temperature. The ancient EF-Tu proteins have temperature optima of 55–65 °C. This value seems to be robust with respect to uncertainties in the ancestral reconstruction. This suggests that the ancient bacteria that hosted these particular genes were thermophiles, and neither hyperthermophiles nor mesophiles. This conclusion can be compared and contrasted with inferences drawn from an analysis of the lengths of branches in trees joining proteins from contemporary bacteria10, the distribution of thermophily in derived bacterial lineages11, the inferred G + C content of ancient ribosomal RNA12, and the geological record combined with assumptions concerning molecular clocks13. The study illustrates the use of experimental palaeobiochemistry and assumptions about deep phylogenetic relationships between bacteria to explore the character of ancient life.

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Figure 1: The two unrooted universal trees used to reconstruct ancestral bacterial sequences.
Figure 2: Comparisons of reconstructed ancestral sequences.
Figure 3: GDP-binding assay to test thermostability of ancestral and modern EF proteins.


  1. Malcolm, B. A., Wilson, K. P., Matthews, B. W., Kirsch, J. F. & Wilson, A. C. Ancestral lysozymes reconstructed, neutrality tested, and thermostability linked to hydrocarbon packing. Nature 345, 86–89 (1990)

    Article  ADS  CAS  Google Scholar 

  2. Stackhouse, J., Presnell, S. R., McGeehan, G. M., Nambiar, K. P. & Benner, S. A. The ribonuclease from an extinct bovid. FEBS Lett. 262, 104–106 (1990)

    Article  CAS  Google Scholar 

  3. Adey, N. B., Tollefsbol, T. O., Sparks, A. B., Edgell, M. H. & Hutchison, C. A. Molecular resurrection of an extinct ancestral promoter for mouse L1. Proc. Natl Acad. Sci. USA 91, 1569–1573 (1994)

    Article  ADS  CAS  Google Scholar 

  4. Jermann, T. M., Opitz, J. G., Stackhouse, J. & Benner, S. A. Reconstructing the evolutionary history of the artiodactyl ribonuclease superfamily. Nature 374, 57–59 (1995)

    Article  ADS  CAS  Google Scholar 

  5. Chandrasekharan, U. M., Sanker, S., Glynias, M. J., Karnik, S. S. & Husain, A. Angiotensin II forming activity in a reconstructed ancestral chymase. Science 271, 502–505 (1996)

    Article  ADS  CAS  Google Scholar 

  6. Golding, G. B. & Dean, A. M. The structural basis of molecular adaptation. Mol. Biol. Evol. 15, 355–369 (1998)

    Article  CAS  Google Scholar 

  7. Miyazaki, J. et al. Ancestral residues stabilizing 3-isopropylmalate dehydrogenase of an extreme thermophile: Experimental evidence supporting the thermophilic common ancestor hypothesis. J. Biochem. 129, 777–782 (2001)

    Article  CAS  Google Scholar 

  8. Chang, B. S., Jonsson, K., Kazmi, M. A., Donoghue, M. J. & Sakmar, T. P. Recreating a functional ancestral archosaur visual pigment. Mol. Biol. Evol. 19, 1483–1489 (2002)

    Article  CAS  Google Scholar 

  9. Zhang, J. Z. & Rosenberg, H. F. Complementary advantageous substitutions in the evolution of an antiviral RNase of higher primates. Proc. Natl Acad. Sci. USA 99, 5486–5491 (2002)

    Article  ADS  CAS  Google Scholar 

  10. Woese, C. R. Bacterial evolution. Microbiol. Rev. 51, 221–271 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Hugenholtz, P., Goebel, B. M. & Pace, N. R. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180, 4765–4774 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Galtier, N., Tourasse, N. & Gouy, M. A nonhyperthermophilic common ancestor to extant life forms. Science 283, 220–221 (1999)

    Article  CAS  Google Scholar 

  13. Cavalier-Smith, T. The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification. Int. J. Syst. Evol. Microbiol. 52, 7–76 (2002)

    Article  CAS  Google Scholar 

  14. Pauling, L. & Zuckerkandl, E. Chemical paleogenetics: molecular “restoration studies” of extinct forms of life. Acta Chem. Scand. A 17, S9–S16 (1963)

    Article  CAS  Google Scholar 

  15. Benner, S. A., Caraco, M. D., Thomson, J. M. & Gaucher, E. A. Planetary biology—paleontological, geological, and molecular histories of life. Science 296, 864–868 (2002)

    Article  ADS  CAS  Google Scholar 

  16. Arai, K.-I., Kawakita, M. & Kaziro, Y. Studies on polypeptide elongation factors from Escherichia coli.. J. Biol. Chem. 37, 7029–7037 (1972)

    Google Scholar 

  17. Nock, S. et al. Properties of isolated domains of the elongation factor Tu from Thermus thermophilus HB8. Eur. J. Biochem. 234, 132–139 (1995)

    Article  CAS  Google Scholar 

  18. Sanangelantoni, A. M., Cammarano, R. & Tiboni, O. Manipulation of the tuf gene provides clues to the localization of sequence element(s) involved in the thermal stability of Thermotoga maritima elongation factor Tu. Microbiol. 142, 2525–2532 (1996)

    Article  CAS  Google Scholar 

  19. Gromiha, M. M., Oobatake, M. & Sarai, A. Important amino acid properties for enhanced thermostability from mesophilic to thermophilic proteins. Biophys. Chem. 82, 51–67 (1999)

    Article  CAS  Google Scholar 

  20. Gaucher, E. A., Miyamoto, M. M. & Benner, S. A. Function-structure analysis of proteins using covarion-based evolutionary approaches: Elongation factors. Proc. Natl Acad. Sci. USA 98, 548–552 (2001)

    Article  ADS  CAS  Google Scholar 

  21. Yang, Z. H., Kumar, S. & Nei, M. A new method of inference of ancestral nucleotide and amino acid sequences. Genetics 141, 1641–1650 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Brochier, C. & Philippe, H. Phylogeny: a non-hyperthermophilic ancestor for bacteria. Nature 417, 244 (2002)

    Article  ADS  CAS  Google Scholar 

  23. Gaschen, B. et al. Diversity considerations in HIV-1 vaccine selection. Science 296, 2354–2360 (2002)

    Article  ADS  CAS  Google Scholar 

  24. Kumar, S., Tamura, K. & Nei, M. MEGA—Molecular evolutionary genetics analysis software for microcomputers. Comput. Appl. Biosci. 10, 189–191 (1994)

    CAS  PubMed  Google Scholar 

  25. Swofford, D. L. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods) Version 4.10 (Sinauer Associates, Sunderland, Massachusetts, 2002)

    Google Scholar 

  26. Adachi, J. & Hasegawa, M. MOLPHY version 2.3: programs for molecular phylogenetics based on maximum likelihood. Comput. Sci. Monogr. 28, 1–150 (1996)

    Google Scholar 

  27. Yang, Z. PAML: a program package for phylogenetic analyses by maximum likelihood. Comput. Appl. Biosci. 13, 555–556 (1999)

    Google Scholar 

  28. Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequence. Comput. Appl. Biosci. 8, 275–282 (1992)

    CAS  PubMed  Google Scholar 

  29. Dayhoff, M. O., Schwartz, R. M. & Orcutt, B. C. Atlas of Protein Sequence and Structure 345–358 (National Biomedical Research Foundation, Washington DC, 1978)

    Google Scholar 

  30. Whelan, S. & Goldman, N. A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol. Biol. Evol. 18, 691–699 (2001)

    Article  CAS  Google Scholar 

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We thank M. Miyamoto, J. Aris, A. Falcon, S. Sassi, C. West and Z. Yang for discussions and assistance with our research. We also thank C. Knudson and A. M. Sanangelantoni for providing EF clones. Funding is provided by the National Research Council and NASA's Astrobiology Institute (E.A.G.), and the NIH and NASA (S.A.B.).

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Correspondence to Eric A. Gaucher.

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Supplementary information


Supplementary Figure: Multiple sequence alignment of the three reconstructed ancestral sequences juxtaposed to EF from modern E. coli. Asterisks, colons, and periods indicate identical, highly conserved, and moderately conserved positions, respectively. Sites are colored according to amino acid side chain physiochemical properties. (DOC 29 kb)

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Gaucher, E., Thomson, J., Burgan, M. et al. Inferring the palaeoenvironment of ancient bacteria on the basis of resurrected proteins. Nature 425, 285–288 (2003).

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