The species of comprise a very heterogeneous assemblage of bacteria that do not form a phylogenetically coherent group. It has been proposed previously that only a subset of the species of that form a distinct cluster in the 16S rRNA tree (cluster I) should be regarded as the true representatives of the genus (i.e. ). However, this cluster is presently defined only in phylogenetic terms, and no biochemical, molecular or phenotypic characteristic is known that is unique to species from this cluster. We report here phylogenomic and comparative analyses based on sequenced clostridial genomes in an attempt to bridge this gap and to clarify the evolutionary relationships among species of clostridia. In phylogenetic trees for species of clostridia based on concatenated sequences for 37 highly conserved proteins, the species of cluster I formed a strongly supported clade that was separated from all other clostridia by a long branch. Several other species that are not part of this cluster grouped reliably with other species of clostridia in a number of well-resolved clades. Our comparative genomic analyses have identified three conserved indels in three highly conserved proteins (a 4 aa insert in DNA gyrase A, a 1 aa deletion in ATP synthase beta subunit and a 1 aa insert in ribosomal protein S2) that are unique to the species of cluster I and are not found in any other bacteria. searches on various proteins in the genomes of and SM101 have also identified more than 10 proteins that are found uniquely in the cluster I species. These results provide evidence that the species of cluster I not only are phylogenetically distinct but also share many unique molecular characteristics. These newly identified molecular markers provide useful tools to define and circumscribe the genus in more definitive terms. We have also identified a 7–9 aa conserved insert in the enzyme phosphoglycerate dehydrogenase that is uniquely found in the , , and homologues, and is absent from all other bacteria. These species form a well-defined clade in the phylogenetic trees and this indel provides a potential molecular marker for this clostridial cluster.


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  1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J.(1997). Gapped blast and psi-blast: a new generation of protein databases search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef] [Google Scholar]
  2. Belda, E., Moya, A. & Silva, F. J.(2005). Genome rearrangement distances and gene order phylogeny in γ-proteobacteria. Mol Biol Evol 22, 1456–1467.[CrossRef] [Google Scholar]
  3. Bettegowda, C., Huang, X., Lin, J., Cheong, I., Kohli, M., Szabo, S. A., Zhang, X., Diaz, L. A., Jr, Velculescu, V. E. & other authors(2006). The genome and transcriptomes of the anti-tumor agent Clostridium novyi NT. Nat Biotechnol 24, 1573–1580.[CrossRef] [Google Scholar]
  4. Binnewies, T. T., Motro, Y., Hallin, P. F., Lund, O., Dunn, D., La, T., Hampson, D. J., Bellgard, M., Wassenaar, T. M. & Ussery, D. W.(2006). Ten years of bacterial genome sequencing: comparative-genomics-based discoveries. Funct Integr Genomics 6, 165–185.[CrossRef] [Google Scholar]
  5. Brown, J. R. & Volker, C.(2004). Phylogeny of γ-proteobacteria: resolution of one branch of the universal tree? Bioessays 26, 463–468.[CrossRef] [Google Scholar]
  6. Brüggemann, H., Bäumer, S., Fricke, W. F., Wiezer, A., Liesegang, H., Decker, I., Herzberg, C., Martinez-Arias, R., Merkl, R. & other authors(2003). The genome sequence of Clostridium tetani, the causative agent of tetanus disease. Proc Natl Acad Sci U S A 100, 1316–1321.[CrossRef] [Google Scholar]
  7. Castresana, J.(2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17, 540–552.[CrossRef] [Google Scholar]
  8. Cato, E. P. & Stackebrandt, E.(1989). Taxonomy and phylogeny. In Clostridia, pp. 1–26. Edited by N. P. Minton & D. J. Clarke. New York: Plenum Press.
  9. Cato, E. P., George, W. L. & Finegold, S. M.(1986). Genus Clostridium Prazmowski 1880, 23AL. In Bergey's Manual of Systematic Bacteriology, vol. 2, pp. 1141–1200. Edited by P. H. A. Sneath, N. S. Mair, M. E. Sharpe & J. G. Holt. Baltimore: Williams & Wilkins.
  10. Ciccarelli, F. D., Doerks, T., von Mering, C., Creevey, C. J., Snel, B. & Bork, P.(2006). Toward automatic reconstruction of a highly resolved tree of life. Science 311, 1283–1287.[CrossRef] [Google Scholar]
  11. Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H., Farrow, J. A. E. & other authors(1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812–826.[CrossRef] [Google Scholar]
  12. Corbett, K. D. & Berger, J. M.(2004). Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu Rev Biophys Biomol Struct 33, 95–118.[CrossRef] [Google Scholar]
  13. Fang, G., Rocha, E. & Danchin, A.(2005). How essential are nonessential genes? Mol Biol Evol 22, 2147–2156.[CrossRef] [Google Scholar]
  14. Finegold, S. M., Song, Y. & Liu, C.(2002). Taxonomy – general comments and update on taxonomy of clostridia and anaerobic cocci. Anaerobe 8, 283–285.[CrossRef] [Google Scholar]
  15. Galperin, M. Y. & Koonin, E. V.(2004). ‘Conserved hypothetical’ proteins: prioritization of targets for experimental study. Nucleic Acids Res 32, 5452–5463.[CrossRef] [Google Scholar]
  16. Gao, B. & Gupta, R. S.(2005). Conserved indels in protein sequences that are characteristic of the phylum Actinobacteria. Int J Syst Evol Microbiol 55, 2401–2412.[CrossRef] [Google Scholar]
  17. Gao, B., Parmanathan, R. & Gupta, R. S.(2006). Signature proteins that are distinctive characteristics of Actinobacteria and their subgroups. Antonie van Leeuwenhoek 90, 69–91.[CrossRef] [Google Scholar]
  18. Griffiths, E. & Gupta, R. S.(2004). Distinctive protein signatures provide molecular markers and evidence for the monophyletic nature of the Deinococcus-Thermus phylum. J Bacteriol 186, 3097–3107.[CrossRef] [Google Scholar]
  19. Griffiths, E., Petrich, A. & Gupta, R. S.(2005). Conserved indels in essential proteins that are distinctive characteristics of Chlamydiales and provide novel means for their identification. Microbiology 151, 2647–2657.[CrossRef] [Google Scholar]
  20. Griffiths, E., Ventresca, M. S. & Gupta, R. S.(2006).blast screening of chlamydial genomes to identify signature proteins that are unique for the Chlamydiales, Chlamydiaceae, Chlamydophila and Chlamydia groups of species. BMC Genomics 7, 14[CrossRef] [Google Scholar]
  21. Gupta, R. S.(1998). Protein phylogenies and signature sequences: a reappraisal of evolutionary relationships among archaebacteria, eubacteria, and eukaryotes. Microbiol Mol Biol Rev 62, 1435–1491. [Google Scholar]
  22. Gupta, R. S.(2006). Molecular signatures (unique proteins and conserved indels) that are specific for the epsilon proteobacteria (Campylobacterales). BMC Genomics 7, 167[CrossRef] [Google Scholar]
  23. Gupta, R. S. & Griffiths, E.(2006). Chlamydiae-specific proteins and indels: novel tools for studies. Trends Microbiol 14, 527–535.[CrossRef] [Google Scholar]
  24. Gupta, R. S. & Lorenzini, E.(2007). Phylogeny and molecular signatures (conserved proteins and indels) that are specific for the Bacteroidetes and Chlorobi species. BMC Evol Biol 7, 71[CrossRef] [Google Scholar]
  25. Gupta, R. S. & Mok, A.(2007). Phylogenomics and signature proteins for the alpha Proteobacteria and its main groups. BMC Microbiol 7, 106[CrossRef] [Google Scholar]
  26. Harris, J. K., Kelley, S. T., Spiegelman, G. B. & Pace, N. R.(2003). The genetic core of the universal ancestor. Genome Res 13, 407–412.[CrossRef] [Google Scholar]
  27. Jeanmougin, F., Thompson, J. D., Gouy, M., Higgins, D. G. & Gibson, T. J.(1998). Multiple sequence alignment with clustal_x. Trends Biochem Sci 23, 403–405.[CrossRef] [Google Scholar]
  28. Johnson, J. L. & Francis, B. S.(1975). Taxonomy of the clostridia: ribosomal ribonucleic acid homologies among the species. J Gen Microbiol 88, 229–244.[CrossRef] [Google Scholar]
  29. Koonin, E. V. & Galperin, M. Y.(1997). Prokaryotic genomes: the emerging paradigm of genome-based microbiology. Curr Opin Genet Dev 7, 757–763.[CrossRef] [Google Scholar]
  30. Lawson, P. A., Llop-Perez, P., Hutson, R. A., Hippe, H. & Collins, M. D.(1993). Towards a phylogeny of the clostridia based on 16S rRNA sequences. FEMS Microbiol Lett 113, 87–92.[CrossRef] [Google Scholar]
  31. Levine, C., Hiasa, H. & Marians, K. J.(1998). DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities. Biochim Biophys Acta 1400, 29–43.[CrossRef] [Google Scholar]
  32. McKitrick, J. C. & Pizer, L. I.(1980). Regulation of phosphoglycerate dehydrogenase levels and effect on serine synthesis in Escherichia coli K-12. J Bacteriol 141, 235–245. [Google Scholar]
  33. Myers, G. S., Rasko, D. A., Cheung, J. K., Ravel, J., Seshadri, R., DeBoy, R. T., Ren, Q., Varga, J., Awad, M. M. & other authors(2006). Skewed genomic variability in strains of the toxigenic bacterial pathogen, Clostridium perfringens. Genome Res 16, 1031–1040.[CrossRef] [Google Scholar]
  34. Nolling, J., Breton, G., Omelchenko, M. V., Makarova, K. S., Zeng, Q., Gibson, R., Lee, H. M., Dubois, J., Qiu, D. & other authors(2001). Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum. J Bacteriol 183, 4823–4838.[CrossRef] [Google Scholar]
  35. Rivera, M. C. & Lake, J. A.(1992). Evidence that eukaryotes and eocyte prokaryotes are immediate relatives. Science 257, 74–76.[CrossRef] [Google Scholar]
  36. Rokas, A. & Holland, P. W.(2000). Rare genomic changes as a tool for phylogenetics. Trends Ecol Evol 15, 454–459.[CrossRef] [Google Scholar]
  37. Rokas, A., Williams, B. L., King, N. & Carroll, S. B.(2003). Genome-scale approaches to resolving incongruence in molecular phylogenies. Nature 425, 798–804.[CrossRef] [Google Scholar]
  38. Rood, J. I.(2006).Clostridium perfringens and histologic disease. In The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 4, pp. 753–770. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H. Schleifer & E. Stackebrandt. New York: Springer.
  39. Schaffer, A. A., Aravind, L., Madden, T. L., Shavirin, S., Spouge, J. L., Wolf, Y. I., Koonin, E. V. & Altschul, S. F.(2001). Improving the accuracy of psi-blast protein database searches with composition-based statistics and other refinements. Nucleic Acids Res 29, 2994–3005.[CrossRef] [Google Scholar]
  40. Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A.(2002).tree-puzzle: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18, 502–504.[CrossRef] [Google Scholar]
  41. Sebaihia, M., Wren, B. W., Mullany, P., Fairweather, N. F., Minton, N., Stabler, R., Thomson, N. R., Roberts, A. P., Cerdeño-Tárraga, A. M. & other authors(2006). The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome. Nat Genet 38, 779–786.[CrossRef] [Google Scholar]
  42. Sebaihia, M., Peck, M. W., Minton, N. P., Thomson, N. R., Holden, M. T., Mitchell, W. J., Carter, A T., Bentley, S. D., Mason, D. R. & other authors(2007). Genome sequence of a proteolytic (group I) Clostridium botulinum strain Hall A and comparative analysis of the clostridial genomes. Genome Res 17, 1082–1092.[CrossRef] [Google Scholar]
  43. Seedorf, H., Fricke, W. F., Veith, B., Brüggemann, H., Liesegang, H., Strittmatter, A., Miethke, M., Buckel, W., Hinderberger, J. & other authors(2008). The genome of Clostridium kluyveri, a strict anaerobe with unique metabolic features. Proc Natl Acad Sci U S A 105, 2128–2133.[CrossRef] [Google Scholar]
  44. Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S. & Hayashi, H.(2002). Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater. Proc Natl Acad Sci U S A 99, 996–1001.[CrossRef] [Google Scholar]
  45. Stackebrandt, E., Kramer, I., Swiderski, J. & Hippe, H.(1999). Phylogenetic basis for a taxonomic dissection of the genus Clostridium. FEMS Immunol Med Microbiol 24, 253–258.[CrossRef] [Google Scholar]
  46. Tamura, K., Dudley, J., Nei, M. & Kumar, S.(2007).mega4: molecular evolutionary genetics analysis (mega) software version 4.0. Mol Biol Evol 24, 1596–1599.[CrossRef] [Google Scholar]
  47. Van de Peer, Y. & De Wachter, R.(1994).treecon for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570. [Google Scholar]
  48. Wiegel, J., Tanner, R. & Rainey, F. A.(2006). An introduction to the family Clostridiaceae. In The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 4, pp. 654–678. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H. Schleifer & E. Stackebrandt. Springer: New York.

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Neighbour-joining distance tree for the set II protein sequences for the clostridia. [PDF](468 KB)


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