There is a need for easy, practical, reliable and robust techniques for the identification and classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence analysis and DNA–DNA hybridization. Here, we demonstrate that multilocus sequence analysis (MLSA) of housekeeping genes is a valuable alternative technique. An MLSA study of 10 housekeeping genes (, , , , , , , , and ) was performed on 34 representatives of the genus . Genetic analysis and comparison with 16S and 23S rRNA gene sequences demonstrated clear species boundaries and a higher discrimination potential for all housekeeping genes. Comparison of housekeeping gene sequence data with DNA–DNA reassociation data revealed good correlation at the intraspecies level, but indicated that housekeeping gene sequencing is superior to DNA–DNA hybridization for the assessment of genetic relatedness between species. Our MLSA data, confirmed by DNA–DNA hybridizations, support the suggestion that is a later heterotypic synonym of .


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  1. Acinas, S. G., Marcelino, L. A., Klepac-Ceraj, V. & Polz, M. F.(2004). Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J Bacteriol 186, 2629–2635.[CrossRef] [Google Scholar]
  2. Adekambi, T. & Drancourt, M.(2004). Dissection of phylogenetic relationships among 19 rapidly growing Mycobacterium species by 16S rRNA, hsp65, sodA, recA and rpoB gene sequencing. Int J Syst Evol Microbiol 54, 2095–2105.[CrossRef] [Google Scholar]
  3. 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 database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef] [Google Scholar]
  4. Amann, R. I., Lin, C. H., Key, R., Montgomery, L. & Stahl, D. A.(1992). Diversity among Fibrobacter strains: towards a phylogenetic classification. Syst Appl Microbiol 15, 23–32. [Google Scholar]
  5. Baele, M., Baele, P., Vaneechoutte, M., Storms, V., Butaye, P., Devriese, L. A., Verschraegen, G., Gillis, M. & Haesebrouck, F.(2000). Application of tRNA intergenic spacer PCR for identification of Enterococcus species. J Clin Microbiol 38, 4201–4207. [Google Scholar]
  6. Charles, L., Carbone, I., Davies, K. G., Bird, D., Burke, M., Kerry, B. R. & Opperman, C. H.(2005). Phylogenetic analysis of Pasteuria penetrans by use of multiple genetic loci. J Bacteriol 187, 5700–5708.[CrossRef] [Google Scholar]
  7. Cho, J. C. & Tiedje, J. M.(2001). Bacterial species determination from DNA-DNA hybridization by using genome fragments and DNA microarrays. Appl Environ Microbiol 67, 3677–3682.[CrossRef] [Google Scholar]
  8. Christensen, H. & Olsen, J. E.(1998). Phylogenetic relationships of Salmonella based on DNA sequence comparison of atpD encoding the beta subunit of ATP synthase. FEMS Microbiol Lett 161, 89–96. [Google Scholar]
  9. Christensen, H., Kuhnert, P., Olsen, J. E. & Bisgaard, M.(2004). Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae. Int J Syst Evol Microbiol 54, 1601–1609.[CrossRef] [Google Scholar]
  10. Coenye, T., Gevers, D., Van de Peer, Y., Vandamme, P. & Swings, J.(2005). Towards a prokaryotic genomic taxonomy. FEMS Microbiol Rev 29, 147–167. [Google Scholar]
  11. de Lajudie, P., Willems, A., Pot, B., Dewettinck, D., Maestrojuan, G., Neyra, M., Collins, M. D., Dreyfus, B., Kersters, K. & Gillis, M.(1994). Polyphasic taxonomy of rhizobia – emendation of the genus Sinorhizobium and description of Sinorhizobium meliloti comb. nov., Sinorhizobium saheli sp. nov. and Sinorhizobium terangae sp. nov. Int J Syst Bacteriol 44, 715–733.[CrossRef] [Google Scholar]
  12. DelVecchio, V. G., Kapatral, V., Redkar, R. J., Patra, G., Mujer, C., Los, T., Ivanova, N., Anderson, I., Bhattacharyya, A. & other authors(2002). The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci U S A 99, 443–448.[CrossRef] [Google Scholar]
  13. Euzéby, J. P. & Tindall, B. J.(2004). Status of strains that contravene Rules 27(3) and 30 of the Bacteriological Code. Request for an Opinion. Int J Syst Evol Microbiol 54, 293–301.[CrossRef] [Google Scholar]
  14. Ezaki, T., Hashimoto, Y. & Yabuuchi, E.(1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[CrossRef] [Google Scholar]
  15. Farris, J. S., Kallersjo, M., Kluge, A. G. & Bult, C.(1995). Constructing a significance test for incongruence. Syst Biol 44, 570–572.[CrossRef] [Google Scholar]
  16. Fox, G. E., Wisotzkey, J. D. & Jurtshuk, P., Jr(1992). How close is close: 16S ribosomal RNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42, 166–170.[CrossRef] [Google Scholar]
  17. Galibert, F., Finan, T. M., Long, S. R., Puhler, A., Abola, P., Ampe, F., Barloy-Hubler, F., Barnett, M. J., Becker, A. & other authors(2001). The composite genome of the legume symbiont Sinorhizobium meliloti. Science 293, 668–672.[CrossRef] [Google Scholar]
  18. Gevers, D., Cohan, F. M., Lawrence, J. G., Spratt, B. G., Coenye, T., Feil, E. J., Stackebrandt, E., Van de Peer, Y., Vandamme, P. & other authors(2005). Re-evaluating prokaryotic species. Nat Rev Microbiol 3, 733–739.[CrossRef] [Google Scholar]
  19. Goodfellow, M., Manfio, G. P. & Chun, J.(1997). Towards a practical species concept for cultivable bacteria. In Species: the Units of Biodiversity, pp. 25–59. Edited by M. F. Claridge & H. A. Dawah. London: Chapman & Hall.
  20. Goodner, B., Hinkle, G., Gattung, S., Miller, N., Blanchard, M., Qurollo, B., Goldman, B. S., Cao, Y., Askenazi, M. & other authors(2001). Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 294, 2323–2328.[CrossRef] [Google Scholar]
  21. Goris, J., Konstantinidis, K. T., Klappenbach, J. A., Coenye, T., Vandamme, P. & Tiedje, J. M.(2007). DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57, 81–91.[CrossRef] [Google Scholar]
  22. Holmes, D. E., Nevin, K. P. & Lovley, D. R.(2004). Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov. Int J Syst Evol Microbiol 54, 1591–1599.[CrossRef] [Google Scholar]
  23. Jaspers, E. & Overmann, J.(2004). Ecological significance of microdiversity: identical 16S rRNA gene sequences can be found in bacteria with highly divergent genomes and ecophysiologies. Appl Environ Microbiol 70, 4831–4839.[CrossRef] [Google Scholar]
  24. Kaneko, T., Nakamura, Y., Sato, S., Asamizu, E., Kato, T., Sasamoto, S., Watanabe, A., Idesawa, K., Ishikawa, K. & other authors(2000). Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti. DNA Res 7, 331–338.[CrossRef] [Google Scholar]
  25. Kaneko, T., Nakamura, Y., Sato, S., Minamisawa, K., Uchiumi, T., Sasamoto, S., Watanabe, A., Idesawa, K., Iriguchi, M. & other authors(2002). Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Res 9, 189–197.[CrossRef] [Google Scholar]
  26. Konstantinidis, K. T. & Tiedje, J. M.(2005). Genomic insights that advance the species definition for prokaryotes. Proc Natl Acad Sci U S A 102, 2567–2572.[CrossRef] [Google Scholar]
  27. Konstantinidis, K. T., Ramette, A. & Tiedje, J. M.(2006). Toward a more robust assessment of intraspecies diversity, using fewer genetic markers. Appl Environ Microbiol 72, 7286–7293.[CrossRef] [Google Scholar]
  28. Kwon, S. W., Park, J. Y., Kim, J. S., Kang, J. W., Cho, Y. H., Lim, C. K., Parker, M. A. & Lee, G. B.(2005). Phylogenetic analysis of the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium on the basis of 16S rRNA gene and internally transcribed spacer region sequences. Int J Syst Evol Microbiol 55, 263–270.[CrossRef] [Google Scholar]
  29. Larimer, F. W., Chain, P., Hauser, L., Lamerdin, J., Malfatti, S., Do, L., Land, M. L., Pelletier, D. A., Beatty, J. T. & other authors(2004). Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris. Nat Biotechnol 22, 55–61.[CrossRef] [Google Scholar]
  30. Maiden, M. C. J., Bygraves, J. A., Feil, E., Morelli, G., Russell, J. E., Urwin, R., Zhang, Q., Zhou, J. J., Zurth, K. & other authors(1998). Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci U S A 95, 3140–3145.[CrossRef] [Google Scholar]
  31. Marmur, J.(1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3, 208–218.[CrossRef] [Google Scholar]
  32. Martens, M., Delaere, M., Coopman, R., De Vos, P., Gillis, M. & Willems, A.(2007). Multilocus sequence analysis of Ensifer and related taxa. Int J Syst Evol Microbiol 57, 489–503.[CrossRef] [Google Scholar]
  33. Mollet, C., Drancourt, M. & Raoult, D.(1997).rpoB sequence analysis as a novel basis for bacterial identification. Mol Microbiol 26, 1005–1011.[CrossRef] [Google Scholar]
  34. Naser, S. M., Thompson, F. L., Hoste, B., Gevers, D., Dawyndt, P., Vancanneyt, M. & Swings, J.(2005). Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151, 2141–2150.[CrossRef] [Google Scholar]
  35. Naser, S. M., Dawyndt, P., Hoste, B., Gevers, D., Vandemeulebroecke, K., Cleenwerck, I., Vancanneyt, M. & Swings, J.(2007). Identification of lactobacilli by pheS and rpoA gene sequence analyses. Int J Syst Evol Microbiol 57, 2777–2789.[CrossRef] [Google Scholar]
  36. Nick, G., Jussila, M., Hoste, B., Niemi, R. M., Kaijalainen, S., de Lajudie, P., Gillis, M., de Bruijn, F. J. & Lindström, K.(1999). Rhizobia isolated from root nodules of tropical leguminous trees characterized using DNA-DNA dot-blot hybridisation and rep-PCR genomic fingerprinting. Syst Appl Microbiol 22, 287–299.[CrossRef] [Google Scholar]
  37. Nierman, W. C., Feldblyum, T. V., Laub, M. T., Paulsen, I. T., Nelson, K. E., Eisen, J., Heidelberg, J. F., Alley, M. R. K., Ohta, N. & other authors(2001). Complete genome sequence of Caulobacter crescentus. Proc Natl Acad Sci U S A 98, 4136–4141.[CrossRef] [Google Scholar]
  38. Nørskov-Lauritsen, N., Bruun, B. & Kilian, M.(2005). Multilocus sequence phylogenetic study of the genus Haemophilus with description of Haemophilus pittmaniae sp. nov. Int J Syst Evol Microbiol 55, 449–456.[CrossRef] [Google Scholar]
  39. Owen, R. J. & Pitcher, D.(1983). Current methods for determining DNA-base composition and levels of DNA-DNA hybridization. J Appl Bacteriol 55, R16–R16. [Google Scholar]
  40. Paulsen, I. T., Seshadri, R., Nelson, K. E., Eisen, J. A., Heidelberg, J. F., Read, T. D., Dodson, R. J., Umayam, L., Brinkac, L. M. & other authors(2002). The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc Natl Acad Sci U S A 99, 13148–13153.[CrossRef] [Google Scholar]
  41. Peng, G. X., Tan, Z. Y., Wang, E. T., Reinhold-Hurek, B., Chen, W. F. & Chen, W. X.(2002). Identification of isolates from soybean nodules in Xinjiang Region as Sinorhizobium xinjiangense and genetic differentiation of S. xinjiangense from Sinorhizobium fredii. Int J Syst Evol Microbiol 52, 457–462. [Google Scholar]
  42. Posada, D. & Buckley, T. R.(2004). Model selection and model averaging in phylogenetics: advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. Syst Biol 53, 793–808.[CrossRef] [Google Scholar]
  43. Posada, D. & Crandall, K. A.(1998).modeltest: testing the model of DNA substitution. Bioinformatics 14, 817–818.[CrossRef] [Google Scholar]
  44. Rokas, A., King, N., Finnerty, J. & Carroll, S. B.(2003). Conflicting phylogenetic signals at the base of the metazoan tree. Evol Dev 5, 346–359.[CrossRef] [Google Scholar]
  45. Rönner, S., Liesack, W., Wolters, J. & Stackebrandt, E.(1991). Cloning and sequencing of a large fragment of the atpD gene of Pirellula marina – a contribution to the phylogeny of Planctomycetales. Endocytobiosis Cell Res 7, 219–229. [Google Scholar]
  46. Rosselló-Mora, R.(2006). DNA-DNA reassociation methods applied to microbial taxonomy and their critical evaluation. In Molecular Identification, Systematics, and Population Structure of Prokaryotes, pp. 23–50. Edited by E. Stackebrandt. Heidelberg: Springer.
  47. Selenska-Pobell, S. & Evguenieva-Hackenberg, E.(1995). Fragmentations of the large-subunit rRNA in the family Rhizobiaceae. J Bacteriol 177, 6993–6998. [Google Scholar]
  48. Stackebrandt, E. & Goebel, B. M.(1994). Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.[CrossRef] [Google Scholar]
  49. Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A., Kämpfer, P., Maiden, M. C. J., Nesme, X., Rosselló-Mora, R., Swings, J. & other authors(2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[CrossRef] [Google Scholar]
  50. Stackebrandt, E., Päuker, O., Steiner, U., Schumann, P., Sträubler, B., Heibei, S. & Lang, E.(2007). Taxonomic characterization of members of the genus Corallococcus: molecular divergence versus phenotypic coherency. Syst Appl Microbiol 30, 109–118.[CrossRef] [Google Scholar]
  51. Sullivan, J. T., Eardly, B. D., van Berkum, P. & Ronson, C. W.(1996). Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus. Appl Environ Microbiol 62, 2818–2825. [Google Scholar]
  52. Swofford, D. L.(2002).paup*: phylogenetic analysis using parsimony (and other methods), version 4. Sunderland, MA: Sinauer Associates.
  53. Tan, Z. Y., Xu, X. D., Wang, E. T., Gag, J. L., Martínez-Romero, E. & Chen, W. X.(1997). Phylogenetic and genetic relationships of Mesorhizobium tianshanense and related rhizobia. Int J Syst Bacteriol 47, 874–879.[CrossRef] [Google Scholar]
  54. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G.(1997). The clustal_x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882.[CrossRef] [Google Scholar]
  55. Thompson, F. L., Gevers, D., Thompson, C. C., Dawyndt, P., Naser, S., Hoste, B., Munn, C. B. & Swings, J.(2005). Phylogeny and molecular identification of vibrios on the basis of multilocus sequence analysis. Appl Environ Microbiol 71, 5107–5115.[CrossRef] [Google Scholar]
  56. Toledo, I., Lloret, L. & Martínez-Romero, E.(2003).Sinorhizobium americanum sp. nov., a new Sinorhizobium species nodulating native Acacia spp. in Mexico. Syst Appl Microbiol 26, 54–64.[CrossRef] [Google Scholar]
  57. Turner, S. L., Zhang, X.-X., Li, F.-D. & Young, J. P. W.(2002). What does a bacterial genome sequence represent? Mis-assignment of MAFF 303099 to the genospecies Mesorhizobium loti. Microbiology 148, 3330–3331. [Google Scholar]
  58. Van Camp, G., Chapelle, S. & De Wachter, R.(1993). Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences. Curr Microbiol 27, 147–151.[CrossRef] [Google Scholar]
  59. Wang, E. T., Tan, Z. Y., Willems, A., Fernández-López, M., Reinhold-Hurek, B. & Martínez-Romero, E.(2002).Sinorhizobium morelense sp nov., a Leucaena leucocephala-associated bacterium that is highly resistant to multiple antibiotics. Int J Syst Evol Microbiol 52, 1687–1693.[CrossRef] [Google Scholar]
  60. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Moore, W. E. C., Murray, R. G. E. & other authors(1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[CrossRef] [Google Scholar]
  61. Wernersson, R. & Pedersen, A. G.(2003). RevTrans: multiple alignment of coding DNA from aligned amino acid sequences. Nucleic Acids Res 31, 3537–3539.[CrossRef] [Google Scholar]
  62. Wertz, J. E., Goldstone, C., Gordon, D. M. & Riley, M. A.(2003). A molecular phylogeny of enteric bacteria and implications for a bacterial species concept. J Evol Biol 16, 1236–1248.[CrossRef] [Google Scholar]
  63. Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., de Lajudie, P., De Vos, P. & Gillis, M.(2001). DNA–DNA hybridization study of Bradyrhizobium strains. Int J Syst Evol Microbiol 51, 1315–1322. [Google Scholar]
  64. Willems, A., Fernández-López, M., Muñoz-Adelantado, E., Goris, J., De Vos, P., Martínez-Romero, E., Toro, N. & Gillis, M.(2003). Description of new Ensifer strains from nodules and proposal to transfer Ensifer adhaerens Casida 1982 to Sinorhizobium as Sinorhizobium adhaerens comb. nov. Request for an Opinion. Int J Syst Evol Microbiol 53, 1207–1217.[CrossRef] [Google Scholar]
  65. Woese, C. R.(1987). Bacterial evolution. Microbiol Rev 51, 221–271. [Google Scholar]
  66. Wood, D. W., Setubal, J. C., Kaul, R., Monks, D. E., Kitajima, J. P., Okura, V. K., Zhou, Y., Chen, L., Wood, G. E. & other authors(2001). The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294, 2317–2323.[CrossRef] [Google Scholar]
  67. Yamamoto, S. & Harayama, S.(1995). PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains. Appl Environ Microbiol 61, 1104–1109. [Google Scholar]
  68. Yamamoto, S., Bouvet, P. J. M. & Harayama, S.(1999). Phylogenetic structures of the genus Acinetobacter based on gyrB sequences: comparison with the grouping by DNA–DNA hybridization. Int J Syst Bacteriol 49, 87–95.[CrossRef] [Google Scholar]
  69. Young, J. M.(2003). The genus name Ensifer Casida 1982 takes priority over Sinorhizobium Chen et al. 1988, and Sinorhizobium morelense Wang et al. 2002 is a later synonym of Ensifer adhaerens Casida 1982. Is the combination ‘Sinorhizobium adhaerens’ (Casida 1982) Willems et al. 2003 legitimate? Request for an Opinion. Int J Syst Evol Microbiol 53, 2107–2110.[CrossRef] [Google Scholar]
  70. Zeigler, D. R.(2003). Gene sequences useful for predicting relatedness of whole genomes in bacteria. Int J Syst Evol Microbiol 53, 1893–1900.[CrossRef] [Google Scholar]

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