Streptomycetes are a complex group of actinomycetes that produce diverse bioactive metabolites of commercial significance. Systematics can provide a useful framework for identifying species that may produce novel metabolites. However, previously proposed approaches to the systematics of have suffered from either poor interlaboratory comparability or insufficient resolution. In particular, the 16S rRNA gene clade is the most challenging and least defined group within the genus in terms of phylogeny. Here we report the results of a multilocus sequence analysis scheme developed to address the phylogeny of this clade. Sequence fragments of six housekeeping genes, , , , , and 16S rRNA, were obtained for 53 reference strains that represent 45 valid species and subspecies. Analysis of each individual locus confirmed the suitability of loci and the congruence of single-gene trees for concatenation. Concatenated trees of three, four, five and all six genes were constructed, and the stability of the topology and discriminatory power of each tree were analysed. It can be concluded from the results that phylogenetic analysis based on multilocus sequences is more accurate and robust for species delineation within . A multilocus phylogeny of six genes proved to be optimal for elucidating the interspecies relationships within the 16S rRNA gene clade. Our multilocus sequence analysis scheme provides a valuable tool that can be applied to other clades for refining the systematic framework of this genus.


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  1. Adiri, R. S., Gophna, U. & Ron, E. Z.(2003). Multilocus sequence typing (MLST) of Escherichia coli O78 strains. FEMS Microbiol Lett 222, 199–203.[CrossRef] [Google Scholar]
  2. Anderson, A. S. & Wellington, E. M.(2001). The taxonomy of Streptomyces and related genera. Int J Syst Evol Microbiol 51, 797–814.[CrossRef] [Google Scholar]
  3. Baldwin, A., Mahenthiralingam, E., Thickett, K. M., Honeybourne, D., Maiden, M. C., Govan, J. R., Speert, D. P., Lipuma, J. J., Vandamme, P. & Dowson, C. G.(2005). Multilocus sequence typing scheme that provides both species and strain differentiation for the Burkholderia cepacia complex. J Clin Microbiol 43, 4665–4673.[CrossRef] [Google Scholar]
  4. Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M., Challis, G. L., Thomson, N. R., James, K. D., Harris, D. E., Quail, M. A., Kieser, H. & other authors(2002). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147.[CrossRef] [Google Scholar]
  5. Berdy, J.(2005). Bioactive microbial metabolites. J Antibiot 58, 1–26.[CrossRef] [Google Scholar]
  6. Clardy, J., Fischbach, M. A. & Walsh, C. T.(2006). New antibiotics from bacterial natural products. Nat Biotechnol 24, 1541–1550.[CrossRef] [Google Scholar]
  7. Coffey, T. J., Pullinger, G. D., Urwin, R., Jolley, K. A., Wilson, S. M., Maiden, M. C. & Leigh, J. A.(2006). First insights into the evolution of Streptococcus uberis: a multilocus sequence typing scheme that enables investigation of its population biology. Appl Environ Microbiol 72, 1420–1428.[CrossRef] [Google Scholar]
  8. Devulder, G., Pérouse de Montclos, M. & Flandrois, J. P.(2005). A multigene approach to phylogenetic analysis using the genus Mycobacterium as a model. Int J Syst Evol Microbiol 55, 293–302.[CrossRef] [Google Scholar]
  9. Dingle, K. E., Colles, F. M., Wareing, D. R., Ure, R., Fox, A. J., Bolton, F. E., Bootsma, H. J., Willems, R. J., Urwin, R. & Maiden, M. C.(2001). Multilocus sequence typing system for Campylobacter jejuni. J Clin Microbiol 39, 14–23.[CrossRef] [Google Scholar]
  10. Egan, S., Wiener, P., Kallifidas, D. & Wellington, E. M.(2001). Phylogeny of Streptomyces species and evidence for horizontal transfer of entire and partial antibiotic gene clusters. Antonie van Leeuwenhoek 79, 127–133.[CrossRef] [Google Scholar]
  11. Enright, M. C. & Spratt, B. G.(1999). Multilocus sequence typing. Trends Microbiol 7, 482–487.[CrossRef] [Google Scholar]
  12. Felsenstein, J.(1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.[CrossRef] [Google Scholar]
  13. Felsenstein, J.(2006).phylip (phylogeny inference package). Version 3.66. Department of Genome Sciences, University of Washington, Seattle, USA.
  14. Grund, E., Knorr, C. & Eichenlaub, R.(1990). Catabolism of benzoate and monohydroxylated benzoates by Amycolatopsis and Streptomyces spp. Appl Environ Microbiol 56, 1459–1464. [Google Scholar]
  15. Hatano, K., Nishii, T. & Kasai, H.(2003). Taxonomic re-evaluation of whorl-forming Streptomyces (formerly Streptoverticillium) species by using phenotypes, DNA–DNA hybridization and sequences of gyrB, and proposal of Streptomyces luteireticuli (ex Katoh and Arai 1957) corrig., sp. nov., nom. rev. Int J Syst Evol Microbiol 53, 1519–1529.[CrossRef] [Google Scholar]
  16. Hilario, E., Buckley, T. R. & Young, J. M.(2004). Improved resolution on the phylogenetic relationships among Pseudomonas by the combined analysis of atpD, carA, recA and 16S rDNA. Antonie Van Leeuwenhoek 86, 51–64.[CrossRef] [Google Scholar]
  17. Hillis, D. M., Moritz, C. & Mable, B. K.(1996).Molecular Systematics. Sunderland, MA: Sinauer Associates.
  18. Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, Y., Hattori, M. & Omura, S.(2003). Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat Biotechnol 21, 526–531.[CrossRef] [Google Scholar]
  19. Ishiyama, D., Vujaklija, D. & Davies, J.(2004). Novel pathway of salicylate degradation by Streptomyces sp. strain WA46. Appl Environ Microbiol 70, 1297–1306.[CrossRef] [Google Scholar]
  20. Iwamoto, T., Tsujii, E., Ezaki, M., Fujie, A., Hashimoto, S., Okuhara, M., Kohsaka, M., Imanaka, H., Kawabata, K. & other authors(1990). FR109615, a new antifungal antibiotic from Streptomyces setonii. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity. J Antibiot 43, 1–7.[CrossRef] [Google Scholar]
  21. Jolley, K. A., Feil, E. J., Chan, M. S. & Maiden, M. C.(2001). Sequence type analysis and recombinational tests (start). Bioinformatics 17, 1230–1231.[CrossRef] [Google Scholar]
  22. Kim, B.-J., Kim, C.-J., Chun, J., Koh, Y.-H., Lee, S.-H., Hyun, J.-W., Cha, C.-Y. & Kook, Y.-H.(2004). Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase β-subunit gene (rpoB) sequences. Int J Syst Evol Microbiol 54, 593–598.[CrossRef] [Google Scholar]
  23. Kimura, M.(1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef] [Google Scholar]
  24. Kumar, S., Tamura, K. & Nei, M.(2004).mega3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform 5, 150–163.[CrossRef] [Google Scholar]
  25. Lanoot, B., Vancanneyt, M., Dawyndt, P., Cnockaert, M., Zhang, J., Huang, Y., Liu, Z. & Swings, J.(2004). BOX-PCR fingerprinting as a powerful tool to reveal synonymous names in the genus Streptomyces. Emended descriptions are proposed for the species Streptomyces cinereorectus, S. fradiae, S. tricolor, S. colombiensis, S. filamentosus, S. vinaceus and S. phaeopurpureus. Syst Appl Microbiol 27, 84–92.[CrossRef] [Google Scholar]
  26. Lanoot, B., Vancanneyt, M., Hoste, B., Vandemeulebroecke, K., Cnockaert, M. C., Dawyndt, P., Liu, Z., Huang, Y. & Swings, J.(2005a). Grouping of streptomycetes using 16S-ITS RFLP fingerprinting. Res Microbiol 156, 755–762.[CrossRef] [Google Scholar]
  27. Lanoot, B., Vancanneyt, M., Van Schoor, A., Liu, Z. & Swings, J.(2005b). Reclassification of Streptomyces nigrifaciens as a later synonym of Streptomyces flavovirens; Streptomyces citreofluorescens, Streptomyces chrysomallus subsp. chrysomallus and Streptomyces fluorescens as later synonyms of Streptomyces anulatus; Streptomyces chibaensis as a later synonym of Streptomyces corchorusii; Streptomyces flaviscleroticus as a later synonym of Streptomyces minutiscleroticus; and Streptomyces lipmanii, Streptomyces griseus subsp. alpha, Streptomyces griseus subsp. cretosus and Streptomyces willmorei as later synonyms of Streptomyces microflavus. Int J Syst Evol Microbiol 55, 729–731.[CrossRef] [Google Scholar]
  28. Liu, Z., Shi, Y., Zhang, Y., Zhou, Z., Lu, Z., Li, W., Huang, Y., Rodriguez, C. & Goodfellow, M.(2005). Classification of Streptomyces griseus (Krainsky 1914) Waksman and Henrici 1948 and related species and the transfer of ‘Microstreptospora cinerea' to the genus Streptomyces as Streptomyces yanii sp. nov. Int J Syst Evol Microbiol 55, 1605–1610.[CrossRef] [Google Scholar]
  29. Maiden, M. C., Bygraves, J. A., Feil, E., Morelli, G., Russell, J. E., Urwin, R., Zhang, Q., Zhou, 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]
  30. Moller, E. M., Bahnweg, G., Sandermann, H. & Geiger, H. H.(1992). A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Res 20, 6115–6116.[CrossRef] [Google Scholar]
  31. Nei, M. & Gojobori, T.(1986). Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 3, 418–426. [Google Scholar]
  32. 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]
  33. Okanishi, M.(1972). An evaluation of taxonomic criteria in streptomycetes on the basis of deoxyribonucleic acid homology. J Gen Microbiol 72, 49–58.[CrossRef] [Google Scholar]
  34. Phelan, M. B., Crawford, D. L. & Pometto, A. L., III(1979). Isolation of lignocellulose-decomposing actinomycetes and degradation of specifically 14C-labeled lignocelluloses by six selected Streptomyces strains. Can J Microbiol 25, 1270–1276.[CrossRef] [Google Scholar]
  35. Piel, J.(2004). Metabolites from symbiotic bacteria. Nat Prod Rep 21, 519–538.[CrossRef] [Google Scholar]
  36. Shirling, E. B. & Gottlieb, D.(1966). Methods for characterization of Streptomyces species. Int J Syst Bacteriol 16, 313–340.[CrossRef] [Google Scholar]
  37. Song, J., Lee, S. C., Kang, J. W., Baek, H. J. & Suh, J. W.(2004). Phylogenetic analysis of Streptomyces spp. isolated from potato scab lesions in Korea on the basis of 16S rRNA gene and 16S–23S rDNA internally transcribed spacer sequences. Int J Syst Evol Microbiol 54, 203–209.[CrossRef] [Google Scholar]
  38. Strohl, W. R.(2003). Antimicrobials. In Microbial Diversity and Bioprospecting, pp. 336–355. Edited by A. T. Bull. ASM Press.
  39. Urwin, R. & Maiden, M. C.(2003). Multi-locus sequence typing: a tool for global epidemiology. Trends Microbiol 11, 479–487.[CrossRef] [Google Scholar]
  40. Watve, M. G., Tickoo, R., Jog, M. M. & Bhole, B. D.(2001). How many antibiotics are produced by the genus Streptomyces?Arch Microbiol 176, 386–390.[CrossRef] [Google Scholar]

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Chromosomal locations of the genes used. The positions of the six genes are shown on a map of the genome sequence of A3(2). The 8,667,507 bp genome is divided into 10 segments, with each segment representing 866,751 bp.


All the following trees were constructed using the neighbour-joing method. H37Rv was used as the outgroup. Numbers at nodes represent levels (%) of bootstrap support from 1000 resampled datasets. L and P indicate branches that were also recovered using maximum-likelihood and maximum-parsimony methods, respectively. The bar indicates 2 % estimated sequence divergence. Strains of clusters I, II, III and IV are highlighted with blue, yellow, green and red backgrounds, respectively. Figs S2-S9 are available in a single PDF file.


vol. , part 1, pp. 149-159

In this paper, the following names have been incorrectly placed in quote marks. Although these names have been later transferred, they are all validly published names and should not therefore appear in quote marks throughout this paper.

An amended version of Table 1, with corrected type strains, is available hereas a PDF file.

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