The species complex is a tight assemblage of closely related species. For many years, it has been recognized that these species cannot be differentiated on the basis of phenotypic characteristics. Recently, it has been shown that phylogenetic analysis of the 16S rRNA gene also fails to differentiate species within the complex due to the highly conserved nature of the gene, yet DNA–DNA hybridization values fall well below 70 % for the same species comparisons. As a complementary approach, we propose that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel taxa. Indeed, our phylogenetic analyses revealed the existence of a previously unknown group of strains closely related to, but distinct from, subsp. . Results of matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses revealed that the group produces a novel surfactin-like lipopeptide with mass / 1120.8 that is not produced by the other currently recognized subspecies. In addition, the group displayed differences in the total cellular content of the fatty acids C and iso-C 10 that distinguish it from the closely related subsp. . Consequently, the correlation of these novel phenotypic traits with the phylogenetic distinctiveness of this previously unknown subspecies group showed that phylogenetic analysis of multiple protein-coding loci can be used as a means to detect and differentiate novel taxa. Therefore, we propose that this new group should be recognized as representing a novel taxon, subsp. subsp. nov., with the type strain NRRL B-23052 (=KCTC 13429=BGSC 3A28).


Article metrics loading...

Loading full text...

Full text loading...



  1. Ash, C., Farrow, J. A. E., Wallbanks, S. & Collins, M. D.(1991). Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Lett Appl Microbiol 13, 202–206. [Google Scholar]
  2. Avise, J. C.(2000).Phylogeography. The History and Formation of Species. Cambridge, MA: Harvard University Press.
  3. Bischoff, K. M., Rooney, A. P., Li, X. L., Liu, S. & Hughes, S. R.(2006). Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnol Lett 28, 1761–1765.[CrossRef] [Google Scholar]
  4. Chun, J. & Bae, K. S.(2000). Phylogenetic analysis of Bacillus subtilis and related taxa based on partial gyrA sequences. Antonie Van Leeuwenhoek 78, 123–127.[CrossRef] [Google Scholar]
  5. Cohan, F. M.(2001). Bacterial species and speciation. Syst Biol 50, 513–524.[CrossRef] [Google Scholar]
  6. Cohan, F. M.(2002). What are bacterial species? Annu Rev Microbiol 56, 457–487.[CrossRef] [Google Scholar]
  7. Cohan, F. M.(2006). Towards a conceptual and operational union of bacterial systematics, ecology, and evolution. Philos Trans R Soc Lond B Biol Sci 361, 1985–1996.[CrossRef] [Google Scholar]
  8. Cohn, F.(1872). Untersuchungen über bakterien. Beitr Biol Pflanz 1, 127–244 (in German). [Google Scholar]
  9. Dykhuizen, D. E. & Green, L.(1991). Recombination in Escherichia coli and the definition of biological species. J Bacteriol 173, 7257–7268. [Google Scholar]
  10. Felsenstein, J.(2006).phylip (phylogeny inference package) version 3.66. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA. http://evolution.gs.washington.edu/phylip.html.
  11. Gatson, J. W., Benz, B. F., Chandrasekaran, C., Satomi, M., Venkateswaran, K. & Hart, M. E.(2006).Bacillus tequilensis sp. nov., isolated from a 2000-year-old Mexican shaft-tomb, is closely related to Bacillus subtilis. Int J Syst Evol Microbiol 56, 1475–1484.[CrossRef] [Google Scholar]
  12. 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]
  13. Gordon, R. E., Haynes, W. C. & Pang, C. H.-N.(1973).The Genus Bacillus. US Department of Agriculture Handbook no. 427. Washington, DC: Agricultural Research Service.
  14. Hou, C. T., Labeda, D. P. & Rooney, A. P.(2005). Evaluation of microbial strains for linoleic acid hydroxylation and reclassification of strain ALA2. Antonie Van Leeuwenhoek 88, 167–171.[CrossRef] [Google Scholar]
  15. Koeppel, A., Perry, E. B., Sikorski, J., Krizanc, D., Warner, A., Ward, D. M., Rooney, A. P., Brambilla, E., Connor, N. & other authors(2008). Identifying the fundamental units of bacterial diversity: a paradigm shift to incorporate ecology into bacterial systematics. Proc Natl Acad Sci U S A 105, 2504–2509.[CrossRef] [Google Scholar]
  16. 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]
  17. Kundrat, W.(1963). Zur differenzierung aerober sporenbildner (Genus Bacillus Cohn). Zentralbl F Veterinaermed Reihe B 10, 418–426 (in German). [Google Scholar]
  18. Lane, D. J.(1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115–175. Edited by E. Stackebrandt & M. Goodfellow. Chichester: Wiley.
  19. Marchesi, J. R., Sato, T., Weightman, A. J., Martin, T. A., Fry, J. C., Hiom, S. J., Dymock, D. & Wade, W. G.(1998). Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl Environ Microbiol 64, 795–799. [Google Scholar]
  20. Nakamura, L. K.(1989). Taxonomic relationship of black-pigmented Bacillus subtilis strains and a proposal for Bacillus atrophaeus sp. nov. Int J Syst Bacteriol 39, 295–300.[CrossRef] [Google Scholar]
  21. Nakamura, L. K., Roberts, M. S. & Cohan, F. M.(1999). Relationship of Bacillus subtilis clades associated with strains 168 and W23: a proposal for Bacillus subtilis subsp. subtilis subsp. nov. and Bacillus subtilis subsp. spizizenii subsp. nov. Int J Syst Bacteriol 49, 1211–1215.[CrossRef] [Google Scholar]
  22. Palmisano, M. M., Nakamura, L. K., Duncan, K. E., Istock, C. A. & Cohan, F. M.(2001).Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona. Int J Syst Evol Microbiol 51, 1671–1679.[CrossRef] [Google Scholar]
  23. Perry, E. B., Krizan, D., Rooney, A. P., Sikorski, J., Nevo, E. & Cohan, F. M.(2007). Identifying the fundamental units of diversity among Bacillus isolates from “Evolution Canyon” III. Isr J Ecol Evol 52, 543–552. [Google Scholar]
  24. Price, N. P. J., Rooney, A. P., Swezey, J. L., Perry, E. & Cohan, F. M.(2007). Mass spectroscopic analysis of lipopeptide for Bacillus strains isolated from diverse geographical locations. FEMS Microbiol Lett 271, 83–89.[CrossRef] [Google Scholar]
  25. Priest, F. G., Goodfellow, M., Shute, L. A. & Berkeley, R. C. W.(1987).Bacillus amyloliquefaciens sp. nov., nom. rev. Int J Syst Bacteriol 37, 69–71.[CrossRef] [Google Scholar]
  26. Roberts, M. S., Nakamura, L. K. & Cohan, F. M.(1994).Bacillus mojavensis sp. nov., distinguishable from Bacillus subtilis by sexual isolation, divergence in DNA sequence, and differences in fatty acid composition. Int J Syst Bacteriol 44, 256–264.[CrossRef] [Google Scholar]
  27. Roberts, M. S., Nakamura, L. K. & Cohan, F. M.(1996).Bacillus vallismortis sp. nov., a close relative of Bacillus subtilis, isolated from soil in Death Valley, California. Int J Syst Bacteriol 46, 470–475.[CrossRef] [Google Scholar]
  28. Rooney, A. P., Swezey, J. L., Wicklow, D. T. & McAtee, M. J.(2005). Bacterial species diversity in cigarettes linked to an investigation of severe pneumonitis in U.S. Military personnel deployed in Operation Iraqi Freedom. Curr Microbiol 51, 46–52.[CrossRef] [Google Scholar]
  29. Ruiz-García, C., Béjar, V., Martínez-Checa, F., Llamas, I. & Quesada, E.(2005a).Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Velez in Malaga, southern Spain. Int J Syst Evol Microbiol 55, 191–195.[CrossRef] [Google Scholar]
  30. Ruiz-García, C., Quesada, E., Martínez-Checa, F., Llamas, I., Urdaci, M. C. & Béjar, V.(2005b).Bacillus axarquiensis sp. nov. and Bacillus malacitensis sp. nov., isolated from river-mouth sediments in southern Spain. Int J Syst Evol Microbiol 55, 1279–1285.[CrossRef] [Google Scholar]
  31. Saitou, N. & Nei, M.(1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425. [Google Scholar]
  32. Schmidt, D. D., Frommer, W., Miller, L. & Truscheit, E.(1979). Glucosidase-inhibitoren aus Bazillen. Naturwissenschaften 66, 584–585 (in German). [Google Scholar]
  33. Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P. A. D., Kämpfer, P., Maiden, M. C. J., Nesme, X., Rossello-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, 1043–1052.[CrossRef] [Google Scholar]
  34. Tamura, K. & Nei, M.(1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10, 512–526. [Google Scholar]
  35. Wang, L. T., Lee, F. L., Tai, C. J., Yokota, A. & Kuo, H. P.(2007). Reclassification of Bacillus axarquiensis Ruiz-Garcia et al. 2005 and Bacillus malacitensis Ruiz-Garcia et al. 2005 as later heterotypic synonyms of Bacillus mojavensis Roberts et al. 1994. Int J Syst Evol Microbiol 57, 1663–1667.[CrossRef] [Google Scholar]
  36. Wang, L. T., Lee, F. L., Tai, C. J. & Kuo, H. P.(2008).Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens. Int J Syst Evol Microbiol 58, 671–675.[CrossRef] [Google Scholar]
  37. Xia, X. & Xie, Z.(2001).dambe: data analysis in molecular biology and evolution. J Hered 92, 371–373.[CrossRef] [Google Scholar]
  38. Zwickl, D. J.(2006). Genetic algorithm approaches for the phylogenetic analysis of large biological sequence datasets under the maximum likelihood criterion. The University of Texas at Austin. www.bio.utexas.edu/faculty/antisense/garli/Garli.html

Data & Media loading...


Maximum-parsimony cladogram of the species complex. [PDF](87 KB)


Mean values of synonymous and non-synonymous substitutions per site among protein-coding genes. [PDF](19 KB)


Geographical origins, isolation substrates and maximum growth temperatures of subsp. subsp. nov., subsp. and subsp. . [PDF](17 KB)

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error