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1 Interdisciplinary Program in Bioinformatics, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
2 School of Biological Sciences and Institute of Microbiology, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea
Correspondence
Jongsik Chun
jchun{at}snu.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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) and that it can be utilized in many ways in various disciplines, notably taxonomy and ecology. Pairwise nucleotide similarity values of 16S rRNA gene sequences have been used routinely for the delineation of prokaryotic species (Stackebrandt & Goebel, 1994) and have been widely accepted. When a 16S rRNA gene sequence is applied for the identification of prokaryotic isolates, the general process includes: (i) similarity search against public domain nucleotide databases, (ii) retrieval of sequences for type strains with validly published names, (iii) calculation of pairwise nucleotide similarity values between sequences of the isolate and phylogenetically neighbouring type strains and (iv) phylogenetic analysis. This process requires expertise in prokaryotic taxonomy and can be complicated and labour-intensive, as public databases have been flooded with sequences of non-type strains including environmental clones and may contain erroneous and mislabelled sequences. Furthermore, there are no curated databases for 16S rRNA gene sequences of type strains of prokaryotic species. In this study, we present a database containing 16S rRNA gene sequences of all prokaryotic type strains and a web-based tool for analysis of 16S rRNA gene sequences, allowing automation of the process indicated above. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Inputs for sequence similarity search.
The formats of input data for sequence similarity searches are either text or tracer files generated from automated sequencers. The PHRED program was used for the base-calling and trimming of uploaded tracer files (Ewing & Green, 1998; Ewing et al., 1998).
Finding phylogenetic neighbours using the EzTaxon server.
The BLAST program (Altschul et al., 1997) was employed for the initial similarity search. The parsed BLAST hits are stored and can be sorted by BLAST scores or e values.
Calculation of pairwise sequence similarity values.
The phylogenetically closest neighbours identified by BLAST search were then selected for the calculation of pairwise nucleotide sequence similarity using the algorithm of Myers & Miller (1988). The alignment gap was not considered in the similarity calculation.
Multiple sequence alignment and phylogenetic inference.
The current version of the EzTaxon server provides multiple sequence alignment by CLUSTAL W (Thompson et al., 1994). The resultant sequence alignment can then be used for the neighbour-joining (Saitou & Nei, 1987), maximum-parsimony (Fitch, 1971), and maximum-likelihood (Felsenstein, 1981) methods using the PHYLIP package (Felsenstein, 2005); all were implemented within the server. The alignment can be exported for use by external programs including jPHYDIT (Jeon et al., 2005), PAUP (Swofford, 2002), PHYLIP, MEGA (Kumar et al., 2004) and MrBayes (Ronquist & Huelsenbeck, 2003).
Operating system and programming languages.
All databases and computer programs were generated using MySQL, JSP and JAVA under Linux operating system.
Availability.
The EzTaxon server is freely accessible over the Internet at http://www.eztaxon.org/
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The overall process for the identification of isolates using the EzTaxon server is given in Fig. 1. The final goal of the system is to find taxonomically meaningful phylogenetic relatives from either sequences or raw tracers (chromatograms). The BLAST search is based on local alignment that cannot be used for the calculation of overall sequence similarity. On the other hand, pairwise global sequence alignment using the algorithm of Myers & Miller (1988) guarantees the identification of the most similar sequence, but requires a higher computing cost. The EzTaxon server utilizes two methods sequentially to obtain accurate search results at reasonable computing cost. Such a combinatory strategy should guarantee the identification of the closest phylogenetic neighbour to the query sequence.
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suggested that a 16S rRNA gene sequence similarity of 97 % should become the boundary for delineation of prokaryotic species, which has been well accepted among microbiologists. More recently, Stackebrandt & Ebers (2006) proposed a more relaxed cut-off value of 98.7–99 %, after inspection of a large amount of recently published data. Even though this new proposal requires further validation and discussion, it is evident that high quality of sequencing should be the prerequisite to the use of lower similarity cut-off values for the identification of prokaryotes.
Similarity values tend to vary depending on the alignment algorithms employed and how gaps are considered. In addition, calculations based on multiple sequence alignment might produce different similarity values depending on the sequences included in the analysis. Therefore, to achieve a reliable and conservative measure of pairwise similarity values, we propose that the calculation should be carried out using rigorous pairwise global alignment algorithms such as that developed by Myers & Miller (1988), and that alignment gaps should not be considered. The calculation of pairwise sequence similarity values from multiple alignments should be avoided.
EzTaxon was successfully developed to include the collection of all reference sequences, and to provide various functions, including a similarity search engine, calculation of pairwise similarity, multiple sequence alignment and phylogenetic treeing algorithms at the server side. This would allow users in places where only poor computing facilities are available to carry out most of the necessary bioinformatic analyses of 16S rRNA gene sequences. It could also be useful in various levels of classes for educational purposes.
In future, the EzTaxon server will be upgraded to include more analysis tools for phylogenetics, taxonomy and ecology of prokaryotes. We also plan to implement parallel computing versions of time-consuming methods such as multiple sequence alignment and maximum-likelihood inference of phylogenetic trees.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Ewing, B. & Green, P. (1998). Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res 8, 186–194.
Ewing, B., Hillier, L., Wendl, M. C. & Green, P. (1998). Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res 8, 175–185.
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
Felsenstein, J. (2005). PHYLIP (Phylogeny Inference Package), version 3.6. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[Abstract]
Jeon, Y. S., Chung, H., Park, S., Hur, I., Lee, J. H. & Chun, J. (2005). jPHYDIT: a JAVA-based integrated environment for molecular phylogeny of ribosomal RNA sequences. Bioinformatics 21, 3171–3173.
Kumar, S., Tamura, K. & Nei, M. (2004). MEGA3: Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5, 150–163.
Myers, E. W. & Miller, W. (1988). Optimal alignments in linear space. Comput Appl Biosci 4, 11–17.
Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574.
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Stackebrandt, E. & Ebers, J. (2006). Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 33, 152–155.
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.
Swofford, D. L. (2002). PAUP*: Phylogenetic analysis using parsimony (*and other methods), version 4. Sunderland, MA: Sinauer Associates.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673–4680.
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Y. Wang, Y.-H. Wu, C.-S. Wang, X.-W. Xu, A. Oren, X.-F. Zhu, and M. Wu Halomonas salifodinae sp. nov., a halophilic bacterium isolated from a salt mine in China Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2855 - 2858. [Abstract] [Full Text] [PDF]
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S. W. Roh, Z.-X. Quan, Y.-D. Nam, H.-W. Chang, K.-H. Kim, S.-K. Rhee, H.-M. Oh, C. O. Jeon, J.-H. Yoon, and J.-W. Bae Marinobacter goseongensis sp. nov., from seawater Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2866 - 2870. [Abstract] [Full Text] [PDF]
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M. C. Gutierrez, A. M. Castillo, M. Kamekura, and A. Ventosa Haloterrigena salina sp. nov., an extremely halophilic archaeon isolated from a salt lake Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2880 - 2884. [Abstract] [Full Text] [PDF]
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K. S. Baik, S. C. Park, M. S. Kim, E. M. Kim, C. Park, J. Chun, and C. N. Seong Luteimonas marina sp. nov., isolated from seawater Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2904 - 2908. [Abstract] [Full Text] [PDF]
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Y.-H. Wu, M. Wu, C.-S. Wang, X.-G. Wang, J.-Y. Yang, A. Oren, and X.-W. Xu Microbacterium profundi sp. nov., isolated from deep-sea sediment of polymetallic nodule environments Int J Syst Evol Microbiol, December 1, 2008; 58(12): 2930 - 2934. [Abstract] [Full Text] [PDF]
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A. Valverde, A. Peix, R. Rivas, E. Velazquez, S. Salazar, I. Santa-Regina, C. Rodriguez-Barrueco, and J. M. Igual Paenibacillus castaneae sp. nov., isolated from the phyllosphere of Castanea sativa Miller Int J Syst Evol Microbiol, November 1, 2008; 58(11): 2560 - 2564. [Abstract] [Full Text] [PDF]
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F.-L. Lee, C.-J. Tien, C.-J. Tai, L.-T. Wang, Y.-C. Liu, and L.-L. Chern Paenibacillus taichungensis sp. nov., from soil in Taiwan Int J Syst Evol Microbiol, November 1, 2008; 58(11): 2640 - 2645. [Abstract] [Full Text] [PDF]
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 S. W. Roh, Y.-D. Nam, H.-W. Chang, K.-H. Kim, M.-S. Kim, J.-H. Ryu, S.-H. Kim, W.-J. Lee, and J.-W. Bae Phylogenetic Characterization of Two Novel Commensal Bacteria Involved with Innate Immune Homeostasis in Drosophila melanogaster Appl. Envir. Microbiol., October 15, 2008; 74(20): 6171 - 6177. [Abstract] [Full Text] [PDF]
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S. H. Ryu, J. H. Park, J. C. Moon, Y. Sung, S.-S. Lee, and C. O. Jeon Flavobacterium resistens sp. nov., isolated from stream sediment Int J Syst Evol Microbiol, October 1, 2008; 58(10): 2266 - 2270. [Abstract] [Full Text] [PDF]
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B.-S. Kim, Y. W. Lim, and J. Chun Sphingopyxis marina sp. nov. and Sphingopyxis litoris sp. nov., isolated from seawater Int J Syst Evol Microbiol, October 1, 2008; 58(10): 2415 - 2419. [Abstract] [Full Text] [PDF]
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S.-K. Tang, X.-P. Tian, X.-Y. Zhi, M. Cai, J.-Y. Wu, L.-L. Yang, L.-H. Xu, and W.-J. Li Haloactinospora alba gen. nov., sp. nov., a halophilic filamentous actinomycete of the family Nocardiopsaceae Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2075 - 2080. [Abstract] [Full Text] [PDF]
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M. Wang, M. Yang, G. Zhou, X. Luo, L. Zhang, Y. Tang, and C. Fang Paenibacillus tarimensis sp. nov., isolated from sand in Xinjiang, China Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2081 - 2085. [Abstract] [Full Text] [PDF]
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J.-H. Qu, H.-F. Li, J.-S. Yang, and H.-L. Yuan Flavobacterium cheniae sp. nov., isolated from sediment of a eutrophic reservoir Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2186 - 2190. [Abstract] [Full Text] [PDF]
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J.-H. Qu and H.-L. Yuan Sediminibacterium salmoneum gen. nov., sp. nov., a member of the phylum Bacteroidetes isolated from sediment of a eutrophic reservoir Int J Syst Evol Microbiol, September 1, 2008; 58(9): 2191 - 2194. [Abstract] [Full Text] [PDF]
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S. Y. Shin, M. Y. Lee, J.-H. Song, and K. S. Ko New Erwinia-Like Organism Causing Cervical Lymphadenitis J. Clin. Microbiol., September 1, 2008; 46(9): 3156 - 3158. [Abstract] [Full Text] [PDF]
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S. G. Dastager, J.-C. Lee, Y.-J. Ju, D.-J. Park, and C.-J. Kim Rubellimicrobium mesophilum sp. nov., a mesophilic, pigmented bacterium isolated from soil Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1797 - 1800. [Abstract] [Full Text] [PDF]
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R. Rivas, P. Garcia-Fraile, J. L. Zurdo-Pineiro, P. F. Mateos, E. Martinez-Molina, E. J. Bedmar, J. Sanchez-Raya, and E. Velazquez Saccharibacillus sacchari gen. nov., sp. nov., isolated from sugar cane Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1850 - 1854. [Abstract] [Full Text] [PDF]
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S. G. Dastager, J.-C. Lee, Y.-J. Ju, D.-J. Park, and C.-J. Kim Frigoribacterium mesophilum sp. nov., a mesophilic actinobacterium isolated from Bigeum Island, Korea Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1869 - 1872. [Abstract] [Full Text] [PDF]
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H.-W. Chang, S. W. Roh, K.-H. Kim, Y.-D. Nam, C. O. Jeon, H.-M. Oh, and J.-W. Bae Vibrio areninigrae sp. nov., a marine bacterium isolated from black sand Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1903 - 1906. [Abstract] [Full Text] [PDF]
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M. C. Marquez, I. J. Carrasco, Y. Xue, Y. Ma, D. A. Cowan, B. E. Jones, W. D. Grant, and A. Ventosa Aquisalibacillus elongatus gen. nov., sp. nov., a moderately halophilic bacterium of the family Bacillaceae isolated from a saline lake Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1922 - 1926. [Abstract] [Full Text] [PDF]
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S. G. Dastager, J.-C. Lee, Y.-J. Ju, D.-J. Park, and C.-J. Kim Leifsonia bigeumensis sp. nov., isolated from soil on Bigeum Island, Korea Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1935 - 1938. [Abstract] [Full Text] [PDF]
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I. J. Carrasco, M. C. Marquez, Y. Xue, Y. Ma, D. A. Cowan, B. E. Jones, W. D. Grant, and A. Ventosa Sediminibacillus halophilus gen. nov., sp. nov., a moderately halophilic, Gram-positive bacterium from a hypersaline lake Int J Syst Evol Microbiol, August 1, 2008; 58(8): 1961 - 1967. [Abstract] [Full Text] [PDF]
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 L. Ponnusamy, N. Xu, S. Nojima, D. M. Wesson, C. Schal, and C. S. Apperson Identification of bacteria and bacteria-associated chemical cues that mediate oviposition site preferences by Aedes aegypti PNAS, July 8, 2008; 105(27): 9262 - 9267. [Abstract] [Full Text] [PDF]
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M. Cai, X.-Y. Zhi, S.-K. Tang, Y.-Q. Zhang, L.-H. Xu, and W.-J. Li Streptomonospora halophila sp. nov., a halophilic actinomycete isolated from a hypersaline soil Int J Syst Evol Microbiol, July 1, 2008; 58(7): 1556 - 1560. [Abstract] [Full Text] [PDF]
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S. W. Roh, Z.-X. Quan, Y.-D. Nam, H.-W. Chang, K.-H. Kim, M.-K. Kim, W.-T. Im, L. Jin, S.-H. Kim, S.-T. Lee, et al. Pedobacter agri sp. nov., from soil Int J Syst Evol Microbiol, July 1, 2008; 58(7): 1640 - 1643. [Abstract] [Full Text] [PDF]
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S. Jit, M. Dadhwal, O. Prakash, and R. Lal Flavobacterium lindanitolerans sp. nov., isolated from hexachlorocyclohexane-contaminated soil Int J Syst Evol Microbiol, July 1, 2008; 58(7): 1665 - 1669. [Abstract] [Full Text] [PDF]
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K. H. Cho, S. G. Hong, H. H. Cho, Y. K. Lee, J. Chun, and H. K. Lee Maribacter arcticus sp. nov., isolated from Arctic marine sediment Int J Syst Evol Microbiol, June 1, 2008; 58(6): 1300 - 1303. [Abstract] [Full Text] [PDF]
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S. G. Dastager, J.-C. Lee, Y.-J. Ju, D.-J. Park, and C.-J. Kim Marmoricola bigeumensis sp. nov., a member of the family Nocardioidaceae Int J Syst Evol Microbiol, May 1, 2008; 58(5): 1060 - 1063. [Abstract] [Full Text] [PDF]
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L.-J. Yuan, Y.-Q. Zhang, Y. Guan, Y.-Z. Wei, Q.-P. Li, L.-Y. Yu, W.-J. Li, and Y.-Q. Zhang Saccharopolyspora antimicrobica sp. nov., an actinomycete from soil Int J Syst Evol Microbiol, May 1, 2008; 58(5): 1180 - 1185. [Abstract] [Full Text] [PDF]
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J.-T. Wang, Y.-J. Chou, J.-H. Chou, C. A. Chen, and W.-M. Chen Tenacibaculum aiptasiae sp. nov., isolated from a sea anemone Aiptasia pulchella Int J Syst Evol Microbiol, April 1, 2008; 58(4): 761 - 766. [Abstract] [Full Text] [PDF]
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M. Pineiro-Vidal, C. G. Carballas, O. Gomez-Barreiro, A. Riaza, and Y. Santos Tenacibaculum soleae sp. nov., isolated from diseased sole (Solea senegalensis Kaup) Int J Syst Evol Microbiol, April 1, 2008; 58(4): 881 - 885. [Abstract] [Full Text] [PDF]
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Y.-J. Chou, J.-H. Chou, K.-Y. Lin, M.-C. Lin, Y.-H. Wei, A. B. Arun, C.-C. Young, and W.-M. Chen Rothia terrae sp. nov. isolated from soil in Taiwan Int J Syst Evol Microbiol, January 1, 2008; 58(1): 84 - 88. [Abstract] [Full Text] [PDF]
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J.-H. Kim, K.-Y. Kim, Y.-T. Hahm, B.-S. Kim, J. Chun, and C.-J. Cha Actibacter sediminis gen. nov., sp. nov., a marine bacterium of the family Flavobacteriaceae isolated from tidal flat sediment Int J Syst Evol Microbiol, January 1, 2008; 58(1): 139 - 143. [Abstract] [Full Text] [PDF]
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M. Okamoto, Y. Benno, K.-P Leung, and N. Maeda Bifidobacterium tsurumiense sp. nov., from hamster dental plaque Int J Syst Evol Microbiol, January 1, 2008; 58(1): 144 - 148. [Abstract] [Full Text] [PDF]
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M. E. Trujillo, R. M. Kroppenstedt, C. Fernandez-Molinero, P. Schumann, and E. Martinez-Molina Micromonospora lupini sp. nov. and Micromonospora saelicesensis sp. nov., isolated from root nodules of Lupinus angustifolius Int J Syst Evol Microbiol, December 1, 2007; 57(12): 2799 - 2804. [Abstract] [Full Text] [PDF]
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S.-C. Chang, W.-M. Chen, J.-T. Wang, and M.-C. Wu Chitinilyticum aquatile gen. nov., sp. nov., a chitinolytic bacterium isolated from a freshwater pond used for Pacific white shrimp culture Int J Syst Evol Microbiol, December 1, 2007; 57(12): 2854 - 2860. [Abstract] [Full Text] [PDF]
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J.-H. Chou, K.-Y. Lin, M.-C. Lin, S.-Y. Sheu, Y.-H. Wei, A. B. Arun, C.-C. Young, and W.-M. Chen Brachybacterium phenoliresistens sp. nov., isolated from oil-contaminated coastal sand Int J Syst Evol Microbiol, November 1, 2007; 57(11): 2674 - 2679. [Abstract] [Full Text] [PDF]
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