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Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of Bacillus species

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Abstract

Restriction fragment length analysis of PCR amplified 16S rDNA with AluI revealed the presence of a 265 bp fragment in all species of Bacillus with the exception of B. cereus and B. thuringiensis, which contains two restriction sites within this fragment which results in three smaller fragments totalling to 265 bp. Some distant species of Bacillus with no evidence of this fragment could be delineated into other genera based on phenotypic and genotypic parameters. BLAST search for homologous sequences of individual species revealed that it is a highly conserved region. Multiple alignment of the fragment suggests that a region between 160 and 265 bp of the 265 bp fragment was a hypervariable region and were highly species-specific. A set of primers was designed for amplification of this hypervariable region. Partial sequencing of the hypervariable region within the 265 bp fragment seems an index for identification of Bacillus species.

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References

  • Ash C, Farrow AE, Wallbanks S, Collins MD (1991) Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit- ribosomal RNA sequences. Lett Appl Microbiol 13:202–206

    Article  CAS  Google Scholar 

  • Ash C, Priest FG, Collins D (1993) Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanksand Collins) using a PCR probe test. Proposal for the creation of a new genus Paenibacillus. Antonie van Leeuwenhoek 64:253–260

    Article  CAS  PubMed  Google Scholar 

  • Daffonchio D, Borin S, Consolandi A, Mora D, Manachini PL, Sorlini C (1998a) 16S–23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group 1 of the genus Bacillus. FEMS Microbiol Lett 163:229–236

    CAS  PubMed  Google Scholar 

  • Daffonchio D, Borin S, Frova G, Manachini PL, Sorlini C (1998b) PCR fingerprinting of whole genome: the spacer between the 16s and 23s rRNA genes and of intergenic tRNA gene regions reveal a different intraspecific genomic variability of Bacillus cereus and Bacillus lichniformis. Int J Syst Bacteriol 48:107–116

    Article  CAS  PubMed  Google Scholar 

  • De Clerck E, Van Mol K, Jannes G, Rossau R, de Vos P (2004) Design of a 5′ exonuclease-based real-time PCR assay for simultaneous detection of Bacillus licheniformis, members of the ‘B. cereus group’ and B. fumarioli in gelatin. Lett Appl Microbiol 39:109–115

    Article  PubMed  Google Scholar 

  • De Silva SM, Petterson B, deMuro MA, Priest FG (2004) A DNA probe for the identification and detection of Bacillus sporothermophilus and using the 16S–23S rDNA spacer region and phylogenetic analysis of some isolates of Bacillus which form highly heat resistant spores. Syst Appl Microbiol 21:398–407

    Google Scholar 

  • Felsenstein J (1989) PHYLIP—phylogeny inference package (version 2). Cladistic 5:164–166

    Google Scholar 

  • Fortina MG, Pukall R, Schumann P, Mora D, Parini C, Manchini P, Stackebrandt E et al (2001) Ureibacillus gen. nov., a new genus to accommodate Bacillus thermosphericus (Anderson et al. 1995) emendation of Ureibacillus thermosphericus and description of Ureibacillus terrreneus sp. nov. Int J Syst Evol Microbiol 51:447–455

    CAS  PubMed  Google Scholar 

  • Goto K, Omura T, Hara Y, Sadaie Y (2000) Application of the partial 16s rDNA sequence as an index for rapid identification of the species in the genus Bacillus. J Gen Appl Microbiol 46:1–8

    Article  CAS  PubMed  Google Scholar 

  • Gurtler V, Stanisich VA (1996) New approaches to typing and identification of bacteria using the 16S–23S rDNA spacer region. Microbiology 142:3–16

    Article  PubMed  Google Scholar 

  • Heyndrickx M, Lebbe L, Kersters K, Scheldeman P et al (1995) Paenibacillus (formerly Bacillus) gordanae (Pichynoty et al. 1986) Ash et al. 1994 is a later synonym of Paenibacillus (formerly Bacillus) validus (Nakamura 1984) Ash et al. 1994: emended description of P. validus. Int J Syst Bacteriol 45:661–669

  • Heyndrickx M, Lebbe L, Kersters K, Devos P, Forsyth G, Logan NA (1998) Virgibacillus: a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus. Int J Syst Bacteriol 48:99–106

    Article  Google Scholar 

  • Joung KB, Cote JC (2002) Evaluation of ribosomal RNA gene patterns for the classification of Bacillus species and related genera. J Appl Microbiol 92:97–108

    Article  CAS  PubMed  Google Scholar 

  • Kalendar R, Lee D, Schulman AH (2009) Fast PCR software for PCR primer and probe design and repeat search. Genes Genomes Genomics 3:1

    Google Scholar 

  • Nakamura LK (1998) Bacillus pseudomycoides sp. Nov. Int J Syst Bacteriol 48:1031–1035

    Article  CAS  PubMed  Google Scholar 

  • Nazina TN, Tourova TP et al (2001) Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov. sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius, Bacillus thermodenitrificans to Geobacillus as Geobacillus stearothermophilus, Geobacillus thermocatenulatus, Geobacillus thermoleovorans Geobacillus kaustophilus, Geobacillus thermoglucosidasius, Geobacillus thermodenitrificans. Int J Syst Evol Microbiol 51:433–446

    CAS  PubMed  Google Scholar 

  • Niimura Y, Koh E, Yanagida F, Suzuki KI, Komagata K, Kozaki M (1990) Amphibacillus xylanus gen. nov., a facultatively anaerobic sporefoming xylin-digesting bacterium which lacks cytochrome, quinone, and catalase. Int J Syst Bacteriol 40:297–301

    Article  CAS  Google Scholar 

  • Priest FG, Goodfellow M, Todd CA (1998) Numerical classification of the genus Bacillus. J Gen Microbiol 134:1847–1882

    Google Scholar 

  • Schlesner H, Lawson PA, Collins MD, Weiss N, Wehmeyer U, Volker H, Thomm M (2001) Filobacillus milensis gen. nov., a new halophilic spore-forming bacterium with Orn-d-Glu-type peptidoglycan. Int J Syst Microbiol 51:425–431

    CAS  Google Scholar 

  • Shida O, Takagi H, Kadowaki K, Yano H, Komagata K (1996) Proposal for two new genera, Brevibacillus gen. Nov. and Aneurinibacillus gen. nov. Int J Syst Bacteriol 46:939–946

    Article  CAS  PubMed  Google Scholar 

  • Sneath PHA, Sokal RR (1973) Numerical taxonomy. Freeman, San Francisco, CA

    Google Scholar 

  • Stackebrandt E, Swiderski J (2002) From phylogeny to systematics: the dissection of the genus Bacillus. In: Berkeley R, Heyndrickx M, Logan N, De Vos P (eds) Applications and systematics of Bacillus and relatives. Blackwell, Malden, p 822

    Google Scholar 

  • Upadhayay SK, Singh DP, Saikia R (2009) Genetic diversity of plant growth promoting rhizobacteria isolated from rhizospheric soil of wheat under saline condition. Curr Microbiol. doi:10.1007/s00284-009-9464-1

  • Waino M, Tindall BJ, Schumann P, Ingvorsen K (1999) Gracillbacillus gen. nov., with description of Graclibacillus holotolerence gen. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. Nov. Int J syst bacteriol 49:821–831

    Article  CAS  PubMed  Google Scholar 

  • Wisotzkey JD, Jurtshuk P Jr, Fox GE, Deinhard G, Poralla K (1992) Comparative sequence analyses on the 16s r RNA (rDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus and proposal for certain of a new genus, Alicyclobacillus gen. nov. Int J Syst Bacteriol 42:263–269

    Article  CAS  PubMed  Google Scholar 

  • Xu D, Cote JC (2003) Phylogenetic relationships between Bacillus species and related genera inferred from comparison of 39 end 16S rDNA and 59 end 16S–23S ITS nucleotide sequences. Int J Syst Evol Microbiol 53:695–704

    Article  CAS  PubMed  Google Scholar 

  • Yoon JH, Weiss N, Lee KC, Lee IS, Kang KH, Park YH (2001) Jeotgalibacillus alimentarius gen. nov., sp. Nov., a novel bacterium isolated from jeotgal with l-lysine in the cell wall, reclassification of Bacillus marinus Ruger 1983 as Marinibacillus marinus gen. nov., comb. Nov. Int J Syst Evol Microbiol 51:2087–2093

    CAS  PubMed  Google Scholar 

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Acknowledgments

The study was supported by the grant from Indian Council of Agricultural Research (ICAR) Network Project, Government of India, New Delhi, India. Authors are grateful to the funding agency and thanks to Mr. R. N. Singh for his technical help.

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Authors and Affiliations

  1. National Bureau of Agriculturally Important Microorganisms, Kusmaur, P. Box No. 6, P.O. Kaithauli, Mau Nath Bhanjan, 275 101, UP, India

    S. Vardhan, R. Kaushik & D. K. Arora

  2. Division of Microbiology, Indian Agricultural Research Institute, New Delhi, 110012, India

    A. K. Saxena

Authors
  1. S. Vardhan
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  2. R. Kaushik
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  3. A. K. Saxena
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  4. D. K. Arora
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Correspondence to S. Vardhan.

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Vardhan, S., Kaushik, R., Saxena, A.K. et al. Restriction analysis and partial sequencing of the 16S rRNA gene as index for rapid identification of Bacillus species. Antonie van Leeuwenhoek 99, 283–296 (2011). https://doi.org/10.1007/s10482-010-9487-4

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