Skip to main content
Log in

Characterization of an Environmental Strain of Bacillus thuringiensis from a Hot Spring in Western Himalayas

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

Bacillus anthracis, the etiological agent of anthrax, is responsible for a serious and often fatal disease of mammalian livestock and humans and is an important biological warfare agent. Bacillus sp. AKG was isolated from a hot spring in western Himalayas and species-specific primers targeting gyrB gene identified the strain as B. anthracis within cereus-group. Cloning, sequencing, and phylogenetic analysis of the partial gyrB sequence from strain AKG indicated a close affiliation with B. anthracis and a few recently isolated strains of B. thuringiensis (e.g., strain Al Hakam and serovar konkukian). Phylogenetic analysis of two other housekeeping genes, clpC and gdpD yielded similar results. This observation is further substantiated by phylogenetic reconstruction using concatenated sequences (1680 bases) of the three genes (gyrB, clpC, and gdpD). Phenotypic features indicated a non-anthracis affiliation for the strain AKG. A novel strategy to distinguish among strains of B. anthracis, B. cereus, and B. thuringiensis based on whole proteome comparison was developed and tested for the identification of this environmental strain. Proteome comparison was used to establish the identity of this unknown environmental strain. Group of replicate 2DE gels for whole cell proteome were generated for each of the three species and strain AKG. Protein spots unique to each group and those showing match between the groups, in a pair-wise comparison, indicated strain AKG as a member of B. thuringiensis. This strategy can be used to assign strains of B. cereus group to their respective species.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Alam SI, Dixit A, Dube S, Reddy GSN, Palit M, Shivaji S, Singh L (2006) Clostridium schirmacheriensis sp. nov., an obligately anaerobic, proteolytic, psychrophilic bacterium isolated from lake sediments of Schirmacher Oasis, Antarctica. Int J Syst Evol Microbiol 56:721–725

    Article  Google Scholar 

  2. Ash C, Farrow JA, Dorsch M, Stackebrandt E, Collins MD (1991) Comparative analysis of Bacillus anthracis, Bacillus cereus and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int J Syst Bacteriol 41:343–346

    Article  CAS  PubMed  Google Scholar 

  3. Blackshear PJ (1984) Systems for polyacrylamide gel electrophoresis. In: Jaeoby WB (ed) Methods in enzymology, vol 104. Academic Press, New York, pp 237–255

    Google Scholar 

  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  5. Carl M, Hawkins R, Coulson N, Lowe J, Robertson DL, Nelson WM, Titball RW, Woody JN (1992) Detection of spores of Bacillus anthracis using the polymerase chain reaction. J Infect Dis 165:1145–1148

    CAS  PubMed  Google Scholar 

  6. Carlson CR, Caugant DA, Kolstø A (1994) Genotypic diversity among Bacillus cereus and Bacillus thuringiensis strains. Appl Environ Microbiol 60:1719–1725

    CAS  PubMed  Google Scholar 

  7. Cherif A, Borin S, Rizzi A, Ouzari H, Boudabous A, Daffonchio D (2003) Bacillus anthracis diverges from related clades of the Bacillus cereus group in 16S–23S rDNA internal transcribed spacers containing tRNA genes. Appl Environ Microbiol 69:33–40

    Article  CAS  PubMed  Google Scholar 

  8. Daffonchio D, Raddadi N, Merabishvili M, Cherif A, Carmagnola L, Brusetti L, Rizzi A, Chanishvili N, Visca P, Sharp R, Borin S (2006) Strategy for identification of Bacillus cereus and Bacillus thuringiensis strains closely related to Bacillus anthracis. Appl Environ Microbiol 72:1295–1301

    Article  CAS  PubMed  Google Scholar 

  9. Dixon TC, Meselson M, Guillemin J, Hanna PC (1999) Anthrax. N Engl J Med 341:815–826

    Article  CAS  PubMed  Google Scholar 

  10. Drobniewski FA (2004) Bacillus cereus and related species. Clin Microbiol Rev 6:324–338

    Google Scholar 

  11. Dwyer KG, Janine ML, Jennifer AS, Leanne EW, Joanne B, Anna L, Rajendra R, Guy P, Vito GD (2004) Identification of Bacillus anthracis specific chromosomal sequences by suppressive subtractive hybridization. BMC Genomics 5:15

    Article  PubMed  Google Scholar 

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

    Article  Google Scholar 

  13. Harford JB, Morris DR (1997) Post-transcriptional gene regulation. Wiley-Liss Inc, New York, NY

    Google Scholar 

  14. Helgason E, Økstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolstø AB (2000) Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: one species on the basis of genetic evidence. Appl Environ Microbiol 66:2627–2630

    Article  CAS  PubMed  Google Scholar 

  15. Hoffmaster AR, Ravel J, Rasko DA, Chapman GD, Chute MD, Marston CK, De BK, Sacchi CT, Fitzgerald C, Mayer LW, Maiden MCJ, Priest FG, Barker M, Jiang L, Cer RZ, Rilstone J, Peterson SN, Weyant RS, Galloway DR, Read TD, Popovic T, Fraser CM (2004) Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proc Natl Acad Sci USA 101:8449–8454

    Article  CAS  PubMed  Google Scholar 

  16. Hoffmaster AR, Hill KK, Gee JE, Marston CK, De BK, Popovic T, Sue D, Wilkins PP, Avashia SB, Drumgoole R, Helma CH, Ticknor LO, Okinaka RT, Jackson PJ (2006) Characterization of Bacillus cereus isolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes. J Clin Microbiol 44:3352–3360

    Article  CAS  PubMed  Google Scholar 

  17. Jackson SG, Goodbrand RB, Ahmed R, Kasatiya S (1995) Bacillus cereus and Bacillus thuringiensis isolated in a gastroenteritis outbreak investigation. Lett Appl Microbiol 21:103–105

    Article  CAS  PubMed  Google Scholar 

  18. Keim P, Kalif A, Schupp J, Hill K, Travis SE, Richmond K, Adair DM, Hugh-Jones M, Kuske CR, Jackson P (1997) Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J Bacteriol 179:818–824

    CAS  PubMed  Google Scholar 

  19. Khaitovich P, Weiss G, Lachmann M, Hellmann I, Enard W, Muetzel B, Wirkner U, Ansorge W, Paabo S (2004) A neutral model of transcriptome evolution. PLOS Biol 2(5):e132

    Article  PubMed  Google Scholar 

  20. Kumar S, Tamura K, Nei M (2004) MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform 5:150–163

    Article  CAS  PubMed  Google Scholar 

  21. Lanyi B (1987) Classical and rapid identification methods for medically important Bacteria. Methods Microbiol 19:1–67

    Article  CAS  Google Scholar 

  22. Mignot T, Denis B, Couture-Tosi E, Kolstø AB, Mock M, Fouet A (2001) Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure. Environ Microbiol 3:493–501

    Article  CAS  PubMed  Google Scholar 

  23. Marmur J (1961) Procedure for the isolation of deoxyribonucleic acid from microorganisms. J Mol Biol 3:208–218

    Article  CAS  Google Scholar 

  24. Mock M, Fouet A (2001) Anthrax. Annu Rev Microbiol 55:647–671

    Article  CAS  PubMed  Google Scholar 

  25. Negoro S, Nakamura S, Okada H (1984) DNA–DNA hybridization analysis of nylon oligomer-degradative plasmid pOAD2: identification of the DNA region analogous to the nylon oligomer degradation gene. J Bacteriol 158(2):419–424

    CAS  PubMed  Google Scholar 

  26. Pelczar MJJr, Chan ECS, Krieg NR (1993) In: Microbiology, concepts and applications, Mc Graw-Hill, New York

  27. Radnedge L, Agron PG, Hill KK, Jackson PJ, Ticknor LO, Keim P, Andersen GL (2003) Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Appl Environ Microbiol 69:2755–2764

    Article  CAS  PubMed  Google Scholar 

  28. Rampersad J, Khan A, Ammons D (2002) Usefulness of Staining Parasporal Bodies when Screening for Bacillus thuringiensis. J Invertebr Pathol 79:203–204

    Article  PubMed  Google Scholar 

  29. Rossnes R, Eidhammer I, Liberles DA (2005) Phylogenetic reconstruction of ancestral character states for gene expression and mRNA splicing data. BMC Bioinformatics 6:127

    Article  PubMed  Google Scholar 

  30. Sacchi CT, Whitney AM, Mayer LW, Morey R, Steigerwalt A, Boras A, Weyant RS, Popovic T (2002) Sequencing of 16S rRNA gene: a rapid tool for identification of Bacillus anthracis. Emerg Infect Dis 8:1117–1123

    CAS  PubMed  Google Scholar 

  31. Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806

    CAS  PubMed  Google Scholar 

  32. Smibert RM, Krieg NR (1994) Phenotypic characterization. In: Gerhardt P (ed) Methods for general and molecular bacteriology. American Society for Microbiology, Washington, pp 607–654

    Google Scholar 

  33. Sorokin S, Candelon B, Guilloux K, Galleron N, Wackerow-Kouzova N, Ehrlich SD, Bourguet D, Sanchis V (2006) Multi-locus sequence typing analysis of Bacillus cereus and Bacillus thuringiensis reveals separate clustering and a distinct population structure of psychrotrophic strains. Appl Environ Microbiol 72:1569–1578

    Article  CAS  PubMed  Google Scholar 

  34. Ticknor LO, Kolstø AB, Hill KK, Keim P, Laker MT, Tonks M, Jackson PJ (2001) Fluorescent amplified fragment length polymorphism analysis of Norwegian Bacillus cereus and Bacillus thuringiensis soil isolates. Appl Environ Microbiol 67:4863–4873

    Article  CAS  PubMed  Google Scholar 

  35. Turnbull PC, Hutson RA, Ward MJ, Jones MN, Quinn CP, Finnie NJ, Duggleby CJ, Kramer JM, Melling J (2007) Bacillus anthracis but not always anthrax. J Appl Bacteriol 72:21–28

    Google Scholar 

  36. Urlinger S, Kuchler K, Meyer TH, Uebel S, Tampe R (1997) Intracellular location, complex formation, and function of the transporter associated with antigen processing in yeast. Eur J Biochem 245:266–272

    Article  CAS  PubMed  Google Scholar 

  37. Varshavsky A (1996) The N-end rule: functions, mysteries, uses. Proc Natl Acad Sci USA 93:12142–12149

    Article  CAS  PubMed  Google Scholar 

  38. Venkateswaran K, Dohmoto N, Harayama S (1998) Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp. Appl Environ Microbiol 64:681–687

    CAS  PubMed  Google Scholar 

  39. Vilas-Boas G, Sanchis V, Lereclus D, Lemos MV, Bourguet D (2002) Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis. Appl Environ Microbiol 68:1414–1424

    Article  CAS  PubMed  Google Scholar 

  40. Yamada S, Ohashi E, Agata N, Venkateswaran K (1999) Cloning and nucleotide sequence analysis of gyrB of Bacillus cereus, B. thuringiensis, B. mycoides, and B. anthracis and their application to the detection of B. cereus in rice. Appl Environ Microbiol 65:1483–1490

    CAS  PubMed  Google Scholar 

Download references

Acknowledgment

We thank Dr R Vijayaraghwan, Director, DRDE Gwalior for providing all facilities and support required for this study.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Syed Imteyaz Alam.

Electronic Supplementary Material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Alam, S.I., Bansod, S., Goel, A.K. et al. Characterization of an Environmental Strain of Bacillus thuringiensis from a Hot Spring in Western Himalayas. Curr Microbiol 62, 547–556 (2011). https://doi.org/10.1007/s00284-010-9743-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-010-9743-x

Keywords

Navigation