Bacillus pakistanensis sp. nov., a halotolerant bacterium isolated from salt mines of the Karak Area in Pakistan

Abstract

A rod shaped, non-motile, endospore forming, Gram-stain positive and moderately halotolerant strain, designated as NCCP-168T, was isolated from salt mines sampled in the Karak district of Khyber Pakhtunkhwa Province in Pakistan. To delineate its taxonomic position, the strain was subjected to polyphasic characterization. Cells of strain NCCP-168T can grow at 10–40 C (optimum at 30–35 C), in a pH range of 5.0–9.0 (optimum at pH 8.0) and in 0–17 % (w/v) NaCl on agar medium. The phylogenetic analysis based on the 16S rRNA gene sequence showed that strain NCCP-168T belongs to the genus Bacillus with the highest similarity to Bacillus seohaeanensis BH724T (97.1 %), and less than 97 % similarity with other closely related taxa (95.6 % with B. subtilis subsp. subtilis NCIB3610T). DNA–DNA relatedness between strain NCCP-168T and the type strains of closely related species was lower than 30 %. Chemotaxonomic data (major menaquinone, MK-7; cell wall peptidoglycan type, A1γ [meso-diaminopimelic acid]; major fatty acids, iso-C15:0 29.9 %, anteiso-C15:0 29.3 %, iso-C16:0 11.4 %, iso-C14:0 8.9 % and anteiso-C17:0 7.0 %; major polar lipids, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine) support the affiliation of strain NCCP-168T with genus Bacillus. On the basis of phenotypic, chemotaxonomic and phylogenetic data, strain NCCP-168T can be distinguished from the closely related taxa and thus represents a novel species in the genus Bacillus, for which the name Bacillus pakistanensis sp. nov. is proposed, with the type strain NCCP-168T (= KCTC 13786T = DSM 24834T = JCM 18975T).

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References

  1. Ahmed I, Yokota A, Fujiwara T (2007a) Gracilibacillus boraciitolerans sp. nov., a highly boron-tolerant and moderately halotolerant bacterium isolated from soil. Int J Syst Evol Microbiol 57 (Pt 4):796–802. doi:10.1099/ijs.0.64284-0

    Article  Google Scholar 

  2. Ahmed I, Yokota A, Fujiwara T (2007b) A novel highly boron tolerant bacterium, Bacillus boroniphilus sp. nov., isolated from soil, that requires boron for its growth. Extremophiles 11:217–224. doi:10.1007/s00792-006-0027-0

    CAS  PubMed  Article  Google Scholar 

  3. Arahal DR, Ventosa A (2002) Moderately halophilic and halotolerant species of Bacillus and related genera, Applications and systematics of bacillus and relatives. Blackwell, Oxford, pp 83–99

    Book  Google Scholar 

  4. Chang YH, Han JI, Chun JS, Lee KC, Rhee MS, Kim YB, Bae KS (2002) Comamonas koreensis sp. nov., a non-motile species from wetland in Woopo Korea. Int J Syst Evol Microbiol 52:377–381. doi:10.1099/ijs.0.01734-0

    CAS  PubMed  Google Scholar 

  5. Chen YG, Liu ZX, Peng DJ, Zhang YQ, Wang YX, Tang SK, Li WJ, Cui XL, Liu YQ (2009) Virgibacillus litoralis sp. nov., a moderately halophilic bacterium isolated from saline soil. Antonie Van Leeuwenhoek 96(3):323–329. doi:10.1007/s10482-009-9349-0

    CAS  PubMed  Article  Google Scholar 

  6. Ezaki T, Hashimoto Y, Yabuuchi E (1989) Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39(3):224–229. doi:10.1099/00207713-39-3-224

    Article  Google Scholar 

  7. Garabito MJ, Márquez C, Ventosa A (1998) Halotolerant Bacillus diversity in hypersaline environments. Can J Microbiol 44:95–102. doi:10.1139/w97-125

    CAS  Article  Google Scholar 

  8. Groth I, Schumann P, Weiss N, Martin K, Rainey FA (1996) Agrococcus jenensis gen. nov., sp. nov., a new genus of actinomycetes with diaminobutyric acid in the cell wall. Int J Syst Bacteriol 46(1):234–239. doi:10.1099/00207713-46-1-234

    CAS  PubMed  Article  Google Scholar 

  9. Ishikawa M, Ishizaki S, Yamamoto Y, Yamasato K (2002) Paraliobacillus ryukyuensis gen. nov., sp. nov., a new Gram-positive, slightly halophilic, extremely halotolerant, facultative anaerobe isolated from a decomposing marine alga. J Gen Appl Microbiol 48(5):269–279. doi:10.2323/jgam.48.269

    CAS  PubMed  Article  Google Scholar 

  10. Jung MY, Kim J-S, Paek WK, Styrak I, Park I-S, Sin Y, Paek J, Park KA, Kim H, Kim HL, Chang Y-H (2012) Description of Lysinibacillus sinduriensis sp. nov., and transfer of Bacillus massiliensis and Bacillus odysseyi to the genus Lysinibacillus as Lysinibacillus massiliensis comb. nov. and Lysinibacillus odysseyi comb. nov. with emended description of the genus Lysinibacillus. Int J Syst Evol Microbiol 62(10):2347–2355. doi:10.1099/ijs.0.033837-0

    CAS  PubMed  Article  Google Scholar 

  11. Kämpfer P, Rossello-Mora R, Falsen E, Busse HJ, Tindall BJ (2006) Cohnella thermotolerans gen. nov., sp. nov., and classification of ‘Paenibacillus hongkongensis’ as Cohnella hongkongensis sp. nov. Int J Syst Evol Microbiol 56:781–786. doi:10.1099/ijs.0.63985-0

    PubMed  Article  Google Scholar 

  12. Kim OS, Cho YJ, Lee K, Yoon SH, Kim M, Na H, Park SC, Jeon YS, Lee JH, Yi H, Won S, Chun J (2012) Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Evol Microbiol 62:716–721. doi:10.1099/Ijs.0.038075-0

    CAS  PubMed  Article  Google Scholar 

  13. Lee JC, Lim JM, Park DJ, Jeon CO, Li WJ, Kim CJ (2006) Bacillus seohaeanensis sp. nov., a halotolerant bacterium that contains l-lysine in its cell wall. Int J Syst Evol Microbiol 56(8):1893–1898. doi:10.1099/ijs.0.64237-0

    CAS  PubMed  Article  Google Scholar 

  14. Logan NA, Berge O, Bishop AH, Busse HJ, De Vos P, Fritze D, Heyndrickx M, Kampfer P, Rabinovitch L, Salkinoja-Salonen MS, Seldin L, Ventosa A (2009) Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria. Int J Syst Evol Microbiol 59(Pt 8):2114–2121. doi:10.1099/ijs.0.013649-0

    CAS  PubMed  Article  Google Scholar 

  15. Minnikin DE, O’Donnell AG, Goodfellow M, Alderson G, Athalye M, Schaal A, Parlett JH (1984) An integrated procedure for the extraction of bacterial isoprenoid quinones and polar lipids. J Microbiol Method 2:233–241. doi:10.1016/0167-7012(84)90018-6

    CAS  Article  Google Scholar 

  16. Oren A (1999) Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 63(2):334–348

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Quesada E, Ventosa A, Rodriguez-Valera F, Ramos-Cormenzana A (1982) Types and properties of some bacteria isolated from hypersaline soils. J Appl Microbiol 53(2):155–161. doi:10.1111/j.1365-2672.1982.tb04671.x

    Google Scholar 

  18. Quesada E, Béjar V, Ferrer M, Calvo C, Llamas I, Martínez-Checa F, Arias S, Ruiz-García C, Páez R, Martinez-Cánovas M, del Moral A (eds) (2004) Moderately halophilic, exopolysaccharide-producing bacteria. Halophilic Microorganisms. Springer-Verlag Berlin

  19. Roohi A, Ahmed I, Iqbal M, Jamil M (2012) Preliminary isolation and characterization of halotolerant and halophilic bacteria from salt mines of Karak, Pakistan. Pak J Bot 44((SI 1)):365–370

    CAS  Google Scholar 

  20. Roohi A, Ahmed I, Khalid N, Iqbal M, Jamil M (2014) Isolation and phylogenetic identification of halotolerant/halophilic bacteria from the salt mines of Karak, Pakistan. Int J Agric Biol (Accepted)

  21. Sasser M (1990) Identification of bacteria by gas chromatography of cellular fatty acids. MIDI Technical Note 101

  22. Schleifer KH, Kandler O (1972) Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev 36(4):407–477

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Shida O, Takagi H, Kadowaki K, Nakamura LK, Komagata K (1997) Transfer of Bacillus alginolyticus, Bacillus chondroitinus, Bacillus curdlanolyticus, Bacillus glucanolyticus, Bacillus kobensis, and Bacillus thiaminolyticus to the genus Paenibacillus and emended description of the genus Paenibacillus. Int J Syst Bacteriol 47(2):289–298. doi:10.1099/00207713-47-2-289

    CAS  PubMed  Article  Google Scholar 

  24. Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Evol Microbiol 44(4):846–849. doi:10.1099/00207713-44-4-846

    CAS  Google Scholar 

  25. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. doi:10.1093/molbev/msr121

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. Tindall BJ (1990) A comparative study of the lipid composition of Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13:128–130. doi:10.1099/ijs.0.65717-0

    CAS  Article  Google Scholar 

  27. Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62(2):504–544

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Ventosa A, Mellado E, Sánchez-Porro C, Marquez M (eds) (2008) Halophilic and halotolerant microorganisms from soils. Microbiology of Extreme Soils, Soil Biology. Springer-Verlag, Berlin

  29. Wainø M, Tindall BJ, Schumann P, lngvorsenl K (1999) Gracilibacillus gen. nov., with description of Gracilibacillus halotolerans gen. nov., sp. nov.; transfer of Bacillus dipsosauri to Gracilibacillus dipsosauri comb. nov., and Bacillus salexigens to the genus Salibacillus gen. nov., as Salibacillus salexigens comb. nov. lnt J Syst Bacteriol 49:821–831. doi:10.1099/00207713-49-2-821

    Article  Google Scholar 

  30. Yoon J-H, Kim I-G, Kang KH, Oh T-K, Park Y-H (2003) Bacillus marisflavi sp. nov. and Bacillus aquimaris sp. nov., isolated from sea water of a tidal flat of the Yellow Sea in Korea. Int J Syst Evol Microbiol 53(5):1297–1303. doi:10.1099/ijs.0.02365-0

    CAS  PubMed  Article  Google Scholar 

  31. Zhang J, Wang J, Fang C, Song F, Xin Y, Qu L, Ding K (2010) Bacillus oceanisediminis sp. nov., isolated from marine sediment. Int J Syst Evol Microbiol 60 (Pt 12):2924–2929. doi:10.1099/ijs.0.019851-0

    Article  Google Scholar 

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Acknowledgments

This work was partially supported by PSDP Project “Research for Agricultural Development Program (RADP)” under a sub-project (Grant No. CS-55/RADP/PARC) entitled “Establishment of Microbial Bio-Resource Laboratories: National Culture Collection of Pakistan (NCCP)” from Pakistan Agricultural Research Council (PARC), Islamabad, Pakistan. The partial financial support of KRIBB to authors, Aneela Roohi and Saira Abbas in the training course at Korean Collection for Type Cultures, Biological Resources Centre, KRIBB under the umbrella of ACM forum is also gratefully acknowledged.

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Correspondence to Iftikhar Ahmed or Young H. Chang.

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Aneela Roohi and Iftikhar Ahmed contributed equally to the experimentation.

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Roohi, A., Ahmed, I., Paek, J. et al. Bacillus pakistanensis sp. nov., a halotolerant bacterium isolated from salt mines of the Karak Area in Pakistan. Antonie van Leeuwenhoek 105, 1163–1172 (2014). https://doi.org/10.1007/s10482-014-0177-5

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Keywords

  • Halotolerant bacterium
  • Karak salt mines
  • Bacillus pakistanensis