Mikrobiol. Z. 2017; 79(6):95-104. Ukrainian.
doi: https://doi.org/10.15407/microbiolj79.06.095

Taxonomic Position of the Strain Bacillus sp. 10.1 as Effective Algicidal Agent

Rybalchenko N.P., Kharkhota M.А., Zelena L.B., Avdeeva L.V.

Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine

Aim. Identifcation of Bacillus sp. 10.1 using phenotype characteristics and sequencing of 16S rRNA gene. Methods. Cultural, morphological, and physiological characteristics of the strain Bacillus sp. 10.1 were studied using guidelines of isolation and identifcation of bacteria of the genus Bacillus. Composition of fatty-acid of the cellular lipids were identifed by the chromatography-mass spectrometry. The 16S rRNA genes were amplifed by PCR using primers 27f and 1492r, according to standard protocol. Phylogenetic tree, constructed using the neighbour-joining method and using the model Kimura 2 parameter. Bootstrap analysis was applied using 100 bootstrap replicas. Results. The strain of Bacillus sp.10.1 inhibited the growth of cyanobacteria Anabaena hassalii, Microcystis aeruginosa, M. pulvereae. Cultural, morphological, and physiological characteristics of the strain Bacillus sp. 10.1 had been studied. It was showed that the studied strain belongs to the species B. amyloliquefaciens. To clarify the taxonomic position of the strain Bacillus sp. 10.1 had been studied the composition of fatty-acid of the cell wall and sequencing of 16S rRNA gene. Conclusions. Based on the analysis of nucleotide sequence of 16S rRNA gene the studied strain Bacillus sp. 10.1 had been classifed as a B. velezensis. The sequence of 16S rRNA gene added to the database GenBank and accession number for the 16S rRNA of Bacillus sp. 10.1 are KH962173.

Key words: genus Bacillus, phylogenetic analysis, B. amyloliquefaciens, B. velezensis.

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  1. Euzeby J. List of Prokaryotic Names with Standing in Nomenclature. 2015.
  2. Guinebretiere M., Auger S., Galleron N., Contzen M., De Sarrau B., De Buyser M., et al. Bacillus cytotoxicus sp. nov. is a new thermotolerant species of the Bacillus cereus group occasionally associated with food poisoning. Int. J. Syst. Evol. Microbiol. 2013; 63:31–40. https://doi.org/10.1099/ijs.0.030627-0
  3. Diomandé S.E., Nguyen-The C., Guinebretière M.-H., Broussolle V., Brillard J. Role of fatty acids in Bacillus environmental adaptation. Front. Microbiol. 2015; 6:813–821. https://doi.org/10.3389/fmicb.2015.00813
  4. Fulbright S.P., Chisholm S., Reardon K.F. Growth inhibition of Nannochloropsis species by Bacillus pumilus. Algal Research. 2016; 20:70–76. https://doi.org/10.1016/j.algal.2016.09.016
  5. Fan B., Blom J., Klenk H.-P., Borriss R. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis Form an "Operational Group B. amyloliquefaciens" within the B. subtilis Species Complex. Front. Microbiol. 2017; 8:22–34. https://doi.org/10.3389/fmicb.2017.00022
  6. Dunlap C.A., Kim S.-J., Kwon S.-W., Rooney A.P. Bacillus velezensis is not a later heterotypic synonym of Bacillus amyloliquefaciens; Bacillus methylotrophicus, Bacillus amyloliquefaciens subsp. plantarum and 'Bacillus oryzicola' are later heterotypic synonyms of Bacillus velezensis based on phylogenomics. International Journal of Systematic and Evolutionary Microbiology. 2016; 66:1212–1217. https://doi.org/10.1099/ijsem.0.000858
  7. Rybalchenko N.P., Kharkhota M.A., Lapa S.V., Avdeeva L.V. Alga-lysing properties of Bacillus sp. Studia Biologica. 2015; 9(2):5–12.
  8. Bergey's Manual of Systematic Bacteriology. D.J. Brenner, N.R. Krieg, J.T.Staley, G.M.Garrity. New York: Springer, 2005. Vol. 2.
  9. Smirnov V.V., Reznik S.R., Sorokulova I.B. Metodicheskie rekomendatsii po vydeleniyu i identifikatsii bakteriy roda Bacillus iz organizma cheloveka i zhivotnykh. Kiev, 1983.
  10. Flores O., Belanche L.A., Blanch A.R. New multiplatform computer program for numerical identification of microorganism. Journal Clinical Microbiology. 2009; 47:4133–4135. https://doi.org/10.1128/JCM.01250-09
  11. Safronova L.A., Zelena L.B., Klochko V.V., Reva O.N. Does the applicability of Bacillus strains in probiotics rely upon their taxonomy. Can. J. Microbiol. 2012; 58(2):212–219. https://doi.org/10.1139/w11-113
  12. Ivanytsya V.O., Horshkova O.H., Korotayeva N.V., Volyuvach O.V., Hudzenko T.V., Ostapchuk A.M. Sklad zhyrnykh kyslot lipidiv shtamu Bacillus sp. OZ-5, vydilenoho iz zabrudnenoho naftoyu hruntu o. Zmiinyi. Mikrobiolohiya i biotekhnolohiya. 2015; 4:28-35.
  13. Lane D.G. Nucleic acids techniques in bacterial systematic. Edited by E. Stackebrandt and M. Goodfellow. Chichester, United Kingdom: John Wiley. 1991. P.115-175.
  14. Reva ON, Sorokulova IB, Smirnov VV. Simplified technique for identification of the aerobic spore-forming bacteria by phenotype. Int J Syst Evol Microbiol. 2001; 51(4):1361–1371. https://doi.org/10.1099/00207713-51-4-1361
  15. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution. 2011; 28:2731–2739. https://doi.org/10.1093/molbev/msr121
  16. De Vos P., Garrity G.M. et al. The Firmicutes. Bergey's manual of systematic bacteriology 3 (2nd ed.). New York: Springer, 2009.
  17. Guinebretiere M., Auger S., Galleron N., Contzen M., De Sarran B., De Buyser H. et al. Bacillus cutotoxicus sp. nov. is a new thermotolerant species of the Bacillus cereus group occasionally associated with food poisoning. Int. J. Syst. Evol. Microbiol. 2013; 63:31–40. https://doi.org/10.1099/ijs.0.030627-0
  18. Kaneda T. Fatty acids of the genus Bacillus; an example of branched-chain preferences. Bacteriol. Rev. 1977; 41(2):391–418.
  19. Song Y., Yang R., Guo Z., Zhang M., Wang X., Zhou F. Distinctness of spore and vegetative cellular fatty acid profiles of some aerobic endospore-forming bacilli. J. Microbiol. Methods. 2000; 39(3):225–241. https://doi.org/10.1016/S0167-7012(99)00123-2
  20. Borriss R., Chen X.-H., Rueckert C. et al. Relationship of Bacillus amiloliquefaciens clades associated with strains DSM7T and FZB42: a proposal for Bacillus amiloliquefaciens subsp. amyloliquefaciens subsp. nov. and Bacillus amiloliquefaciens subsp. plantarum subsp. nov. based on their discriminating complete genome sequences. Int. J. Syst. Evol. Microbiol. 2011; P. 232–267.
  21. Ruiz-Garcia C., Bejar V., Martinez-Checa F., Llamas I., Quesada E. Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Velez in Malaga, southern Spain. Int J Syst Evol Microbiol. 2005; 55:191–195. https://doi.org/10.1099/ijs.0.63310-0
  22. Connor N., Sikorski J., Rooney P. et al. Ecology of speciation of the genus Bacillus. Appl. Environ. Microbiol. 2010; 76:1349–1358. https://doi.org/10.1128/AEM.01988-09
  23. Ehrhardt C.J., Chu V., Brown T.C. et al. Use of fatty acid methyl ester profiles for discrimination of Bacillus cereus T-strain spores grown on different media. Appl. Environ. Microbiol. 2010; 76(6):1902–1912. https://doi.org/10.1128/AEM.02443-09