Biopolym. Cell. 2016; 32(1):41-48.
http://dx.doi.org/10.7124/bc.00090B
Genomics, Transcriptomics and Proteomics
Antifungal activity and gene expression of lipopeptide antibiotics in strains of genus Bacillus
1Grabova A. Yu., 1Dragovoz I. V., 1Zelena L. B., 2Tkachuk D. M., 1Avdeeva L. V.
  1. D. K. Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
    154, Academika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Educational and Scientific Center "Institute of Biology",
    Taras Shevchenko National University of Kyiv
    64/13, Volodymyrska Str., Kyiv, Ukraine, 01601

Abstract

Aim. To research the antifungal activity and gene expression of lipopeptide antibiotics in strains of genus Bacillus. Methods. Deferred antagonism method, PCR, qRT-PCR, MALDI-TOF mass spectrometry. Results. It was revealed that Bacillus sp. strains C6 and Lg37s out of five tested strains had the highest antifungal activity. Based on the molecular genetic methods, it was shown that the expression of genes of lipopeptide antibiotics, related to the fengycin family, occurred in all these strains. At the same time, the gene expression of cyclolipopeptide iturin was found in the Bacillus sp. strains C6 and Lg37s. It was determined that Bacillus sp. C6 strain had the highest level of expression of the fengycin operon`s genes, whereas the lowest level was observed in Bacillus sp. C10 strain. By means of MALDI-TOF mass spectrometry, the presence of fengycins in the cell-free cultural fluid of Bacillus sp. C6 strain was detected. Conclusion. The direct correlation between the level of antifungal activity and the fengycin synthetases expression has not been disclosed. A higher level of antagonism detected for two Bacillus strains is more likely associated with the expression and subsequent synthesis of fengycin and iturin.
Keywords: bacteria of genus Bacillus, antifungal activity, MALDI-TOF mass spectrometry, lipopeptide antibiotics
Full text: (PDF, in English)

References

[1] Cazorla FM, Romero D, Pérez-García A, Lugtenberg BJ, Vicente Ad, Bloemberg G. Isolation and characterization of antagonistic Bacillus subtilis strains from the avocado rhizoplane displaying biocontrol activity. J Appl Microbiol. 2007;103(5):1950–9.
[2] Ostrikova MYa, Konstantinov AV, Balandina IM. Influence rhizosphere microorganisms, with nitrogen fixing and phosphate-ability, on the growth and development of pine seedlings. Trudy Bel Gos Tekh Univ. 2014; 1(165):159–62.
[3] Ongena M, Jacques P. Bacillus lipopeptides: versatile weapons for plant disease biocontrol. Trends Microbiol. 2008;16(3):115-25.
[4] Vater J, Kablitz B, Wilde C, Franke P, Mehta N, Cameotra SS. Matrix-assisted laser desorption ionization--time of flight mass spectrometry of lipopeptide biosurfactants in whole cells and culture filtrates of Bacillus subtilis C-1 isolated from petroleum sludge. Appl Environ Microbiol. 2002;68(12):6210-9.
[5] Tao Y, Bie XM, Lv FX, Zhao HZ, Lu ZX. Antifungal activity and mechanism of fengycin in the presence and absence of commercial surfactin against Rhizopus stolonifer. J Microbiol. 2011;49(1):146-50.
[6] Koumoutsi A, Chen XH, Henne A, Liesegang H, Hitzeroth G, Franke P, Vater J, Borriss R. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. J Bacteriol. 2004;186(4):1084-96.
[7] Peypoux F, Bonmatin JM, Wallach J. Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol. 1999;51(5):553-63.
[8] Maget-Dana R, Thimon L, Peypoux F, Ptak M. Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A. Biochimie. 1992;74(12):1047-51.
[9] Grabova AY, Dragovoz IV, Kruchkova LA, Pasichnik LA, Avdeeva LV. [Bacillus strains's screening--active antagonists of bacterial and fungal phytopathogens]. Mikrobiol Z. 2015;77(6):47-54.
[10] Burova YA, Ibragimova SA, Revin VV. Production a bacterial suspension Pseudomonas aureofaciens 2006 on molasses medium and study of its several properties. Vestn Orenburg Gos Univ. 2012;146(10):61–5.
[11] Nechypurenko O, Kharhota M, Avdeeva L. Safety of carotene-producing strains Bacillus sp. 1.1 and B. amyloliquefaciens UCM B-5113 for homoiothermal animals. Bulletin of Taras Shevchenko National University of Kyiv. Series: Biology. 2014;68(3):21–4.
[12] McKeen CD, Reilly CC, Pusey PL. Production and partial characterization of antifungal substances antagonistic to Monilinia fructicola from Bacillus subtilis. Phytopatology.1986;76(2):136–9.
[13] Malfanova N, Kamilova F, Validov S, Shcherbakov A, Chebotar V, Tikhonovich I, Lugtenberg B. Characterization of Bacillus subtilis HC8, a novel plant-beneficial endophytic strain from giant hogweed. Microb Biotechnol. 2011;4(4):523-32.
[14] Dragovoz IV, Leonova NO, Zelena LB, Rebriyev AV, Avdeeva LV. Identification of Bacillus amyloliquefaciens subsp. plantarum IMV B-7404 strain exometabolites with antifungal activity. Dopov Nac Akad Nauk Ukr.2015;(7):129–35.
[15] Hsieh FC, Lin TC, Meng M, Kao SS. Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A. Curr Microbiol. 2008;56(1):1-5.
[16] Zelena L, Gretsky I, Gromozova E. Influence of ultrahigh frequency irradiation on Photobacterium phosphoreum luxB gene expression. Cent Eur J Biol. 2014;9(10):1004–10.
[17] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.
[18] Hofemeister J, Conrad B, Adler B, Hofemeister B, Feesche J, Kucheryava N, Steinborn G, Franke P, Grammel N, Zwintscher A, Leenders F, Hitzeroth G, Vater J. Genetic analysis of the biosynthesis of non-ribosomal peptide- and polyketide-like antibiotics, iron uptake and biofilm formation by Bacillus subtilis A1/3. Mol Genet Genomics. 2004;272(4):363-78.
[19] Vanittanakom N, Loeffler W, Koch U, Jung G. Fengycin--a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot (Tokyo). 1986;39(7):888-901.
[20] Lin TP, Chen CL, Chang LK, Tschen JS, Liu ST. Functional and transcriptional analyses of a fengycin synthetase gene, fenC, from Bacillus subtilis. J Bacteriol. 1999;181(16):5060-7.
[21] Ramarathnam R, Bo S, Chen Y, Fernando WG, Xuewen G, de Kievit T. Molecular and biochemical detection of fengycin- and bacillomycin D-producing Bacillus spp., antagonistic to fungal pathogens of canola and wheat. Can J Microbiol. 2007;53(7):901-11.
[22] Wu CY, Chen CL, Lee YH, Cheng YC, Wu YC, Shu HY, Götz F, Liu ST. Nonribosomal synthesis of fengycin on an enzyme complex formed by fengycin synthetases. J Biol Chem. 2007;282(8):5608-16.
[23] Athukorala SN, Fernando WG, Rashid KY. Identification of antifungal antibiotics of Bacillus species isolated from different microhabitats using polymerase chain reaction and MALDI-TOF mass spectrometry. Can J Microbiol. 2009;55(9):1021-32.
[24] Stein T. Whole-cell matrix-assisted laser desorption/ionization mass spectrometry for rapid identification of bacteriocin/lantibiotic-producing bacteria. Rapid Commun Mass Spectrom. 2008;22(8):1146-52.
[25] Pabel CT, Vater J, Wilde C, Franke P, Hofemeister J, Adler B, Bringmann G, Hacker J, Hentschel U. Antimicrobial activities and matrix-assisted laser desorption/ionization mass spectrometry of Bacillus isolates from the marine sponge Aplysina aerophoba. Mar Biotechnol (NY). 2003;5(5):424-34.
[26] Madonna AJ, Voorhees KJ, Taranenko NI, Laiko VV, Doroshenko VM. Detection of cyclic lipopeptide biomarkers from Bacillus species using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry. Anal Chem. 2003;75(7):1628-37.
[27] Wei YH, Wang LC, Chen WC, Chen SY. Production and characterization of fengycin by indigenous Bacillus subtilis F29-3 originating from a potato farm. Int J Mol Sci. 2010;11(11):4526-38.