Co-production of Multiple Antimicrobial Compounds by Bacillus amyloliquefaciens WY047, a Strain with Broad-Spectrum Activity

Abstract

A bacterial strain WY047 was isolated from fermented grains and the bacterium was identified as Bacillus amyloliquefaciens, based on morphological, biochemical, and physiological tests, and analysis of 16S rRNA and gyrA sequences. The culture supernatant of WY047 demonstrated inhibition of a wide spectrum of bacteria (Gram positive and Gram negative) and fungi. Nine pairs of primers were designed and six genes (bmyD, fenA, hag, ituA, mrsA, and tasA) of antimicrobial substances were detected by PCR, one of which was isolated by 80% ammonium sulfate precipitation, D201 resin anion-exchange chromatography, and Sephadex G-75 filtration column. The purified peptide was estimated to be 35,207 Da and identified as flagellin by MALDI-TOF mass spectrometry. Another four antimicrobial substances were extracted with methanol and identified as iturin A, fengycin, bacillomycin D, and mersacidin through the liquid chromatography–mass spectrometry (LC–MS) method. The sixth possible peptide encoded by tasA could not be isolated in this study; however, the broader spectrum suggested huge application prospects.

Keywords

Antimicrobial substances / Purification / Identification / Broad spectrum

References

[1] Li Y, Xiang Q, Zhang Q, et al. Overview on the recent study of antimicrobial peptides: origins, functions, relative mechanisms and application. Peptides, 2012, 37(2): 207-215.

[2] Touré Y, Ongena M, Jacques P, et al. Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. J Appl Microbiol, 2004, 96: 1151-1160.

[3] Chen XH, Koumoutsi A, Scholz R, et al. Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens. J Biotechnol, 2009, 140(1–2): 27-37.

[4] Wang G, Feng G, Snyder AB, et al. Bactericidal thurincin H causes unique morphological changes in Bacillus cereus F4552 without affecting membrane permeability. FEMS Microbiol Lett, 2014, 357(1): 69-76.

[5] Koberl M, Ramadan EM, Adam M, et al. Bacillus and Streptomyces were selected as broad-spectrum antagonists against soilborne pathogens from arid areas in Egypt. FEMS Microbiol Lett, 2013, 342(2): 168-178.

[6] Lee YK, Senthilkumar M, Kim JH, Annapurna K, et al. Purification and partial characterization of antifungal metabolite from Paenibacillus lentimorbus WJ5. World J Microbiol Biotechnol, 2008, 24: 3057-3062.

[7] 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): 1021-1032.

[8] Mora I, Cabrefiga J, Montesinos E. Antimicrobial peptide genes in Bacillus strains from plant environments. Int Microbiol, 2011, 14(4): 213-223.

[9] Deng H, Han Y, Liu Y, et al. Identification of a newly isolated erythritol-producing yeast and cloning of its erythrose reductase genes. J Ind Microbiol Biotechnol, 2012, 39(11): 1663-1672.

[10] Roberts MS, Nakamura LK, Cohan FM. Bacillus mojavensis sp. nov., distinguishable from Bacillus subtilis by sexual isolation, divergence in DNA sequence, and differences in fatty acid composition. Int J Syst Bacteriol, 1994, 44(2): 256-264.

[11] Zhao X, Zhou ZJ, Han Y, et al. Isolation and identification of antifungal peptides from Bacillus BH072, a novel bacterium isolated from honey. Microbiol Res, 2013, 168(9): 598-606.

[12] Chun J, Goodfellow M. A phylogenetic analysis of the genus Nocardia with 16S rRNA gene sequences. Int J Syst Bacteriol, 1995, 45(1): 240-245.

[13] Koumoutsi A, Chen XH, Henne A, et al. Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens Strain FZB42. J Bacteriol, 2004, 186(4): 1084-1096.

[14] Bizani D, Brandelli A. Characterization of a bacteriocin produced by a newly isolated Bacillus sp Strain 8A. J Appl Microbiol, 2002, 93(3): 512-519.

[15] Laemmli UK, Favre M. Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol, 1973, 80(4): 575-599.

[16] Tan Z, Lin B, Zhang R, et al. A novel antifungal protein of Bacillus subtilis B25. SpringerPlus, 2013, 2: 543

[17] Price NP, Rooney AP, Swezey JL, et al. Mass spectrometric analysis of lipopeptides from Bacillus strains isolated from diverse geographical locations. FEMS Microbiol Lett, 2007, 271(1): 83-89.

[18] Hiradate S, Yoshida S, Sugie H, et al. Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2. Phytochemistry, 2002, 61: 693-698.

[19] He P, Hao K, Blom J, et al. Genome sequence of the plant growth promoting strain Bacillus amyloliquefaciens subsp. plantarum B9601-Y2 and expression of mersacidin and other secondary metabolites. J Biotechnol, 2012, 164(2): 281-291.

[20] Herzner AM, Dischinger J, Szekat C, et al. Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42. PLoS ONE, 2011, 6(7): e22389

[21] Ben Ayed H, Hmidet N, Béchet M, et al. Identification and biochemical characteristics of lipopeptides from Bacillus mojavensis A21. Process Biochem, 2014, 49(10): 1699-1707.

[22] Roy A, Mahata D, Paul D, et al. Purification, biochemical characterization and self-assembled structure of a fengycin-like antifungal peptide from Bacillus thuringiensis strain SM1. Front Microbiol, 2013, 4: 332.

[23] Romero D, de Vicente A, Rakotoaly Rivo H, et al. The iturin and fengycin families of lipopeptides are key factors in antagonism of Bacillus subtilis toward Podosphaera fusca. Mol Plant Microbe Interact, 2007, 20(4): 430-440.

[24] Alvarez F, Castro M, Principe A, et al. The plant-associated Bacillus amyloliquefaciens strains MEP218 and ARP23 capable of producing the cyclic lipopeptides iturin or surfactin and fengycin are effective in biocontrol of sclerotinia stem rot disease. J Appl Microbiol, 2012, 112(1): 159-174.

[25] Denning N, Morgan JAW, Whipps JM, et al. The flagellin gene as a stable marker for detection of Pseudomonas fluorescens SBW25. Lett Appl Microbiol, 1997, 24: 198-202.

[26] Raaijmakers JM, de Bruijn I, Nybroe O, et al. Natural functions of lipopeptides from Bacillus and Pseudomonas: more than surfactants and antibiotics. FEMS Microbiol Rev, 2010, 34(6): 1037-1062.

[27] Ma Z, Hu J. Production and characterization of iturinic lipopeptides as antifungal agents and biosurfactants produced by a marine pinctada martensii-derived Bacillus mojavensis B0621A. Appl Biochem Biotechnol, 2014, 173(3): 705-715.

[28] McCormick SP. Microbial detoxification of mycotoxins. J Chem Ecol, 2013, 39(7): 907-918.

[29] Hu LB, Shi ZQ, Zhang T, et al. Fengycin antibiotics isolated from B-FS01 culture inhibit the growth of Fusarium moniliforme Sheldon ATCC38932. FEMS Microbiol Lett, 2007, 272: 91-98.

[30] Hu LB, Zhang T, Yang ZM, et al. Inhibition of fengycins on the production of fumonisin B1 from Fusarium verticillioides. Lett Appl Microbiol, 2009, 48: 84-89.

[31] Falardeau J, Wise C, Novitsky L, et al. Ecological and mechanistic insights into the direct and indirect antimicrobial properties of Bacillus subtilis lipopeptides on plant pathogens. J Chem Ecol, 2013, 39(7): 869-878.

[32] Gong Q, Zhang C, Lu F, et al. Identification of bacillomycin D from Bacillus subtilis fmbJ and its inhibition effects against Aspergillus flavus. Food Control, 2014, 36(1): 8-14.

[33] Appleyard AN, Choi S, Read DM, et al. Dissecting structural and functional diversity of the lantibiotic mersacidin. Chem Biol, 2009, 16(5): 490-498.

[34] Stover AG, Driks A. Regulation of synthesis of the Bacillus subtilis transition-phase, spore-associated antibacterial protein TasA. J Bacteriol, 1999, 181(17): 5476-5481.