Characterization and Antimicrobial Studies of Iturin-Like and Bogorol-Like Lipopeptides From Brevibacillus spp. Strains GI9 and SKDU10

Abstract

Accession numbers for whole-genome sequence of Brevibacillus sp. strain GI9 and SKDU10 are CAGD01000001 to CAGD01000061 and LSSO00000000, respectively. Members of the genus Brevibacillus have been demonstrated to produce a variety of bioactive compounds including polyketides, lipopeptides and bacteriocins. Lipopeptides are non-ribosomally synthesized surface-active compounds with antimicrobial, antitumor, and immune-stimulatory activities. They usually exhibit strong antifungal and antibacterial activities and are considered as promising compounds in controlling fungal diseases. In this study, we have characterized two lipopeptides from Brevibacillus sp. strains GI9 and SKDU10. The corresponding lipopeptides were purified by reverse-phase high-performance liquid chromatography. Mass analysis and characterization by MALDI-TOF-MS (Matrix-assisted laser desorption ionization time-of-flight mass spectrometry) analysis revealed production of an iturin-like lipopeptide by strain GI9 and bogorol-like lipopeptide by strain SKDU10. Both lipopeptides exhibited broad spectrum antibacterial activity and inhibited the growth of various fungi. They showed minimum inhibitory concentration (MIC) values between 90 and 300 μg/ml against indicator strains of bacteria and drug-resistant Candida indicator strains. The lipopeptides did not show phytotoxic effect in seed germination experiments but caused hemolysis. Further, both lipopeptides inhibited the growth of fungi on fruits and vegetables in in vitro experiments, thereby exhibited potential use in biotechnological industry as effective biocontrol agents.

Keywords: BLL; Brevibacillus; ILL; bogorol; iturin; lipopeptide.

FIGURE 1

Purification and characterization of antimicrobial compound from strain GI9. (A) MALDI of crude extract from strain GI9 showing secretion of different compounds. (B) HPLC chromatogram of purified antimicrobial peptide at retention time 9 min. Inset shows phosphomolybdic acid stained TLC of pure compound and bioautogram with inhibition zone against S. aureus MTCC 1430. (C) MS/MS fragmentation pattern of iturin-like lipopeptide (ILL) at m/z 966.1 Da.

FIGURE 2

Purification and characterization of antimicrobial compound from strain SKDU10. (A) HPLC chromatogram purified peptide at retention time 3 min. Inset shows phosphomolybdic acid-stained TLC of pure compound and bioautogram with inhibition zone against S. aureus MTCC 1430. (B) MALDI spectrum showing intact mass of peptide m/z 1604 Da. (C) MS/MS fragmentation pattern of bogorol like lipopeptide (BLL) at m/z 1604 Da. (D) Biosynthetic gene cluster from strain SKDU10 consisting of modules responsible for secretion of BLL in comparison to bogorol biosynthetic cluster of B. laterosporus strain DSM25.

FIGURE 3

Killing kinetics of antimicrobial lipopeptides. (A) ILL against S. aureus MTCC 1430. (B) ILL against V. cholerae MTCC 3904. (C) ILL against C. albicans MTCC 1430. (D) BLL against S. aureus MTCC 1430. (E) BLL against V. cholerae MTCC 3904. (F) BLL against C. albicans MTCC 1430.

FIGURE 4

Microscopic examination of indicator strains after treatment with lipopeptides. (A) TEM micrographs of untreated and treated cells of S. aureus MTCC 1430. (B) TEM micrographs of untreated and treated cells of V. cholerae MTCC 3904. (C) SEM micrographs of untreated and treated cells of C. albicans MTCC 183. (D) Phase-contrast images of untreated and treated spores of A. brassicicola MTCC 2102. (Cell damage is marked by arrows).

FIGURE 5

Toxicity testing of antimicrobial lipopeptides. Seed germination-based phytotoxicity of (A) ILL (B) BLL using Vigna radiata. Water and 0.1% mercuric chloride were used as negative and positive control, respectively. (C) Hemolysis assay showing lysis of erythrocytes by both lipopeptides.

FIGURE 6

Protective ability of lipopeptides against infection of A. brassicicola MTCC 2102 in in vitro assay. (A) Inhibition of A. brassicicola MTCC 2102 on sliced tomato. (B) Inhibition of A. brassicicola MTCC 2102 on grapes.