Structural Elucidation, Mode of Action, Fermentation and Application in Commercial Apple Juice

Brevibacillin, an Antimicrobial Lipopeptide Discovered from Genus Brevibacillus: Structural Elucidation, Mode of Action, Fermentation and Application in Commercial Apple Juice

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Xu Yang, B.S.

Graduate Program in Food Science and Technology

The Ohio State University

2017

Dissertation Committee:

Dr. Ahmed E. Yousef, Advisor

Dr. Farnaz Maleky

Dr. Hua Wang

Dr. Monica Giusti

Copyrighted by

Xu Yang

2017

Abstract

Bacteria are capable of causing food spoilage and foodborne illness which lead to substantial economic losses and public health problems. All food processing steps cannot completely eliminate these microbial contaminations; therefore, antimicrobial agents are usually added into foods for post-processing control. However, resistance has been developed by some bacteria to the widely-used antimicrobial agents, such as potassium sorbate and sodium benzoate. In addition, consumers are now more concerned about the safety of chemical preservatives used in foods, and are more in favor of clean label products. Considering this dilemma, new safe and natural antimicrobial agents are needed in the food industry. On the other hand, the resistance of pathogenic bacteria has become a worldwide threat: in early 2016, a woman in

Pennsylvania was reported to carry a “superbug” — a strain of Escherichia coli resistant to colistin. The emergence of this “superbug” induced a global health concern. In an earlier report provided by the US Centers of Disease Control and Prevention (CDC), more than two million people suffered from antibiotic-resistant bacteria with 23,000 deaths annually, and these estimates didn’t even include people who die from other diseases exacerbated by antibiotic-resistant bacteria. Considering all these risks, both in food industry and clinical field, the need for new antimicrobial agents is vital.

The genus Brevibacillus was reclassified from Bacillus brevis cluster in 1996, based on 16S rRNA gene sequences and further phylogenetic analyses. A variety of antimicrobial compounds were produced from the former B. brevis cluster and the latter genus Brevibacillus, including gramicidin S and tyrocidine, which are applied for antibiotic treatment. However, few papers reviewed antimicrobial compounds produced from the genus Brevibacillus and its previous B. brevis cluster, despite the fact that the genus is a rich source for novel antimicrobial compounds.

In the current study, we summarized antimicrobial compounds produced both from the genus Brevibacillus and its previous classification B. brevis cluster. These bioactive compounds belong to diverse structural groups: bacteriocin, lipopeptide, cyclic peptide and polyketides. The major focus was on the structure of these antimicrobial compounds, since some of these agents produced by Brevibacillus spp. share structural similarities that were overlooked by researchers. For example, BT peptide, BL-A60, and bogorol are made up of peptides with structural similarity that was revealed only recently. We also summarized the potential applications of antimicrobial compounds from Brevibacillus in various biological systems as antibiotics, feed additives or biological control agents.

Brevibacillin is a new antimicrobial compound discovered in this study. It is produced by Brevibacillus laterosporus OSY-I1 and combats antibiotic-resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant strains of Enterococcus faecalis (VRE) and Lactobacillus plantarum. Brevibacillin was produced from isopropanol extracts of OSY-I1 cells, purified by high-performance liquid chromatography (HPLC), and structurally analyzed by mass spectrometry (MS) and nuclear magnetic resonance (NMR). MS and NMR results uncovered brevibacillin as a linear lipopeptide consisting of 13 amino acids, with an N terminus C6-fatty acid

iii chain (FA), 2-hydroxy-3-methylpentanoic acid. The lipopeptide has a molecular mass of 1583.0794 Da and contains three modified amino acid residues: α,ß- didehydrobutyric acid, ornithine, and valinol. The minimum inhibitory concentrations of brevibacillin against a selected panel of Gram-positive bacteria were comparable to that of vancomycin, and these indicators included foodborne pathogenic and spoilage bacteria. Brevibacillin demonstrated high stability under heat or various pH condition: it showed no sign of degradation at 80°C for 60 min and retained at least 50% antimicrobial activity when held at room temperature for 22 hours in acidic or alikaline condition (pH 3.0 and pH 9.0 buffer, respectively).

In addition to structural elucidation and minimum inhibitory concentration (MIC) analysis of brevibacillin, mode of action of the novel antimicrobial compound was also investigated. Results of the study proved that brevibacillin disrupts S. aureus ATCC

6538 cytoplasmic membrane through depolarization, increasing membrane permeability and leakage of intracellular potassium. Therefore, cytoplasmic membrane is determined as one of the major targets. Additionally, results have also shown that brevibacillin can bind to lipoteichoic acid (LTA), a cell wall component, before disrupting cell membrane. In summary, we propose that the electrostatic attraction between anionic LTA and cationic brevibacillin induced the accumulation of the antimicrobial agent at cell surface. The accumulation of brevibacillin was then translocated into the cytoplasmic membrane to disrupt its integrity. In addition to LTA binding and membrane disruption, the possibility of one or multiple intracellular targets for brevibacillin may still exist.

Production of brevibacillin in liquid medium with subsequent scale-up production in laboratory-scale fermenter was tested. The optimization of liquid medium was first experimented in test tubes to compare the yield of brevibacillin in liquid M9 medium

iv supplemented with various nitrogen sources. A semi-synthetic medium was chosen and prepared in a laboratory-scale bioreactor to produce brevibacillin. Antimicrobial activity was detected after 8 hours of incubation in the fermenter and the presence of brevibacillin was proved by matrix-assisted laser desorption/ionization time-of-flight

(MALDI-TOF) MS analysis.

The application of brevibacillin was tested in commercial apple juice to inhibit the growth of Alicyclobacillus acidoterrestris, both vegetative cells and spores. A. acidoterrestris is famous for causing spoilage and producing guaiacol, a smoky and medicinal compound to deteriorate product quality in a variety of fruit juices.

Brevibacillin containing fermentate generated from the laboratory-scale bioreactor was applied into commercial apple juice for spoilage inhibition: the fermentate which contained brevibacillin at concentration of 0.089 μg/ml exhibited significant (p < 0.05) inhibition against A. acidoterrestris vegetative cells. In comparison, 0.81 μg/ml brevibacillin containing fermentate was required to inhibit the spoilage caused by spores of A. acidoterrestris. The capability of producing brevibacillin in semi-synthetic liquid medium and potent antimicrobial activity of the compound both indicated brevibacillin can potentially be applied as a juice additive to combat spoilage caused by A. acidoterrestris.

Acknowledgement

My sincerest thanks go to my advisor, Dr. Ahmed Yousef. He provided the opportunity for me to work with the best group of researchers, and trained me from an individual who knows nothing about food microbiology, to a scientist capable of designing, conducting and publishing food microbiology related researches. I am proud to say that I have spent four and a half wonderful years in Dr. Yousef’s lab and have improved all aspects of my life. I would also like to offer my thanks to my committee members, Dr. Hua Wang, Dr. Farnaz Maleky and Dr. Monica Giusti, for their patience, guidance and encouragement.

I am also grateful for my lab associates who have helped me through the years: Dr.

Jennifer Perry, Dr. En Huang, Dr. Jin-Gab Kim, Dr. Baosheng Liu, Ebrahim Elkhtab,

Dr. Ismet öztürk, Dr. Rui Li, David Kasler, Greg Culbertson, Mustafa Yesil, Nathan

Morrison, Michelle Gerst, Yang Song, Emily Holman, Walaa Hussein, Joshua Kim,

Alexandra Chirakos and Clifford Park. Special thanks to Dr. En Huang for being a great mentor to guide my research, provide valuable suggestion and feedback. Together we have conquered many difficult projects. Special thanks go to Greg Culbertson for training me all aspectic technique and looking over my shoulder during the lab course.

Last but not the least, I would like to thank Dr. Liwen Zhang and Dr. Chunhua Yuan, the two of the best chemists on campus, who make my research a lot easier.

I would also like to thank my family members. My parents, Jun Yang and Yanxia

He who raised me and provided all they can to help me finish my degree. And my beloved grandmother, Jinying Dong, I will miss you for a lifetime. I would also like to say thank you to my wife Jingxin Guo, for putting up with me and encourage me when

I was down. Thank you for your unconditional support!

vii

Vita

July 17, 1989…………………………………………Born, Zhengzhou, Henan, China

2012…………………………………………...………B.S., Food Safety and Nutrition China Agricultural University, China

2012-present……………………………………………..Graduate Research Associate The Ohio State University

Publications

Yang, X., Huang, E., & Yousef, A. E. (2017). Brevibacillin, a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus. Microbiological Research, 195, 18-23.

Yang, X., Huang, E., Yuan, C., Zhang, L., & Yousef, A. E. (2016). Isolation and Structural Elucidation of Brevibacillin, an Antimicrobial Lipopeptide from Brevibacillus laterosporus That Combats Drug-Resistant Gram-Positive Bacteria. Applied and environmental microbiology, 82, 2763-2772.

Guo, Y., Huang, E., Yang, X., Zhang, L., Yousef, A. E., & Zhong, J. (2016). Isolation and characterization of a Bacillus atrophaeus strain and its potential use in food preservation. Food Control, 60, 511-518.

Fields of Study

Major Field: Food Science and Tehcnology

viii

Table of Contents Abstract ...... ii Acknowledgement ...... vi Vita ...... viii List of Tables ...... xiii List of Figures ...... xiv Chapter 1: Literature Review ...... 1 1.1 Introduction ...... 2 1.2 Antimicrobial compounds produced from previous B. brevis cluster ...... 3 1.2.1 Gramicidin S ...... 3 1.2.2 Linear gramicidin (gramicidin A-C) ...... 4 1.2.3 Tyrocidine ...... 4 1.2.4 Brevin ...... 5 1.2.5 Edeine ...... 5 1.2.6 Brevistin ...... 5 1.2.7 Spergualin and laterosporamine ...... 6 1.3 Antimicrobial compounds produced from the genus Brevibacillus ...... 8 1.3.1 Lipopeptide ...... 8 1.3.2 Bacteriocins...... 11 1.3.3 Other peptide based structures ...... 12 1.3.4 Non-peptide small molecules (< 500 Da) ...... 14 1.4 Application of antimicrobial compounds from both previous B. brevis cluster and the genus Brevibacillus...... 14 1.5 Conclusion ...... 18 References ...... 20 Chapter 2 Isolation and Structural Elucidation of Brevibacillin, an Antimicrobial Lipopeptide from Brevibacillus laterosporus That Combats Drug-Resistant Gram- positive Bacteria...... 26 Abstract ...... 26 2.1 Introduction ...... 26 2.2 Materials and Methods ...... 28 2.2.1 Strain screening ...... 28 2.2.2 Cultures and media ...... 29 2.2.3 Strain identification ...... 29

2.2.4 Isolation and purification of the antimicrobial agent from OSY-I1...... 30 2.2.5 MALDI-TOF MS analysis ...... 31 2.2.6 LC-MS/MS ...... 32 2.2.7 NMR analysis...... 33 2.2.8 Sensitivity to heat and pH ...... 34 2.2.9 In vitro antimicrobial activity determination ...... 34 2.2.10 16S rRNA sequence accession number ...... 35 2.3 Results ...... 35 2.3.1 Isolation and identification of an antimicrobial-producing strain ...... 35 2.3.2 Extraction, isolation and purification of the antimicrobial agent produced by OSY-I1 ...... 36 2.3.3 MIC determination and stability test...... 36 2.3.4 MALDI-TOF MS and LC-MS/MS analyses ...... 37 2.3.5 Peptide sequence determination and terminal structural elucidation by NMR ...... 39 2.4 Discussion ...... 47 References ...... 53 Chapter 3 Brevibacillin, a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus ...... 56 Abstract ...... 56 3.1 Introduction ...... 57 3.2 Materials and Methods ...... 58 3.2.1 Bacterial cultures and growth conditions ...... 58 3.2.2 Lipoteichoic acids binding assay ...... 59 3.2.3 Membrane permeability assay by fluorescence microscope ...... 60 3.2.4 Membrane depolarization study ...... 61 3.2.5 Potassium leakage analysis ...... 62 3.2.6 Scanning electron microscopy analysis ...... 62 3.2.7 Statistical analysis ...... 63 3.3 Results ...... 63 3.3.1 Brevibacillin binds to lipoteichoic acids before interacting with membrane ...... 63 3.3.2 Brevibacillin disrupts phospholipid bilayer membrane ...... 65 3.3.3 Depolarization of cell membrane by brevibacillin...... 66

3.3.4 Brevibacillin releases intracellular components ...... 67 3.3.5 The interaction of brevibacillin with cell envelope components does not cause morphological lesions ...... 68 3.4 Discussion ...... 69 References ...... 73 Chapter 4 The optimization of brevibacillin production in semi-synthetic medium and its use for controlling Alicyclobacillus acidoterrestris in a commercial fruit juice ..... 76 Abstract ...... 76 4.1 Introduction ...... 77 4.2 Materials and methods ...... 79 4.2.1 Bacterial strains and cultivation conditions ...... 79 4.2.2 Preparation of A. acidoterrestris ATCC 49025 spore suspension ...... 79 4.2.3 Liquid medium optimization for brevibacillin production in test tube system ...... 80 4.2.4 Production of brevibacillin in a laboratory-scale fermenter ...... 81 4.2.5 Confirmation of brevibacillin production by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis...... 81 4.2.6 Quantification of brevibacillin concentration in fermentate ...... 82 4.2.7 Effect of brevibacillin containing fermentate on controlling vegetative cells and spores of A. acidoterrestris ATCC 49025 in commercial apple juice ...... 82 4.2.8 Statistical analysis ...... 83 4.3 Results ...... 83 4.3.1 Liquid medium optimization for brevibacillin production in test tubes ...... 83

4.3.2 Growth and production of brevibacillin by OSY-I1 in the laboratory-scale fermenter ...... 85 4.3.3 Confirmation of brevibacillin production by MALDI-TOF MS analysis.... 86 4.3.4 Quantification of brevibacillin concentration in fermentate ...... 87 4.3.5 Effect of brevibacillin containing fermentate on controlling A. acidoterrestris ATCC 49025 vegetative cells and spores in commercial apple juice ...... 88 4.4 Discussion ...... 90 References ...... 93 Appendix A ...... 96 Structural elucidation of an antifungal lipopeptide from Pseudomonas fluorescence isolated from shiitake mushroom ...... 96 Abstract ...... 96

Introduction ...... 97 Materials and Methods ...... 100 Results ...... 103 Discussion ...... 108 References ...... 110 Bibliography ...... 112

xii

List of Tables

Table 1. Antimicrobial compounds produced from previous B. brevis cluster……...... 7 Table 2. Minimum inhibitory concentration (μg/ml) of brevibacillin (from Brevibacillus laterosporus OSY-I1), vancomycin, and nisin against selected bacteria including antibiotic-resistant strains...... 51 Table 3. Chemical shift assignments of lipopeptide in DMSO-d6, 298.0 K ...... 52 Table 4. Diameters of inhibition zones from modified M9 medium supplemented with various nitrogen sources. Different letter represents significant difference (p = 0.05) 84 Table 5. Representative bacteria isolates producing antifungal agent with their strain identification and spectrum...... 104

xiii

List of Figures

Figure 1. Vegetative cells and spores of Brevibacillus laterosporus under phase contrast microscope ...... 2 Figure 2. Chemical structures of selected antimicrobial compounds produced from Brevibacillus after genetic reclassification ...... 9 Figure 3. HPLC chromatogram at different stages of purification of antimicrobial agent from Brevibacillus laterosporus OSY-I1; antimicrobial activity of fractions was tested against Listeria innocua ATCC 33090...... 37 Figure 4. MALDI-TOF MS analysis of the antimicrobial agent isolated from

Brevibacillus laterosporus OSY-I1...... 38 Figure 5. Fragmentation of brevibacillin by tandem MS to generate b and y ion ...... 38 Figure 6. NMR spectra signifying the existence of a polypeptide and the acylated N- terminal cap ...... 40 Figure 7. NMR spectra exemplifying the residue and sequential assignment of the lipopeptide...... 42 Figure 8. NMR data showing the critical evidence for the assignment of FA, Dhb-1, and valinol-13 ...... 45 Figure 9. Chemical structure of brevibacillin and related cationic peptide antibiotics, bogorol A, BT peptide and BL-A60 (BL-A60 is presented in reversed sequence order)...... 50 Figure 10. Changes in populations of Staphylococcus aureus after seven hours of incubation at 37 °C as affected by the presence of brevibacillin and lipoteichoic acid (LTA)...... 65 Figure 11. Fluorescence microscope pictures of Staphylococcus aureus treated with brevibacillin (32 μg/ml) for two hours and stained with membrane-permeable SYTO-9 and membrane-impermeable propidium iodide stains...... 66 Figure 12. Changes in membrane potential of Staphylococcus aureus expressed by the fluorescent DiSC3(5) probe after treatment with 1-8 µg/ml brevibacillin...... 67 Figure 13. Potassium leakage, as detected by the fluorescent of the PBFI probe, after addition of 32 µg/ml brevibacillin...... 68 xiv

Figure 14. Scanning electron microscopy pictures of Staphylococcus aureus after treatment with brevibacillin (32 μg/ml) for two hours...... 69 Figure 15. Spot-on-lawn assay with L. innocua ATCC 33090 as indicator...... 85 Figure 16. Growth curve and antimicrobial activity test...... 86 Figure 17. MALDI-TOF MS spectrum of fermentate acquired after 14 hour incubation in laboratory-scale fermenter...... 87 Figure 18. Production of brevibacillin at different time points in fermentate...... 88 Figure 19. Effect of brevibacillin on Alicyclobacillus acidoterrestris ATCC 49025. . 89 Figure 20. Effect of brevibacillin on Alicyclobacillus acidoterrestris ATCC 49025 spore germination in commercial shelf-stable apple juice at 37 oC...... 90 Figure 21. Crude extract in hexane and water...... 105 Figure 22. HPLC chromatogram of DMSO dissolved antifungal compound from OSY-

M12 (top) and re-injection of fraction #42 into HPLC chromatogram (bottom)...... 106 Figure 23. Antifungal activity of HPLC purified fractions against A. niger...... 107 Figure 24. MALDI-TOF MS spectra for HPLC purified antifungal compound. m/z at 1354.9, 1376.9 and 1392.9 represented [M + H]+, [M + Na]+ and [M + K]+, respectively...... 108 Figure 25. MS/MS analysis for the antifungal compound structure elucidation...... 108

Chapter 1: Literature Review

Antimicrobial compounds produced by Brevibacillus spp.: structural diversity and potential application—a mini review Abstract

Brevibacillus spp. can be found in a variety of environments including plant leaves, intestinal tracts of animals, sea water, and soil. It is one of the most widely-spread genera of Gram-positive bacteria. Many antimicrobials have been isolated from

Brevibacillus cultures; these include antibacterial, antifungal and anti-invertebrate agents produced from Brevibacillus before and after genetic reclassification. These bioactive compounds belong to diverse structural groups including lipopeptide, cyclic peptide, bacteriocins and polyketides. This diversity makes Brevibacillus a rich source for novel, natural and potent antimicrobial agents. However, some of the antimicrobial compounds produced by Brevibacillus spp. share structural similarities that were overlooked by researchers. For instance, BT peptide, BL-A60, and bogorol are made up of peptides with structural similarity that was revealed only recently. Here we review the antimicrobial compounds produced by members of the genus Brevibacillus, and its previous genetic group, Bacillus brevis clusters, focusing on structural diversity and correlating structures with antimicrobial activity. The review also summarizes the potential applications of antimicrobial compounds from Brevibacillus in various biological systems. These applications include usage in the therapeutic field, animal feed and biological control agent.

1.1 Introduction

The genus Brevibacillus is a collection of rod-shaped Gram-positive or Gram-variable bacteria, mostly strict aerobic spore-formers (Figure. 1). The genus was reclassified from Bacillus brevis cluster in 1996, based on 16S rRNA gene sequences and further phylogenetic analyses. According to Shida et al. (1996), the established Brevibacillus genus included ten previous Bacillus species (B. brevis cluster), which were B. agri, B. borstelensis, B. brevis, B. centrosporus, B. choshinensis, B. formosus, B. laterosporus,

B. parabrevis, B. reuszeri, and B. thermoruber. Currently, there are twenty species under the genus Brevibacillus and all species have been reviewed carefully by Panda et al. (2014) except for some recent updates: Br. texasporus was removed from the existing species since it shares 98.5% 16S rRNA identity with Br. laterosporus (Wu et al., 2005); and a novel species Br. fulvus was added (Hatayama et al., 2014). The genus

Brevibacillus is ubiquitous and worldwide distributed in almost all environmental habitats, including plant leaves, intestinal tracts of animals, sea water, soil and many food products (Ruiu, 2013).

Figure 1. Vegetative cells and spores of Brevibacillus laterosporus under phase contrast microscope

Before genetic reclassification, B. brevis cluster served as a rich source for producing antimicrobial compounds, including tyrothricin and gramicidin S, which are used in antibiotic therapy (Katz and Demain, 1977). The genus Brevibacillus, after genetic reclassification, remained to be a decent source for novel antimicrobial compounds production. This mini-review summarizes, integrates and reviews antimicrobial agents produced both from previous B. brevis cluster and the genus

Brevibacillus.

1.2 Antimicrobial compounds produced from previous B. brevis cluster

The previous B. brevis cluster has been responsible for producing many antimicrobial compounds as listed in Table 1, along with producer strain (old and new names), chemical structure, antimicrobial spectrum and references.

1.2.1 Gramicidin S

Gramicidin S (GS, S stands for Soviet) was first discovered in the year of 1942 by

Gause and Brazhnikova (1944) from a B. brevis strain. The producer of GS is still under debate: based on genetic reclassification, GS producer shall be concluded as Br. brevis, as agreed by several researchers (Edwards and Seddon, 2001; Chandel et al., 2010;

Wang et al., 2010; Li et al., 2005). However, according to other group of researchers,

GS producer was reclassified into a new species Bacillus migulanus in 1993 (Takagi et al. 1993) and further reclassified as Aneurinibacillus migulanus (Shida et al., 1996;

Berditsch et al., 2007; Mogi et al., 2009). In this review article, in spite of the debate, we still consider GS is produced from Brevibacillus sp..

GS is a cyclic decapeptide which is formed by two identical pentapeptides (Table

1). It is effective against Gram-positive bacteria on solid agar medium and becomes inhibitory against Gram-positive, Gram-negative and fungi in the liquid medium

(Prenner et al., 1999). The proposed mode of action of GS is to interact with bacterial

3 cells to destruct the integrity of cytoplasmic membrane, especially the lipid bilayer

(Prenner et al., 1999). The biosynthesis of GS was reported to be accomplished through non-ribosomal peptide synthetase (Conti et al., 1997).

1.2.2 Linear gramicidin (gramicidin A-C)

The linear gramicidins are pentadecapeptide produced from B. brevis (Katz and

Demain, 1977) and are not structurally related to gramicidin S, even though with similar names. The linear gramicidins (Table 1 with detailed structure) were discovered in 1939 by Hotchkiss and Dubois (1940). The antimicrobial compounds have been reported to cause membrane lysis by creating transmembrane channels as a main antimicrobial mode of action (Wang et al., 2012): intracellular component (potassium ions) diffusing out of the cell leads to cell dysfunction. Linear gramicidins are effective against mainly

Gram-positive bacteria with no inhibitory effect against Gram-negative microorganisms (Wang et al., 2012).

1.2.3 Tyrocidine

Tyrocidine shares similarities in structure to GS as a cyclic decapeptide, for instance, tyrocidine A is also a cyclic decapeptide: cyclo (Phe-Pro-Phe-Phe-Asn-Gln-

Tyr-Val-Orn-Leu) (Munyuki et al., 2013). The producer of tyrocidine, coincidentally, was also under debate: some researchers reported tyrocidine was produced from B. brevis which later classified as B. aneurinolyticus (Spathelf and Rautenbach, 2009;

Munyuki et al., 2013) but in reality, the genus Brevibacillus also serves as a tyrocidine producer, for instance, Br. parabrevis ATCC 8185 is capable of producing both linear gramicidin and tyrocidine (American Type Cculture Collection). Tyrocidine has been reported to have bactericidal effect and antifungal activity (Munyuki et al., 2013;

Vosloo et al., 2013) and it has been used as one of the first antibiotics in clinical field for topical treatment (Van Epps, 2006). Tyrocidine is a membrane active antimicrobial

4 peptide which can disrupt cytoplasmic membrane structure to exert its bactericidal efficacy (Munyuki et al., 2013) and tyrocidine biosynthetic pathway is also through

NRPS, similarly to biosynthetic pathways of GS. Unlike a lot of NRPS domains, the thioesterase (TE) domain from tyrocidine NRPS can be independently engineered to cyclize novel substrates and to generate new compounds (Trauger et al., 2000).

1.2.4 Brevin

Brevin was produced from the previous B. brevis but without fully-elucidated structural information. Brevin, as an anti-Gram-positive compound, was isolated in the year of 1953 with structural novelties comparing to GS and tyrocidine. The structure of brevin contains aspartic acid, tyrosine, serine, glycine and an unknown basic component (Barnes and Newton, 1953).

1.2.5 Edeine

Edeine is produced by the previous B. brevis TT02-8 and is composed of five amino acid residues along with an organic moiety: a spermidine structure in edeine A1

(Table 1) and a guanylspermidine structure in edeine B1. Edeine has a broad antimicrobial spectrum as a protein synthesis inhibitor and is active against Gram- positive bacteria, Gram-negative bacteria and fungi (Shimotohno et al., 2000).

1.2.6 Brevistin

Brevistin was discovered in the year of 1975 and is a peptide antibiotic isolated from the previous B. brevis 342-14. Brevistin has a chemical formula of C63H91N15O18, and is consisted of aspartic acid, threonine, glycine, valine, isoleucine, phenylalanine, tryptophan and 2, 4-diaminobutyric acid. Brevistin is effective against Gram-positive bacteria including Staphylococcus aureus and Streptococcus pneumoniae, and it was reported to have low toxicity to mice (Shoji et al., 1976).

1.2.7 Spergualin and laterosporamine

Spergualin is an antimicrobial compound produced from the previous B. laterosporus with activity against Gram-positive bacteria. Spergualin is not a peptide antibiotic but a polyamine compound with the molecular mass of 403.53 (C17H37N7O4)

(Umezawa et al., 1981). Besides its antimicrobial activity, spergualin is more under investigated as an antitumor compound which significantly prolonged the survival time of mice inoculated with leukemia cells (Takeuchi et al., 1981). In addition, spergualin was reported to have low toxicity: 80 mg/kg of intravenous injection did not correspond to death of mice (Takeuchi et al., 1981).

Laterosporamine has an empirical chemical formula of C17H35N7O4 with a non- peptidic structure which shares high similarity with spergualin. Laterosporamine was also produced from the previous B. laterosporus with antibacterial activity (mainly

Gram-positive bacteria). The high similarity between laterosporamine and spergualin indicates that they may be closely related analogues (Umezawa et al., 1981).

Table 1. Antimicrobial compounds produced from previous B. brevis cluster. Name Producer strain Chemical structure References Old name New name Gramicidin S B. brevis or Br. Brevis or Cyclo (Leu-Phe-Pro-Val-Orn-Leu- Conti et al., 1997 (GS) B. migulanus Aneurinibacillus Phe-Pro-Val-Orn-) Prenner et al., 1999 migulanus Katz and Demain, 1977 Linear B. brevis Br. brevis HCO-Val-Gly-Ala-Leu-Ala-Val- Dzikovski et al., 2004 gramicidin A Val-Val-Trp-Leu-Trp-Leu-Trp- Katz and Demain, 1977 Leu-Trp-NHCH2CH2OH Wang et al., 2012 Tyrocidine B. brevis or Br. brevis or Tyrocidine A: cyclo (Phe-Pro-Phe- Munyuki et al., 2013 B. aneurinolyticus Br. parabrevis Phe-Asn-Gln-Tyr-Val-Orn-Leu) Vosloo et al., 2013 Van Epps, 2006 Brevin B. brevis Br. brevis Tyrosine, aspartic acid, serine, Barnes and Newton, 1953 glycine, and an unknown basic substance Edeine A B. brevis TT02-8 Br. brevis TT02-8 Spermidine-Gly-isoserine-(β- Shimotohno et al., 2000 Tyrosine)-(2, 3-diaminopropionic Wojciechowska et al., 1972 acid)-(2, 6-diamino-7-hydroxy- azelaic acid) Brevistin B. brevis 342-14 Br. brevis 342-14 Contains aspartic acid, threonine, Shoji et al., 1975 glycine, valine, isoleucine, phenylalanine, tryptophan and 2, 4-diaminobutyric acid spergualin B. laterosporus Br. laterosporus (15S)-1-amino-19-guanidino- 11, Umezawa et al., 1981 15 - dihydroxy -4, 9, 12- triazanonadecane-10, 13-dione Laterosporamine B. laterosporus Br. laterosporus Potential similar structure with Umezawa et al., 1981 spergualin

1.3 Antimicrobial compounds produced from the genus Brevibacillus

After genetic reclassification of B. brevis cluster into Brevibacillus, discovery of novel antimicrobial compounds have been continuously reported by many literatures.

On the basis of their chemical structures, the mini-review summarizes and integrates them into several categories: lipopeptide, bacteriocin, other peptide-based structures and non-peptide small molecules. Detailed chemical structures of selected compounds are shown in Figure. 2.

1.3.1 Lipopeptide

Lipopeptide is usually composed of a lipid chain attached to a peptide structure

(Makovitzki et al., 2006). The antimicrobial lipopeptide surfactin has been reported to be a biocontrol agent that has been produced mostly from B. subtilis (Seydlová and

Svobodová, 2008). Recently, a Br. brevis strain was discovered to produce a surfactin isoforms (Wang et al., 2010). Through electrospray ionization mass spectrometry (ESI-

MS), gas chromatography-mass spectrometry (GC/MS), and electrospray ionization tandem mass spectrometry (ESI-MS/MS), the compound was determined to have a structure of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C, the same as surfactin. This was the first time that Br. brevis was reported to be a new species to produce surfactin.

Tauramamide (Figure. 2) (Desjardine et al., 2007) is a lipopeptide antimicrobial agent produced from Br. laterosporus PNG276 in the year of 2007. Tauramamide is a linear lipopeptide composed of a 7-methyloctanoic acid esterified on a pentapeptide chain as C7-Tyr-Ser-Leu-Trp-Arg; the entire structure has a molecular formula of

C45H67N9O9. Tauramamide was synthesized to confirm the deduced chemical structure and its broad antimicrobial activity: tauramamide was reported to be antimicrobial against methicillin-resistant S. aureus (MRSA), Candida albicans, vancomycin- resistant Enterococcus (VRE), Mycobacterium tuberculosis and Escherichia coli.

Tauramamide

Brevibacillin

A C Q C P D A I S G W T H T D Y Q C H G L E N K M Y R H V Y A I C M N G T Q V Y C R T E W G S S C

laterosporulin

Basiliskamides A

Loloatin A

FIG 2 Chemical structures of selected antimicrobial compounds produced from genums Brevibacillus after genetic reclassification. Figure 2. Chemical structures of selected antimicrobial compounds produced from Brevibacillus after genetic reclassification

A group of lipopeptide antimicrobial compounds share similar structure and thus are most investigated lipopeptide compounds produced from the genus Brevibacillus.

In the year of 2000, Basby et al. first reported bogorol A, a lipopeptide that consists of a C6 fatty acid chain (2-hydroxy-3-methylpentanoic acid) and 13 amino acid residues:

FA-Dhb-Leu-Orn-Ile-Val-Val-Lys-Val-Leu-Lys-Tyr-Leu-valinol. Bogorol A was first 9 announced to be isolated from a marine Bacillus spp. but then revised to Br. laterosporus by the same group of researchers. Bogorol A is effective against MRSA,

VRE and has moderate antimicrobial activity against E. coli (35 μg/ml). Bogorol B-E structures were later published by the same research group and all bogorol compounds consisted of the family of bogorol antimicrobial agents (Barsby et al., 2006). In 2005,

Wu et al. reported the discovery of BT peptide, an antimicrobial agent isolated from genus Brevibacillus. BT peptide shares high similarity with bogorol A with the only difference at N terminus structure hypothesized as Bmt: 4-methyl-4-[(E)-2-butenyl]-4,

N-methyl-threonine. However, the ambiguity structural information about Bmt indicated the resemblances between BT peptide and bogorol A. In the year of 2012,

Zhao et al. reported the discovery of a new antimicrobial peptide, BL-A60. The structure at both C and N terminus was not elucidated by the authors, but the partial peptide sequence shares high similarity with bogorol C, with the exception that this sequence order is reversed (Zhao et al., 2012). Bogorol B was rediscovered from Br. laterosporus JX-5 three years later (Jiang et al., 2015). The bacterium was isolated and demonstrated strong antifungal activity and produced a heat-stable, pH and UV- resistant compound. The compound was later analyzed through tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) as bogorol B. Novel lipopeptide brevibacillin (Figure. 2) was discovered and structurally elucidated in the year of 2016 (Yang et al., 2016) and it also shares structure similarity with bogorol A but with 2 amino acids difference: valine and leucine structure at position 5 and 9 in bogorol A was replaced by isoleucine and valine in brevibacillin. Brevibacillin demonstrated comparable antimicrobial activity with that of vancomycin against a panel of selected Gram-positive bacteria, including several foodborne pathogens/spoilage indicators such as Listeria monocytogenes, Alicyclobacillus

10 acidoterrestris and S. aureus (Yang et al., 2016).

1.3.2 Bacteriocins

Bacteriocins are antimicrobial peptides which are ribosomally synthesized by bacteria (Martinez et al., 2013). In recent years, the genus Brevibacillus has been reported as a rich source to produce novel bacteriocins. Brevibacilus sp. strain GI-9 was reported to produce a bacteriocin named laterosporulin (Figure. 2). Laterosporulin was claimed to be effective against both Gram-positive and Gram-negative bacteria and had been purified using reverse-phase high performance liquid chromatography (HPLC) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-

TOF). The molecular mass of laterosporulin was reported to be 5.6 kDa. Based on published draft genome of GI9 (Sharma et al., 2012), laterosporulin biosynthetic gene cluster was identified, along with the open reading frames (ORFs) of the structural gene.

Laterosporulin structure was determined by protein sequencer and structural gene ORFs, with the sequence as: N-

ACQCPDAISGWTHTDYQCHGLENKMYRHVYAICMNGTQVYCRTEWGSSC-C

(Singh et al., 2012). Laterosporulin was later reported as a class IId bacteriocin which contained disulfide bonds intramolecularly: six cysteine residues formed three pairs of disulfide-bonds. The mode of action of laterosporulin was proved through creating holes in cytoplasmic membrane of target bacteria which leads to bactericidal activity

(Singh et al., 2015).

Another potential bacteriocin was produced from Br. laterosporus strain SA14 isolated from Thailand. The molecular weight was first reported to be 116 kDa and the compound has antimicrobial activity against both Gram-positive and Gram-negative bacteria (Choopan et al., 2011). The antimicrobial compound was then partially purified and analyzed to have a molecular mass of 6.9 kDa. Purification of this antimicrobial

11 compound was achieved through both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and reverse phase-HPLC, and a single peak from HPLC chromatogram was obtained (Somsap et al., 2015). The compound was claimed to be a bacteriocin but further MS and NMR experiments may be needed to confirm its entire structure.

Another potential bacteriocin was produced from a Brevibacillus strain in

“marcha”, an herbal cake used for wine fermentation. After 16S rRNA gene sequence analysis, the producer strain was designated Br. borstelensis. The potential bacteriocin compound was purified by gel exclusion chromatography with a molecular weight of

12 kDa. The compound showed high heat resistance, and remained bioactive activity at various pH conditions, but was sensitive to trypsin digestion. Even though structural information was inconclusive, the author claimed that this antimicrobial compound could potentially be used as a food biopreservative due to its antimicrobial efficacy against several foodborne indicators including L. monocytogenes, Bacillus subtilis,

Clostridium perfringens and Lactobacillus plantarum (Sharma et al., 2014).

Bac-GM100 was another potential bacteriocin produced from Br. brevis strain

GM100 isolated from Ononis angustissima. The compound Bac-GM100 has a molecular weight of 4375.66 Da analyzed by MALDI-TOF MS. A protein sequencer was used to partially analyze the structure of Bac-GM100 at N-terminal which contains

21 amino acid residues: N-DWTFANWSCLVCDDCSVNLTY. The partially deduced sequence shared 65% similarity with thurincin. Bac-GM100 was also reported to be active against gram-negative (Pseudomonas aeruginosa), gram-positive (Enterococcus faecalis) and fungi (Candida tropicalis) (Mouloud et al., 2013).

1.3.3 Other peptide based structures

Tupuseleiamides were discovered from Br. laterosporus as acyldipeptides with

12 antimicrobial activity against E. coli. The molecular weight of tupuseleiamides A and

B were both 408.2262, with a chemical formula of C21H32N2O6. Even though shared with the same chemical formula, they differed in a methyl group position: a methyl group at C-19 for tupuseleiamide A and a methyl group at C-18 for tupuseleiamide B

(Barsby et al., 2002).

The loloatin family antimicrobial compounds are cyclic decapeptide isolated from

Br. laterosporus and contain four members: loloatin A-D. The most abundant family member, loloatin B, has a molecular weight of 1295.63403 and chemical formula of

C67H86N13O14 (Gerard et al., 1996). All loloatins were reported effective against Gram- positive bacteria, especially drug-resistant strains, including MRSA, VRE, and penicillin-resistant Streptococcus pneumoniae. Interestingly, the structure of loloatin A shares high similarity with tyrocidine A, the antimicrobial agent produced from previous B. brevis. The structural differences are colored as shown in Figure. 2: tyrocidine and aspartic acid residues in loloatin A while phenelalanine and glutamine residues in tyrocidine A, respectively (Gerard et al., 1999).

Tostadin is a novel small antimicrobial peptide which was effective against both

Gram-positive and Gram-negative bacteria isolated from Br. brevis XDH. The peptide has a molecular weight of 1102.35 Da and is consisted of nine amino acids: Ser-Leu-

Tyr-Lys-Leu-Thr-Cys-Lys-Phe. Even though authors claimed the novel structure was analyzed by MS and NMR but no detailed methods or results were available in the paper (Song et al., 2012).

Chitinases were also isolated from a Br. laterosporus strain used against a Fusarium indicator. Of the two chitinases produced, one belonged to a four domain chitodextrinase and the other belongs to a two domain enzyme designated ChiA1

(Prasanna et al., 2013).

1.3.4 Non-peptide small molecules (< 500 Da)

The genus Brevibacillus has also been reported to produce non-peptide small molecules which have a molecular weight less than 500 Da.

Basiliskamides (Figure. 2) are a group of non-peptide polyketides produced by Br. laterosporus, a marine bacterium. Based on MS and NMR results, both basiliskamide

A and B had a molecular weight of 385.2256 and chemical formula of C23H31NO4. Both compounds are differed in a cinnamoyl ester: it attached at C-7 position in basiliskamide A and at C-9 in basiliskamide A. The basiliskamides were reported to inhibit C. albicans and Aspergillus fumigatus. The antifungal activity of basiliskamide

A was comparable to an antifungal drug, amphotericin B, but with four-fold less cytotoxicity against human fibroblast cells (Barsby et al., 2002).

Br. brevis FJAT-0809-GLX was described to produce antimicrobial compound ethylparaben that can inhibit both bacteria and fungi. The purification process of ethylparaben was achieved by using column chromatographic technique by an Agilent

GC system. Several bacterial and fungal indicators were tested including Ralstonia solanacearum, Fusarium oxysporum, Aspergillus and E. coli. The author claimed this was the first report describing Br. brevis as an ethylparaben producer (Che et al., 2014).

1.4 Application of antimicrobial compounds from both previous B. brevis cluster and the genus Brevibacillus.

The genus Brevibacillus has been applied as probiotics for long times (Sanders et al. 2003). A Br. Laterosporus (BOD) strain, for instance, has been reported to be used as a human probiotic since 1989 by Flora Banlance Montana, USA, with patent

(O’donnell 1995). Recently, another probiotic Br. brevis strain has been suggested for its probiotic properties in sea bass (Dicentrarchus labrax). The strain was described to produce antimicrobial activity against Vibrio, a fish pathogen and according to the

14 literature, treatment of sea bass larvae with B. brevis does not affect its survival rate but improve the specific growth, comparing to non-probiotic treatment control group

(Mahdhi et al. 2012).

The antimicrobial compounds isolated from previous B. brevis cluster have long been applied in clinical filed. In the same year of GS discovery, Soviet military hospitals had applied this antibiotic to treat infected wounds, and by 1943 it was being used at the front (Gause and Brazhnikova, 1944; Gall and Konashev, 2001). GS nowadays has been used mainly as an antibiotic to treat topical infections due to high hemolytic activity (Waki and Izumiya, 1990; Xu et al., 1995).

The linear gramicidin family compound (gramicin A-C) has very low solubility in water (<50 nM) and could lead to hemolytic reaction to human cells at bactericidal concentration. As a result, similar to GS, the clinical use for this group of compound has also been limited to topical applications: the linear gramicidin drugs has been commercialized in Canada as an antibiotic ointment Polysporin (Want et al., 2012). In addition to Polysporin, linear gramicidin family antibiotics are also applied as one of the major active ingredients in several clinical drugs according to FDA, including “Neo- polycin”, “Neomycin and polymyxin B sulfates and gramicidin” and “Neomycin sulfate and polymyxin B sulfate gramicidin” (FDA).

The application of tyrocidine is combined with the treatment of linear gramicidin in the form of tyrothricin (katz et al., 1977). The application of tyrothricin component in a wound gel system (Tyrosur® gel) was reported to significantly improve the wound healing in thirty-three healthy volunteers, especially with an earlier healing onset time

(Wigger-Alberti et al., 2012).

Antimicrobial compounds produced from genus Brevibacillus have been applied into many biological systems, as feed antibiotic, biocontrol agent or food additives. BT

15 peptide, as mentioned earlier, has been applied as a feed antibiotic. In the year of 2005,

BT peptide was used to prevent a colibacillosis natural outbreak in broiler chickens.

The experiments initiated to mimic the conditions that are conducive to an outbreak of colibacillosis and BT peptide was used as a medication treatment, along with unmedicated negative control. There was a significant increase regarding weight gain and feed conversion of using BT peptide as a feed antibiotic comparing to unmedicated negative control. The mortality rate of using BT peptide as medication was reduced to

0.051% comparing to the unmedicated treatment 13.402% (Jiang et al., 2005). The following research was conducted two years later to use BT peptide as an immunostimulator of the innate immune system for young chickens, especially chicken that were born within a week. The use of BT peptide significantly increased the protection of boiler chicken against Salmonella Enteritidis by improving the innate response of the new-born chicken: immunological factors were up-regulated including phagocytosis, oxidative burst and degranulation (Kogut et al., 2007). Further research in 2012 proved that the improving of boiler chickens’ health does not correspond to

BT’s antimicrobial activity: the concentrations applied were below the MIC of BT against S. Enteritidis; also, BT peptide was not absorbed in the intestine but enhanced immunological performance of the tested new-born chickens. As a result, agreed upon

Kogut et al. in 2007, BT served as an immune modulator which improved leukocyte function, leukocyte proinflammatory cytokine production and chemokine mRNA transcription in vitro (Kogut et al., 2012).

The bacteriocin Bac-GM100 mentioned earlier has been used to extend the shelf- life of tomato paste through challenge studies by inoculating L. monocytogenes

MTCC839, B. subtilis CRI or Clostridium perfringens MTCC1739 into the product. All treatment groups (partially purified Bac-GM100, purified Bac-GM100, nisin and

16 sodium benzoate) showed significant effect of extending shelf-life of tomato paste in the first five days of incubation at refrigeration temperature: at least 1 log difference comparing to negative control (no preservative added). As a result, authors claimed that

Bac-GM100 (purified or partially purified) can be applied in an acid food at refrigeration to effectively extend their shelf-life (Gupta et al., 2015).

Br. laterosporus ZQ2 which produces tostadin was used as a biocontrol agent according to Song et al. (2013). Apple seeds were surface sterilized and sown. Bacterial strain Br. laterosporus ZQ2 and plant pathogen Rhizoctonia solani were inoculated 40 days later. The disease severity was evaluated by the area of lesion on the leaves. The maximum protection was achieved by inoculating the biocontrol microorganisms 3 days before the introduction of the plant pathogen and the disease severity was only

17.55±0.32% comparing to the negative control 64.44±2.05% (Song et al., 2013).

The application of several strains of Brevibacillus as biocontrol agent has been reported, even though without clear information in terms of their bioactive compounds produced. One Br. laterosporus strain BPM3 was isolated from the mud of a natural hot water spring in India, the strain could inhibit the growth of fungi including F. oxysporum,

Magnaporthe grisea and Rhizotonia oryzae, along with Gram-positive bacteria S. aureus. Partial structural analysis indicated that the compound contained C-H, carbonyl group, dimethyl group, -CH2 and methyl group. Br. laterosporus strain BPM3 had been used to control rice blast diseases caused by Magnapothe grisea. According to Saikia et al. (2011), the maxium protection of BPM3 was when the plant pathogen fungus was inoculated two days after the biocontrol agent inoculation, which provides 67% protection and 56.5% weight loss protection.

Br. laterosporus B4 was also used as a biological control agent to inhibit

Acidovorex avenae and to prevent rice from brown strip diseases. Result indicated that

17 maximum protection of rice against bacterial brown strip in vivo was achieved when soaking seedlings in B4 suspension for 24 hours before inoculation of A. avenae, in which condition provided 71±3.9% protection of rice after 24 hours of seed treatment comparing to negative control which had no effect on rice disease control (0%) (Kakar et al., 2014).

A Br. reuszeri strain R2a23 was discovered to have antifungal activity and had been used to control Fusarium growth on full-grown maize ears. Different from the previous bio-control experiment, the culture filtrate instead of live culture was applied to the ear cuts. The results showed that the strain R2a23 significantly inhibit the growth of F. proliferaturn and F. verticillioides by calculating the ear rot percentage (affected kernels/total kernels) (Joo et al., 2015).

The genus Brevibacillus has not been reported to pose any health hazard to human beings, but has been associated with food spoilage, especially milk spoilage (Gopel et al. 2015). Two Br. agri (LMG 19651, LMG 19652) were isolated from ‘commercially sterilized’ milk sample (Logan et al. 2002) and according to a molecular-based investigation, the presence (percentage of the total samples under investigation) of Br. agri, Br. borstelensis and other Brevibacilus sp. from 17 dairy farms across Belgium is

4.8%, 7.2% and 4.8%, respectively (Scheldeman et al. 2005). In addition, the presence of Br. borstelensis was also detected from whole milk and skim milk powders in China

(Yuan et al. 2012).

1.5 Conclusion

The genus Brevibacillus is ubiquitous and contains 20 different species. The genus

Brevibacillus has been known as a rich source of antimicrobial compounds before and after genetic reclassification. These antimicrobial agents have diverse structures which include cyclic peptide, linear lipopeptide, bacteriocin and polyketides. Several of these

18 antimicrobial compounds are structurally correlated and have been clarified in this mini review. Antimicrobial agents produced from Brevibacillus have been applied as clinical drugs, biocontrol agent, feed additives and potential food preservatives.

References

Barnes, E. M., & Newton, G. G. F. (1953). Brevin: an antibiotic produced by Bacillus brevis. Antibiotics & chemotherapy, 3(9), 866-72.

Barsby, T., Kelly, M. T., & Andersen, R. J. (2002). Tupuseleiamides and basiliskamides, new acyldipeptides and antifungal polyketides produced in culture by a Bacillus laterosporus isolate obtained from a tropical marine habitat. Journal of natural products, 65(10), 1447-1451.

Barsby, T., Kelly, M. T., Gagné, S. M., & Andersen, R. J. (2001). Bogorol A produced in culture by a marine Bacillus sp. reveals a novel template for cationic peptide antibiotics. Organic letters, 3(3), 437-440.

Barsby, T., Warabi, K., Sørensen, D., Zimmerman, W. T., Kelly, M. T., & Andersen, R. J. (2006). The bogorol family of antibiotics: template-based structure elucidation and a new approach to positioning enantiomeric pairs of amino acids. The Journal of organic chemistry, 71(16), 6031-6037.

Berditsch, M., Afonin, S., & Ulrich, A. S. (2007). The ability of Aneurinibacillus migulanus (Bacillus brevis) to produce the antibiotic gramicidin S is correlated with phenotype variation. Applied and environmental microbiology, 73(20), 6620-6628.

Chandel, S., Allan, E. J., & Woodward, S. (2010). Biological control of Fusarium oxysporum f. sp. lycopersici on tomato by Brevibacillus brevis. Journal of phytopathology, 158(7‐8), 470-478.

Choopan, A., Nakbud, K., Dawveerakul, K., Chawawisit, K., & Lertcanawanichakul, M. (2011). Anti-methicillin resistant Staphylococcus aureus activity of Brevibacillus laterosporus strain SA14. Walailak Journal of Science and Technology (WJST), 5(1), 47-56.

Conti, E., Stachelhaus, T., Marahiel, M. A., & Brick, P. (1997). Structural basis for the activation of phenylalanine in the non‐ribosomal biosynthesis of gramicidin S. The EMBO Journal, 16(14), 4174-4183.

Desjardine, K., Pereira, A., Wright, H., Matainaho, T., Kelly, M., & Andersen, R. J. (2007). Tauramamide, a lipopeptide antibiotic produced in culture by Brevibacillus laterosporus isolated from a marine habitat: structure elucidation and synthesis. Journal of natural products, 70(12), 1850-1853.

Dzikovski, B. G., Borbat, P. P., & Freed, J. H. (2004). Spin-labeled gramicidin A: channel formation and dissociation. Biophysical journal, 87(5), 3504-3517.

Edwards, S. G., & Seddon, B. (2001). Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro. Journal of applied Microbiology, 91(4), 652- 659.

Gall, Y. M., & Konashev, M. B. (2001). The discovery of gramicidin S: the intellectual transformation of GF gause from biologist to researcher of antibiotics and on its meaning for the fate of russian genetics. History and philosophy of the life sciences, 137-150.

Gause, G. F., & Brazhnikova, M. G. (1944). Gramicidin S and its use in the treatment of infected wounds. Nature, 154, 703.

Gerard, J. M., Haden, P., Kelly, M. T., & Andersen, R. J. (1999). Loloatins AD, cyclic decapeptide antibiotics produced in culture by a tropical marine bacterium. Journal of natural products, 62(1), 80-85.

Gerard, J., Haden, P., Kelly, M. T., & Andersen, R. J. (1996). Loloatin B, a cyclic decapeptide antibiotic produced in culture by a tropical marine bacterium. Tetrahedron letters, 37(40), 7201-7204.

Ghadbane, M., Harzallah, D., Laribi, A. I., Jaouadi, B., & Belhadj, H. (2013). Purification and biochemical characterization of a highly thermostable bacteriocin isolated from Brevibacillus brevis strain GM100. Bioscience, biotechnology, and biochemistry, 77(1), 151-160.

Gopal, N., Hill, C., Ross, P. R., Beresford, T. P., Fenelon, M. A., & Cotter, P. D. (2015). The Prevalence and Control of Bacillus and Related Spore-Forming Bacteria in the Dairy Industry. Frontiers in microbiology, 6.

Gupta, A., Sharma, N., & Gautam, N. (2015). Preservative potential of purified bacteriocin produced from Brevibacillus borstelensis AG1 isolated from marcha - a traditional wine starter culture cake in tomato paste. The Journal of Microbiology, Biotechnology and Food Sciences, 4(5), 448.

Hotchkiss, R. D., & Dubos, R. J. (1940). Fractionation of the bactericidal agent from cultures of a soil Bacillus. Journal of Biological Chemistry, 132(2), 791-792.

Jiang, H., Wang, X., Xiao, C., Wang, W., Zhao, X., Sui, J., Sa, R., Guo, T. L., & Liu, X. (2015). Antifungal activity of Brevibacillus laterosporus JX-5 and characterization of its antifungal components. World Journal of Microbiology and Biotechnology, 31(10), 1605-1618.

Jiang, Y. W., Sims, M. D., & Conway, D. P. (2005). The efficacy of TAMUS 2032 in preventing a natural outbreak of colibacillosis in broiler chickens in floor pens. Poultry science, 84(12), 1857-1859.

Jianmei, C., Bo, L., Zheng, C., Huai, S., Guohong, L., & Cibin, G. (2015). Identification of ethylparaben as the antimicrobial substance produced by Brevibacillus brevis FJAT-0809-GLX. Microbiological research, 172, 48-56.

Joo, H. J., Kim, H. Y., Kim, L. H., Lee, S., Ryu, J. G., & Lee, T. (2015). A Brevibacillus sp. antagonistic to mycotoxigenic Fusarium spp. Biological Control, 87, 64-70. 21

Kakar, K. U., Nawaz, Z., Cui, Z., Almoneafy, A. A., Zhu, B., & Xie, G. L. (2014). Characterizing the mode of action of Brevibacillus laterosporus B4 for control of bacterial brown strip of rice caused by A. avenae subsp. avenae RS-1. World Journal of Microbiology and Biotechnology, 30(2), 469-478.

Katz, E., & Demain, A. L. (1977). The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bacteriological Reviews, 41(2), 449.

Kogut, M. H., Genovese, K. J., He, H., Li, M. A., & Jiang, Y. W. (2007). The effects of the BT/TAMUS 2032 cationic peptides on innate immunity and susceptibility of young chickens to extraintestinal Salmonella enterica serovar Enteritidis infection. International immunopharmacology, 7(7), 912-919.

Kogut, M. H., Genovese, K. J., He, H., Swaggerty, C. L., & Jiang, Y. W. (2012). BT cationic peptides: small peptides that modulate innate immune responses of chicken heterophils and monocytes. Veterinary immunology and immunopathology, 145(1), 151-158.

Li, H., Tanikawa, T., Sato, Y., Nakagawa, Y., & Matsuyama, T. (2005). Serratia marcescens gene required for surfactant serrawettin W1 production encodes putative aminolipid synthetase belonging to nonribosomal peptide synthetase family. Microbiology and immunology, 49(4), 303-310.

Logan, N. A., Forsyth, G., Lebbe, L., Goris, J., Heyndrickx, M., Balcaen, A., & De Vos, P. (2002). Polyphasic identification of Bacillus and Brevibacillus strains from clinical, dairy and industrial specimens and proposal of Brevibacillus invocatus sp. nov.. International journal of systematic and evolutionary microbiology, 52(3), 953-966.

Mahdhi, A., Kamoun, F., Messina, C., & Bakhrouf, A. (2012). Probiotic properties of Brevibacillus brevis and its influence on sea bass (Dicentrarchus labrax) larval rearing. African Journal of Microbiology Research, 6(35), 6487-6495.

Makovitzki, A., Avrahami, D., & Shai, Y. (2006). Ultrashort antibacterial and antifungal lipopeptides. Proceedings of the National Academy of Sciences, 103(43), 15997-16002.

Martinez, F. A. C., Balciunas, E. M., Converti, A., Cotter, P. D., & de Souza Oliveira, R. P. (2013). Bacteriocin production by Bifidobacterium spp. A review. Biotechnology advances, 31(4), 482-488.

Mogi, T., & Kita, K. (2009). Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics. Cellular and molecular life sciences, 66(23), 3821- 3826.

Munyuki, G., Jackson, G. E., Venter, G. A., Kö vér, K. E., Szilágyi, L., Rautenbach, M., Barbara, M. S., Bhaswati B., & van der Spoel, D. (2013). β-Sheet structures and dimer models of the two major tyrocidines, antimicrobial peptides from Bacillus aneurinolyticus. Biochemistry, 52(44), 7798-7806.

O'donnell, B. J. (1995). U.S. Patent No. 5,455,028. Washington, DC: U.S. Patent and Trademark Office. 22

Prasanna, L., Eijsink, V. G., Meadow, R., & Gåseidnes, S. (2013). A novel strain of Brevibacillus laterosporus produces chitinases that contribute to its biocontrol potential. Applied microbiology and biotechnology, 97(4), 1601-1611.

Prenner, E. J., Lewis, R. N., & McElhaney, R. N. (1999). The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes. Biochimica et Biophysica Acta (BBA)-Biomembranes, 1462(1), 201-221.

Saikia, R., Gogoi, D. K., Mazumder, S., Yadav, A., Sarma, R. K., Bora, T. C., & Gogoi, B. K. (2011). Brevibacillus laterosporus strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam, India. Microbiological research, 166(3), 216-225.

Sanders, M. E., Morelli, L., & Tompkins, T. A. (2003). Sporeformers as human probiotics: Bacillus, Sporolactobacillus, and Brevibacillus. Comprehensive reviews in food science and food safety, 2(3), 101-110.

Scheldeman, P., Pil, A., Herman, L., De Vos, P., & Heyndrickx, M. (2005). Incidence and diversity of potentially highly heat-resistant spores isolated at dairy farms. Applied and Environmental Microbiology, 71(3), 1480-1494.

Seydlová, G., & Svobodová, J. (2008). Review of surfactin chemical properties and the potential biomedical applications. Open Medicine, 3(2), 123-133.

Sharma, N., Gupta, A., & Gautam, N. (2014). Characterization of bacteriocin like inhibitory substance produced by a new strain Brevibacillus borstelensis AG1 isolated from 'Marcha'. Brazilian Journal of Microbiology, 45(3), 1007-1015.

Sharma, V., Singh, P. K., Midha, S., Ranjan, M., Korpole, S., & Patil, P. B. (2012). Genome sequence of Brevibacillus laterosporus strain GI-9. Journal of bacteriology, 194(5), 1279-1279.

Shimotohno, K. W., Hiraishi, Y., Miura, Y., & Endo, T. (2000). Molecular cloning and sequencing of the edeine B1 amidinohydrolase gene of Bacillus brevis TT02-8 and its expression in Escherichia coli. The Journal of antibiotics, 53(12), 1363- 1372.

Shoji, J., Sakazaki, R., Wakisaka, Y., Koizumi, K., & Mayama, M. (1976). Isolation of brevistin, a new peptide antibiotic. Studies on antibiotics from the genus Bacillus. IX. The Journal of antibiotics, 29(4), 375-379.

Singh, P. K., Sharma, V., Patil, P. B., & Korpole, S. (2012). Identification, purification and characterization of laterosporulin, a novel bacteriocin produced by Brevibacillus sp. strain GI-9. PloS one, 7(3), e31498.

Singh, P. K., Solanki, V., Sharma, S., Thakur, K. G., Krishnan, B., & Korpole, S. (2015). The intramolecular disulfide‐stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin‐like structural module. FEBS Journal, 282(2), 203-214.

Somsap, O. A., Bangrak, P., Bhoopong, P., & Lertcanawanichakul, M. (2015). 23

Antibacterial activity and purification of bacteriocin produced by Brevibacillus laterosporus SA14. Walailak Journal of Science and Technology (WJST), 13(1), 55-65.

Song, Z., Guo, H., Fu, W., Zhang, G., Jiang, H., Liu, P., Zhou, B., Bao, Z., & Liu, X. (2013). Medium optimization for production of antifungal active substances from antagonistic Brevibacillus laterosporus and its antifungal activity analysis. African Journal of Microbiology Research, 7(19), 1933-1939.

Song, Z., Liu, Q., Guo, H., Ju, R., Zhao, Y., Li, J., & Liu, X. (2012). Tostadin, a novel antibacterial peptide from an antagonistic microorganism Brevibacillus brevis XDH. Bioresource technology, 111, 504-506.

Spathelf, B. M., & Rautenbach, M. (2009). Anti-listerial activity and structure–activity relationships of the six major tyrocidines, cyclic decapeptides from Bacillus aneurinolyticus. Bioorganic & medicinal chemistry, 17(15), 5541-5548.

Takagi, H., Shida, O., Kadowaki, K., Komagata, K., & Udaka, S. (1993). Characterization of Bacillus brevis with descriptions of Bacillus migulanus sp. nov., Bacillus choshinensis sp. nov., Bacillus parabrevis sp. nov., and Bacillus galactophilus sp. nov. International Journal of Systematic and Evolutionary Microbiology, 43(2), 221-231.

Takeuchi, T., Iinuma, H., Kunimoto, S., Masuda, T., Ishizuka, M., Takeuchi, M., Masa, H., Hiroshi, N., Shinichi, K. & Umezawa, H. (1981). A new antitumor antibiotic, spergualin: isolation and antitumor activity. The Journal of antibiotics, 34(12), 1619-1621.

Trauger, J. W., Kohli, R. M., Mootz, H. D., Marahiel, M. A., & Walsh, C. T. (2000). Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature, 407(6801), 215-218.

Umezawa, H., Kondo, S., Iinuma, H., Kunimoto, S., Ikeda, Y., Iwasawa, H., Daishiro, I., & Takeuchi, T. (1981). Structure of an antitumor antibiotic, spergualin. The Journal of antibiotics, 34(12), 1622-1624.

Van Epps, H. L. (2006). René Dubos: unearthing antibiotics. Journal of Experimental Medicine, 203(2), 259.

Vosloo, J. A., Stander, M. A., Leussa, A. N.-N., Spathelf, B. M., & Rautenbach, M. (2013) Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus Microbiology DOI: 10.1099/mic.0.068734-0

Waki, M., & Izumiya, N. (1990). Recent advances in the biotechnology of β-lactams and microbial bioactive peptides. Biochemistry of Peptide Antibiotics, 205-240.

Wang, F., Qin, L., Pace, C. J., Wong, P., Malonis, R., & Gao, J. (2012). Solubilized gramicidin A as potential systemic antibiotics. ChemBioChem, 13(1), 51-55.

Wang, J., Haddad, N. I., Yang, S. Z., & Mu, B. Z. (2010). Structural characterization of lipopeptides from Brevibacillus brevis HOB1. Applied biochemistry and biotechnology, 160(3), 812-821. 24

Wang, J., Haddad, N. I., Yang, S. Z., & Mu, B. Z. (2010). Structural characterization of lipopeptides from Brevibacillus brevis HOB1. Applied biochemistry and biotechnology, 160(3), 812-821.

Wigger-Alberti, W., Stauss-Grabo, M., Grigo, K., Atiye, S., Williams, R., & Korting, H. C. (2012). Efficacy of a tyrothricin-containing wound gel in an abrasive wound model for superficial wounds. Skin pharmacology and physiology, 26(1), 52-56.

Wojciechowska, H., Ciarkowski, J., Chmara, H., & Borowski, E. (1972). The antibiotic edeine IX: the isolation and the composition of edeine D. Experientia, 28(12), 1423-1424.

Wu, X., Ballard, J., & Jiang, Y. W. (2005). Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus. Applied and environmental microbiology, 71(12), 8519-8530.

Xu, Y., Sugár, I. P., & Krishna, N. R. (1995). A variable target intensity-restrained global optimization (VARTIGO) procedure for determining three-dimensional structures of polypeptides from NOESY data: Application to gramicidin S. Journal of biomolecular NMR, 5(1), 37-48.

Yuan, D. D., Liu, G. C., Ren, D. Y., Zhang, D., Zhao, L., Kan, C. P., & Zhang, L. B. (2012). A survey on occurrence of thermophilic bacilli in commercial milk powders in China. Food Control, 25(2), 752-757.

Zhao, J., Guo, L., Zeng, H., Yang, X., Yuan, J., Shi, H., Xiong, Y., Chen, M., Han, L., & Qiu, D. (2012). Purification and characterization of a novel antimicrobial peptide from Brevibacillus laterosporus strain A60. Peptides, 33(2), 206-211.

Chapter 2* Isolation and Structural Elucidation of Brevibacillin, an Antimicrobial Lipopeptide from Brevibacillus laterosporus That Combats Drug- Resistant Gram-positive Bacteria

*Reprint from Applied and Environmental Microbiology

Abstract

A new environmental bacterial strain exhibited strong antimicrobial characteristics against methicillin-resistant Staphylococcus aureus, vancomycin-resistant strains of

Enterococcus faecalis and Lactobacillus plantarum, and other Gram-positive bacteria.

The producer strain, designated OSY-I1, was determined to be Brevibacillus laterosporus via morphological, biochemical and genetic analyses. The antimicrobial agent was extracted from cells of OSY-I1 with isopropanol, purified by high- performance liquid chromatography, and structurally analyzed using mass spectrometry

(MS) and nuclear magnetic resonance (NMR). The MS and NMR results, taken together, uncovered a linear lipopeptide consisting of 13 amino acids and an N-terminal C6-fatty acid chain (FA) chain, 2-hydroxy-3-methylpentanoic acid. The lipopeptide (FA- Dhb -

Leu - Orn - Ile - Ile - Val - Lys - Val - Val - Lys - Tyr - Leu – valinol, where Dhb is ,- didehydrobutyric acid and valinol is 2-amino-3-methyl-1-butanol) has a molecular mass of 1583.0794 Da and contains three modified amino acid residues; ,- didehydrobutyric acid, ornithine, and valinol. The compound, designated brevibacillin, was determined to be a member of a cationic lipopeptide antibiotic family. In addition to its potency against drug-resistant bacteria, brevibacillin also exhibited low MICs (1-

8 µg/ml) against selected foodborne pathogenic and spoilage bacteria, such as Listeria monocytogenes, Bacillus cereus and Alicyclobacillus acidoterrestris. Purified

26 brevibacillin showed no sign of degradation when it was held at 80°C for 60 min, and it retained at least 50% of its antimicrobial activity when it was held for 22 h under acidic or alkaline conditions. On the basis of these findings, brevibacillin is a potent antimicrobial lipopeptide which is potentially useful to combat drug-resistant bacterial pathogens and foodborne pathogenic and spoilage bacteria.

2.1 Introduction

Unregulated access to antibiotics is one of the main reasons for the spread of antibiotic-resistant pathogens and their resistance genes through migration, travel and trade (Grundmann et al., 2001). It was reported that in Europe alone, 25,000 patients die annually because of bacterial infections which cannot be treated with common antibiotics (Cars et al., 2001). Examples of antibiotic-resistant bacteria are methicillin- resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus spp., carbapenem-resistant Mycobacterium tuberculosis and highly virulent multidrug resistant Clostridium difficile strains (Cars et al., 2001; Vergidis and Falagas, 2008;

Wright, 2005; Kumarasamy et al., 2010). Therefore, the discovery and development of new antimicrobial agents are of paramount importance. Despite their natural scarcity, new antimicrobial agents can be discovered by subjecting microorganisms that potentially produce such agents to screening and isolating processes (Guo et al., 2012;

He et al., 2007).

The current study led to the discovery of a new strain of Brevibacillus sp. with promising antimicrobial activity. The genus Brevibacillus was established in 1996 on the basis of a genetic reclassification of strains previously recognized to be Bacillus brevis (Panda et al., 2014). Many bioactive compounds have been isolated from

Brevibacillus spp. since then, including new antibacterial, antifungal and anti- invertebrate agents (Panda et al., 2014; Ruiu, 2013). For instance, extracellular neutral

27 protease that controls nematode growth was obtained from Brevibacillus sp. (De

Oliveira et al., 2004); a Brevibacillus laterosporus strain was reported to inhibit the growth of a number of fungi, including Fusarium, Aspergillus and Alternaria (Saikia et al., 2011); and a B. laterosporus strain, A60, was found to be active against Gram- negative bacteria of the genus Pseudomonas and the Gram-positive bacterium Bacillius subtilis (Zhao et al., 2012). Recently, a lipopeptide antibiotic, designated tauramamide, was discovered in a culture of B. laterosporus strain (Desjardine et al., 2007). A lipopeptide is generally composed of a specific lipophilic moiety attached to a peptide chain. This category includes antimicrobials that are potentially useful as antibacterial, antifungal, and antiviral agents (Evans et al., 1999; Denning, 2003; Steenbergen et al.,

2005; Huang et al., 2006). A series of lipopeptides synthesized by Makovitzki et al.

(2006) was found to interact with target pathogens, leading to cell membrane permeation and disintegration. The current study was initiated to unveil new natural antimicrobial agents effective against human pathogens, preferably drug-resistant strains, along with some foodborne pathogenic and spoilage microorganisms.

2.2 Materials and Methods

2.2.1 Strain screening. Soil and food samples were collected and screened for antimicrobial-producing bacteria. Most soil samples were collected from the vicinity of

The Ohio State University campus, Columbus, OH. Fermented foods were purchased from local grocery stores; these included vegetables (kimchi, pickles and sauerkraut), meat (salami and sausage), dairy products (yogurt, kefir, and imported cheeses) and soybean products. Soil or food subsamples (10 g each) were homogenized in 0.1% peptone water using a stomacher. Tenfold dilutions were made from the homogenate, and a 100-μl aliquot from each dilution was spread-plated onto Trypticase soy agar

(TSA; BD Diagnostic Systems, Sparks, MD). The plates were incubated at 37°C for 48

28 h. Hundreds of colonies were screened for their abilities to produce antimicrobial agents following the method described by Guo et al. (2012) with slight modification. Briefly, portions of colonies on the TSA plates were transferred by the use of sterile toothpicks onto new TSA plates and incubated at 37 °C for 48 h. The incubated plates were then overlaid with soft agar medium (TSA with 0.75% agar) that had been pre-inoculated with Listeria innocua ATCC 33090 or Escherichia coli K-12. After further incubation at 37°C overnight, the overlaid plates were inspected for any zones of inhibition of the indicator strain. Among a few isolates showing antimicrobial activity, one isolate

(designated OSY-I1) produced a clear zone of inhibition against L. innocua with a diameter greater than 3.0 cm. Considering its strong activity against the indicator organism, this isolate was subjected to further analysis.

2.2.2 Cultures and media. The new isolate, OSY-I1, was propagated on TSA, and a subculture was stocked in the Yousef laboratory’s culture collection. For stock preparation, an overnight culture of OSY-I1 in Trypticase soy broth (TSB) was mixed with 80% sterile glycerol in a 1:1 ratio and stored at -80°C. Selected bacterial strains were tested for sensitivity to the newly-discovered antimicrobial agent (Table 2). These bacterial strains were obtained from Yousef laboratory stock cultures at The Ohio State

University, unless stated otherwise.

2.2.3 Strain identification. The morphology of OSY-I1 was examined by Gram staining of cells and malachite green staining of spores. The biochemical characteristics of the isolate were examined using a commercial biochemical testing kit (API 50 CH test strips; bioMérieux, Inc., Durham, NC) following the kit manufacturer’s instructions.

Briefly, overnight colonies of OSY-I1 were transferred into the medium provided with the kit (API 50 CHB/E) to prepare a cell suspension with a turbidity equivalent to an optical density at 600 nm of 0.242 (Mcfarland Standard No. 2). The prepared

29 suspension was inoculated into the wells of the biochemical strips; this was followed by incubation at 30 or 37°C, and readings were taken after 24 and 48 hours.

Biochemical reactions were recorded, and the isolate’s identity was determined by comparing the results to the data in the database provided by the kit manufacturer.

For genetic identification, the isolate (OSY-I1) was analyzed using the 16S rRNA gene sequencing technique (Drancourt et al., 2000). The genomic DNA of OSY-I1, was extracted using a commercial kit (DNeasy blood and tissue kit; Qiagen, Valencia, CA).

Universal primers (16S Forward Primer: 5’-

CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3’ and 16S Reverse

Primer: 5’-CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3’) were used to amplify the 16S rRNA gene by the use of Taq DNA polymerase (Taq PCR core kit;

Qiagen, Valencia, CA). The PCR was conducted in a thermocycler under the following conditions: an initial incubation at 94 °C for 3 minutes and 30 cycles of denaturition at

94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 2 min. The final extension step was conducted at 72°C for 10 min. The amplicon of the 16S rRNA genes was purified using a commercial DNA extraction kit (QIAquick gel extraction kit;

Qiagen, Valencia, CA). The purified PCR product was then sequenced from both 5’ and

3’ ends with an automated DNA analyzer (Applied Biosystems, Foster City, CA). The resultant 16S rRNA sequence (1450 bp) was compared to known sequences in a national database (Ribosomal Database Project, release 11; http://rdp.cme.msu.edu) using the Seqmatch algorithm.

2.2.4 Isolation and purification of the antimicrobial agent from OSY-I1. An overnight liquid culture of OSY-I1 in TSB was aliquoted (aliquots of 100 µl each) and spread plated onto TSA plates. After incubation at 37°C for 72 h, bacterial cells were scraped into centrifuge tubes by use of a microscopic slide and mixed with isopropanol

30 in a 1:4 (wt/vol) ratio. The contents of the centrifuge tubes were then agitated at 200 rpm for 4 h, followed by centrifugation at 7,710 × g for 15 min. The supernatant was held in a chemical hood at 25°C for 48 hours to allow solvent evaporation. The dry residues were then suspended in water to prepare the crude extract, and its antimicrobial activity against L. innocua ATCC 33090 was examined. The crude extract was purified with a high-performance liquid chromatography (HPLC) system (Hewlett Packard

1050; Agilent Technologies, Palo Alto, CA) equipped with an analytical reverse-phase column (particle size, 5 μm; 250 by 4.6 mm; Biobasic C18; Thermo Electron Corp.,

Bellefonte, PA). The purification process was achieved by elution with a linear gradient consisting of two mobile phases: HPLC-grade water with 0.1% trifluoroacetic acid

(TFA) and HPLC-grade acetonitrile with 0.1% TFA. In each run, 40 μl crude extract was injected and separated by use of a linear gradient from 0 to 66.6% acetonitrile for

20 min at a flow rate of 1 ml/min, and the effluent was monitored by a UV-visible detector set at 220 nm. Fractions were collected at a rate of one fraction per minute, and fractions from multiple runs were combined and placed into glass beakers, followed by air drying in a chemical hood. Each dry fraction was dissolved in 50% acetonitrile, and the antimicrobial activity was assayed by spot-on-lawn method (He et al., 2007).

Purified antimicrobial agent (obtained from fractions with antimicrobial activity) from multiple runs was pooled and reinjected into the HPLC, using the same conditions described above, to check the purity. The fraction with the strongest antimicrobial activity and the highest purity was then subjected to analysis by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.

2.2.5 MALDI-TOF MS analysis. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS analysis was conducted on a Bruker UltrafleXtreme MALDI-

TOF/TOF MS (Bruker Daltonics Inc., Billerica, MA) operated in reflection positive-

31 ion mode and accelerated at a voltage of 28 kV. The HPLC-purified sample was mixed with a matrix in a ratio of 1:5 (vol/vol). The matrix used was α-cyano-4- hydroxycinnamic acid (Bruker Daltonics Inc.), which was dissolved in 50% acetonitrile with 0.1% TFA in water. A nitrogen laser was set at a threshold level to minimize fragmentation but that was adequate to generate signals.

2.2.6 LC-MS/MS. The sequence of the target antimicrobial agent was determined using capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS). A Thermo

Fisher LTQ orbitrap XL mass spectrometer equipped with a microspray source

(Michrom Bioresources Inc, Auburn, CA) was operated in positive ion mode. A capillary column (Magic C18AQ [Bruker Daltonics]; 0.2 by 150 mm; particle size, 3

µm; 200Å,) was used for sample separation on an HPLC (UltiMate™ 3000; Thermo

Scientific). The sample was dissolved in acetonitrile-H2O (50:50, vol:vol) and was loaded to the column bypassing the desalting trap. Mobile phase A was 50 mM acetic acid in water and mobile phase B was acetonitrile. The flow rate was set at 2 µl/min.

Typically, mobile phase B was increased from 2% to 50% in 30 min, and from 50% to

90% in 5 min, then kept at 90% for another 2 min before being quickly brought back to

2% in 1 min. The column was equilibrated at 2% mobile phase B (and 98% mobile phase A) for 20 min before the injection of a subsequent sample. A spray voltage of 2.2

KV and a capillary temperature of 175°C were used to acquire the MS/MS data. The scan sequence of the mass spectrometer was based on the preview mode data-dependent

TopTen method, as follows. For the analysis, the mass spectrometer was programmed for a full scan, recorded between m/z 350 – 2000, and an MS/MS scan to generate product ion spectra to determine amino acid sequence from consecutive scans of the 10 most abundant peaks in the spectrum. To achieve high mass accuracy by MS, the full scan was performed at Fourier transform (FT) mode, and the resolution was set at

60,000. The automatic gain control (AGC) target ion number for the full scan in the FT mode was set at 1×106 ions, the maximum ion injection time was set at 1,000 ms, and microscan number was set at 1. MS2 was performed using the ion trap mode to ensure the highest signal intensity of the MSn spectra. The AGC target ion number for the ion trap MSn scan was set at 10,000 ions, the maximum ion injection time was set at 50 ms, and microscan number was set at 1. The collision-induced dissociation fragmentation energy was set to 35%.

2.2.7 NMR analysis. For NMR analysis, a standard protocol was followed to determine the constituents and then their sequential arrangement (Wüthrich, 1986) as have been performed previously (Guo et al., 2012; He et al., 2007). The HPLC-purified dry sample

(approximately, 1 mg) was dissolved in 99.9% perdeuterated DMSO-d6 (Cambridge

Isotope Lab, Tewksbury, MA). Experiments were conducted at 298K on Bruker

Avance-III HD-600 and -700 spectrometers (Bruker, Karlsruhe, Germany), both equipped with a 5-mm (1H, 13C, 15N) triple-resonance cryoprobe (TXI and TXO, respectively) and z-axis gradient. The following data sets were recorded: 1D 1H NMR,

1D 13C NMR, 1D 13C DEPT90 and DEPT135, 2D 1H-homonulcear DQF-COSY,

TOCSY (60 ms DIPSI2 mixing time), and NOESY (200 and 400 ms mixing time), 2D heteronuclear 1H-13C HSQC, multiplicity-edited HSQC, HMBC, HSQC-NOESY (350 ms mixing time), HSQC-TOCSY (60 ms DIPSI2), HMQC-COSY (Nyberg et al., 2005), and 2D heteronuclear 1H-15N HSQC, all using standard Bruker pulse sequences and the natural abundance. Data were processed using NMRPipe (Delaglio, 1995), which were typically zero-filled prior to application of window functions followed by Fourier transform. Chemical shifts were referenced directly (1H and 13C) or indirectly (15N) to the internal residual solvent peak. The spectra were visualized by NMRView (Johnson and Blevins, 1994) for the assignments. All of the NMR spectral figures were prepared

33 using TopSpin 3.2 software (Bruker, Karlsruhe, Germany).

2.2.8 Sensitivity to heat and pH. Pure brevibacillin, prepared as described previously, was subjected to heat and acid treatment. For the thermal stability test, brevibacillin was diluted with dimethyl sulfoxide (DMSO) and heated in a water bath at 80°C simulating a pasteurization temperature. Samples of the antimicrobial agent solution were taken at designated intervals during heating, and changes in antimicrobial activity against MRSA as an indicator bacterium were monitored using a 2-fold dilution scheme in a 96-well microtiter plate. For the pH stability test, pure brevibacillin was dissolved in citrate buffer (0.1 M, pH 3.0), phosphate buffer (0.1 M, pH 7.0) and Tris buffer (0.1

M, pH 9.0) to achieve a concentration of 16 µg/ml. The pH range (pH 3 to 9) was chosen to encompass the pH values of most food products. The pH-adjusted buffer containing brevibacillin was then incubated at 25°C for 22 hours. Treated brevibacillin buffers were then neutralized to pH 7.0 and tested for changes in antimicrobial activity using the same method used to test for the effects of heat treatment. Both the thermal and pH treatment experiments were done in triplicate.

2.2.9 In vitro antimicrobial activity determination. The MICs of the HPLC-purified antimicrobial agent prepared from the OSY-I1 isolate for selected Gram-positive and

Gram-negative bacteria (Table 2) were determined by the broth microdilution method following the protocol of the Clinical and Laboratory Standards Institute (CLSI, 2007).

Specifically, purified antimicrobial agent was collected from the HPLC, weighed, and dissolved in DMSO at a concentration of 3,200 μg/ml (stock solution). Working solutions were prepared from the stock solution through 2-fold serial dilution using

DMSO as a diluent. The MIC study was conducted in 96-well microtiter plates. Each well contained 178 μl of medium, 20 μl indicator bacteria (~2.0×104 CFU/well) and 2

μl of diluted antimicrobial agent. Therefore, the total volume was 200 μl/well, and

DMSO concentration was 1%. In addition, vancomycin (Sigma, St. Louis, MO) and nisin (Aplin and Barrett Ltd., Trowbridge, United Kingdom), each of which was dissolved individually in DMSO, were used as positive controls, and 1% DMSO was used as a negative control. In order to test the MICs of all three antimicrobial agents under the same condition, DMSO was used as the solvent to avoid variations in the solubilities of these agents in water. The MIC represented the lowest concentration (in micrograms per milliliter) of a certain antimicrobial agent that led to no visible growth of the indicator bacteria after incubation at 35°C for 20 h (CLSI, 2007). The MIC experiments were done in triplicate.

2.2.10 16S rRNA sequence accession number. The DNA sequence of the 16S rRNA gene of OSY-I1 (1,450 bp) has been deposited in the GenBank database under accession no. KR919625.

2.3 Results

2.3.1 Isolation and identification of an antimicrobial-producing strain. A total of approximately 2,500 isolates from tested samples were screened for their antimicrobial activity against L. innocua ATCC 33090 and E. coli K-12. A soil isolate showed strong activity against L. innocua, with the area of the diameter of inhibition being greater than

3.0 cm. The isolate was given the strain designation OSY-I1. The new isolate is a Gram- positive, spore-forming bacterium. Biochemical test results showed that OSY-I1 is positive for catalase and oxidase; fermentation of glucose, fructose, mannose, and mannitol; and esculin hydrolysis but that it is negative for sorbitol and lactose fermentation. The results of biochemical tests (API strips) indicated 99.9% similarity between OSY-I1 and Brevibacillus laterosporus. Genetic analysis by sequencing of the

16S rRNA gene (1,450 bp) also showed that isolate OSY-I1 shares a high degree of identity (97.7%) with B. laterosporus. Therefore, the new isolate was identified as B.

35 laterosporus on the basis of both biochemical and genetic tests.

2.3.2 Extraction, isolation and purification of the antimicrobial agent produced by

OSY-I1. Analysis of crude extract by HPLC (Figure. 3A) revealed that fraction A (at a retention time of 16.4 min) had activity against L. innocua ATCC 33090. The purity of the active fraction was tested by reinjection onto the HPLC, and a single peak was observed (Figure. 3B), suggesting that the compound representing the fraction was purified to homogeneity. The pure compound was designated brevibacillin.

2.3.3 MIC determination and stability test. Purified brevibacillin was used for MIC determination. Targeted microorganisms include pathogenic and nonpathogenic bacteria. The antimicrobial agent was active only against the Gram-positive bacteria tested, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant

Lactobacillus plantarum and vancomycin-resistant Enterococcus faecalis (Table 2).

This antimicrobial agent had antimicrobial activity more potent than that of nisin for most of the tested microorganisms, and its potency was often comparable to that of vancomycin (Table 2). In the heat stability test, brevibacillin showed no sign of degradation when it was heated at 80°C for up to 60 min (data not shown). For pH stability test, 50% and 62.5% of the antimicrobial activity remained in pH 3.0 and 9.0 buffer, respectively, compared to the amount of antimicrobial activity of brevibacillin that remained when it was incubated at pH 7.0 for 22 h.

Figure 3. HPLC chromatogram at different stages of purification of the antimicrobial agent from Brevibacillus laterosporus OSY-I1. The antimicrobial activity of the different fractions was tested against Listeria innocua ATCC 33090. [A] Chromatogram of the crude extract. [B] Chromatogram of purified antimicrobial agent after reinjection of fraction A of crude extract.

2.3.4 MALDI-TOF MS and LC-MS/MS analyses. The purified active fraction was analyzed using MALDI-TOF MS to determine the molecular mass of the antimicrobial compound. As shown in Figure. 4, three peaks were measured for the fraction at m/z values of 1584.1, 1606.1 and 1622.1, corresponding to the singly protonated antimicrobial agent [M + H]+, its sodium-cationized ion [M + Na]+ and its potassium- cationized ion [M + K]+. The active fraction was also subjected to LC-MS/MS analysis to deduce an accurate molecualr mass and provide a preliminary peptide sequence for the antimicrobial agent. As shown in Figure 5, the b and y ions were generated by

MS/MS analysis, and the preliminary sequence was proposed to be X – Dhb – Leu –

Orn – Ile – Ile – Val – Lys – Val – Val – Lys – Tyr – Leu – W (where Dhb is α,ß- didehydrobutyric acid, X is C6H11O, and W is C5H13NO) from the N terminus to the C

37 terminus. The observed m/z (z = 2) was equal to 792.54772+; the theoretical m/z was equal to 792.54682+, which indicated a mass error of 1.13 ppm; and the observed molecular mass was 1583.0794 Da.

1606.1

1622.1

1584.1

1500 1520 1540 1560 1580 1600 1620 1640 1660 1680 1700 m/z Figure 4. MALDI-TOF MS analysis of the antimicrobial agent isolated from Brevibacillus laterosporus OSY-I1. The ions at m/z (z = 1) 1584.1, 1606.1 and 1622.1 represent the singly protonated [M + H]+, sodium-cationized [M + Na]+, and potassium cationized [M + K]+.

y12 y11 y10 y9 y8 y7 y6 y5 y4 y3 X=C6H11O1 X Dhb L Orn I I V K V V K Y L W W=C5H13N1O1 b2 b3 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13

Intensity X 5

380.25 508.35 607.42 706.49 834.58 933.65 1046.74 1159.82 1273.90 y3 y4 y5 y6 y7 y8 y9 y10 y11

128.10 99.07 99.07 128.09 99.07 113.09 113.08 114.08 Lys Val Val Lys Val Ile Ile Orn

2+ 467.33 580.412+ 637.452+ 693.992+ y8 y10 y11 y12

Intensity X 5

425.28 538.36 651.44 750.51 878.61 977.68 1076.75 1204.84 1367.90 1480.99 311.20 b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b3 114.08 113.08 113.08 99.07 128.10 99.07 99.07 128.09 163.06 113.09 Orn Ile Ile Val Lys Val Val Lys Tyr Leu

741.002+ b13 602.922+ 684.452+ b13 b12

300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z Figure 5. Fragmentation of brevibacillin by tandem MS to generate b and y ion

2.3.5 Peptide sequence determination and terminal structural elucidation by NMR.

In this study, DMSO-d6 was used to dissolve the compound as it offers higher solubility than water or CDCl3, two other commonly used NMR solvents. This choice also facilitates the observation of labile protons and their associated cross-peaks in 2D NMR.

The spectral assignments were performed by simultaneous analysis of multiple data sets through cross-checking in an effort to rule out alternative explanations. The representative and critical observations are summarized in Figure 6, 7 and 8, particularly those regarding the elucidation of N- and C-terminal structures.

Figure 6. NMR spectra signifying the existence of a polypeptide and the acylated N-terminal cap. [A] 1D 1H NMR showing the spectral region from 5 to 10 ppm. The peaks of “a1” to N  N +   “a7” are attributed to Dhb-1 H , Tyr-11 H , Leu-2 H , NH3 (Orn-3 H /Lys-7 H /Lys- 10 H), Valinol-13 HN, Dhb-1 H, and FA OH. [B] Partial 2D 1H DQF-COSY 3   spectrum showing the JHH correlations with the following peaks labeled: Orn H -H or Lys H-H (“b4”), Valinol-13 HN-H (“b5”), Dhb-1 H-H (“b6”), and FA H-H (“b7”). Also Dhb-1 HN is unique without showing any COSY peak. [C] 2D 1H-13C HMBC showing the 1H-13C multiple-bond correlations including Dhb-1 C´ to Dhb-1 HN (“c1”), Leu-2 HN (“c3”), and Dhb-1 H (“c6”), and FA C´ to Dhb-1 HN (“c1´”). The left projection is the 1D 13C NMR spectrum of the carbonyl region.

A quick 1D 1H NMR revealed resonances between 6.5 and 9.5 ppm (Figure 6A), 40 the typical amide and aromatic spectral region. Subsequently, 2D 1H-15N HSQC confirmed a total of 13 amide cross-peaks (Figure 7A), signifying the existence of a peptidyl fragment. Since an equal number of carbonyl resonances were observed in 1D

13C NMR (Figure 6C), it was plausible to assume that the polypeptide likely consists of

13 amino acids. The majority of the amides, i.e. peak “2” through “12” in Figure 7A, have chemical shifts between 7.77 and 8.22 ppm in 1H dimension. The lack of dispersion, together with the chemical shifts close to random coil values, is indicative of a flexible conformation, likely linear and unstructured in nature. Signal overlapping also occurs in the side chains, presumably attributed to the same cause, rendering the unequivocal and complete assignment somewhat challenging for this molecular size.

Nevertheless, the problem was alleviated by collecting high resolution data sets as well as by exploiting heteronuclear NMR techniques as further elaborated.

The above amide protons were subsequently correlated with the scalar-coupled aliphatic side chains via 2D COSY and TOCSY experiments. For example, the HN of residue “3” displays through-bond correlations in 2D 1H-13C HSQC-TOCSY (Figure

7B) to four protonated carbons, including one CH and three CH2 as determined in 2D multiplicity-edited 1H-13C HSQC. Their chemical shifts and the scalar connectivity revealed in COSY further suggest an aliphatic side chain resembling the one of an arginine residue. However, two lines of NMR evidence led to the assignment of an ornithine, in agreement with the MS observation. Firstly, no H cross-peak was detected in the above 2D 1H-15N HSQC. Secondly, the broad proton resonance at 7.67

 + ppm (“a4” in Figure 6A), initially assigned to Lysine N H3 groups (“b4” in Figure 6B), appears to contain close to 9 protons from peak integration, indicating three lysine-like residues in this compound.

Figure 7. NMR spectra exemplifying the residue and sequential assignment of the lipopeptide. [A] 2D 1H-15N HSQC spectrum and the insert showing the 13 amide cross-peaks. The numbering from 1 to 13 is the actual final sequence number. [B] 2D 1H-13C HSQC- TOCSY spectrum illustrating the identification of side chain spin network associated with amide protons. Peaks marked by pound (“#”) and asterisk (“*”) are scalar- correlated to Orn-3 HN and Valinol-13 HN, respectively. Their multiplicities were 1 13 determined in 2D multiplicity-edited H- C HSQC as CH2, CH2, CH2, and CH for Orn- 3, and CH3, CH3, CH, CH, and CH2 for Valinol-13, both from up to bottom. [C] Partial 2D 1H-homonuclear NOESY and the insert showing the example NOE assignments of Dhb-1 HN to FA H (“c1”), Lys-7 HN to Val-6 H (“c7”), and Valinol-13 HN to Leu12 H (“c13”). Ambiguities arising from signal overlapping such as the region close to “c7” were resolved by 2D 1H-13C HSQC-NOESY.

Ten other residues in the center of 2D 1H-15N HSQC were readily determined in a

42 similar fashion, including 1 Tyr, 2 Ile, 2 Lys, 2 Leu, and 3 Val. The two well-dispersed peaks “1” and “13” (Figure 7A) however appear unusual and warrant in-depth investigation. Nevertheless, the peptide sequence was subsequently deduced by analyzing 2D 1H NOESY and 2D 1H-13C HSQC-NOESY spectra. This is exemplified by the assignment of H(i)-HN(i+1) sequential NOE “c7” between residues “6” and “7”, and “c13” between “12” and “13” (Figure 7C). It was concluded that the sequence determined at this stage is in full agreement with the above MS results and the undetermined residues “1” and “13” are positioned at the N- and C-terminus, respectively.

The HN of the residue “13” shows scalar correlations to five protonated carbons

(Figure 7B), four of which (two -CH and two -CH3) form a spin network reminiscent

3 of a valine side chain on the basis of JHH correlations and the chemical shifts. The remaining methylene moiety, on the other hand, demonstrates 13C/1H chemical shifts

61.04/3.340 ppm (“b7” in Figure 8B) characteristic of a hydroxymethyl group. Its degenerate proton resonance (“a7” in Figure 8A), however, is masked by the intense

3 and broad residual water signal, hampering observation of the JHH COSY peaks to the vicinal protons that would otherwise provide direct evidence to unravel the covalent bonding. This problem was overcome by 2D 1H-13C HMQC-COSY or the so-called

H2BC (Nyberg et al., 2005) that employs heteronuclear technique to correlate 1H and

13 3 1 C spins separated by two covalent bonds via JHH and JCH while significantly

2 suppressing unwanted signals. Figure 8D shows the reciprocal JCH correlations “d6” and “d7” between this CH2 moiety and one of the aforementioned methanetriyl groups.

The latter with 13C/1H of 55.24/3.530 ppm (“b6” in Figure 8B) is analogues to CH in

3 N a valine residue that is JHH correlated to the H proton (“b5” in Figure 6B). Moreover, a hydroxyl proton at 4.503 ppm (“a3” in Figure 8A), barely resolved from Tyr-11 H

(“a4” in Figure 8A and “b4” in Figure 8B), was uncovered in the same H2BC to engage

2 in a JCH correlation with the above methylene carbon (“d3” in Figure 8D). All of these observations taken together unequivocally identified a valinol residue (2-amino-3- methyl-1-butanol) at C-terminus, which can be viewed as a modified valine residue with its carboxylic group replaced by a hydroxymethyl group.

Figure 8. NMR data showing the critical evidence for the assignment of FA, Dhb- 1, and valinol-13. [A] 1D 1H NMR showing the spectral region 3.00 – 6.00 ppm with the following peaks marked from “a1” to “a7”: Dhb-1 H, FA OH, Valinol-13 OH, Tyr-11 H, FA H, Valinol-13 H, and Valinol-13 H. [B] 2D multiplicity-edited 1H-13C HSQC showing the 1H-13C one-bond correlations with the following peaks labeled: Tyr-11 CH (“b4”),    FA CH (“b5”), Valinol-13 CH (“b6”), and Valinol-13 CH2 (“b7”). In this experiment, the cross-peaks of CH2 (red) and those of CH and CH3 (black) are of opposite signs. 13 The CH and CH3 can be further distinguished by 1D C DEPT experiments. The noise marked inside a rectangular box arose from the strong residual H2O resonance. [C] Partial 2D 1H-13C HSQC-TOCSY highlighting the spin networks associated with Dhb- 1 H (indicated by the box “c1”) and FA H (“c5”). The former only consists of 1 CH (aliased in this spectrum) and 1 CH3, while the latter has five protonated carbons including FA CHbeyond the figure range. [D] 2D HMQC-COSY or H2BC showing 2  13    the exclusive JCH correlations: FA H to C (“d2”), Valinol-13 H to C (“d3”), Valinol-13 H to C (“d6”), and Valinol-13 H to C (“d7”).

Unlike natural amino acids, the amide proton of unknown peak “1” does not show a typical COSY peak to a Hα proton (Figure 8B). An extremely weak peak however was visible in 2D 1H homonuclear TOCSY (data not shown) between this amide proton and the protons of a methyl group at 13C/1H of 12.65/1.742 ppm. The latter in turn exhibits

3 13 1 JHH correlation (“b6” in Figure 8B) to a CH moiety with C/ H of 117.35/5.812 ppm.

It was found that the spin network only consists of these CH and CH3 (box “c1” in

Figure 8C), and their distinctive 1H and 13C chemical shifts further imply the existence of an alkene group with one methyl substituent. Since the above methanetriyl proton and the amide protons of peak “1” and Leu-2 all have an HMBC peak to the same carbonyl resonance at 164.43 ppm (“c6”, “c1”, and “c3”, respectively, in Figure 6C), the N-terminal residue was unequivocally determined to be a Dhb (He et al., 2007).

Evidently the peptide is N-terminal acylated as Dhb-1 HN has HMBC peak to another carbonyl resonance at 172.55 ppm (“c1'” in Figure 6C). It also shows several

NOEs that could only be rationalized by the through-space interactions to the N- terminal capping, most notably to a CH group (“c1” in Figure 7C), whose distinctive

13C/1H chemical shifts of 74.61/3.815ppm (“b5” in Figure 8B) suggests hydroxylation on this site. This group can be scalar-correlated with four other protonated carbons (box

3 2 “c5” in Figure 8C), including 1 CH, 1 CH2, and 2 CH3. On the basis of JHH and JCH correlations extracted from the 2D DQF-COSY and H2BC, respectively, the aliphatic chain comprising these five protonated carbons was delineated being identical to that of an isoleucine residue. Since a hydroxyl proton at 5.613 ppm (“a2” in Figure 8A)

3 α 2 shows JHH (“b7” in Figure 6B) to the H and JCH correlation (“d2” in Figure 8D) to the Cα, this N-terminal fatty acyl chain was unambiguously identified as 2-hydroxy-3- methyl-pentanoyl group.

In conclusion, the structure of brevibacillin was elucidated by MS and NMR to be

46 a linear lipopeptide (Figure 9) comprising 13 amino acids (identified in order by the numbers after the amino acids in parentheses below) that is acylated to a C6 fatty acid

(FA) tail, 2-hydroxy-3-methylpentanoic acid: FA- (Dhb-1) – (Leu-2) – (Orn-3) – (Ile-

4) – (Ile-5) – (Val-6) – (Lys-7) – (Val-8) – (Val-9) – (Lys-10) – (Tyr-11) – (Leu-12) –

(Valinol-13). The sequence includes three modified amino acid residues, two of which are at the termini. The chemical shift assignments are summarized in Table 3. It should be noted that the stereospecific assignments are tentative with regard to the methyl groups of Val, Leu, and valinol.

2.4 Discussion

A new bacterial strain, B. laterosporus OSY-I1, was found to produce a potent lipopeptide with activity against Gram-positive bacteria. This species has no history of posing health hazards to animals or humans. In fact, B. laterosporus (BOD strain) has been used as a probiotic (Sanders et al., 2003) and this strain has been patented

(O'Donnell BJ, 1995). The product which contains the BOD probiotic strain has been commercially available since 1989 (Flora Balance). According to Chawawisit &

Lertcanawanichakul (2008), bioactive agents from probiotic bacteria may serve as promising candidates for the treatment of infections caused by antibiotic-resistant pathogens.

The newly discovered antimicrobial agent, brevibacillin, has a relatively novel structure, which is shown in Figure 9. Brevibacillin is a cationic lipopeptide with three positively charged residues, which results in a net positive charge at neutral pH

(Chawawisit and Lertcanawanichakul, 2008). After careful consideration, we concluded this new compound is a member of a linear cationic antimicrobial lipopeptide family for which scattered information is available in the published literature. In 2001, Barsby et al. reported the isolation of the first known compound in

47 this family from a marine Bacillus sp. The producer organism was later reclassified as

B. laterosporus by the same researchers (Barsby et al., 2006). The lipopeptide was named bogorol A, and is different from our brevibacillin in two amino acid resides at positions 5 and 9; isoleucine and valine in brevibacillin are replaced by valine and leucine in bogorol A, respectively. In 2005, Wu et al., reported the isolation of an antimicrobial peptide, named BT peptide, from Brevibacillus texasporus. The peptide is structurally similar to bogorol A but differs in the residue at the N terminus (Figure

9). The authors did not report this similarity, and their ambiguity about the N terminus warranted description of the compound as a lipopeptide. Seven years after the publication of BT peptide structure, Zhao et al. (2012) reported the discovery of a new antimicrobial peptide, BL-A60. Although neither C terminus nor the N terminus was fully elucidated by the authors, the sequence of eight of the peptide’s amino acid residues are similar to that of their counterparts in bogorol, except that the sequence order was reversed (Figure 9). The BL-A60 pseudopeptide shares the highest similarity with bogorol C (Barsby et al., 2006), another member of this family of antibiotics with a methionine residue at position 2, replacing the leucine residue in bogorol A (data not shown).

Brevibacillin carries three cationic amino acids: an ornithine and two lysines.

These positively charged residues contribute to the hydrophilicity of the antimicrobial agent, whereas the aliphatic amino acids (Leu-3, Ile-4, Ile-5, Val-7, Val-8, Leu-9, Leu-

12, Val-13) and an aromatic amino acid (Tyr-11) contribute to its hydrophobicity. In addition to its net positive charge and amphipathic nature, the lipophilic chain enhances the antimicrobial activity by increasing the hydrophobicity of compound’s N-terminal.

Based on the mechanism of action of other lipopeptides (Huang and Yousef, 2014), it is presumed that brevibacillin may accumulate at the anionic surface of bacterial cell

48 membrane. The integrity of the indicator cell membrane could then be disrupted by this antimicrobial agent based on its amphipathic nature. This amphipathic trait is often considered a prerequisite for the lytic activity of cationic antimicrobial peptides against targeted microorganisms (Shai and Oren, 2001; Tossi and Sandri, 2002). Based on these observations, it is presumed that brevibacillin damages cell membrane, probably by creating holes, which depolarize cell membrane potential and lead to the leakage of intracellular contents. Further research is underway to confirm these hypotheses.

Brevibacillin showed strong inhibitory activity against MRSA, and the vancomycin-resistant strains of E. faecalis and Lactobacillus sp. This is the first time

L. plantarum ATCC 8014 has been reported as a vancomycin-resistant species.

Brevibacillin also showed potent antimicrobial activity against two Clostridium difficile strains, indicating its potency to serve as a good antibiotic against anaerobic microorganisms. Brevibacillin also showed high inhibitory effect against foodborne spoilage and pathogenic microorganisms and with MICs as low as that of nisin for many chosen strains. The antimicrobial agent was very effective against Alicyclobacillus spp., indicating the possibility of future use as a food additive (Grande et al., 2005).

Brevibacillin 1 5 9

β1 β γ 1 1 β2 α α γ 1 α δ1 β2 γ β 2 γ 1 γ 2 γ 3

Bogoral A 5 9

BT peptide 1

BL-A60

Figure 9. Chemical structure of brevibacillin and related cationic peptide antibiotics, bogorol A, BT peptide and BL-A60 (BL-A60 is presented in reversed sequence order). The structural differences are highlighted in boxes.

Table 2. Minimum inhibitory concentration (μg/ml) of brevibacillin (from Brevibacillus laterosporus OSY-I1), vancomycin, and nisin against selected bacteria including antibiotic-resistant strains. Bacterial strainsa Brevibacillin Vancomycin Nisin Gram-positives Alicyclobacillus acidoterrestrisb 1.0 <0.5 <0.5 A. acidoterrestris ATCC 49025b 0.5-1.0 <0.5 <0.5 Bacillus cereus ATCC 11778 2.0-4.0 2.0-4.0 8.0 B. cereus ATCC 14579 1.0 1.0 2.0 Clostridium difficile A515c 4.0-8.0 2.0 4.0-8.0 C. difficile CL148d 4.0-8.0 2.0 4.0-8.0 Enterococcus faecalis ATCC 29212e 2.0 2.0 >16.0 E. faecalis ATCC 51299, vancomycin 4.0-8.0 >16.0 >16.0 resistante Lactobacillus plantarum ATCC 8014f 1.0 >16.0 <0.5 Lactococcus lactis ATCC 11454g 2.0 <0.5 >16.0 Listeria innocua ATCC 33090h 1.0-2.0 1.0 2.0-4.0 L. monocytogenes OSY-8578h 1.0-2.0 1.0 <0.5 L. monocytogenes Scott Ah 1.0 1.0 4.0 Staphylococcus aureus ATCC 6538 1.0-2.0 1.0 1.0 S. aureus, methicillin resistant 1.0 1.0-2.0 2.0-4.0 (MRSA)i Gram-negatives Escherichia coli K-12 >32 >16 >16 E. coli O157:H7 EDL 933 32 >16 >16 Pseudomonas aeruginosa ATCC >32 >16 >16 27853e Salmonella Typhimurium DT 109 >32 >16 >16 aUnless stated, strains were incubated in cation-adjusted Mueller–Hinton II broth (MHB; Becton, Dickinson & Co., Sparks, MD), at 37°C for 20 hours. bA highly-spoilage isolate provided by a food processor; the strain was cultured in yeast starch glucose broth at 37°C for 48 hours. c Provided by W. A. Gebreyes, Department of Veterinary Preventive Medicine, The Ohio State University; the strain was cultured in brain heart infusion, supplemented with 5% yeast extract, and incubated anaerobically at 37°C for 24 hours. dProvided by J.T. Lejeune, College of Veterinary Medicine, The Ohio State University; the strain was cultured under the same conditions used with the A515 strain. eIncubated in trypticase soy broth (TSB), at 37°C for 20 hours fIncubated in de Man, Rogosa and Sharpe (MRS) broth, at 30°C for 20 hours, antibiotic- resistant strain gIncubated in MRS broth, at 30°C for 20 hours hIncubated in TSB at 37°C for 20 hours iMIC for oxacillin is more than 32 μg/ml

Table 3. Chemical shift assignments of lipopeptide in DMSO-d6, 298.0 K

Residue 15N/1HN 13C/1H 13C/1H (ppm) Others 13C/1H, C´ and OH (ppm) (ppm)

     FA 74.61/3.815  5.613; CH CH2 22.94/1.383, 1.119; CH3 15.10/0.875; CH3 37.94/1.700 11.36/0.807; C´ 172.55

 Dhb(1) 128.19/9.278 131.23/~ 117.35/5.812 CH3 12.65/1.742; C´ 164.43

  Leu(2) 122.88/8.219 51.29/4.288 39.84/1.557, 1.546 CH 23.94/1.657; CH3 22.93/0.860, 20.64/0.846

   Orn(3) 115.69/8.013 51.50/4.362 28.57/1.679, 1.650 CH2 23.28/1.551, 1.507; CH2 38.01/2.755; H 7.670

   ILe(4) 115.45/7.774 56.03/4.265 36.74/1.710 CH2 23.78/1.353, 1.009; CH3 15.01/0.768; CH3 10.72/0.752

   ILe(5) 120.69/8.000 4.167/56.70 35.64/1.708 CH2 24.13/1.427, 1.043; CH3 14.94/0.764; CH3 10.58/0.772

 Val(6) 118.58/7.808 4.184/57.64 30.43/1.928 CH3 18.88/0.806, 17.57/0.802

   Lys(7) 119.20/7.938 4.372/51.58 31.73/1.630, 1.482 CH2 21.85/1.267; CH2 26.22/1.491; CH2 38.39/2.693; H 7.670; N 33.3

 Val(8) 115.77/7.883 4.224/57.15 30.23/1.907 CH3 18.89/0.790, 17.89/0.757

 Val(9) 118.72/7.890 4.107/57.45 29.94/1.937 CH3 18.93/0.775, 17.83/0.774

   Lys(10) 120.14/7.788 4.231/51.79 31.39/1.433, 1.321 CH2 21.58/1.040; CH2 26.28/1.419 ; CH2 38.39/2.662; H 7.670; N 33.3

Tyr(11) 118.75/8.095 4.471/54.06 37.17/2.840, 2.603 CHCH C 155.81; H 9.189

  Leu(12) 121.00/8.095 4.226/50.82 40.56/1.400, 1.383 CH 23.65/1.331; CH3 22.87/0.818, 21.21/0.764

  , Valinol(13) 118.27/7.405 3.530/55.24 CH2 61.04/3.340;   4.507; CH3 19.31/0.814, 17.71/0.786 CH 27.90/1.801

References

Cars, O., Hedin, A., & Heddini, A. (2011). The global need for effective antibiotics— moving towards concerted action. Drug Resistance Updates, 14(2), 68-69.

Chawawisit, K., & Lertcanawanichakul, M. (2008). Minimum inhibitory concentration (MIC) of crude preparations of Brevibacillus laterosporus SA14 bioactive material compared to vancomycin and oxacillin, against clinical isolates of methicillin-resistant Staphylococcus aureus. World Journal of Microbiology and Biotechnology, 24(10), 2199-2204.

CLSI; formerly NCCLS. 2009. Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial disc susceptibility tests; approved standard. CLSI document M02-A10. Clinical and Laboratory Standards Institute, Wayne, PA.

De Oliveira, E. J., Rabinovitch, L., Monnerat, R. G., Passos, L. K. J., & Zahner, V. (2004). Molecular characterization of Brevibacillus laterosporus and its potential use in biological control. Applied and environmental microbiology, 70(11), 6657-6664.

Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., & Bax, A. D. (1995). NMRPipe: a multidimensional spectral processing system based on UNIX pipes. Journal of biomolecular NMR, 6(3), 277-293.

Denning, D. W. (2003). Echinocandin antifungal drugs. The Lancet, 362(9390), 1142- 1151.

Drancourt, M., Bollet, C., Carlioz, A., Martelin, R., Gayral, J. P., & Raoult, D. (2000). 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. Journal of clinical microbiology, 38(10), 3623-3630.

Evans, M. E., Feola, D. J., & Rapp, R. P. (1999). Polymyxin B sulfate and colistin: old antibiotics for emerging multiresistant gram-negative bacteria. Annals of Pharmacotherapy, 33(9), 960-967.

Grande, M. J., Lucas, R., Abriouel, H., Omar, N. B., Maqueda, M., Martínez-Bueno, M., Valdivia, E., & Gálvez, A. (2005). Control of Alicyclobacillus acidoterrestris in fruit juices by enterocin AS-48. International journal of food microbiology, 104(3), 289-297.

Grundmann, H., Klugman, K. P., Walsh, T., Ramon-Pardo, P., Sigauque, B., Khan, W., Laxminarayan, R., Heddini, A., & Stelling, J. (2011). A framework for global surveillance of antibiotic resistance. Drug Resistance Updates, 14(2), 79- 87.

Guo, Y., Huang, E., Yuan, C., Zhang, L., & Yousef, A. E. (2012). Isolation of a Paenibacillus sp. strain and structural elucidation of its broad-spectrum lipopeptide antibiotic. Applied and environmental microbiology, 78(9), 3156- 3165.

Hancock, R. E. (2001). Cationic peptides: effectors in innate immunity and novel antimicrobials. The Lancet infectious diseases, 1(3), 156-164.

He, Z., Kisla, D., Zhang, L., Yuan, C., Green-Church, K. B., & Yousef, A. E. (2007). Isolation and identification of a Paenibacillus polymyxa strain that coproduces a novel lantibiotic and polymyxin. Applied and Environmental Microbiology, 73(1), 168-178.

Huang, E., & Yousef, A. E. (2014). The lipopeptide antibiotic paenibacterin binds to the bacterial outer membrane and exerts bactericidal activity through cytoplasmic membrane damage. Applied and environmental microbiology, 80(9), 2700-2704.

Huang, X., Lu, Z., Zhao, H., Bie, X., Lü, F., & Yang, S. (2006). Antiviral activity of antimicrobial lipopeptide from Bacillus subtilis fmbj against pseudorabies virus, porcine parvovirus, newcastle disease virus and infectious bursal disease virus in vitro. International Journal of Peptide Research and Therapeutics, 12(4), 373- 377.

Johnson, B. A., & Blevins, R. A. (1994). NMR View: A computer program for the visualization and analysis of NMR data. Journal of biomolecular NMR, 4(5), 603-614.

Kumarasamy, K. K., Toleman, M. A., Walsh, T. R., Bagaria, J., Butt, F., Balakrishnan, R., Chaudhary, U., Doumith, M., Giske, C. G., Irfan, S., & Krishnan, P. (2010). Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. The Lancet infectious diseases, 10(9), 597-602.

Makovitzki, A., Avrahami, D., & Shai, Y. (2006). Ultrashort antibacterial and antifungal lipopeptides. Proceedings of the National Academy of Sciences,103(43), 15997-16002.

Nyberg, N. T., Duus, J. Ø., & Sørensen, O. W. (2005). Editing of H2BC NMR spectra. Magnetic Resonance in Chemistry, 43(12), 971-974.

O'donnell, B. J. (1995). U.S. Patent No. 5,455,028. Washington, DC: U.S. Patent and Trademark Office.

Panda, A. K., Bisht, S. S., DeMondal, S., Kumar, N. S., Gurusubramanian, G., & Panigrahi, A. K. (2014). Brevibacillus as a biological tool: a short review. Antonie van Leeuwenhoek, 105(4), 623-639.

Ruiu, L. (2013). Brevibacillus laterosporus, a pathogen of invertebrates and a broad- spectrum antimicrobial species. Insects, 4(3), 476-492.

Shai, Y., & Oren, Z. (2001). From “carpet” mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides. Peptides, 22(10), 1629- 1641.

Steenbergen, J. N., Alder, J., Thorne, G. M., & Tally, F. P. (2005). Daptomycin: a lipopeptide antibiotic for the treatment of serious Gram-positive infections. Journal of antimicrobial Chemotherapy, 55(3), 283-288.

Tossi, A., & Sandri, L. (2002). Molecular diversity in gene-encoded, cationic antimicrobial polypeptides. Current pharmaceutical design, 8(9), 743-761.

Vergidis, P. I., & Falagas, M. E. (2008). New antibiotic agents for bloodstream infections. International journal of antimicrobial agents, 32, S60-S65.

Wright, G. D. (2005). Bacterial resistance to antibiotics: enzymatic degradation and modification. Advanced drug delivery reviews, 57(10), 1451-1470.

Wu, X., Ballard, J., & Jiang, Y. W. (2005). Structure and biosynthesis of the BT peptide antibiotic from Brevibacillus texasporus. Applied and environmental microbiology, 71(12), 8519-8530.

Wuthrich, K. (1986). NMR of proteins and nucleic acids. Wiley.

Chapter 3* Brevibacillin, a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus

*Reprint from Microbiological Research

Abstract

Brevibacillin is a newly-discovered antimicrobial lipopeptide produced by

Brevibacillus laterosporus OSY-I1. It is active against Gram-positive bacteria, including antibiotic resistant strains. This research was initiated to investigate the mechanism of action of brevibacillin against an indicator strain, Staphylococcus aureus ATCC 6538.

Results of the study proved that brevibacillin disrupts S. aureus cytoplasmic membrane by increasing its permeability, depolarization and potassium leakage. Therefore, cytoplasmic membrane serves as a major target for brevibacillin. Additionally, brevibacillin binds to lipoteichoic acid (LTA) on cell wall before interacting with cell membrane. Despite the presence of multiple sites on S. aureus cell envelope, scanning electron microscope observation didn’t reveal evidence of cell lysis or any morphological defects in cells treated with brevibacillin. Based on the results of this study, we propose that the electrostatic interaction between the cationic brevibacillin and the anionic LTA helped the accumulation of the antimicrobial agent at cell surface; this was followed by translocation of the lipopeptide to the cytoplasmic membrane and disrupting its vital functions.

3.1 Introduction

The US Centers of Disease Control and Prevention (CDC) classified antibiotic- resistant bacteria, based on threat level, into urgent, serious, or concerning threats (CDC,

2013). Clostridium difficile is an example of urgent threat, vancomycin-resistant

Enterococcus spp. (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) are serious threats, whereas vancomycin-resistant S. aureus (VRSA) is considered a concerning threat. CDC estimates that antibiotic-resistant bacteria affect more than two million people, causing 23,000 deaths, annually. These estimates do not include people who die from other diseases exacerbated by antibiotic-resistant bacteria. Considering these risks, the need for new antimicrobial agents that are effective against antibiotic- resistant strains is vital.

The newly-discovered brevibacillin is one of the emerging natural antimicrobial agents that can be useful in combating drug-resistant bacteria. Brevibacillin is a lipopeptide produced by a strain of Brevibacillus laterosporus and contains 13 amino acids and an N terminus C6-fatty acid chain (Yang et al., 2016). The compound has a molecular mass of 1583 Da, and includes three modified amino acid residues, valinol, ornithine and α,ß-didehydrobutyric acid. The lipopeptide was found effective against

MRSA, VRE and vancomycin-resistant Lactobacillus plantarum. Brevibacillin also showed antimicrobial activity against several C. difficile strains, raising the prospects of using the lipopeptide to treat anaerobic bacterial pathogens. The potency of this new lipopeptide against selected bacterial strains was comparable to that of vancomycin.

This study was initiated to reveal the mechanism of antimicrobial action of brevibacillin against S. aureus ATCC 6538 as an indicator strain. Three of brevibacillin’s amino acids are positively-charged at cell’s physiological pH (Yang et al., 2016). These residues (an ornithine and two lysine) contribute to the overall cationic

57 as well as amphiphilic nature of the lipopeptide. The N-terminal lipid chain and neutral amino acid residues including isoleucine, leucine and valine provide hydrophobicity to the compound. Collectively, brevibacillin is amphipathic in nature. One of the hypotheses for this study is that brevibacillin can bind to the phospholipids of cytoplasmic membrane through electrostatic and amphipathic interaction, followed by disruption of cell membrane which leads to cell death. Many membrane-active peptides show this characteristic interaction (Shai, 1999). However, before interacting with cell membrane, brevibacillin needs to travel through the peptidoglycan layer, which covers the phospholipid bilayer membrane (Silhavy et al., 2010). This raises the prospect for the presence of target or binding site in the peptidoglycan layer that can accumulate brevibacillin (or other cationic antimicrobial peptides), thus helping such compound traverse the peptidoglycan layer. This is an important concept but is rarely investigated

(Brogden, 2005). It was reported that lipoteichoic acids (LTA), the negatively-charged component of gram-positive cell wall, can bind to cationic artificial chimeric peptides in vitro (Scott et al., 1999). In current study, we hypothesized that brevibacillin interacts with LTA before targeting and disrupting the cytoplasmic membrane, and that signs of the interaction with these cell envelope components are detectable by bioassay and morphological examination.

3.2 Materials and Methods

3.2.1 Bacterial cultures and growth conditions. Staphylococcus aureus ATCC 6538 was obtained from American Type Culture Collection (Manassas, VA). Brevibacillus laterosporus OSY-I1 was obtained from the culture collection of food safety laboratory,

The Ohio State University. Both cultures were grown in tryptic soy broth (Becton

Dickinson, Sparks, MD) at 37 °C with shaking at 200 rpm.

Brevibacillin preparation. Overnight liquid culture of B. laterosporus OSY-I1 was

58 spread-plated on tryptic soy agar (TSA; Becton Dickinson, Sparks, MD). After incubation at 37 °C for 72 hrs, cells were collected and mixed with isopropanol. The mixture was centrifuged and supernatant was collected and dried in a chemical hood.

The dried residue was resuspended in 50% acetonitrile and purified by a high performance liquid chromatography (HPLC) system (Hewlett Packard 1050; Agilent

Technologies, Palo Alto, CA). Brevibacillin was collected at the retention time of 16.38 min (Yang et al., 2016), followed by evaporation of the mobile phase. The brevibacillin residues were dissolved in dimethyl sulfoxide (DMSO) at 3,200 μg/ml as stock solution and kept at -20 °C until use.

Brevibacillin was compared to two known antimicrobial agents, nisin and vancomycin. Pure nisin (A gift from the former Aplin and Barrett Ltd., Trowbridge,

United Kingdom) was used in the study. Purity of the nisin sample was checked by

HPLC using water-acetonitrile mobile phase. A single, sharp, isolated peak was observed in the HPLC chromatogram (figure not shown) indicating the integrity and purity of the compound. Nisin and vancomycin (Sigma, St. Louis, MO) were dissolved individually in DMSO to prepare stock solutions containing 1,600 μg /ml. Working solutions were later prepared from the stock solutions. Solubility of the three antimicrobial agents (brevibacillin, nisin and vancomycin) in water is likely different; hence, these compounds were dissolved in DMSO.

3.2.2 Lipoteichoic acids binding assay. Gram-positive bacteria cell wall contains LTA, which includes a single lipid chain that anchors at the outer layers of cytoplasmic membrane (Fischer et al., 1990). To measure possible interaction between LTA and brevibacillin, commercial S. aureus LTA (Sigma, St. Louis, MO) was used. A stock of

LTA was prepared in filter-sterilized HPLC-grade water at 1 mg/ml and kept at -20 °C.

The minimum inhibitory concentration (MIC) of brevibacillin, nisin and vancomycin

59 was determined, as described by the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS, 2007), with or without the presence of LTA at a final concentration of 100 μg/ml. The difference in MIC value between brevibacillin and brevibacillin+LTA treatments indicates the interaction between brevibacillin and LTA.

An alternative approach was designed to measure brevibacillin-LTA interaction in

96-well titer plate. Mueller–Hinton II broth aliquots (178 μl) were dispensed in the titer plate wells and mixed with S. aureus (2.0×104 CFU/well) cell suspension (20 μl) and 2

μl brevibacillin (final concentration of 4 μg/ml). Other wells of the plate received LTA at a final concentration of 100 μg/ml. A negative control was also prepared with no brevibacillin added. The inoculated wells were incubated at 37 °C for 12 hours to construct an OD600nm growth curve over time. The experiment was conducted in triplicates.

3.2.3 Membrane permeability assay by fluorescence microscope. Membrane- permeable (SYTO-9) and membrane-impermeable (propidium iodide, PI), UV- fluorescent DNA-binding stains (LIVE/DEAD® BacLight™ Bacterial Viability Kit;

Thermo Fisher Scientific, Columbus, OH) were used for analysis of cell membrane permeability, followed by observation under fluorescence microscope. The assay was performed according to Huang and Yousef (2014) with some modifications. Overnight culture of S. aureus was inoculated at 1% level into cation-adjusted Mueller–Hinton II broth (MHB; Becton, Dickinson & Co., Sparks, MD). The inoculated medium was incubated with shaking (200 rpm) at 37 °C for 5 hours so that the population reached the exponential phase. Cells were then harvested by centrifugation at 16,100 × g and washed twice with saline solution. Portions of ells suspension (400 μl) were treated with brevibacillin, at 32 μg/ml, using DMSO-dissolved stock solution (3,200 μg/ml).

Therefore, the final DMSO concentration was 1% and a negative control containing 1%

DMSO also was tested. After treatment at 37 °C for 2 hours, cells from both brevibacillin treatment and negative control were harvested by centrifugation, washing and resuspension in saline solution; this was followed by the addition of SYTO-9 and

PI stains, at 7.5 μM and 30 μM, respectively. The SYTO-9 stain can be internalized into cell cytoplasm regardless of membrane condition (healthy or damaged) whereas PI can traverse disrupted cytoplasmic membranes only. The stain-treated cells were incubated in the dark for 15 minutes followed by washing in saline solution and centrifugation.

Resuspended stained cells were spotted (5 μl) on a microscopic slide and smeared using a pipette tip. After the slide was air-dried, it was visualized under a fluorescence microscope (BX61 Olympus, Melville, NY) without a cover slip. A camera (Olympus,

U-L100HGAPO) equipped with fluorescence optical filters were used to take digital images at both wavelength: excitation/emission 480/500 nm for SYTO-9, and excitation/emission 490/635 nm for PI. The experiment was run in triplicate.

3.2.4 Membrane depolarization study. Cytoplasmic membrane depolarization was determined by using a cyanine dye, DiSC3(5) (ThermoFisher Scientific, Columbus,

OH). DiSC3(5) is a cationic fluorescent probe which accumulates at the lipid layer of healthy cell membrane and become self-quenched, presumably due to the formation of dye-aggregate structure. Antimicrobial agents, which form pores or disrupt the integrity of the membrane, dissipate DiSC3(5), causing the fluorescence to increase (Wu et al.,

1999). The experiment was performed as described by Zhang et al. (2000), with some modifications. Exponential-phase cells of S. aureus were prepared as described earlier.

Cells were rinsed twice and resuspended, to OD600nm of 0.05, in HEPES buffer (Sigma) supplemented with 5 mM glucose. Subsequently, DiSC3(5) probe was added to a final concentration of 0.4 μM. After equilibration at ambient temperature for 20 min, potassium chloride was added to the cell suspension to a final concentration of 100 mM.

Aliquots of prepared cell suspensions were transferred into a white non-binding surface

96-well plate (ThermoFisher Scientific, Columbus, OH), followed by addition of brevibacillin at various final concentrations (1-8 μg/ml). Nisin was prepared in DMSO at a final concentration of 16 μg/ml (with 1% DMSO) was applied as positive control, while 1% DMSO aqueous solution was used as a negative control. The fluorescence reading of mixtures, after treated with brevibacillin at different concentrations, along with positive and negative controls, were monitored by a luminescence spectrometer

(LS55; Perkin-Elmer, Melville, NY), at an excitation and emission wavelengths of 622 and 670 nm, respectively. Experiments were performed in triplicates.

3.2.5 Potassium leakage analysis. Potassium ion release was analyzed using a potassium-sensitive fluorescent dye, PBFI (ThermoFisher Scientific), following the method of Herranz et al. (2001). Exponential-phase cells of S. aureus were prepared, washed and resuspended in HEPES buffer (with 5 mM glucose) as described earlier, and the cell density was adjusted to OD600nm of 0.3. The PBFI fluorescent dye was added to the cell suspension to a final concentration of 2 μM. Cell suspensions were aliquoted in a black non-binding surface 96-well plate (ThermoFisher Scientific), followed by the addition of brevibacillin at various final concentrations (16-64 μg/ml). Nisin at 32

μg/ml was used as a positive control while 2% DMSO aqueous solution was used as a negative control. The fluorescence reading was also monitored by the luminescence spectrometer at an excitation/emission wavelength of 346/505 nm. The experiment was performed in triplicate.

3.2.6 Scanning electron microscopy analysis. The scanning electron microscopy

(SEM) analysis was performed according to Singh et al. (2011) with some modifications. Cells of S. aureus, at their exponential phase, were prepared and washed

3 times in saline solution (0.85% NaCl). Cells were treated with brevibacillin at final

62 concentrations of 4 and 32 µg/ml. The brevibacillin solvent, DMSO, at 1% (vol/vol) in water, was used to prepare cells that serve as control [A]. Cells not treated with brevibacillin or DMSO were used as control [B]. Treated and untreated cells were held at 37 °C for 2 hours in a shaking incubator at 200 rpm. Cells were separated by centrifugation, rinsed in saline solution three times and fixed by adding 2.5% glutaraldehyde in 0.1 M phosphate buffer. The fixation was carried out by holding the cells in fixation buffer at ambient temperature for 2 hours, then at 4 °C for 72 hrs.

Cells were rinsed and dehydrated in a series of ethanol solutions from 50% to 100%.

The dehydrated cells were then mounted on aluminum stubs and sputter-coated with a thin sheet of gold/palladium using a sputter coater (Cressington 108; Ted Pella Inc.,

Redding, CA). The coated cells were visualized under a scanning electron microscope

(Nova NanoSEM 400; FEI, Hillsboro, OR). Ten SEM images, at least, were taken from different locations of each specimen.

3.2.7 Statistical analysis. For membrane depolarization and potassium leakage study, the last time points on dose-response curves were compared statistically using analysis of variance (SAS Institute Inc., Cary, N.C.). In the alternative approach for LTA binding assay, OD600nm values after 12 hour of incubation were compared statistically. Tukey’s honest significant difference (HSD) test was used to determine the significance of the difference among means.

3.3 Results

3.3.1 Brevibacillin binds to lipoteichoic acids before interacting with membrane.

Minimum inhibitory concentration of brevibacillin against S. aureus, determined in the current study, was 2 μg/ml, which is consistent with the value reported previously (Yang et al., 2016). However, after addition of LTA to a concentration of 100 μg/ml, brevibacillin MIC increased to 16 μg/ml, an 8-fold increase. Nisin was used as a

63 positive control and its MIC changed from 1 μg/ml, initially, to 8 μg/ml after the addition of LTA. In comparison, vancomycin, which was used as negative control, showed no sign of MIC variation after LTA addition. This result was expected because nisin is a cationic peptide; therefore, it can bind to negatively charged teichoic acid through electrostatic force (Peschel et al., 1999). Vancomycin, on the other hand, is not a cationic peptide, and no electrostatic interaction with LTA is expected.

Interaction between brevibacillin and LTA was also tested in a competitive inhibition assay. When compared to brevibacillin alone, the LTA-brevibacillin treatment and the negative control showed significant higher (p < 0.05) OD600nm value, after 12 hours of incubation at 37 °C (Figure 10). Additionally, brevibacillin treatment exhibited a lag phase that lasted the whole 12 hours of the test, whereas the other treatments showed 6-hr lag period. Therefore, brevibacillin, at 4 μg/ml, was inhibitory to S. aureus but it lost its antimicrobial efficacy when the cell suspension was supplemented with LTA. Interestingly, OD600nm was significantly higher in LTA- brevibacillin treatment than it was for the negative control (p < 0.05). Considering the importance of LTA synthesis for cell division, separation and multiplication (Gründling and Schneewind, 2007), it may be plausible that the presence of this compound in the medium enhanced the growth of S. aureus.

0.17 Brevibacillin + LTA 0.15 Brevibacillin, no LTA 0.13

0.11 No brevibacillin, no LTA

0.09

0.07

Optical nm 600 density at Optical 0.05

0.03 0 2 4 6 8 10 12 14 Time (h)

Figure 10. Growth of Staphylococcus aureus, measured as OD600nm, during incubation at 37 °C, as affected by the presence of brevibacillin and lipoteichoic acid (LTA). Experiments were conducted in triplicates. OD600nm values after 12 hours of incubation were statistically different (P < 0.05)

3.3.2 Brevibacillin disrupts phospholipid bilayer membrane. Untreated cells of S. aureus, which were stained with CYTO 9 and propidium iodide and examined under

UV-microscope, exhibited good membrane integrity as indicated by their green fluorescence (Figure 11). On the contrary, cells treated with brevibacillin (32 μg/ml for

2 hrs) and stained appeared red; indicating that the antimicrobial agent compromised cell membrane, allowing PI to reach the DNA and exhibit its red fluorescence.

Therefore, untreated S. aureus cells showed no sign of membrane disruption; however,

S. aureus cells became permeable after treatment with brevibacillin.

A B

Figure 11. Fluorescence microscope pictures of Staphylococcus aureus treated with brevibacillin (32 μg/ml) for two hours and stained with membrane-permeable SYTO-9 and membrane-impermeable propidium iodide stains. Panel A: Untreated; Panel B: Brevibacillin-treated.

3.3.3 Depolarization of cell membrane by brevibacillin. The electrochemical gradient due to proton flux creates a potential difference across bacterial cell membrane; this is described as membrane potential, ΔΨ (Yeaman and Yount, 2003). The fluorescence probe, DiSC3(5), can bind to the healthy cell membrane and become self- quenched. Upon cell membrane disruption by membrane active antimicrobial agent

(e.g., gramicidin S), the probe is dequenched (Chung and Hancock, 2000). Changes in the fluorescence of probe-treated S. aureus after brevibacillin supplement, at 1-8 μg/ml levels, are shown in Figure 12. For the negative control, presence of 1% DMSO didn’t depolarize the cytoplasmic membrane. All brevibacillin concentrations tested showed significant difference from the negative control (p < 0.05). A clear dose-response relationship between brevibacillin concentration and membrane potential, as indicated by the florescence of DiSC3(5), was demonstrated (Figure 12). In addition, nisin at

16 μg/ml as a positive control, also showed significant difference, when compared to the negative control.

Figure 12. Changes in membrane potential of Staphylococcus aureus expressed by the fluorescent DiSC3(5) probe after treatment with 1-8 µg/ml brevibacillin. Experiments were conducted in triplicates and positive standard error bars were indicated. Final data points marked with different letters are significantly different at p = 0.05.

3.3.4 Brevibacillin releases intracellular components. Early stages of interaction between cationic peptide and negatively charged target membrane may form transient holes. These can increase the passage of small intracellular components, especially potassium ions, to cell’s exterior environment (Shai, 1999). In this study, a bacterial cell impermeable potassium sensitive fluorescent probe, PBFI, was used to detect the release of potassium ions (Huang and Yousef, 2014). As shown in Figure 13, the addition of brevibacillin increased PBFI fluorescence. Brevibacillin concentrations tested (32 μg/ml and 64 μg/ml) showed significant difference (p < 0.05), when compared to the negative control (2% DMSO), indicating the intracellular potassium was released from the indicator microorganism. Nisin, at 32 μg/ml, was used as a positive control and it also showed significant difference (p < 0.05) from the negative control.

Figure 13. Potassium leakage, as detected by the fluorescent of the PBFI probe, after addition of 32 µg/ml brevibacillin. Experiments were conducted in triplicates and positive standard error bars are indicated. Final data points marked with different letters are significantly different at p = 0.05.

3.3.5 The interaction of brevibacillin with cell envelope components does not cause morphological lesions. The SEM analysis may help reveal any morphological defects caused by interactions of brevibacillin with LTA and cell membrane. Based on the results of this study (Figure 14), the majority of the cells in SEM picture for both negative control and brevibacillin treatment (32 μg/ml) had similar appearance. This indicates that the binding of brevibacillin to LTA and membrane didn’t cause detectable morphological lesions in S. aureus cells. Additionally, no evidence of cell lysis was detected. The conclusion was drawn based on observation of 10 images, at least, taken from each treatment or control.

A B

Figure 14. Scanning electron microscopy pictures of Staphylococcus aureus after treatment with brevibacillin (32 μg/ml) for two hours. Panel A: Untreated; Panel B: Brevibacillin-treated. No distinct morphological differences were observed in treated and non-treated cells. Images were chosen representatively from at least ten pictures.

3.4 Discussion

Results of this study supports the hypothesis that brevibacillin interacts with

Gram-positive target cells by binding initially to LTA and subsequently disrupting the cytoplasmic membrane. The initial interaction between brevibacillin and S. aureus

ATCC 6538 surface may influence the subsequent membrane-disrupting effect. It is assumed that initial interaction is due to electrostatic binding between cationic peptides

(brevibacillin) and anionic component existed on target cell (S. aureus ATCC 6538) envelope (Yeaman and Yount, 2003). Examples of such initial interaction have been reported in Gram-negative bacteria. Hancock (1997) provided a hypothesis that cationic antimicrobial peptide can vigorously replace LPS-related divalent cations, which are known to stabilize the outer membrane structure. The binding affinity between LPS and cationic peptide is approximately 3 orders of magnitude greater than it is between LPS and the divalent cations. This hypothesis is termed “self-promoted uptake pathway” and many cationic antimicrobial peptides later have been shown to follow this mechanism of action, including polymyxin and paenibacterin (Hancock, 1997; Huang

69 and Yousef, 2014). In comparison, the Gram-positive bacteria lack outer membrane, but contain negatively charged teichoic acids, including LTA and wall teichoic acid

(WTA). WTA is covalently linked to peptidoglycan and LTA binds to cytoplasmic membrane through a single lipid chain (Scott et al., 1999; Xia et al., 2010). Both LTA and WTA are negatively charged and together regulate the susceptibility of Gram- positive bacteria (e.g. S. aureus) to many cationic antimicrobial peptides such as definsin and nisin, through electrostatic interaction. According to Peschel et al. (1999),

S. aureus mutant, with higher teichoic acids contents than the parent strain, exhibited increased sensitivity to cationic antimicrobial peptide. Interestingly, the same researchers reported that by changing the surface charge of S. aureus mutant more negatively, helped to increase the susceptibility of the mutant strain to vancomycin

(Peschel et al., 2000), which is not predicted by our current findings. Other researchers reported that the synthetic cationic antimicrobial peptide can bind to negatively charged

LTA in vitro (Scott et al., 1999). The current study provides evidence that addition of the negatively charged LTA to S. aureus culture decreased the MIC of both brevibacillin and nisin, which proved that lipoteichoic acid can serve as one of the primary binding sites for these cationic peptides.

The amphipathic nature of brevibacillin and LTA may be a good indication of their high binding affinity, in addition to electrostatic interaction. The binding of brevibacillin with LTA and cell membrane, in the current study, does not appear to damage indicator’s cell wall (Figure 14). Results reported by Scott et al. (1999) showed that the binding to LTA does not correlate with the ability of cationic antimicrobial peptide to kill bacteria which is consistent with our study.

Lipoteichoic acid is a putative pro-inflammatory structure on Gram-positive cell envelopes which can lead to sepsis. When injected to animals, LTA was reported to

70 increase cytokine production, organ dysfunction and increased mortality rate (Morath,

2001; Scott et al., 1999). The binding of cationic antimicrobial peptide with LTA can reduce its pro-inflammatory effect (Scott et al., 1999), since the overall negative charge on LTA was highly related to cytokine induction in human blood cells (Morath, 2001;

Peschel et al., 1999; Scott et al., 1999). Considering the inflammatory reaction induced by LTA, several studies recently were initiated to screen for antimicrobial compounds with LTA neutralization effect. For example, human antimicrobial peptides LL-37 derivatives were developed to have equal or better LTA neutralization effect than original LL-37 peptide (Nell et al., 2006). Another study evaluated the LTA neutralization effect of chlorhexidine and alexidine; both compounds suppressed inflammatory responses induced by LTA (Zorko and Jerala, 2008).

In addition to initial binding to LTA and translocation, antimicrobial peptides are known to target cytoplasmic membrane for two reasons: high amphipathic similarity with phospholipids and electrostatic attraction (Yeaman and Yount, 2003). Taking daptomycin as an example, its hydrophobic moieties (decanoyl aliphatic chain and tryptophan side chain) are grouped at one side of its “hairpin” structure, while the anionic hydrophilic residues are positioned closely at the other side of the hairpin, contributing to its amphipathic nature (Rotondi and Gierasch, 2005). This characteristic amphipathic structure helps daptomycin bind to cytoplasmic membrane as a major target (Silverman et al., 2003). Additionally, cell membrane structure of bacteria is more negatively charged than mammalian cells. The phospholipid bilayer of bacterial cytoplasmic membrane contains more phosphatidylglycerol (PG), cardiolipin (CL) or phosphatidylserine (PS), which are highly electronegative, compared to phospholipids of mammalian cytoplasmic membrane (Yeaman and Yount, 2003). As a result, the mechanism of action based on the electrostatic interaction between cationic

71 antimicrobial peptide and anionic cell membrane of bacteria has been widely accepted

(Bessalle et al., 1992; Dathe et al., 2001; Matsuzaki et al., 1997; Vaz Gomes et al., 1993).

In conclusion, brevibacillin is a cationic lipopeptide which binds to LTA on the bacteria surface. The binding interaction provides initial attachment and potential further translocation through peptidoglycan. Brevibacillin then targets bacterial cytoplasmic membrane to exert its bactericidal effect. These findings do not preclude the existence of additional intracellular targets for brevibacillin, or the possible contribution of these targets to its mechanism of action.

References

Bessalle, R., Haas, H., Goria, A., Shalit, I., & Fridkin, M. (1992). Augmentation of the antibacterial activity of magainin by positive-charge chain extension. Antimicrobial agents and chemotherapy, 36(2), 313-317.

Brogden, K. A. (2005). Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology, 3(3), 238-250.

Centers for Disease Control and Prevention. 2013. Antibiotic Resistance Threats in the United States, 2013. Available at: www.cdc.gov/drugresistance/threat-report- 2013/ Accessed August 24, 2016.

Chung, W., & Hancock, R. E. (2000). Action of lysozyme and nisin mixtures against lactic acid bacteria. International Journal of Food Microbiology, 60(1), 25-32.

Dathe, M., Nikolenko, H., Meyer, J., Beyermann, M., & Bienert, M. (2001). Optimization of the antimicrobial activity of magainin peptides by modification of charge. FEBS letters, 501(2-3), 146-150.

Fischer, W., Mannsfeld, T., & Hagen, G. (1990). On the basic structure of poly(glycerophosphate) lipoteichoic acids. Biochemistry and Cell Biology, 68(1), 33-43.

Gründling, A., & Schneewind, O. (2007). Synthesis of glycerol phosphate lipoteichoic acid in Staphylococcus aureus. Proceedings of the National Academy of Sciences, 104(20), 8478-8483.

Hancock, R. E. (1997). Peptide antibiotics. The Lancet, 349(9049), 418-422.

Herranz, C., Cintas, L. M., Hernández, P. E., Moll, G. N., & Driessen, A. J. (2001). Enterocin P causes potassium ion efflux from Enterococcus faecium T136 cells. Antimicrobial agents and chemotherapy, 45(3), 901-904.

Matsuzaki, K., Sugishita, K. I., Harada, M., Fujii, N., & Miyajima, K. (1997). Interactions of an antimicrobial peptide, magainin 2, with outer and inner membranes of Gram-negative bacteria. Biochimica et Biophysica Acta (BBA)- Biomembranes, 1327(1), 119-130.

Morath, S., Geyer, A., & Hartung, T. (2001). Structure–function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus. The Journal of experimental medicine, 193(3), 393-398.

Nell, M. J., Tjabringa, G. S., Wafelman, A. R., Verrijk, R., Hiemstra, P. S., Drijfhout, J. W., & Grote, J. J. (2006). Development of novel LL-37 derived antimicrobial peptides with LPS and LTA neutralizing and antimicrobial activities for therapeutic application. Peptides, 27(4), 649-660.

Peschel, A., Otto, M., Jack, R. W., Kalbacher, H., Jung, G., & Götz, F. (1999). Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. Journal of Biological Chemistry, 274(13), 8405-8410.

Peschel, A., Vuong, C., Otto, M., & Götz, F. (2000). The d-Alanine residues of Staphylococcus aureus teichoic acids alter the susceptibility to vancomycin and the activity of autolytic enzymes. Antimicrobial agents and chemotherapy, 44(10), 2845-2847.

Rotondi, K. S., & Gierasch, L. M. (2005). A well‐defined amphipathic conformation for the calcium‐free cyclic lipopeptide antibiotic, daptomycin, in aqueous solution. Peptide Science, 80(2‐3), 374-385.

Scott, M. G., Gold, M. R., & Hancock, R. E. (1999). Interaction of cationic peptides with lipoteichoic acid and gram-positive bacteria. Infection and immunity, 67(12), 6445-6453.

Shai, Y. (1999). Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by α-helical antimicrobial and cell non- selective membrane-lytic peptides. Biochimica et Biophysica Acta (BBA)- Biomembranes, 1462(1), 55-70.

Silhavy, T. J., Kahne, D., & Walker, S. (2010). The bacterial cell envelope. Cold Spring Harbor perspectives in biology, 2(5), a000414.

Silverman, J. A., Perlmutter, N. G., & Shapiro, H. M. (2003). Correlation of daptomycin bactericidal activity and membrane depolarization in Staphylococcus aureus. Antimicrobial agents and chemotherapy, 47(8), 2538-2544.

Singh, M., & Mukhopadhyay, K. (2011). C-terminal amino acids of alpha-melanocyte- stimulating hormone are requisite for its antibacterial activity against Staphylococcus aureus. Antimicrobial agents and chemotherapy, 55(5), 1920- 1929.

Vaz Gomes, A., De Waal, A., Berden, J. A., & Westerhoff, H. V. (1993). Electric potentiation, cooperativity, and synergism of magainin peptides in protein-free liposomes. Biochemistry, 32(20), 5365-5372.

Wu, M., Maier, E., Benz, R., & Hancock, R. E. (1999). Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli. Biochemistry, 38(22), 7235- 7242.

Xia, G., Kohler, T., & Peschel, A. (2010). The wall teichoic acid and lipoteichoic acid polymers of Staphylococcus aureus. International Journal of Medical Microbiology, 300(2), 148-154.

Yang, X., Huang, E., Yuan, C., Zhang, L., & Yousef, A. E. (2016). Isolation and structural elucidation of brevibacillin, an antimicrobial lipopeptide from Brevibacillus laterosporus that combats drug-resistant gram-positive bacteria. Applied and environmental microbiology, 82(9), 2763-2772.

Yeaman, M. R., & Yount, N. Y. (2003). Mechanisms of antimicrobial peptide action and resistance. Pharmacological reviews, 55(1), 27-55.

Zhang, L., Dhillon, P., Yan, H., Farmer, S., & Hancock, R. E. (2000). Interactions of bacterial cationic peptide antibiotics with outer and cytoplasmic membranes of Pseudomonas aeruginosa. Antimicrobial agents and chemotherapy, 44(12), 3317-3321.

Zorko, M., & Jerala, R. (2008). Alexidine and chlorhexidine bind to lipopolysaccharide and lipoteichoic acid and prevent cell activation by antibiotics. Journal of antimicrobial chemotherapy, 62(4), 730-737.

Chapter 4 The optimization of brevibacillin production in semi-synthetic medium and its use for controlling Alicyclobacillus acidoterrestris in a commercial fruit juice

Abstract

Brevibacillin is a linear natural lipopeptide antimicrobial agent produced by

Brevibacillus laterosporus OSY-I1. This study aimed to identify and optimize a low- cost liquid medium to produce brevibacillin in laboratory-scale fermenter. Seven M9 based semi-synthetic media with different nitrogen sources were compared for their ability of supporting brevibacillin production. An M9 medium supplemented with 0.2% peptone water was finally selected for brevibacillin production in a laboratory-scale fermenter because of its simplicity and high level of production. Brevibacillin production reached to its maximum concentration at 8.89 μg/ml after 12 to 14 hours incubation in this semi-synthetic medium in a 2-liter fermentor. Presence of brevibacillin in the fermentate was further confirmed by mass spectrometry analysis using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF).

Brevibacillin containing fermentate was used to control vegetative cells or spores of

Alicyclobacillus acidoterrestris ATCC 49025 in a juice product. A. acidoterrestris has been reported to cause spoilage of fruit juice through production of guaiacol, a smoky and medicinal compound. Brevibacillin at 0.089 μg/ml resulted in 5-log reduction of A. acidoterrestris vegetative cells while 0.81 μg/ml brevibacillin was required to completely inhibit the outgrowth of A. acidoterrestris spores in an apple juice. The capability of producing brevibacillin in laboratory-scale fermenter and potent antimicrobial activity of the compound both indicated brevibacillin can potentially be

76 applied as a juice additive to combat spoilage caused by A. acidoterrestris.

4.1 Introduction

A linear antimicrobial lipopeptide brevibacillin is produced from a soil microorganism Brevibacillus laterosporus OSY-I1, and is consisted of 13 amino acids and an N-terminal C6-fatty acid chain (Yang et al., 2016). Brevibacillin is active against

Gram-positive bacteria with potency comparable to vancomycin, and is also effective against drug-resistant microorganisms such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant (VR) Enterococcus and VR Lactobacillus.

According to our previous research, brevibacillin can survive pasteurization process: no sign of antimicrobial activity degradation after incubating at 80 °C for 60 min (Yang et al., 2016).

Current production of brevibacillin is from a solid medium, tryptic soy agar (TSA;

Becton Dickinson, Sparks, MD). Briefly, producer strain OSY-I1 was spread-plated on

TSA and cells were scraped-off and extracted after incubation. This scrapping-plate method from agar plate was labor-intensive for scale-up production which means liquid fermentation medium is necessary for higher yield of brevibacillin production. Ideally, the selected liquid medium contains defined components and can replace expensive laboraty medium. According to several studies, antimicrobial agents can be produced from industrial waste in replacement of laboratory media that are high in cost: nisin and pediocin can be produced from muscle-processing waste (Guerra and Pastrana, 2002); nisin can also be produced from raw milk whey mixed with wash water (de Arauz et al., 2009).

Alicyclobacillus acidoterrestris is a spore-forming aerobic Gram-positive thermophilic and acidophilic bacterium (Goto et al., 2007), which can be found in soil

(Groenewald et al., 2009) and fruit surfaces (Parish and Goodrich, 2005). The optimum

77 growth temperature and pH for Alicyclobacillus spp. were reported between 40 and

60 °C and pH 3.5 to 4.5, respectively (Yokota et al., 2007). Thermophilic nature of

Alicyclobacillus has made fruit juices produced from tropical region countries more susceptible: it was estimated that 11.4% of apple juice concentrate in Argentina is contaminated with Alicyclobacillus (Oteiza et al., 2011). The low pH growth range of this microorganism also indicates its ability to spoil a variety of commercial fruit juices including apple, orange, grape, and pineapple juices (Chang and Kang, 2004).

Spores of Alicyclobacillus spp. are even more likely to cause spoilage in fruit juice: these spores cannot be inactivated under standard juice pasteurization (90-95 °C, 15-20 s) without causing quality deterioration (Yokota et al., 2007; Silva 2016). One example of A. acidoterrestris AB-1 spores had a D89°C=10.9-13.7 min and D95°C=2.1-3.2 min;

D-value refers to the time required to reduce the population by 90% at specified temperature (Yokota et al., 2007). Heat-resistant spores after pasteurization can germinate and outgrow in high-acid fruit juices when storage and distribution are at spring and summer times (Spllttstoesser et al., 1994).

To reduce the surviving Alicyclobacillus spores in fruit juices, several non-thermal technologies and their combination with heat were investigated. As summarized by

Silva (2016), high hydrostatic pressure combined with heat, high pressure CO2, radiation and power ultrasound have been applied to inactivate Alicyclobacillus spores in fruit juices. However, to implement these technologies may require high capital expenditure and/or significant changes in current processing protocols. An alternative way of controlling spoilage induced by Alicyclobacillus spp. is to supplement novel antimicrobial compound into fruit juices. Nisin, for instance, has been greatly investigated to control the outgrowth of Alicyclobacillus spores (Yamazaki et al., 2000;

Komitopoulou et al., 1999). However, with the emergence of nisin resistant strains

(Harris et al., 1991; Mantovani et al., 2001), novel antimicrobial compounds need to be tested for their efficacy in controlling Alicyclobacillus vegetative cells growth and spore germination in fruit juices application.

The first goal of this study was to find a suitable liquid medium for brevibacillin production, especially with low cost and defined components in a laboratory-scale fermenter. Another goal of the study was to apply the fermentate containing brevibacillin to combat A. acidoterrestris ATCC 49025 (both vegetative cells and spores) in commercial apple juice.

4.2 Materials and methods

4.2.1 Bacterial strains and cultivation conditions. Brevibacillus laterosporus OSY-I1 and MRSA was acquired from food safety laboratory culture collection, The Ohio State

University. Listeria innocua ATCC 33090 and A. acidoterrestris ATCC 49025 was purchased from American Type Culture Collection (Manassas, VA). B. laterosporus

OSY-I1 and L. innocua ATCC 33090 were cultured in tryptic soy broth (Becton

Dickinson, Sparks, MD) unless otherwise stated; MRSA was cultured in cation- adjusted Mueller–Hinton II broth (MHB; Becton, Dickinson & Co., Sparks, MD) and

A. acidoterrestris ATCC 49025 was cultured in yeast starch glucose broth (YSG). All cultures were incubated at 37 °C.

4.2.2 Preparation of A. acidoterrestris ATCC 49025 spore suspension. Sporulation medium was formulated according to Silva and Gibbs (2001). Agar and other nutrients were prepared separately into two bottles. The first bottle included 1 g yeast extract, 0.2

g (NH4)2SO4, 0.25 g CaCl2, 0.5 g MgSO4, 1g dextrose, 0.6 g KH2PO4 and 500 ml distilled water, with pH adjusted to 3.8 by 6 M HCl. The other bottle contained 15 g of agar dissolved in 500 ml distilled water. Both bottles were sterilized at 121 °C for 15 min, and mixed together after cooling to 45 °C in a water bath, and dispensed into petri-

79 dishes. After agar plates were completely solidified, 100 μl overnight culture of A. acidoterrestris ATCC 49025 was spread-plated and incubated at 43 °C for 5 days.

Bacterial cells were collected into a microcentrifuge tube (1 plate/tube) after incubation and supplemented with 1 ml of filter-sterilized distilled water. The spore crop was harvested by centrifuging at 16100 × g for 15 min followed by mixing with 50%

(vol/vol) ethanol for 60 min to destroy any vegetative cells. The spore crop was then centrifuged again using the same condition, rinsed three times and resuspended in 1 ml of sterile water before the entire mixture was heat treated in a water bath at 80 °C for

10 min to further inactivate vegetative cells. The spore suspension was stored at 4 °C before using.

4.2.3 Liquid medium optimization for brevibacillin production in test tube system.

Brevibacillus laterosporus OSY-I1 was first inoculated into TSB to evaluate the production of antimicrobial agents, since brevibacillin was readily produced from TSA.

Modified M9 medium (11.28 g M9 medium base [Becton Dickinson, Sparks, MD],

4 g dextrose, 0.12 g MgSO4, 0.011 g CaCl2 and 1000 ml distilled water) was used as basal medium for testing the effect of various nitrogen sources with different concentrations on production of brevibacillin. The following media with different nitrogen sources were prepared: (a) modified M9 with 1 and 2% TSB; (b) modified M9 with 0.05, 0.1, 0.2 and 0.4% peptone water and (c) modified M9 with 0.4% (NH4)2SO4.

All media were autoclaved and dispensed (10 ml) into 50 ml centrifuge tubes before inoculation was achieved at 1% (vol/vol) from overnight OSY-I1 culture in TSB. After incubation at 37 °C with 200 rpm shaking, aliquot (1 ml) from each 50 ml centrifuge tube was centrifuged at 16100 × g for 5min, and tested for antimicrobial activity in the supernatant against L. innocua ATCC 33090 by soft-agar overlay method. Briefly, filter-sterilized supernatant from each 50ml centrifuge tube was spotted on the TSA soft

80 agar (0.75%) premixed with L. innocua indicator. Inhibition zones were observed and diameter was measured after incubation at 37 °C for 16 hrs.

4.2.4 Production of brevibacillin in a laboratory-scale fermenter. Modified M9 medium with 0.2% peptone water (referred as semi-synthetic medium) was selected to produce brevibacillin in laboratory-scale fermenter (VirTis omniculture; The Virtis

Company, Inc., Gardiner, N.Y., U.S.A.) with a 2-liter glass fermenter vessel with 1 liter working volume. The lid of the vessel was equipped with several different ports including inoculation, water and gas circulation coil, along with a motor at the lid connected to an agitator inside of the glass fermenter vessel. A Neslab RTE-110 water bath (Neslab Instruments, Newington, NH, USA) was used to maintain the temperature at 37 °C during fermentation. The detailed experimental design for OSY-I1 growth and brevibacillin production was carried out as follows: semi-synthetic medium was autoclaved at 121 °C for 25 min before transferring into the glass fermenter vessel. The medium was then cooled to 37 °C by water bath circulation pipes and stirring at 200 rpm for 30 min on a Virtis Omni-Culture base before inoculation (1%, vol/vol) of overnight OSY-I1 culture in M9 with 0.2% peptone water. Filter-sterilized air was then circulated into the vessel at 0.2-0.4 liter per minute (LPM) and fermentate was taken at

0, 4, 8, 12 and 14 hour time points to measure pH, bacterial cell population and antimicrobial efficacy.

4.2.5 Confirmation of brevibacillin production by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis. Fermentate at 14 hour incubation was analyzed by MALDI-TOF MS analysis to verify the production of brevibacillin. Briefly, the analysis was conducted on a Bruker UltrafleXtreme MALDI-

TOF/TOF MS (Bruker Daltonics Inc., Billerica, MA), operated in reflection-positive

81 ion mode with acceleration at the voltage of 28 kV. Filter-sterilized fermentate sample was mixed with α-cyano-4-hydroxycinnamic acid (Bruker Daltonics Inc.) dissolved in

50% acetonitrile with 0.1% trifluoroacetic acid in a ratio of 1:5 (vol/vol). A nitrogen laser was used to excite the molecules at a threshold level in order to minimize fragmentation but adequate to generate signals.

4.2.6 Quantification of brevibacillin concentration in fermentate. Brevibacillin concentration in fermentate was calculated by the following method. To start with, antimicrobial activity of brevibacillin in fermentate was measured in 96-well plate.

Each well contained 90 μl of 2-times concentrated MHB medium, 90 μl of fermentate

(or its 2-fold serial dilution 2-n) and 20 μl of MRSA indicators (~2.0×104 CFU/well).

The highest fermentate dilution with no turbidity after incubation at 37 °C for 16-20 hours was determined minimum inhibitory concentration (MIC), which was reported as 1 μg/ml (Yang et al., 2016). According to previously determine MIC value, concentration of brevibacillin in fermentate (μg/mL) can be calculated as:

[Brevibacillin] × 90 × 2- n/200 = 1 μg/ml. Experiments were performed in triplicate.

4.2.7 Effect of brevibacillin containing fermentate on controlling vegetative cells and spores of A. acidoterrestris ATCC 49025 in commercial apple juice.

Brevibacillin containing fermentate was applied to inhibit both vegetative cells and spores of A. acidoterrestris ATCC 49025 in commercial apple juice. For vegetative cells inactivation study, the experiment was carried out in 500 ml Pyrex® glass bottle

(ThermoFisher Scientific, Columbus, OH). Briefly, two glass bottles were labeled as negative control and treatment, respectively. Each bottle contained 100 ml of commercial apple juice followed by inoculation of 1 ml overnight culture of A. acidoterrestris in YSG. Then, 1 ml of filter-sterilized brevibacillin containing fermentate was inoculated into treatment bottle while 1 ml of semi-synthetic medium

82 was inoculated into the negative control bottle. Both bottles were incubated at 37 °C and aliquots were taken at 0, 1, 2, 3, 5, 7, 14 days to monitor pH and survivor population.

Experiments were done in triplicate.

For spore inactivation study, suspension of A. acidoterrestris ATCC 49025 spores suspension was prepared as described previously. Experiments were carried out in two

2 ml micro-centrifuge tubes labeled as negative control and treatment, respectively.

Each microcentrifuge tube contained 1 ml of commercial apple juice with 10 μl of spore suspension inoculation. Treatment tube was inoculated with 100 μl filter-sterilized fermentate while negative control was inoculated with the same amount of semi- synthetic medium. Both microcentrifuge tubes were incubated at 37 °C and time points were taken at 0 h, 6 h, 1, 2, 4 and 7 days to measure survival cell population.

4.2.8 Statistical analysis. Diameters of inhibition zones from each medium were compared statistically using analysis of variance (ANOVA) (SAS Institute Inc., Cary,

N.C.). Tukey’s honest significant difference (HSD) test was applied to compare the significance of the difference among means. For inactivation of Alicyclobacillus vegetative cells and spore study, the last time points on both curves were compared statistically using ANOVA by t-test to determine the significance of the difference between negative control and treatment.

4.3 Results

4.3.1 Liquid medium optimization for brevibacillin production in test tubes. As shown in Figure 15, no zone of inhibition was detected from TSB medium, indicating no brevibacillin was produced in this complex liquid medium. As shown in Table 4, diameters of inhibition zones from modified M9 media supplemented with various nitrogen sources were summarized: the larger diameter represents the stronger antimicrobial activity. Based on the size of inhibition zones, we chose modified M9

83 supplemented with 0.2% peptone water for scale-up production of brevibacillin: this medium combination showed significantly (p < 0.05) higher antimicrobial activity compared to others, with no difference in activity compares to modified M9 supplemented with 0.4% peptone water (p > 0.05). We referred the modified M9 medium with 0.2% peptone as semi-synthetic medium since all medium components except for peptone are chemically-defined.

Table 4. Diameters of inhibition zones from modified M9 medium supplemented with various nitrogen sources. Different letter represents significant difference (p = 0.05)

Nitrogen source Concentration (w/w) Inhibition zone diameter (cm)

Peptone water 0.4% 1.64±0.03a

0.2% 1.63±0.05a

0.1% 1.22±0.06cd

0.05% 0.76±0.01e

TSB 1% 1.31±0.08bc

2% 1.41±0.03b

d (NH4)2 SO4 0.4% 1.17±0.02

Figure 15. Spot-on-lawn assay with L. innocua ATCC 33090 as indicator. Spot 1 is the supernatant of B. laterosporus OSY-I1 inoculated into TSB for brevibacillin production; Zone 2 was the supernatant of B. laterosporus OSY-I1 inoculated into semi- synthetic medium for brevibacillin production. No antimicrobial activity can be observed from spot 1 indicating no brevibacillin was produced from TSB.

4.3.2 Growth and production of brevibacillin by OSY-I1 in the laboratory-scale fermenter. Growth curve of B. laterosporus OSY-I1 in 2-liter fermenter supplemented with semi-synthetic medium was shown in Figure 16a. Population of OSY-I1 increased from 102 to 107 CFU/ml during the incubation period of 14 hours at 37 ºC. In addition, pH value of the fermentate during incubation maintained between pH 6.8 and pH 7.0.

The semi-synthetic medium during fermentation had a clean odor without any major defect. Fermentate acquired from all time points were tested for antimicrobial activity against L. innocua by soft-agar overlay method, the results were shown in Figure 16b.

Figure 16. Growth curve and antimicrobial activity test. (a, left) Growth curve of B. laterosporus OSY-I1 in 2-liter laboratory-scale fermenter inoculated into semi-synthetic medium; fermentate pH was also monitored at each time points. (b, right) Spot-on-lawn assay with L. innocua ATCC 33090 as indicator. Spot 1, 0 h fermentate; Spot 2, 4 h fermentate; Zone 3, 8 h fermentate; Zone 4, 12 h fermentate; and Zone 5, 14 h fermentate.

4.3.3 Confirmation of brevibacillin production by MALDI-TOF MS analysis.

Figure 17 represents the mass spectrum of fermentate at 14 hours from m/z 500 to 5000.

Three peaks were detected at m/z 1583.9, 1605.9 and 1621.9 (z = 1), which corresponded to the singly protonated brevibacillin [M + H]+, its sodium-cationized ion

[M + Na]+ and potassium-cationized ion [M + K]+ (Yang et al. 2016). The presence of these molecular masses confirmed the existence of brevibacillin in the fermentate.

Figure 17. MALDI-TOF MS spectrum of fermentate acquired after 14 hour incubation in semi-synthetic medium. Ions at m/z (z = 1) 1583.9, 1605.9 and 1621.9 represented the singly protonated [M + H]+, sodium-cationized [M + Na]+, and potassium cationized [M + K]+ adducts

4.3.4 Quantification of brevibacillin concentration in fermentate. As reported from our previous research, MIC of brevibacillin against MRSA is 1 μg/ml (Yang et al., 2016), which equals to the highest dilution of fermentate showed no turbidity in the 96-well plate. Under optimal fermentation conditions, the production of brevibacillin reached to the maximum concentration at 8.89 μg/ml in semi-synthetic medium (Figure 18).

10.0

8.0

6.0

4.0

Brevibacillin concentrationBrevibacillin (ug/ml) 2.0

0.0 0 2 4 6 8 10 12 14 Time (hr)

Figure 18. Production of brevibacillin at different time points in fermentate.

4.3.5 Effect of brevibacillin containing fermentate on controlling A. acidoterrestris

ATCC 49025 vegetative cells and spores in commercial apple juice. Brevibacillin containing fermentate acquired as described previously was applied to treat A. acidoterrestris vegetative cells at final concentration of 0.089 μg/ml in commercial apple juice. Both untreated negative control and treatment group were hold at 37 ºC, mimicking the temperature in tropical region countries during spring/summer times. As shown in Fig. 19a, population of A. acidoterrestris steadily increased from 106 CFU/ml to 107 CFU/ml over 14 days of incubation in untreated negative control. In comparison, population of A. acidoterrestris in treated apple juice was decreasing: from 106 CFU/ml to 102 CFU/ml over the same period of time. To sum up, brevibacillin at 0.089 μg/ml successfully achieved 5-log reduction in treated apple juice comparing to negative control after 14 days of incubation (P < 0.05). Presence of brevibacillin in commercial apple juice not only controlled the microbial growth, but also maintained the quality of the juice. As shown in Fig. 19b, brevibacillin treated commercial apple juice maintained

88 pH at 3.35 while an increase of pH from 3.35 to 4.02 was observed in negative control, indicating its quality loss.

5a 5b

9 4.2

8 Control 4.1 Control Treatment 7 4 Treatment 3.9 6 3.8 5 3.7 4 pH 3.6

Log (CFU/ml) Log 3 3.5 2 * 3.4 1 3.3 * 0 3.2 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Time (days) Time (days)

Figure 19. Effect of brevibacillin on Alicyclobacillus acidoterrestris ATCC 49025. (a, left) and pH change (b, right) in commercial shelf-stable apple juice incubated at 37 °C. Treated commercial apple juice contained brevibacillin at a final concentration of 0.089 μg/ml. * indicated significant difference (p = 0.05) comparing to negative control.

In comparison, higher concentration of brevibacillin (0.81 μg/ml) was required to inhibit spore germination and amplification of Alicyclobacillus in the same juice

(Figure 20). The untreated negative control, during the first day of incubation, decreased its population from 104.5 CFU/ml to 104 CFU/ml, but increased to 106

CFU/ml after 4 days of incubation. In comparison, the treated commercial apple juice with brevibacillin decreased its population to 103 CFU/ml and maintained the population during the 7 days of incubation. Brevibacillin treated commercial apple juice achieved 3-log microbial reduction comparing to untreated group with significant difference (p < 0.05).

7 Negative control

6 Treatment

4 Log (CFU/ml) Log

3 *

2 0 1 2 3 4 5 6 7 8 Time (days)

Figure 20. Effect of brevibacillin on Alicyclobacillus acidoterrestris ATCC 49025 spore germination in commercial shelf-stable apple juice at 37 oC. Treated commercial apple juice contained brevibacillin at a final concentration of 0.81 μg/ml. * indicated significant difference comparing to negative control (p = 0.05)

4.4 Discussion

In this study, a semi-synthetic medium was developed and applied to produce brevibacillin in a laboratory-scale fermenter. Then, the fermentate was confirmed for brevibacillin production and used to control the spoilage induced by A. acidoterrestris

ATCC 49025 vegetative cells/spores. Brevibacillin was readily produced from the solid complex medium TSA, but not TSB. Similar scenarios have also been observed in many other antimicrobial compounds, such as paenibacterin, a cyclic lipopeptided from

Paenibacillus sp. (Guo et al., 2012), and a lantibiotic from Bifidobacterium longum 191

DJO10A (Lee et al., 2011). Moreover, it has been recently reported that certain antibiotics produced from fungi can only be produced from solid media (Bigelis et al.,

2006; Hölker et al., 2004).

Stress may be one of the major reasons brevibacillin can be produced from the semi-synthetic medium. To start with, despite the fact that TSB is not suitable for

90 brevibacillin production, 1% TSB supplemented in modified M9 as nitrogen source was capable of culturing OSY-I1 to produce brevibacillin. Secondly, antimicrobial compound production has long been related with stress which the producer strains are exposed to (Robison et al., 2001). According to De Vuyst et al. (1996), at non-optimum pH and temperature, antimicrobial agent production was enhanced. Another example of stress-induced production of antifungal compound is YvgO antifungal protein. It was isolated from Bacillus thuringiensis SF361 and produced only under stress condition: the same stress-response promoter σB-dependent commonly found in Gram-positive bacteria (Haldenwang and Losick, 1979) was shared by this antifungal protein expression. Also, no trace of YvgO production can be detected if optimal nutrients were supplemented. It had been proposed that the B. thuringiensis SF361 must recognize the nutrient stress during exponential phase to produce YvgO, once the stress has not been perceived by the producer strain before stationary phase, the antifungal compound expression will never be achieved (Manns et al., 2012). Similar trending was discovered for brevibacillin production, as shown in Fig. 16a, brevibacillin was also produced from exponential phase at 8 hour time point: indicating the production of brevibacillin and

YvgO compounds followed similar stress-regulated production.

M9 minimal medium has been widely used for culturing recombinant E. coli strains and as far as we know, this is the first report using M9 minimal medium to produce antimicrobial compounds. All components of the semi-synthetic medium are known except for peptone water, thus the medium is also referred to as semi-defined medium.

Although the application of small antimicrobial peptide in model food system to inhibit the growth of spoilage microorganisms is rising (Gerst et al., 2015; Guo et al.,

2016), the use of these small peptide in fruit juice preservation has seldom been studied

(Yue et al., 2013). Nisin, as one of the most famous antimicrobial food additives, was applied to treat Alicyclobacillus in commercial apple juice. Nisin was reported to decrease the D-value of Alicyclobacillus by up to 40% during pasteurization with potent activity against Alicyclobacillus spores: the MIC for nisin against spores was only 5 international units/ml. Enterocin AS-48 was another antimicrobial small peptide used to treat Alicyclobacillus cells. Concentration of enterocin AS-48 at 2.5 μg/ml can successfully inactivate the vegetative cells of Alicyclobacillus with no growth in 14 days. In addition, enterocin was reported remarkably for its fast antimicrobial effect: as early as 15 min after incubation, no viable cells can be detected (Grande et al., 2005).

The use of natural small antimicrobial peptide has advantage over using food preservatives because people are now more pursuing preservative-free all natural clean label products (Zink, 1997).

In conclusion, brevibacillin was produced from a semi-synthetic modified M9 medium supplemented with 0.2% peptone water as nitrogen source. The production of brevibacillin reached to its maximum to 8.89 μg/ml after 12 hours of fermentation.

Fermentate containing brevibacillin successfully inhibit the spoilage induced by either vegetative cells or spores of A. acidoterrestris ATCC 49025.

References

Bigelis, R., He, H., Yang, H. Y., Chang, L. P., & Greenstein, M. (2006). Production of fungal antibiotics using polymeric solid supports in solid-state and liquid fermentation. Journal of Industrial Microbiology and Biotechnology, 33(10), 815- 826.

Chang, S. S., & Kang, D. H. (2004). Alicyclobacillus spp. in the fruit juice industry: history, characteristics, and current isolation/detection procedures. Critical Reviews in Microbiology, 30(2), 55-74. de Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Penna, T. C. V. (2009). Nisin biotechnological production and application: a review. Trends in Food Science & Technology, 20(3), 146-154.

De Vuyst, L., Callewaert, R., & Crabbe, K. (1996). Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiology, 142(4), 817-827.

Gerst, M. M., Huang, E., Zhang, L., & Yousef, A. E. (2015). Development of a New Paenibacillin‐Producing Strain and Testing its Usability in Improving Food Safety. Journal of food science, 80(7), M1538-M1543.

Goto, K., Mochida, K., Kato, Y., Asahara, M., Fujita, R., An, S. Y., Kasai, H., & Yokota, A. (2007). Proposal of six species of moderately thermophilic, acidophilic, endospore-forming bacteria: Alicyclobacillus contaminans sp. nov., Alicyclobacillus fastidiosus sp. nov., Alicyclobacillus kakegawensis sp. nov., Alicyclobacillus macrosporangiidus sp. nov., Alicyclobacillus sacchari sp. nov. and Alicyclobacillus shizuokensis sp. nov. International journal of systematic and evolutionary microbiology, 57(6), 1276-1285.

Groenewald, W. H., Gouws, P. A., & Witthuhn, R. C. (2009). Isolation, identification and typification of Alicyclobacillus acidoterrestris and Alicyclobacillus acidocaldarius strains from orchard soil and the fruit processing environment in South Africa. Food Microbiology, 26(1), 71-76.

Guerra, N. P., & Pastrana, L. (2002). Nisin and pediocin production on mussel‐ processing waste supplemented with glucose and five nitrogen sources. Letters in applied microbiology, 34(2), 114-118.

Harris, L. J., Fleming, H. P., & Klaenhammer, T. R. (1991). Sensitivity and resistance of Listeria monocytogenes ATCC 19115, Scott A, and UAL500 to nisin. Journal of Food Protection®, 54(11), 836-840.

Hölker, U., Höfer, M., & Lenz, J. (2004). Biotechnological advantages of laboratory- scale solid-state fermentation with fungi. Applied Microbiology and Biotechnology, 64(2), 175-186.

Komitopoulou, E., Boziaris, I. S., Davies, E. A., Delves‐Broughton, J., & Adams, M. R. (1999). Alicyclobacillus acidoterrestris in fruit juices and its control by nisin. International journal of food science & technology, 34(1), 81-85.

Lee, J. H., Li, X., & O'Sullivan, D. J. (2011). Transcription analysis of a lantibiotic gene cluster from Bifidobacterium longum DJO10A. Applied and environmental microbiology, 77(17), 5879-5887.

Liao, C. C., Yousef, A. E., Richter, E. R., & Chism, G. W. (1993). Pediococcus acidilactici PO2 bacteriocin production in whey permeate and inhibition of Listeria monocyfogenes in foods. Journal of food science, 58(2), 430-434.

Manns, D. C., Churey, J. J., & Worobo, R. W. (2012). Functional assignment of YvgO, a novel set of purified and chemically characterized proteinaceous antifungal variants produced by Bacillus thuringiensis SF361. Applied and environmental microbiology, 78(8), 2543-2552.

Mantovani, H. C., & Russell, J. B. (2001). Nisin resistance of Streptococcus bovis. Applied and environmental microbiology, 67(2), 808-813.

Oteiza, J. M., Ares, G., Sant'Ana, A. S., Soto, S., & Giannuzzi, L. (2011). Use of a multivariate approach to assess the incidence of Alicyclobacillus spp. in concentrate fruit juices marketed in Argentina: results of a 14-year survey. International journal of food microbiology, 151(2), 229-234.

Parish, M. E., & Goodrich, R. M. (2005). Recovery of presumptive Alicyclobacillus strains from orange fruit surfaces. Journal of Food Protection®, 68(10), 2196- 2200.

Robinson, T., Singh, D., & Nigam, P. (2001). Solid-state fermentation: a promising microbial technology for secondary metabolite production. Applied microbiology and biotechnology, 55(3), 284-289.

Silva, F. V. (2016). High pressure processing pretreatment enhanced the thermosonication inactivation of Alicyclobacillus acidoterrestris spores in orange 94

juice. Food Control, 62, 365-372.

Silva, F. V., & Gibbs, P. (2001). Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes. Trends in food science & technology, 12(2), 68-74.

Spllttstoesser, D. F., Churey, J. J., & Lee, C. Y. (1994). Growth characteristics of aciduric sporeforming bacilli isolated from fruit juices. Journal of Food Protection®, 57(12), 1080-1083.

Yamazaki, K., Murakami, M., Kawai, Y., Inoue, N., & Matsuda, T. (2000). Use of nisin for inhibition of Alicyclobacillus acidoterrestris in acidic drinks. Food microbiology, 17(3), 315-320.

Yokota, A., Fujii, T., & Goto, K. (Eds.). (2007). Alicyclobacillus: Thermophilic Acidophilic Bacilli. Springer.

Yue, T., Pei, J., & Yuan, Y. (2013). Purification and characterization of anti- Alicyclobacillus bacteriocin produced by Lactobacillus rhamnosus. Journal of Food Protection®, 76(9), 1575-1581.

Zink, D. L. (1997). The impact of consumer demands and trends on food processing. Emerging infectious diseases, 3(4), 467.

Appendix A

Structural elucidation of an antifungal lipopeptide from Pseudomonas fluorescence isolated from shiitake mushroom

Abstract

Due to their low requirement for nutrients and capability of growing under harsh conditions, yeasts and molds can cause serious food spoilage and deterioration. The damage to rice, maize and wheat alone costs $60 billion loss every year. This research was initiated to search for novel antifungal compounds which can be useful in controlling foodborne fungi. A new bacterial strain was isolated from shiitake mushroom which produces a potent antifungal compound against a fungus indicator,

Aspergillus niger. The new producer strain was analyzed by 16S rDNA gene sequencing approach and was recognized as Pseudomonas fluorescence. The antifungal compound collected from the cells was extracted using acetone; the air-dried crude extract was resuspended in hexane/water, which resulted in an interface with strongest antifungal activity. Further purification was achieved by high performance liquid chromatography

(HPLC). The mass spectrometry (MS) results showed this compound has a molecular weight of 1353.81 Dalton and the formula of C64H111N11O20. The antifungal compound structure was analyzed and determined by MS as a cyclic lipopeptide with 11 amino acids acylated with a C10 fatty acid (FA) tail. This compound showed an identical structure with lokisin produced by a Pseudomonas strain which was used as a biocontrol agent for crop protection. However, the application of this strong antimicrobial agent in food to control foodborne fungi has not been explored before. Considering the high productivity of the new bacterial strain, the potent antifungal agent is very promising

96 to be used in food for the reduction of fungal growth and mycotoxin production.

Introduction

There is never enough emphasis on how important it is to inhibit fungi from ruining our food supply. There was some hope that mankind is on the verge of winning the battle against problematic fungi but it later turned out that it was an everlasting battle. Despite the fact that important antifungal agents were introduced to control fungi growth, we are still far from a breakthrough (Jucker, 2003).

Fungi cause serious food spoilage problems. By secreting digestive enzyme, fungi can easily break down food components. Some fungi are known to resist harsh environmental conditions and can be found in foods with low water activity or high acidity (Webster and Weber, 2007). In addition, xerophilic fungi like

Zygosaccharomyces rouxii can grow at water activity below 0.85 and

Zygosaccharomyces bailli is famous for its problematic preservative-resistant character in beverage, bakery and dairy industries (Yousef & Carlstrom, 2003). In addition, the presence of patulin in apple juice had brought European authorities’ attention and the regulation of this mycotoxin was in force since 2003 (EC, 2004).

What’s even worse, foodborne molds are capable of producing carcinogenic/acute mycotixns in food and feed including aflatoxin, ochratoxin, fumonisin, deoxynivalenol and zearalenone. An outbreak associated with aflatoxin intoxication happened in Kenya, a decade ago, causing 125 deaths within 317 reported illnesses (CDC, 2004). In addition to mycotoxins, some fungi are capable of causing mycosis which is actual growth of a fungus on a human host. Some effective antifungals have been used for treating deep mycoses or mycoses involved with immune-compromised patients. For instance, amphotericin B, the conventional antifungal drug, has an extremely broad antifungal spectrum which includes Candida spp., Aspergillus spp., and Cryptococcus neoformans.

However, serious problems have also been observed. This antifungal drug was reported causing chill, fever, nausea and rigor and some unpleasant feeling in treating mycoses in some patients (Jucker, 2003). In addition, a lot of emergences of antifungal drugs resistance in the clinical field involving Candida spp. have been reported. It was stated that Candida spp. were resistant to most of the widely used antifungal drugs such as azoles, polyenes and allylamines (Ghannoum and Rice, 1999).

Many antifungal compounds have been discovered and applied both in food and clinical field. For instance, benzoic, sorbic, and acetic acids are Generally Recognized as Safe (GRAS) agents that have been approved by Food and Drug Administration

(FDA) for use as antifungal food additives. Antifungal agents currently used in the food industry have several drawbacks: (i) weak activity at neutral pH; (ii) potential negative impact on food flavor; and (iii) lack of stability (the widely used potassium sorbate may be degraded by preservative-resistant fungi such as Penicillium roqueforti) (Pitt and

Hocking, 2009). Also, many chemical preservatives are known to lead to allergic reactions in sensitive individuals; and there is no nutritional value for most of the chemical preservatives. In addition, after becoming aware of health concerns for chemical preservatives, consumer nowadays are more in favor of those ‘natural’ foods processed without chemical preservatives (Rai & Chikindas, 2011). Natamycin and nystatin are the only two natural antifungal food additives approved by FDA in the

United States (FDA, 2013). Even though natamycin and nystatin can be applied as food additives, they are not very effective against some foodborne fungi; natamycin can even be degraded when stored at refrigeration temperature (Pedersen, 1992). These and other antifungal compounds, including amphotericin, flucytosine and ketoconazole, are applied as antifungal drugs (Jucker, 2003). Antifungal drugs which have been widely used in the clinical field have side effects which cannot be ignored. For example,

98 ketoconazole has been reported to cause nausea, vomiting and even death to patients

(Hoeprich, 1995; Lyman & Walsh, 1992; Como & Dismuskes, 1993).

Antifungal compounds produced from bacteria may be the solution to combat against fungi. For instance, Lactobacillus coryniformis subsp. coryniformis strain Si3 was found to produce an agent active against Aspergillus fumigatus, Penicillium roqueforti and Fusarium poae. The target compound was produced in liquid culture, by fermentation, and partial purification was conducted to verify the compound is a protein with approximately 3 kDa molecular weight (Magnusson and schnürer, 2001).

Pseudomonas spp. were found to produce cyclic lipopeptides (CLPs) which are potential antifungal biocontrol agents. Pholypeptin is a case in point. This compound was first isolated from Pseudomonas as a phosphatidylinositol-specific phospholipase

C inhibitor and its structure was confirmed by 13C-NMR (Ui et al., 1997). Lokisin was later isolated from Psedomonas fluorescens sp. strain DSS41 and its antifungal activity was observed. The structure of lokisin was very similar to pholipeptin with only conformational differences of several amino acids (Sørensen et al., 2002).

Paenibacillus spp. also are capable of producing antifungal compound, but not as many as Pseudomonas spp. It was reported by Beatty and Jensen (2002) that

Paenibacillus polymyxa can produce fusaricidin which is active against a blackleg disease-causing Leptosphaeria maculans. The antifungal compound was produced upon sporulation of producer strain in potato dextrose broth. Methanol was used to extract the compound and HPLC was further applied to purify it. Later on, partially elucidated structure was conducted by Edman degradation sequence and this compound was determined as a relative compound of fusaricidin family (Beatty and Jensen, 2002).

The antifungal agents produced by Streptomyces spp. are heavily investigated and applied in both food and medical field. Natamycin, which is used to be called pimaricin,

99 was found in 1957 produced by Streptomyces natalensis. It belongs to polyene antifungal group and has the molecular formula of C33H47NO13 (Struyk et al., 1958).

The application of natamycin has long been researched. Pederson pointed out that natamycin does not affect the count of bacteria but it suppressed fungal contamination remarkably. Even though it degraded with time at refrigerator temperature, the addition of 21.6 µg/ml of natamycin showed desirable result (Pedersen 1992). As a food additive approved by FDA (FDA, 2013), natamycin has also been used to extend shelf-life of foods. As pointed out by Fajado et al., natamycin was applied to a chitosan-based coating material to inhibit microbial growth in semi-hard cheese. According to Fajado et al., after 27 days of storage, natamycin-coated cheese had 1.1 log colony forming unit per gram (CFU/g) reduction of molds and yeasts less than control group (Fajado et al., 2010). Another polyene antifungal compound, amphotericin, which is also produced by Streptomyces has been used in clinical filed for very long time to treat mycoses caused by Candida albicans and Cryptococcus neoformans (Jucker, 2003).

The current research initiated to search for novel antifungal compound to fight against Aspergillus niger, a commonly used fungi indicator. Ideally, the antifungal compound would have high activity with low toxicity.

Materials and Methods

Bacterial strains and culture condition. A. niger was kept on a potato dextrose agar

(PDA) slant and stored in 4 °C fridge. Bacteria were cultured in tryptic soy broth (TSB) or on tryptic soy agar (TSA) unless stated otherwise.

Fungal spore suspension preparation. A. niger was first streaked on PDA and incubated at room temperature (25 °C) for 7 days with white mycelia covered with black spores. Peptone water (0.1%) with 0.1% tween 80 was autoclaved and added to cover the mycelia/spores with gentle agitation to collect spores.

100

Screening and isolation of antifungal compound producing bacteria from food and soil samples. Fermented food samples (kimchi, pickle, imported cheese, natto, soy sauce and others) were bought from local grocery stores. Soil samples were collected from campus of The Ohio State University, Columbus, Ohio. Food and environmental samples were first diluted in 0.1% peptone water and 10-fold serial diluted. Selected dilutions (10-3, 10-4 and 10-5) were plated on TSA agar followed by incubation at 37 °C for 48 hours. After incubation, colony with characteristic morphology was picked onto a new set of agar plate and incubated at the same condition for another 48 hours. Then,

10 µl of A. niger spore suspension was spotted in the middle of the plate and incubated at 25 °C for 5-7 days. The interaction between fungi and bacteria can be seen by the pattern of A. niger growth: if no antifungal compound being produced from bacteria, the mycelia will keep growing until it covers the bacterial isolate. In comparison, if antifungal compound was produced, an elliptical shape of fungi indicator can be observed.

16S rRNA gene sequencing of antifungal agent producing bacteria. Selected bacteria with antifungal activity were chosen to genetic analyze their species. Briefly, genomic DNA of these microorganisms was extracted respectively using Qiagen kit

(DNeasy blood and tissue kit; Qiagen, Valencia, CA) as DNA template. To PCR amplify the 16S rRNA gene sequences, universal primers (Forward: 5’-

CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3’ and Reverse: 5’-

CCCGGGATCCAAGCTTAAGGAGGTGATCCAGCC-3’) were chosen to amplify the targeted region by Taq DNA polymerase (Taq PCR core kit; Qiagen, Valencia, CA).

Amplified PCR product was then purified using a gel-purification kit (QIAquick gel extraction kit; Qiagen, Valencia, CA), followed by Sanger sequencing. The retrieved

16S rRNA sequence was compared to the National Center for Biotechnology

101

Information database (NCBI GenBank) using the Basic Local Alignment Search Tool

(BLAST) algorithm to the known 16S rRNA gene sequences from other bacteria provided in this database.

Extraction of antifungal compound from OSY-M12 isolate. One Pseudomonas fluorescens OSY-M12 strain with potent antifungal activity was selected for further analysis. Briefly, overnight culture of OSY-M12 was spread-plated on TSA followed by incubation at 30 ºC for 72 hours. Then, the culture was scraped into 50 ml centrifuge tubes using a glass slide. Acetone was added to extract the antifungal compound with shaking at room temperature (200 rpm) for 5 hours before centrifugation at 7710 × g for 20 min. Collected supernatant was air-dried in a chemical hood. Dried pellet at bottom of the beaker was first resuspended with water, followed by adding the same volume of hexane in a glass test tube. After agitation and settle down at room temperature, a three layer mixture was observed with hexane layer at top, an interface in between and water layer at the bottom. The interface layer was then air-dried again and resuspended in DMSO before HPLC purification.

Purification of antifungal compound from OSY-M12 using HPLC. Antifungal compound crude extract dissolved in DMSO was purified by HPLC (Agilent 1050 series) equipped with a reverse-phase column (Biobasic C18, 250×4.6mm, 5-µm particle size). Mobile phase A was HPLC-grade water with 0.1% trifluoroacetic acid

(TFA) and mobile phase B consists of isopropanol and acetonitrile (2:1) with 0.1% TFA.

Each run included 50 µl of crude extract and running from 0 to 30 min with mobile phase B from 0 to 100% lineally and from 30 to 50 min with mobile phase B constant at 100%. Fraction of every minute of multiple runs was collected and antifungal activity was tested.

MALDI-TOF MS analysis and LC-MS/MS analysis. A single peak purified from

102

HPLC run with potent antifungal activity was collected for further analysis. MALDI-

TOF MS (Matrix-Assisted Laser Desorption Ionization - Time of Flight Mass

Spectrometry) and LC-MS/MS was applied to determine the molecular weight and partial structure of the compound. MALDI-TOF MS analysis was performed by combining antifungal fraction with α-cyano-4-hydroxycinnamic at a ratio of 1:5 followed by spotting on the target plate and let air dry. The MS was operated at an accelerating voltage of 28 kv in reflection-positive ion mode with a N2 laser set at minimum threshold level to produce signal and avoid fragmentation.

In terms of LC-MS/MS study, a micromass Q-Tof II apparatus equipped with an orthogonal electrospray source was applied for tandem MS fragmentation analysis by positive ion mode. The first quadrupole Q1 was set to pass ions between 200 and 2500 m/z; target ion was fragmented within the second quadrupole Q2. TOF was used as the detector to analyze the m/z of ions fragmented in Q2.

Results

Isolate screening for antifungal compound producing bacteria. Hundreds of bacterial colonies were screened using traditional culture media method for antifungal activity. Twenty-three promising bacterial isolates were obtained in the screening process and nine isolates which demonstrate strong activity and broad spectrum are listed in Table 5. One bacterial species which was capable of producing antifungal agent was isolated from shiitake mushroom was designated OSY-M12. Identification of this strain was conducted using 16s rRNA gene sequence amplification and the species was most likely Pseudomonas fluorescens.

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Table 5. Representative bacteria isolates producing antifungal agent with their strain identification and spectrum. Isolates Strain I.D. Spectrum of Source antifungal activity a b c S4 2 Streptomyces sp. A Z P Soil S2 2 Paenibacillus AP Soil polymyxa Asp 2 Pseudomonas AZP Asparagus putida d S7’ 10 Streptomyces sp. AZS P Soil S7’ 14 Streptomyces sp. AZSP Soil SA3 6 Paenibacillus sp. AZSP Soil M12 Pseudomonas AZSP Shiitake mushroom fluorescens RP 6 Bacillus pumillus AZP Red pepper S10 11 Bacillus safensis AP Soil aAspergillus niger bZygosaccharomyces bailii cPenicillium chrysogenum dSaccharomyces cerevisiae

Extraction and concentration of antifungal compound from OSY-M12. Multiple organic solvent was tried to extracted the antifungal compound from OSY-M12 and acetone demonstrated the best recovery rate. Air-dried pellet of acetone fraction was resuspended in water/hexane mixture and as shown in Figure 21, the interface layer demonstrated the strongest antifungal activity, followed by less potent activity in hexane layer and no activity in water fraction.

104

B A A B C

C C

Figure 21. Crude extract in hexane and water. Hexane layer (A), interface (B) and water layer (C). Interface demonstrated strongest antifungal activity.

Antifungal compound purification through reverse-phase HPLC. Antifungal compound dissolved in DMSO was purified through HPLC as shown the chromatogram in Figure 22a. Fractions of every minute were collected and tested against A. niger by resuspension in DMSO. Activity shown in Figure 23 indicated that fraction #42 demonstrated the strongest activity. Re-injection of fraction #42 into HPLC under the same condition demonstrated a single, isolated peak (Figure 22b) comparing to Figure 22a. The fraction was subjected to further MS analysis.

105

DAD1 A, Sig=220,4 Ref =550,100 (M12\11-03002.D) mAU

20.245 20.311 30.309 30.372 40.721

15.812

40.682

19.761

15.725

19.737

15.325 16.133 19.994 40.646

16.089

15.975 16.347

16.256 16.406 17.914

16.174 16.488 16.822

19.850

15.245 16.674 18.030 19.665

16.043 16.642 25.027

15.131 17.334 17.553 20.068

16.564 19.924

14.906

14.844 16.515 17.430

14.626 15.085 15.464 19.880

15.008 16.747 18.085 20.164

15.532

15.862 17.483

14.758 14.804 15.191 15.404 16.721 20.124

17.159 17.853 18.267 19.560

14.696 15.617 16.920

14.572 19.204 19.628

15.902 17.966 19.404

17.294 18.503

19.366

17.025 17.088

19.067

14.520 17.244 18.197 18.751

14.450 14.958 17.695 17.764 18.148 18.321

16.956

18.705 19.312

14.347

18.38718.408 18.668

19.483

18.829

19.436

18.936

18.558

18.97118.999

14.278

2500 21.654

2000

23.930 1500 22.056

23.148

27.452

1000 24.769

21.162

27.755

21.372

28.591

41.377

26.837

26.520

23.432

26.073

500 29.652

10.986

31.956

29.241

32.151

31.240

33.489

30.935

39.107

34.372

13.654

13.363

35.324

39.634

33.000

36.897

42.370

36.207 37.376 42.620 0 43.823

5 10 15 20 25 30 35 40 min

DAD1 A, Sig=220,4 Ref =550,100 (DATA\M12\12-19003.D) mAU

40.501 1750

1500

1250

1000

750

500

250

12.075

22.028

21.062

8.193

43.947

37.572

36.724

19.576

0 32.147

12.656

14.202

13.761 16.987

17.983

14.994

16.274

5 10 15 20 25 30 35 40 min

Figure 22. HPLC chromatogram of DMSO dissolved antifungal compound from OSY-M12 (top) and re-injection of fraction #42 into HPLC chromatogram (bottom).

106

Figure 23. Antifungal activity of HPLC purified fractions against A. niger.

MALDI-TOF MS and LC-MS/MS analysis. Purified antifungal compound from

HPLC was subjected for MALDI-TOF MS and LC-MS/MS analysis. As shown in

Figure 24, multiple m/z peaks were detected by MALDI. The major peaks were m/z at

1354.9, 1376.9 and 1392.9, each correlate to [M + H]+, [M + Na]+ and [M + K]+. The molecular weight for the antimicrobial compound can thus be calculated as 1353.9 Da.

Detailed LC-MS/MS fragmentation was shown in Figure 25 as a cyclic lipopeptide with a molecular mass of 1353.8132 Dalton and the formula of C64H111N11O20. The antifungal compound structure was analyzed as a cyclic lipopeptide with 11 amino acids acylated with a C10 fatty acid (FA) tail. The deduced sequence is C10 FA - Leu - Asp -

Thr - Leu - Leu - Ser - Leu - Ser - Leu - Ile - Asp; and the Thr was esterified with the

Asp at the carboxylic terminus. The compound shares the same structure as lokisin, which was discovered in 2002 (Sørensen et al., 2002).

107

Figure 24. MALDI-TOF MS spectra for HPLC purified antifungal compound. m/z at 1354.9, 1376.9 and 1392.9 represented [M + H]+, [M + Na]+ and [M + K]+, respectively.

728.15 841.24 954.32 1041.36 1154.42 1241.47 M-LSLSLI M-LSLSL M-LSLS M-LSL M-LS M-L

113.09 113.08 87.04 113.06 87.05 Leu Leu Ser Leu Ser

728.15 841.24 928.31 1041.36 1128.40 1241.47 M-LSLSLI M-SLSLI M-LSLI M-SLI M-LI M-I

113.09 87.07 113.05 87.04 113.07 Leu Ser Leu Ser Leu

284.01 399.04 956.47 1071.44 b2 b3 y9 y10

300 400 500 600 700 800 900 1000 1100 1200

Figure 25. MS/MS analysis for the antifungal compound structure elucidation.

Discussion

In this study, an antifungal compound was successfully extracted from the producer strain, purified and structural elucidated by MS. The purified compound had very low

108 water solubility and can only be dissolved in acetone and DMSO. According to

Raaijmakers et al. (2006), many antifungal compounds produced from Pseudomonas spp. are hydrophobic with cyclic structure. In addition, another research reported that the higher the hydrophobicity of antimicrobial compounds, the stronger the antagonistic efficacy (Yeaman and Yount, 2003). However, the current antifungal compound produced from OSY-M12 was not only containing hydrophobic moiety: the fact that it accumulated at the interface of water and hexane indicated the compounds was amphipathic in nature.

In conclusion, an antifungal compound sharing the same structure as lokisin was isolated from a P. fluorescens strain with potent antifungal activity against A. niger. The compound has a molecular weight of 1353.81 Dalton and the formula of C64H111N11O20.

Further application of this compound may be needed to test its efficacy in a food/biocontrol system.

109

References

Beatty, P. H., & Jensen, S. E. (2002). Paenibacillus polymyxa produces fusaricidin-type antifungal antibiotics active against Leptosphaeria maculans, the causative agent of blackleg disease of canola. Canadian Journal of Microbiology, 48(2), 159-169.

Centers for Disease Control, and Prevention. (2004). Outbreak of Aflatoxin Poisoning --- Eastern and Central Provinces, Kenya, January--July 2004. Retrieved Sep. 18, 2016, from https://www.cdc.gov/mmwr/preview/mmwrhtml/mm5334a4.htm

Como, J. A., & Dismukes, W. E. (1994). Oral azole drugs as systemic antifungal therapy. New England Journal of Medicine, 330(4), 263-272.

Fajardo, P., Martins, J. T., Fuciños, C., Pastrana, L., Teixeira, J. A., & Vicente, A. A. (2010). Evaluation of a chitosan-based edible film as carrier of natamycin to improve the storability of Saloio cheese. Journal of Food Engineering, 101(4), 349-356.

FDA. (2013). Food Additive Status List. Retrieved 9/18, 2016, from http://www.fda.gov/food/ingredientspackaginglabeling/foodadditivesingredients/ ucm091048.htm#ftnN

Ghannnoum, M. and Rice, L.B. (1999). Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clinical Microbiology Reviews, 12(4), 501-517.

Hartman, P. G., & Sanglard, D. (1997). Inhibitors of ergosterol biosynthesis as antifungal agents. Current Pharmaceutical Design, 3(2), 177-208.

Jucker, E. M. (Ed.). (2003). Antifungal agents: advances and problems (Vol. 1). Springer Science & Business Media.

Lyman, C. A., & Walsh, T. J. (1992). Systemically administered antifungal agents. Drugs, 44(1), 9-35.

Magnusson J. and Schnürer J. (2001). Lactobacillus coryniformis subsp. coryniformis Strain Si3 Produces a Broad-Spectrum Proteinaceous Antifungal Compound. Applied Environmental Microbiology, 67(1), 1-5.

Pedersen J. C. (1992). Natamycin as a Fungicide in Agar Media. Applied and Environmental Microbiology, 58(3), 1064-1066.

Pedersen, J. C. (1992). Natamycin as a fungicide in agar media. Applied and Environmental Microbiology, 58(3), 1064-1066.

110

Pitt, J. I., & Hocking, A. D. (2009). Fungi and food spoilage (Vol. 519). New York: Springer.

Raaijmakers, J. M., de Bruijn, I., & de Kock, M. J. (2006). Cyclic lipopeptide production by plant-associated Pseudomonas spp.: diversity, activity, biosynthesis, and regulation. Molecular Plant-Microbe Interactions, 19(7), 699-710.

Rai, M. (2011). Natural antimicrobials in food safety and quality. CABI.

Sørensen, D., Nielsen, T. H., Sørensen, J., & Christophersen, C. (2002). Cyclic lipoundecapeptide lokisin from Pseudomonas sp. strain DSS41.Tetrahedron letters, 43(25), 4421-4423.

Struyk, A. P., Drost, G., Haisvisz, J. M., van Eek, T., & Hoogerheide, J. C. (1958). Pimaricin, a new antifungal antibiotic. Antibiot. Ann. 1957-1958, 878-885.

Ui, H., Miyake, T., Iinuma, H., Imoto, M., Naganawa, H., Hattori, S., Hamada, M., Takeuchi, T., Umezawa, S. & Umezawa, K. (1997). Pholipeptin, a novel cyclic lipoundecapeptide from Pseudomonas fluorescens. Journal of Organic Chemistry, 62(1), 103-108.

Webster, J., & Weber, R. (2007). Introduction to fungi. Cambridge University Press.

Yeaman, M. R., & Yount, N. Y. (2003). Mechanisms of antimicrobial peptide action and resistance. Pharmacological reviews, 55(1), 27-55.

Yousef, A. E., & Carlstrom, C. (2003). Food microbiology: a laboratory manual. John Wiley & Sons.

111

Bibliography

Barnes, E. M., & Newton, G. G. F. (1953). Brevin: an antibiotic produced by Bacillus brevis. Antibiotics & chemotherapy, 3(9), 866-72.

Brogden, K. A. (2005). Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology, 3(3), 238-250.

Cars, O., Hedin, A., & Heddini, A. (2011). The global need for effective antibiotics— moving towards concerted action. Drug Resistance Updates, 14(2), 68-69.

112

Centers for Disease Control and Prevention. 2013. Antibiotic Resistance Threats in the United States, 2013. Available at: www.cdc.gov/drugresistance/threat-report- 2013/ Accessed August 24, 2016.

Chandel, S., Allan, E. J., & Woodward, S. (2010). Biological control of Fusarium oxysporum f. sp. lycopersici on tomato by Brevibacillus brevis. Journal of phytopathology, 158(7‐8), 470-478.

Chung, W., & Hancock, R. E. (2000). Action of lysozyme and nisin mixtures against lactic acid bacteria. International Journal of Food Microbiology, 60(1), 25-32.

Como, J. A., & Dismukes, W. E. (1994). Oral azole drugs as systemic antifungal therapy. New England Journal of Medicine, 330(4), 263-272.

Dathe, M., Nikolenko, H., Meyer, J., Beyermann, M., & Bienert, M. (2001). Optimization of the antimicrobial activity of magainin peptides by modification of charge. FEBS letters, 501(2-3), 146-150. de Arauz, L. J., Jozala, A. F., Mazzola, P. G., & Penna, T. C. V. (2009). Nisin biotechnological production and application: a review. Trends in Food Science & Technology, 20(3), 146-154.

113

Denning, D. W. (2003). Echinocandin antifungal drugs. The Lancet, 362(9390), 1142- 1151.

Dzikovski, B. G., Borbat, P. P., & Freed, J. H. (2004). Spin-labeled gramicidin A: channel formation and dissociation. Biophysical journal, 87(5), 3504-3517.

Edwards, S. G., & Seddon, B. (2001). Mode of antagonism of Brevibacillus brevis against Botrytis cinerea in vitro. Journal of applied Microbiology, 91(4), 652- 659.

Evans, M. E., Feola, D. J., & Rapp, R. P. (1999). Polymyxin B sulfate and colistin: old antibiotics for emerging multiresistant gram-negative bacteria. Annals of Pharmacotherapy, 33(9), 960-967.

FDA. (2013). Food Additive Status List. Retrieved 9/18, 2016, from http://www.fda.gov/food/ingredientspackaginglabeling/foodadditivesingredien ts/ucm091048.htm#ftnN

Fischer, W., Mannsfeld, T., & Hagen, G. (1990). On the basic structure of poly(glycerophosphate) lipoteichoic acids. Biochemistry and Cell Biology, 68(1), 33-43.

Gall, Y. M., & Konashev, M. B. (2001). The discovery of gramicidin S: the intellectual 114

transformation of GF gause from biologist to researcher of antibiotics and on its meaning for the fate of russian genetics. History and philosophy of the life sciences, 137-150.

Gause, G. F., & Brazhnikova, M. G. (1944). Gramicidin S and its use in the treatment of infected wounds. Nature, 154, 703.

Gerard, J., Haden, P., Kelly, M. T., & Andersen, R. J. (1996). Loloatin B, a cyclic decapeptide antibiotic produced in culture by a tropical marine bacterium. Tetrahedron letters, 37(40), 7201-7204.

Gründling, A., & Schneewind, O. (2007). Synthesis of glycerol phosphate lipoteichoic

115

acid in Staphylococcus aureus. Proceedings of the National Academy of Sciences, 104(20), 8478-8483.

Guerra, N. P., & Pastrana, L. (2002). Nisin and pediocin production on mussel‐ processing waste supplemented with glucose and five nitrogen sources. Letters in applied microbiology, 34(2), 114-118.

Hancock, R. E. (1997). Peptide antibiotics. The Lancet, 349(9049), 418-422.

Hancock, R. E. (2001). Cationic peptides: effectors in innate immunity and novel antimicrobials. The Lancet infectious diseases, 1(3), 156-164.

Hartman, P. G., & Sanglard, D. (1997). Inhibitors of ergosterol biosynthesis as antifungal agents. Current Pharmaceutical Design, 3(2), 177-208.

Hölker, U., Höfer, M., & Lenz, J. (2004). Biotechnological advantages of laboratory- scale solid-state fermentation with fungi. Applied Microbiology and Biotechnology, 64(2), 175-186.

Hotchkiss, R. D., & Dubos, R. J. (1940). Fractionation of the bactericidal agent from cultures of a soil Bacillus. Journal of Biological Chemistry, 132(2), 791-792. 116

Jiang, Y. W., Sims, M. D., & Conway, D. P. (2005). The efficacy of TAMUS 2032 in preventing a natural outbreak of colibacillosis in broiler chickens in floor pens. Poultry science, 84(12), 1857-1859.

Johnson, B. A., & Blevins, R. A. (1994). NMR View: A computer program for the visualization and analysis of NMR data. Journal of biomolecular NMR, 4(5), 603-614.

Joo, H. J., Kim, H. Y., Kim, L. H., Lee, S., Ryu, J. G., & Lee, T. (2015). A Brevibacillus sp. antagonistic to mycotoxigenic Fusarium spp. Biological Control, 87, 64-70.

Jucker, E. M. (Ed.). (2003). Antifungal agents: advances and problems (Vol. 1). Springer Science & Business Media.

Katz, E., & Demain, A. L. (1977). The peptide antibiotics of Bacillus: chemistry, biogenesis, and possible functions. Bacteriological Reviews, 41(2), 449.

117

Lee, J. H., Li, X., & O'Sullivan, D. J. (2011). Transcription analysis of a lantibiotic gene cluster from Bifidobacterium longum DJO10A. Applied and environmental microbiology, 77(17), 5879-5887.

Lyman, C. A., & Walsh, T. J. (1992). Systemically administered antifungal agents. Drugs, 44(1), 9-35.

Makovitzki, A., Avrahami, D., & Shai, Y. (2006). Ultrashort antibacterial and antifungal lipopeptides. Proceedings of the National Academy of Sciences, 103(43), 15997-16002.

Makovitzki, A., Avrahami, D., & Shai, Y. (2006). Ultrashort antibacterial and antifungal lipopeptides. Proceedings of the National Academy of Sciences,103(43), 15997-16002.

118

microbiology, 78(8), 2543-2552.

Mantovani, H. C., & Russell, J. B. (2001). Nisin resistance of Streptococcus bovis. Applied and environmental microbiology, 67(2), 808-813.

Martinez, F. A. C., Balciunas, E. M., Converti, A., Cotter, P. D., & de Souza Oliveira, R. P. (2013). Bacteriocin production by Bifidobacterium spp. A review. Biotechnology advances, 31(4), 482-488.

Mogi, T., & Kita, K. (2009). Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics. Cellular and molecular life sciences, 66(23), 3821- 3826.

Nyberg, N. T., Duus, J. Ø., & Sørensen, O. W. (2005). Editing of H2BC NMR spectra. Magnetic Resonance in Chemistry, 43(12), 971-974.

O'donnell, B. J. (1995). U.S. Patent No. 5,455,028. Washington, DC: U.S. Patent and Trademark Office.

Panda, A. K., Bisht, S. S., DeMondal, S., Kumar, N. S., Gurusubramanian, G., & Panigrahi, A. K. (2014). Brevibacillus as a biological tool: a short review. Antonie van Leeuwenhoek, 105(4), 623-639.

Parish, M. E., & Goodrich, R. M. (2005). Recovery of presumptive Alicyclobacillus strains from orange fruit surfaces. Journal of Food Protection®, 68(10), 2196- 2200.

Pedersen, J. C. (1992). Natamycin as a fungicide in agar media. Applied and 119

Environmental Microbiology, 58(3), 1064-1066.

Pitt, J. I., & Hocking, A. D. (2009). Fungi and food spoilage (Vol. 519). New York: Springer.

Rai, M. (2011). Natural antimicrobials in food safety and quality. CABI.

Robinson, T., Singh, D., & Nigam, P. (2001). Solid-state fermentation: a promising microbial technology for secondary metabolite production. Applied microbiology and biotechnology, 55(3), 284-289.

Ruiu, L. (2013). Brevibacillus laterosporus, a pathogen of invertebrates and a broad- spectrum antimicrobial species. Insects, 4(3), 476-492.

120

Scott, M. G., Gold, M. R., & Hancock, R. E. (1999). Interaction of cationic peptides with lipoteichoic acid and gram-positive bacteria. Infection and immunity, 67(12), 6445-6453.

Seydlová, G., & Svobodová, J. (2008). Review of surfactin chemical properties and the potential biomedical applications. Open Medicine, 3(2), 123-133.

Shai, Y., & Oren, Z. (2001). From “carpet” mechanism to de-novo designed diastereomeric cell-selective antimicrobial peptides. Peptides, 22(10), 1629- 1641.

Sharma, V., Singh, P. K., Midha, S., Ranjan, M., Korpole, S., & Patil, P. B. (2012). Genome sequence of Brevibacillus laterosporus strain GI-9. Journal of bacteriology, 194(5), 1279-1279.

Silhavy, T. J., Kahne, D., & Walker, S. (2010). The bacterial cell envelope. Cold Spring Harbor perspectives in biology, 2(5), a000414.

Silva, F. V. (2016). High pressure processing pretreatment enhanced the thermosonication inactivation of Alicyclobacillus acidoterrestris spores in orange juice. Food Control, 62, 365-372.

Silva, F. V., & Gibbs, P. (2001). Alicyclobacillus acidoterrestris spores in fruit products and design of pasteurization processes. Trends in food science & technology, 12(2), 68-74.

121

Somsap, O. A., Bangrak, P., Bhoopong, P., & Lertcanawanichakul, M. (2015). Antibacterial activity and purification of bacteriocin produced by Brevibacillus laterosporus SA14. Walailak Journal of Science and Technology (WJST), 13(1), 55-65.

Sørensen, D., Nielsen, T. H., Sørensen, J., & Christophersen, C. (2002). Cyclic lipoundecapeptide lokisin from Pseudomonas sp. strain DSS41.Tetrahedron letters, 43(25), 4421-4423.

Spllttstoesser, D. F., Churey, J. J., & Lee, C. Y. (1994). Growth characteristics of aciduric sporeforming bacilli isolated from fruit juices. Journal of Food Protection®, 57(12), 1080-1083.

Struyk, A. P., Drost, G., Haisvisz, J. M., van Eek, T., & Hoogerheide, J. C. (1958). Pimaricin, a new antifungal antibiotic. Antibiot. Ann. 1957-1958, 878-885.

122

Microbiology, 43(2), 221-231.