Send Orders for Reprints to reprints@benthamscience.ae Current Pharmaceutical Biotechnology, Volume, Page Enation 1 °°LETTER ARTICLE Construction and functional analysis of the recombinant bacteriocins Weissellicin-MBF from Weissella confusa MBF8-1 Running title: Recombinant bacteriocins from Weissella confusa Amarila Malika*, Elita Yuliantiea, Nisa Yulianti Suprahmana, Theresa Linardia, Angelina Wening Widiyantia, Jeanita Haldya, Catherine Tjiaa, and Hiroshi Takagi**b a Division of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, Universitas Indonesia, UI Depok Campus, Depok 16424, Indonesia. bDivision of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. 1389-2010 /19 $58.00+.00 © 2019 Bentham Science Publishers 2 Current Pharmaceutical Biotechnology, 2019, Vol. 0, No. 0 Principle Author et al. Abstract:  ARTICLE HISTORY Received: Revised: Accepted: DOI:     Background: Bacteriocins (Bac1, Bac2, and Bac3) from Weissella confusa MBF8-1, weissellicinMBF, have been reported as potential alternative substances as well as complements to the existing antibiotics against many antimicrobial-resistant pathogens. Previously, the genes encoded in the large plasmid, pWcMBF8-1, and the spermicidal activity of their synthetic peptides, have been studied in originally discovered Indonesia. Three synthetic bacteriocins peptides of this weissellicin-MBF have been reported as well for their potential activities, i.e. antibacterial and spermicidal. Objective: The aim of this study was to construct the recombinant Bacteriocin (r-Bac) genes, as well as to investigate the genes expressions and their functional analysis. Method: Here, we constructed the recombinant Bacteriocin (r-Bac) genes and produced the recombinant peptides (r-Bac1, r-Bac2, and r-Bac3) in B. subtilis DB403 cells in a large scale. After purification using the His-tag affinity column, their potential bioactivities measured as antibacterial minimum inhibitory concentrations against Leuconostoc mesenteroides and Micrococcus luteus were determined. Results: Pure His-tag-recombinant Bac1, Bac2, and Bac3 were obtained and they could inhibit the growth of L. mesenteroides and M. luteus. Conclusion: The recombinant bacteriocin could be obtained although with weak activity in inhibiting gram-positive bacterial growth. Keywords: Bacillus subtilis; bacteriocin; recombinant peptide; synthetic peptide; Weissella confusa, weissellicin Title of the Article Antimicrobial resistance (AMR) is a major global health problem. It has been predicted that AMR will cause an additional 10 million deaths per year and a loss of up to US$100 trillion from global GDP by 2050 1. In Indonesia, AMR cases are remarkably high; in 2009, the World Health Organization stated that Indonesia was on the 8th rank of 27 countries with multidrug resistance 2. Redesign of antimicrobial compounds from animals, plants, insects, and bacteria is one of the favorable alternative ways 3. Bacteriocin, which is a bioactive peptide produced naturally by lactic acid bacteria (LAB) 4, has been commonly used as food preservative. However, its mechanism of killing other bacteria is mostly done by disruption of membrane integrity and thus thought to be less likely to induce resistance. In addition, bacteriocin is more active than conventional antibiotics against the resistant Gram-positive bacteria 5; it acts in micro to nanomolar concentration, hence is relatively safe either for human body and environment 3, 6. Consequently, bacteriocin is extensively evaluated as a potential novel antimicrobial drug or as a conventional antibiotics complement 7. For example, nisin has been reported since decades for its potential use in combination with peptidoglycan modulating antibiotics, such as chloramphenicol and bacitracin 8. New bacteriocins have been identified widely and the genes encoding have been deposited in DNA database; this could serve as potential and promising source for the exploitation of new antimicrobial peptides. However, the low yields, the *Address correspondence to this author at the Division of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, Universitas Indonesia, UI Depok Campus, Depok 16424, Indonesia.Tel: +62-217270031 / Fax: 62-21-7863433 / E-mail: amarila.malik@ui.ac.id ** Address correspondence to this author at the Division of Biological Science, Graduate School of Science and Technology, Nara lnstitute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan. Tel: +81-743-72-5420 / Fax: +81-743-72-5429 / E-mail: hiro@bs.naist.jp generation of potential virulence factors from natural producers and the high cost of synthetic synthesis drives numerous researchers to utilize genetic engineering to induce heterologous expression of the bacteriocin. Yet their potential is difficult to evaluate in the absence of suitable expression systems; E. coli has been used widely as a heterologous host to produce recombinant bacteriocins and has an extensive set of the different expression systems in an attempt to guarantee successful production of a range of bacteriocins 9-13. A LAB Weissella confusa MBF8-1, which was previously isolated from soya waste in Indonesia 14, showed bacteriocinlike inhibitory substance (BLIS) activity 15. This activity can inhibit growth of several pathogenic bacteria by performing deferred antagonism assay (DAA), but its spectrum of activity is fairly narrow, and was limited to species very closely related to W. confusa, i.e. Micrococcus luteus T18 and Lactococcus lactis T-21 16. Recently the synthetic bacteriocin also showed activity to inhibit sperm motility 17. The genetic locus comprising three putative bacteriocinencoding bac genes, i.e. bac1, bac2, and bac3, and their related transport complex (bacT and bacE) as well as putative immunity protein (bacI) for weissellicin MBF, the MBF8-1 BLIS, is harbored by the plasmid pWcMBF8-1 (AN KR350502). This study aimed to construct a suitable Current Pharmaceutical Biotechnology, 2019, Vol. 0, No. 0 3 expression vector to produce large-scale and functional recombinant bacteriocins (r-Bacs). W. confusa MBF8-1 used in this study was taken from the collection of the Laboratory of Pharmaceutical Microbiology and Biotechnology, Faculty of Pharmacy, Universitas Indonesia, Depok, Indonesia. W. confusa MBF8-1 was cultivated at 32C for 18 h in MRS (Difco, Franklin Lakes, NJ) medium without shaking 16. Specific oligonucleotides primer pairs targeting three putative bacteriocin (bac) encoding gene bac1, bac2 and bac3 in the plasmid pWcMBF8-1 15 were designed from the annotated draft genome sequence of W. confusa MBF8-1 (AN KR350502) and were analyzed by using OligoAnalyzer 3.1 [http://sg.idtdna.com/calc/analyzer], and Primer BLAST [http://www.ncbi.nlm.nih.gov/tools/primer-blast] and Sequence Manipulation Suite (SMS): Primer Map [www.bioinformatics.org/sms2/primer_map.html]. These primers were designed to give final recombinant genes as follows: the start codon ATG upstream of the first nucleotide as well as the Shine-Dalgarno (SD) sequence with 7-bp space to ATG of the bacteriocin gene were placed at the 5’ end and the 3’ end should not have the stop codon but be directly tagged with 7×His (Fig. 1). The tag sequence was added for the purpose of cloning, isolation, expression and purification by GatewayR system (Invitrogen, La Jolla, Ca. USA). All oligonucleotides used in this study are listed in Table 1. The plasmid pWcMBF8-1 was isolated from W. confusa MBF8-1 according to the method as described previously 16, 18 , and was stored at -20 º C. Polymerase chain reaction (PCR) was performed for generating pEntry, which was conducted in two steps according to the GatewayR manufacturer protocol. Product obtained from the first PCR was verified by running on a 2% agarose gel in 1×TAE. The PCR product for further cloning purpose was purified with PrepEaseR Gel Extraction Kit (Affymetrix USB, USA) according to manufacturer’s protocol. By using pDONR221 (Invitrogen, La Jolla, Ca. USA) to construct pEntry of each bac gene, we performed BP-ClonaseIIR according to manufacturer’s manual. Transformation to E. coli DH5α was carried out by heat shock method with CaCl2 as described for GatewayR system (Invitrogen, La Jolla, Ca. USA). To obtain entry clone, transformant was positively selected on LB agar medium supplemented with kanamycin (50 μg/ml). To confirm and verify the correct constructed recombinant plasmid, randomly chosen clones were subjected to plasmid purification according to the alkaline lysis method 19, followed by PCR using pairs of universal pDONRTM primer as listed in Table 1. The amplicon DNAs were sequenced and analyzed by using Clustal Omega (https://www.ebi. ac.uk/Tools/msa/clustalo/) to confirm that no error was introduced during the recombinatorial cloning process. Correct entry clones were further processed for LRrecombinatorial cloning using LR-ClonaseR according to manufacturer’s manual. For the expression of the recombinant peptide in B. subtilis DB403, we used a shuttle vector as destination vector, pOXGW 20-21. The LR- reaction was transformed to competent E. coli DH5α by performing the same method as mentioned above, and was positively selected on LB agar plates containing tetracyclin (10 μg/ml). Transformation of recombinant plasmids pMBF8-1bac1, pMBF8-1bac2 and pMBF8-1bac3 to B. subtilis DB403 was 4 Current Pharmaceutical Biotechnology, 2019, Vol. 0, No. 0 carried out by electroporation 22. Before the B. subtilis DB403 transformants were used for the expression, each gene was confirmed by PCR by using internal oligonucleotide primers listed in Table 1, and they showed the expected size of inserts, i.e. 186 bp, 153 bp and 138 bp for bac1, bac2, and bac3, respectively (Fig. 2). The B. subtilis transformants harboring recombinant plasmids of each bac gene were obtained after PCR recombinatorial cloning, and designated pMBF8-1bac1, pMBF8-1bac2 and pMBF8-1bac3. To produce the recombinant bacteriocins in large scale, use of less expensive inducer than isopropyl β-D-thiogalactopyranoside would be preferred. Thus, pOXGW was used as the overexpression vector in which cloned gene can be induced by xylose. It was also designed for easy purification by adding 7×His-tag, and efficient expression in B. subtillis by using the artificial ATG start codon and the ribosome-binding site. Fig. 1 shows the structure of the construct. For the production of the recombinant bacteriocin peptides, confirmed clones of B. subtilis DB403 were pre-cultured. Recombinant peptides were produced by heterologous expression in a 3-L bioreactor flask (New Brunswick BioFloR/CelliGenR 115, Eppendorf, Hamburg, Germany). One-liter of LB broth medium containing tetracycline (10 μg/ml) was used and was cultured at 30 °C with agitation 100 rpm to reach OD600 = 0.6-0.8, followed by adding xylose (0.5% w/v) to the culture to induced the production of the recombinant peptides by the strain. Cells were harvested by centrifugation at 8,000 x g at 4 °C, and cell debris was resuspended in 20 mM Imidazole (20 mM Phosphate buffer, 0.3 M NaCl, 20 mM Imidazole, pH 7.5) and disrupted by repetitive ultrasonication (Labsonic M Cell disrupter, Sartorius, Germany) at 20 kHz for 15 s, interval 5 s and for 5 min with anti-protease PMSF was added to the culture. The supernatant (crude protein preparation) was subjected to further purification by employing HisTrap FF column (GE, USA) according to the manufacturer’s protocol, and the final elution harboring the His-tag recombinant peptide was released by using 500 mM imidazole (20mM phosphate buffer, 0.3 M NaCl, 500 mM imidazole, pH 7.5) and 900 mM imidazole (20mM phosphate buffer, 0.3M NaCl, 900 mM imidazole, pH 7.5). The purity of recombinant peptides was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a 15% acrylamide gel. The protein concentration was measured spectrophotometrically using the Bicinchoninic Acid (BCA) Protein Assay kit (Pierce, USA) using bovine serum albumin (BSA) as the standard. For subsequent biochemical and bioassay studies, which the latter was to establish whether the three peptides are the sole agent responsible for the antimicrobial activity of W. confusa MBF8-1, the recombinant peptides were produced in a large scale. The purity of peptides designated r-Bac1, r-Bac2 and r-Bac3 was confirmed by SDS-PAGE as a single band, but some bands with unexpected molecular weight were observed (Fig. 3). This result might be explained due to the misfolding of recombinant peptides with 7×His-tag when forming aggregate, or the hydrophobic/cationic feature of the peptides23-24. However, some pure peptides can be obtained by the method used. Functional assays were carried out including standard dilution method and MTT assay for minimum inhibitory concentration (MIC) determination, and disc diffusion assay. Principle Author et al. The desalted 900 mM fraction of r-Bac1, r-Bac2 and r-Bac3 were tested against Leuconostoc mesenteroides TISTR 120. Each r-Bac was prepared by two folds serial dilution in the growth medium to make six point concentrations. Bacteria were added 1×105 CFU/tube and the tubes were then incubated at 32 °C in aerobic condition for 24 hours. Standard MIC assay was employed to determine r-Bacs activity, r-Bac 1 and r-Bac2 could inhibit the growth of indicator bacteria but not r-Bac3 (Fig. 4A). All r-Bacs could inhibit growth of L. mesenteroides cells, but had very weak activity. Judging from the turbidity by MIC, we categorized three types, i.e., well observed, slightly observed and none. MTT assay, a colorimetric assay for measuring the activity of enzymes that reduce 3-(4,5-dimethylthiazol-2-yl)-2,3diphenyltetrazolium bromidefor, has been widely used as an additional method to confirm the activity of antibacterial compounds 25. Each r-Bacs was prepared in serial dilution, in the growth medium before addition of L. mesenteroides TISTR 120 5×104 CFU/well and were grown overnight at 35±2 °C. Subsequently, 10 μL of 5 mg/mL MTT reagent was added into each well and color change was observed after 3 hours incubation at 37 °C. With this method, all the r-Bacs component could actively inhibit L. meseteroides (Fig. 4B). Agar diffusion assay by using paper disc was done as described 17 with Micrococcus luteus T18 as indicator bacteria, diluted to 1×107 CFU/mL. Nisin was used as positive control and BLIS fractions were also prepared as comparison. As a single component, r-Bacs did not show activity against M. luteus; however, combination of r-Bac1 and 2 as well as r-Bac 1 and 3 showed small inhibition. Combination of the three bacteriocins altogether could distinctly inhibit test bacteria (Fig. 5). Taken together (Table 2), these results aligned with our previous report of synthetic bacteriocin tested against L. mesenteroides 17. Interestingly, while the recombinant peptide showed activity, the BLIS fractions which also were tested did not show any effect against M. luteus. Based on their DNA sequences characterization, bacteriocin of gram-positive W. confusa MBF8-1 was proposed as bacteriocin class IIb 3. The bacteriocin class IIb consists of 2 or more peptides that synergistically act to inhibit other bacterial growth. To analyze the characteristic, each bacteriocin peptide was studied as single component and in combination with other peptides. To achieve this goal, a construction of recombinant peptide was attempted by employing GatewayR system based on high-efficient recombinatorial technique compared to conventional method using restriction endonuclease and ligase 26. This is the first study reported on the construction of recombinant bacteriocins of W. confusa MBF8-1. The well diffusion agar method has been carried out to test whether the peptides possessed growth inhibition activity on agar plates observed as a clear zone. The recombinant peptides exhibited very limited a clear zone against M. luteus but previously reported the synthetic peptides showed a better clear zone 17, in line with the results, which exhibited only by s-Bac in combination mixture, i.e. s-Bac1+2, sBac1+3 and s-Bac1+2+3; the strongest inhibitory activity was shown by s-Bac1+2+3. BLIS supernatant from W. confusa MBF8-1 as the bioactive reference, comprise of fractions <3 kDa, >3 kDa, <10 kDa and >10 kDa, showed Title of the Article that fraction <3 kDa is the strongest inhibitory activity as indicated by the largest clear zone on agar plates 17. Although it was weak, the recombinant peptides showed antimicrobial activity that inhibits the growth of the grampositive L. mesenteroides. It can be assumed that the weak activity results from the bacteriostatic nature of bacteriocin itself other than the possibility of aggregation of small peptides. This result suggests that the design of recombinant peptides could affect the peptide folding 27. The poly His-tag might interfere the folding process 28. The recombinant bacteriocin obstructs bacterial growth, but does not kill bacteria. Moreover, Bac peptides are expected as type IIb of bacteriocin, which cannot work as antimicrobial agents as a single component but must be in a combination with other two Bac peptides 15. This could serve the fact with speculation that the antimicrobial activity cannot be preserved as a single Bac peptide only. The AMP structure, which is simple poly-amino acids, is relatively easy to modify (including library construction and screening) and as well as to immobilize on surfaces 29. Since it is small, AMP can be chemically made as synthetic peptides 30. Several studies employed recombinant expression systems to produce AMP 31-32. Therefore, it is useful to modify existing AMPs for designing new synthetic peptides. Such modifications have potential to change the targets of AMPs and improve the stability of AMPs against proteases 33. The construction of the recombinant bacteriocin peptides from W. confusa MBF8-1 for the development of AMP to be combined with existing antibiotics might be a good strategy to combat the “superbugs”. Further studies to construct and modify such AMP molecules are necessary in the future. Nevertheless, even though the cost of chemical synthesis for AMP is not inexpensive, it can be highly guaranteed as a pure form, and therefore could be potential to develop rapidly as compare to the recombinant one. LIST OF ABBREVIATIONS aa – amino acid, AMP – antimicrobial peptide, AMR – antimicrobial resistance, BCA - bicinchoninic acid, BSA – bovine serum albumin, dNTP - deoxyribonucleotide triphosphate, LAB – lactic acid bacteria, MW – molecular weight, PCR – polymerase chain reaction, r-Bac – recombinant bacteriocin, s-Bac – synthetic bacteriocin CONSENT FOR PUBLICATION If the manuscript has an individuals’ data, such as personal detail, audio-video material etc., consent should be obtained from that individual. In case of children, consent should be obtained from the parent or the legal guardian. A specific declaration of such approval must be made in the copyright letter and in a stand-alone paragraph at the end of the Methods section especially in the case of human studies where inclusion of a statement regarding obtaining the written informed consent from each subject or subject's guardian is a must. The original should be retained by the guarantor or corresponding author. Editors may request to provide the original forms by fax or email. All such case reports should be followed by a proper consent prior to publishing. Current Pharmaceutical Biotechnology, 2019, Vol. 0, No. 0 5 CONFLICT OF INTEREST The authors declare no existing conflict of interest. This work was supported by the World Academy of Sciences (TWAS), Italy, basic science research grant [14–094 RG/BIO/AS G-UNESCO FR: 324028606] (2015) to AM and partly by Global Collaborative Program (2016-2017), Nara Institute of Science and Technology, Nara, Japan, to HT. E.Y. is currently a PhD student of Shanghai Institute of Materia Medica, University of Chinese Academy of Sciences under the CAS-TWAS Presedential Fellowship Program. ACKNOWLEDGEMENTS We greatly appreciate valuable scientific discussions with Dr. Nick C.K. Heng, Sir John Walsh Research Institute, University of Otago, Dunedin, New Zealand. We also thank Dr. Shu Ishikawa, Kobe University, Nara Institute of Science and Technology, Nara, Japan, for his generous support in the manipulation of B. subtillis DB403. FUNDING This work was supported partly by the World Academy of Sciences (TWAS), Italy, basic science research grant [14– 094 RG/BIO/AS G-UNESCO FR: 324028606] (2015) to AM and partly by Global Collaborative Program (20162017), Nara Institute of Science and Technology, Nara, Japan, to HT, and Hibah Penelitian Dasar Unggulan Perguruan Tinggi (PDUPT) 2018-2019 from the Ministry of Research, Technology and Higher Education of the Republic of Indonesia to A.M (No. 261/UN2.R3.1/HKP05.00/2018 and NKB-1469/UN2.R3.1/HKP.05.00/2019). REFERENCES 1. Sugden, R.; Kelly, R.; Davies, S., Combatting antimicrobial resistance globally. Nat. Microbiol., 2016, 1, 16187. 2. Sosa, A. d. J.; Amábile-Cuevas, C. F.; Byarugaba, D. K.; Hsueh, P.-R.; Kariuki, S.; Okeke, I. N., Antimicrobial resistance in developing countries. 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Oligonucleotides used for cloning in this study Current Pharmaceutical Biotechnology, 2019, Vol. 0, No. 0 7 Title of the Article No. Name Sequence 1 SD-start-attB1 5'- GGG GAC AAG TTT GTA CAA AAA AGC AGG CTC GAA GGG AGG TGT CAT AAA For recombinatorial cloning TG-3' 2 7his-attB2 5'- GGG GAC CAC TTT GTA CAA GAA AGC TGG GTC TCA TCT ATT TAA TGG TGG TGG TGA TGG TGA TGC GAT CC-3' For recombinatorial cloning 3 Bac8-1a_ Gw_Fw 5'- CGA AGG GAG GTG TCA TAA ATG GAT GAT AAG TTT AGT GC -3' For recombinatorial cloning of bac1 4 Bac8-1a_ Gw_Rv 5'- GAT GGT GAT GGT GAT GCG ATC CAT GCT GTG GAC GGT AC-3' For recombinatorial cloning of bac1 5 Bac8-1b_ Gw_Fw 1. 5’-CGA AGG GAG GTG TCA TAA ATG GAT AAA AAA TTA ATT TTA G -3’ For recombinatorial cloning of bac2 6 Bac8-1b_ Gw_Rv 2. 5’-GAT GGT GAT GGT GAT GCG ATC CCC CAA AGG CAC TGC AAA TCC -3’ For recombinatorial cloning of bac2 7 Bac8-1c_ Gw_Fw 5’- CGA AGG GAG GTG TCA TAA ATG AAT AAT AAT TTA AAG -3’ For recombinatorial cloning of bac3 8 Bac8-1c_ Gw_Rv 5’- GAT GGT GAT GGT GAT GCG ATC CAC GAACACGTCGCATAATTC-3’ For recombinatorial cloning of bac3 9 pDONR-F 5'-TCGCGTTAACGCTAGCATGGATCT C-3' Specific primer for recombinatorial cloning confirmation 10 pDONR-R 5'-GTAACATCAGAGATTTTGAGACAC-3' Specific primer for recombinatorial cloning confirmation 11 Bac8-1_aFw 5’-ATGGATGATAAGTTTAGTGCTTTAAG-3’ Internal primer for recombinant strain confirmation carrying bac1 12 Bac8-1_aRv 5’-ATGCTGTGGACGGTACTTTG-3’ Internal primer for recombinant strain confirmation carrying bac1 13 Bac8-1_bFw 5'-ATGGATAAAAAATTAATTTTAGATAAG-3’ Internal primer for recombinant strain confirmation carrying bac2 14 Bac8-1_bRv 5’-CCCAAAGGCACTTGCAAATC-3’ Internal primer for recombinant strain confirmation carrying bac2 15 Bac8-1_cFw 16 Bac8-1_cRv 5’-ATGAATAATAATTTAAAGATATTAAATG-3’ 5’-ACGAACACGTCGCATAATTC-3’ Description Internal primer for recombinant strain confirmation carrying bac3 Internal primer for recombinant strain confirmation carrying bac3 Table 2. Antibacterial assay performing MIC and inhibition zones of crude and r-Bacs Peptides MIC by MTT assay (µg/mL) (L. mesenteroides TISTR120) Disc diffusion assay (diameter in mm) (M. luteus T18) 8 Current Pharmaceutical Biotechnology, 2019, Vol. 0, No. 0 Principle Author et al. Nisin ND 22.13 BLIS ˃30 kDa ND NA BLIS ˂30 kDa ND NA BLIS ˃10 kDa ND NA BLIS ˂10 kDa ND NA r-Bac1 10,416.7 NA r-Bac2 12,500 NA r-Bac3 12,500 NA r-Bac1,2 ND 8 r-Bac1,3 ND 7.9 r-Bac2,3 ND NA r-Bac1,2,3 ND 9.15 NA: not active; ND: not determined FIGURE LEGENDS Fig. 1. Construction of the recombinant three putative bacteriocin genes in B. subtilis. pWcMBF8-1, plasmid harboring the genes encoding bacteriocins from W. confusa MBF8-1; bac, bacteriocin encoding gene; attB1 and attB2, GatewayR recombinatorial sites; SD, a Shine-Dalgarno sequence; 7his, 7×histidine tag; pDONR221, the entry vector; pOXGW, the expression vector; r-Bac1/2/3, recombinant bacteriocin 1/2/3/. Fig. 2. Confirmation of B. subtilis DB403 transformants. Genomic PCR was done using internal primer pairs with plasmid extracted from B. subtilis DB403 transformants. Amplicons with primer pairs for: bac1 (expected size 186 bp), bac2 (expected size 153 bp), and bac3 (expected size 158 bp) from cells harboring pMBF8-1bac1, pMBF8-1bac2, and pMBF8-1bac3, respectively. DNA marker used was 100 bp DNA ladder (HyperLadder™, Bioline, London, UK). PCR products were loaded duplicate on 2% agarose gel with 1×TAE buffer. Fig. 3. Purification of recombinant bacteriocins. Recombinant bacteriocins r-Bac1, r-Bac2, and r-Bac3 were obtained as mostly pure peptides after purification step with final elution using 500 mM Imidazole. Fig. 4. Bioactivity of r-Bacs against Leuconostoc mesenteroides. MIC by MTT assay results of r-Bacs. WT: wild type B. subtilis DB403; Blank: growth medium only; Positive control: 50 µg/mL ampicillin in growth medium with bacteria; Negative control: growth medium with bacteria; each done in triplicate. Fig. 5. Bioactivity of r-Bacs against Micrococcus luteus. Single and combined r-Bacs were tested with nisin as control. As comparison, fractions of MBF8-1 BLIS were also tested. Fig. 1 Fig. 2 Fig. 3 Fig. 4 A. L. mesenteroides B. - + + + + + + + [Conc.] (µg/mL) 100,000 50,000 [r-Bac1] (µg/mL) - 150 75 37.5 18.8 9.4 4.7 - 25,000 12,500 6,250 [r-Bac2] (µg/mL) - 175 87.5 43.8 21.9 10.9 5.7 - [r-Bac3] (µg/mL) - 150 - 75 37.5 18.8 9.4 4.7 3,125 Blank KM KK Fig. 5