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Beneficial Microbes, 2015; 6(4): 603-613

Plantaricin IIA-1A5 from Lactobacillus plantarum IIA-1A5 displays bactericidal activity http://www.wageningenacademic.com/doi/pdf/10.3920/BM2014.0064 - Monday, October 30, 2017 5:30:52 AM - Göteborgs Universitet IP Address:130.241.16.16

against Staphylococcus aureus I. Isnafia Arief1*#, C. Budiman1,2,3#, B. Sri Laksmi Jenie4, E. Andreas1 and A. Yuneni1 1Department of Animal Production and Technology, Faculty of Animal Science, Bogor Agricultural University (IPB), Jl. Agatis,

IPB Darmaga Campus, Bogor 16680, Indonesia; 2Okinawa Institute of Science and Technology, 1919-1 Tancha, Kunigami, Onna son, Okinawa 904-0495, Japan; 3Biotechnology Research Institute, Universiti Malaysia Sabah (UMS), Jl. UMS 88400, Kota Kinabalu, Sabah, Malaysia; 4Food Microbiology Laboratory, Department of Food Science and Technology, Faculty of Agricultural Engineering and Technology, Bogor Agricultural University (IPB). P.O. Box 220, Bogor 16680, Indonesia; [email protected]; # both authors contributed equally to this work Received: 14 May 2014 / Accepted: 16 December 2014 © 2015 Wageningen Academic Publishers

RESEARCH ARTICLE Abstract Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5 isolated from Indonesian beef. This research aimed to identify the genes involved in plantaricin IIA-1A5 production and examine its mode of action against Staphylococcus aureus. It has been reported that a bacteriocin structural gene, plnW, is present in genome of L. plantarum IIA-1A5. Here, we reported the presence of additional genes responsible for plantaricin precursor (plnA and plnEF) and a gene encoding the quorum sensor of histidine kinase (plnB). It indicates that genes involved in production of plantaricin IIA-1A5 are organized in at least two bacteriocin operons (plnABCD, plnEFI) and a structural plnW gene. Purified plantaricin IIA-1A5 yielded a single band in SDS-PAGE with apparent size of 6.4 kDa. Amino acid composition of purified plantaricin IIA-1A5 was mainly composed of cationic glutamic acid and cysteine that allowed the formation of disulphide bonds, suggesting plantaricin IIA-1A5 belongs to the pediocinsubclass of class II bacteriocins. Plantaricin IIA-1A5 displayed remarkable antibacterial activity against S. aureus, which was initiated by the adsorption of plantaricin IIA-1A5 onto the cell membrane of S. aureus. The adsorption is hypothesised to be facilitated by non-ionic interactions as it is reduced by the presence of organic solvents or detergents. This adsorption promoted leakage of cellular metabolites through the cell membrane of S. aureus, as indicated by the release of genetic and proteinaceous material of S. aureus observed at 260 and 280 nm, respectively. The leakage also promoted the release of divalent (Ca2+, Mg2+) and monovalent (K+) cations. The release of these intracellular components might be due to pores formed in the cell membrane of S. aureus by plantaricin IIA-1A5 as shown by scanning electron microscopy. Altogether, the mode of action of plantaricin IIA-1A5 against S. aureus seems to be bactericidal as indicated by lysis of the cell membrane. Keywords: bacteriocin, plantaricin IIA-1A5, Lactobacillus plantarum, mode of action, bactericidal

1. Introduction The increase of consumer awareness on food safety has attracted researchers to meet the need in producing safe foods. Nowadays, the trends of consumer demand on food safety include, among others, fresher food, lower salt and no synthetic preservatives. Natural preservatives are considered good alternative to synthetic ones. Therefore, exploration of natural preservative should be done to

fulfil this need. Lactic acid bacteria (LAB) have been reported to produce potential antimicrobial substances known as bacteriocins. Nisin is an example of a bacteriocin produced by Lactococcus lactis which has been successfully commercialised as natural food preservative. Bacteriocins are ribosomally synthesised antimicrobial peptides or proteins (Masuda et al., 2012). The bacteriocins from LAB have attracted significant attention because of

ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2014.0064603

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I. Isnafia Arief et al.

their potential use as non-toxic and safe additives for food preservation and prevention of food spoilage by foodborne pathogenic bacteria (Bauer et al., 2005; Hata, et al., 2010; Rashid et al., 2009; Savadogo et al., 2004). The bacteriocins of LAB have been classified into four classes, with most of the bacteriocins belonging to class I or II (Klaenhammer, 1993; Savadogo et al., 2006; Tiwari and Srivastava, 2008). Most bacteriocins are produced as protein precursors, in which their N-terminal leader peptides are removed during secretion, yielding mature bacteriocin peptides. Recently, two novel types of class II bacteriocins have been reported to possess unique characteristics. One class is composed of circular bacteriocins that contain a head-to-tail structure in the mature form, and the other is a leaderless bacteriocin without an N-terminal extension in the precursor peptide (Masuda et al., 2012). Several strains of Lactobacillus plantarum have been reported to produce bacteriocins, usually called plantaricins (El-Naggar, 2004; Holo et al., 2001; Navarro et al., 2000), although not in all cases (Essid et al., 2009). Explorations of plantaricins have been done by many researchers from many countries (Ben-Omar et al., 2006; Hata et al., 2010; Holo et al., 2001; Ogunbanwo et al., 2003; Sáenz et al., 2009; Todorov and Dicks, 2005; Todorov et al., 2010). However, to our knowledge, there is no report so far on isolation and application of plantaricins from Indonesian isolates. Suarsana et al. (2001) isolated a bacteriocin from Streptococcus lactis in Indonesia, however, this is not classified as a plantaricin. Indonesian L. plantarum IIA-1A5 was previously isolated from Indonesian cattle, Peranakan Ongole, and was shown to produce a bacteriocin, called plantaricin IIA-1A5 (Arief et al., 2013). It displays remarkable antimicrobial activity against pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Bacillus cereus and Salmonella typhimurium. Further characterisation showed that it was heat stable at 80 °C and 121 °C for 30 and 15 min, respectively, and digestible by trypsin. Therefore, plantaricin IIA-1A5 showed promising function as an antimicrobial substance (Arief et al., 2013). However, the mode of action of this plantaricin against pathogenic bacteria remained unknown. The genes for production of plantaricin precursor in some strains were reported to be organised in at least five operons, plnABCD, plnEFI, plnJKLR, plnGHSTUV, and plnMNOP (Ben-Omar et al., 2006; Diep et al., 1996), and three structural genes of plantaricin were reported: plantaricin NC8, plantaricin S, and plantaricin W (Holo et al., 2001; Maldonado et al., 2003; Stephens et al., 1998). The genes of the operons are not only plantaricin precursors, but also other proteins involved in plantaricin production, including: quorum sensor of histidine kinase (HK), bacteriocin immunity proteins, ABC transporters and other accessory proteins (Diep et al., 1996). While the structural gene of plantaricin W has been identified 604

in L. plantarum IIA-1A5 (Arief et al., 2013), we have not confirmed the presence of other genes encoding plantaricin IIA-1A5 precursors and other accessory proteins involved in plantaricin IIA-1A5 production. The objectives of this research were to identify the genes involved in plantaricin IIA-1A5 production and their organisation in chromosomal DNA of L. plantarum IIA-1A5 and to determine the mode of action of plantaricin IIA-1A5 against pathogenic bacteria.

2. Materials and methods Isolation of plantaricin genes The screening and amplification of the plantaricin genes from genomic DNA of strain IIA-1A5 was performed according to Holo et al. (2001) and Sáenz et al. (2009). Briefly, total genomic DNA was isolated from 10 ml of overnight culture grown at 37 °C in De Man, Rogosa and Sharpe (MRS; Oxoid, Basingstoke, UK) broth according to Sambrook et al. (1989). The target genes for identifications were the three genes encoding plantaricin precursor that were reported to be located in three different operons: plnA, plnEF, and plnJ from plnABCD, plnEFI, and plnJKLR operons, respectively (Ben-Omar et al., 2006; Diep et al., 1996). In addition, we also targeted a gene encoding histidine kinase protein (plnB) that has been reported to be located in plnABCD operon (Ben-Omar et al., 2006; Diep et al., 1996), as a representative of non-plantaricin precursor genes in the operon system. The amplifications were performed through PCR using Taq DNA polymerase (GoTaq PCR core system, Promega, Madison, WI, USA) with primers designed according to Ben-Omar et al. (2006) (Table 1), and the protocol as provided by Promega. PCR products were separated by electrophoresis on a 2% (w/v) agarose gel, which was stained with ethidium bromide and visualised using UV light.

Purification of plantaricin Plantaricin was purified based on modified methods of Tiwari and Srivastava (2008) and Hata et al. (2010). L. plantarum IIA-1A5 was grown in MRS broth, supplemented with 3% yeast extract at 37 °C without shaking for 20 h. Cells were removed by centrifugation (10,000 rpm for 20 min, 4 °C), followed by filter-sterilisation (0.2 µm cut-off membrane) of the supernatant. The bacteriocin was purified from the cell-free supernatant by ammonium sulphate precipitation followed by cationexchange chromatography as described below:

Ammonium sulphate precipitation Ammonium sulphate was added to 90% saturation at 4 °C under constant stirring, after which the mixture was stirred for a further 2 h at 4 °C. The protein precipitate was Beneficial Microbes 6(4)

Bactericidal activity of plantaricin IIA-1A5 against Staphylococcus aureus



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Table 1. Primer sequences used for amplification of genes responsible for plantaricin production. Target

Primer

Sequence (5 → 3)

References

plnA

forward reverse forward reverse forward reverse forward reverse

GTA CAG TAC TAA TGG GAG CTT ACG CCA ATC TAT ACG TTC AGA GCA AGC CTA AAT GAC GCC ACT GTA ACA CCA TGA C GGC ATA GTT AAA ATT CCC CCC CAG GTT GCC GCA AAA AAA G TAA CGA CGG ATT GCT CTG AAT CAA GGA ATT ATC ACA TTA GTC

Ben-Omaret al., 2006; Diep et al., 1996, Remiger et al., 1996

plnB plnEF plnJ

centrifuged at 20,000×g for 30 min at 4 °C. The resulting pellet was re-suspended in 20 mM sodium phosphate buffer, pH 6.0 (SPB). The samples were desalted by dialyzing (2.0 kDa cut-off membrane) against SPB prior to cationexchange chromatography.

Cation-exchange chromatography Samples were applied at a flow rate of 1 ml/min to a SP sepharose fast flow cation-exchange column (GE Healthcare, Pittsburgh, PA, USA) equilibrated with SPB. The column was washed with 4 bed volumes of SPB. Protein elution was performed by employing a 0 to 500 mM sodium chloride linear gradient and monitored at 280 nm with a UV VIS spectrophotometer (GeneQuant Pro, Cambridge, UK) to detect the presence of proteins in each fraction. Protein containing fractions were collected and tested for bacteriocin activity and further analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).

SDS-PAGE electrophoresis In order to determine the molecular mass and confirm the purity, selected fractions were pooled and analysed by SDS-PAGE (Bio-Rad, Hercules, CA, USA) (Laemmli, 1970) using 15% polyacrylamide gel, followed by staining with Coomassie Brilliant Blue R250 (Sigma, St. Louis, MO, USA). Protein concentrations of purified plantaricin IIA-1A5 were determined based on the Lowry method (Lowry et al., 1951).

Amino acid composition Amino acid composition of plantaricin IIA-1A5 was determined by hydrolysing the peptide into free amino acids by heating it in 6 N HCl at 110 °C for 24 h. The liberated amino acids were determined using liquid chromatography carried out on an Agilent 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA). Amino acids were separated using an ZORBAX SB RRHT C18 column (21×50 mm) with a 1.8 µm particle size (Agilent Technologies). Buffer A was 0.5 mM tridecafluoroheptanoic acid in HPLCBeneficial Microbes 6(4)

Ben-Omaret al., 2006; Diep et al., 1996 Ben-Omaret al., 2006; Diep et al., 1996; Anderssen et al., 1998 Ben-Omaret al., 2006; Diep et al., 1996; Anderssen et al., 1998

grade water, and buffer B was 100% acetonitrile. The initial flow rate was 2.4 ml/min. Separation was accomplished using a gradient recommended by the company (Agilent Technologies) maintained at 95 °C under 550 bar. The chromatograph was detected using the Agilent 1100 series diode-array detector SL, 80 Hz data rate.

Effects of organic solvents and detergents on adsorption of plantaricin IIA-1A5 to Staphylococcus aureus cells This assay was performed based on Atrih et al. (2001) with slight modifications. Cells of S. aureus ATCC 25923 were grown in Nutrient Difco medium (BD, Franklin Lakes, NJ, USA) for 24 h at 37 °C, centrifuged as above for L. plantarum and finally suspended in 1% sodium dodecyl sulphate (SDS), 2% Triton X-100, and heated at 60 °C for 15 min. The suspensions were centrifuged again as above, followed by three times pellet washing with 5 mM phosphate buffer (pH 7.2). To test the effect of organic solvents on subsequent adsorption of plantaricin IIA-1A5, cells were suspended in methanol, ethanol, butanol, hexane or chloroform, all used at 80% (v/v) in water. The mixtures were incubated at 30 °C for 1 h and the solvents were removed by centrifugation and drying; the residual pellets were washed and thereafter resuspended in 5 mM phosphate buffer (pH 7.0). Suspensions obtained after detergent or organic solvent treatments were mixed with plantaricin IIA-1A5 (20 AU/ml) and incubated at 30 °C for 30 min. After centrifugation, the residual (unbound) plantaricin IIA1A5 was measured based on Yang et al. (1992). The results were compared to the controls of cells without detergent or organic solvent treatment in the presence of plantaricin IIA-1A5. The adsorption of plantaricin IIA-1A5 in control was set to 100% for comparison.

Effect of organic solvents, detergents and plantaricin IIA1A5 on leakage of Staphylococcus aureus To examine the lysis ability of plantaricin IIA-1A5 against S. aureus ATCC 25923 over the time of incubation, the following experiment was performed based on Atrih et al. 605

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I. Isnafia Arief et al.

(2001). A total of 10 ml of S. aureus ATCC 25923 culture was centrifuged for 10 min. The pellet was resuspended in 5 ml physiological saline (0.85% NaCl). Plantaricin IIA-1A5 was then added (20 AU/ml) and left for 24 h. For comparison, the same cells without the addition of the plantaricin were used. After the 24 h incubation, the suspension was centrifuged at 10,000 rpm for 10 min, and the supernatant was filtered (0.2 µm). Leakage was observed through absorbance of the supernatant, using a UV-vis spectrophotometer (GeneQuant Pro) at 260 and 280 nm to measure the presence of genetic and proteinaceous materials, respectively, released from the cells. Further analysis on the effect of the presences of organic solvents or detergents treatments on the leakage of S. aureus by plantaricin was based on Atrih et al. (2001) and Padilla et al. (2002) with slight modifications. S. aureus ATCC 25923 cells were prepared and treated with organic solvents and detergents as described above. After a heating treatment at 60 °C for 15 min, the suspension was centrifuged and supernatant was collected. The absorbance of supernatant was monitored by UV-spectrophotometer (GeneQuant Pro) at 260 and 280 nm to measure the presence of genetic and proteinaceous materials, respectively, released from the cells.

Analysis of inorganic components (Ca2+, Mg2+ and K+) from Staphylococcus aureus The experiment was performed based on Pham et al. (2004) with slight modifications. Suspension of S. aureus ATCC 25923 cells obtained as described above was incubated with plantaricin IIA-1A5 up to 20 AU/ml final concentration for 24 h at 37 °C, and then centrifuged. Pellets were suspended in 10 ml deionised water and 1 ml HNO3 was added for determination of total ion Ca2+, Mg2+ and K+ by the use of an atomic absorption spectrometer (Perkin-Elmer 3100; Perkin-Elmer Optoelectronics, Fremont, CA, USA). Each treatment was performed in triplicate with suspension of S. aureus cells in the absence of plantaricin IIA-1A5 as a control.

Analysis of changes in bacterial cell morphology by scanning electron microscopy Analysis of cell morphology changes were performed to study changes in cell structure due to the interaction with plantaricin IIA-1A5. At first, the bacteria were suspended with plantaricin IIA-1A5 (20 AU/ml), and then incubated on a rocking incubator at 100 rpm. Subsequently, the liquid was centrifuged and the supernatant removed, after which the pellet was fixed with 2% (w/v) glutaraldehyde in a filtersterilised 0.1 M sodium cocadylate buffer (pH 7.4) at room temperature for 2 h and then rinsed 3 times for 15 min in a 0.15 M sodium cacodylate buffer (pH 7.4). Post fixation step was performed for 1 h with 0.2% (w/v) OsO4 in a 0.1 606

M sodium cacodylate buffer, followed by a quick rinse in distilled water. The samples were dehydrated with a graded ethanol series, including en bloc staining with 3% uranyl acetate in 30% ethanol, and then air dried. For washing and dilution of fixing reagents, 0.15 M sodium phosphate buffer (pH 7.2) was used. The dried sample was then observed by using a JEOL scanning electron microscope JSM 5300 LV (JEOL, Peabody, MA, USA) at 20,000 magnification as described before (Hartmann et al., 2010).

Statistical analysis The data are presented as mean ± standard deviation from at least triplicate of experiments. The data were analysed by analysis of variance (Anova) under complete randomised experimental design. Significant differences were tested using Tukey`s HSD test (Steel and Torie, 1995).

3. Results and discussion Identification of genes involved in production of plantaricin IIA-1A5 Previously, we have identified the presence of the structural gene for plantaricin W, plnW, in chromosomal DNA of L. plantarum IIA-1A5 (Arief et al., 2013). In this work, we observed the presence of genes encoding plantaricin precursors, plnA and plnEF of plnABCD and plnEFI operons, respectively, in the chromosomal DNA (Figure 1). The amplicons showed that the apparent size of plnA and plnEF amplicons were approximately 450 bp, which are comparable to those previously reported (Sáenz et al., 2009). This result might imply that at least three genes encoding plantaricin precursors are present in the chromosomal DNA, including plnA, plnEF and plnW. Given that plnA and plnEF genes are located in different operons (Ben-Omar et al., 2006; Diep et al., 1996), our result might indicate that the genes involved in bacteriocin production in L. plantarum IIA-1A5 are organised at least in two different operons: plnABCD and plnEFI. However, we have not confirmed the presence of gene(s) located in plnGHSTUV and plnMNOP operons. Figure 1 shows the presence of the quorum sensor of histidine kinase (HK) encoded by the plnB gene confirming the presence of the plnABCD operon. The apparent size of plnB is about 175 bp, which is close to that of the amplified plnB reported by Ben-Omar et al. (2006). The role of the HK protein in bacteriocin production is well documented (Brurberg et al., 1997; Diep et al., 1995; Eijsink et al., 1996); basically it senses the pheromone peptide, so-called ‘inducing peptide’ (IP), triggering an autoinduction loop, resulting in the increased transcription of genes involved in bacteriocin production. The presence of plnB implies that L. plantarum IIA-1A5 possesses an autoinduction mechanism involving a secreted peptide pheromone encoded by a gene in its chromosomal DNA. Complete identification of genes in Beneficial Microbes 6(4)

Bactericidal activity of plantaricin IIA-1A5 against Staphylococcus aureus



plnB A

plnA B

A

B

plnEF A

B

plnJ A

B

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100 bp 500 bp 300 bp 100 bp

Figure 1. Amplification of precursor plantaricin genes (plnB, plnEF, plnA, plnJ) in Lactobacillus plantarum IIA-1A5. The amplifications were performed in two independent reactions (A and B). The marker (M) lane is shown as reference of the amplicon size.

all operons involved in bacteriocin production is needed to find the exact gene. The absence of amplified plnJ fragments in our result might suggest three following possibilities: 1. plnJKLR is absent in the genomic DNA of L. plantarum IIA-1A5. This possibility was shown in L. plantarum 2.4.1, 2.6.1 and 3.9.1, which were found to contain no plnJKLR operon in their genomes (Ben-Omar et al., 2006). 2. The operon is present but without the plnJ gene. In this respect, this operon might be responsible solely for production of immunity proteins (Diep et al., 1996). L. plantarum 2.9 has previously been shown to carry the plnK gene, but not plnJ. 3. The plnJ gene is replaced by another gene encoding a different type of bacteriocin. The operon then still produces bacteriocin in addition to immunity proteins. These assumptions, however, need to be more clarified by further experiments.

Plantaricin IIA-1A5 was precipitated with 90% saturated ammonium sulphate from the cell free culture supernatant. Plantaricin IIA-1A5 containing-fractions from ammonium precipitation were then applied to ion exchange column chromatography. Plantaricin binds strongly to cationic resin at pH 6.8. The total amount of purified plantaricin IIA-1A5 using SP Sepharose fast flow binding was 4.5 mg (from 1 litre of culture). This yield is considerably higher to that of plantaricin ASM1 (0.7 mg) and plantaricin LR14 (59.21 µg) (Hata et al., 2010; Tiwari and Srivastava, 2008). This discrepancy in yield of plantaricins is likely to be caused by differences in L. plantarum strains. Functional characteristics of antibacterial activity of plantaricins produced by L. plantarum depend on the strains used (Sáenz et al., 2009).

Purification of plantaricin

A single band in SDS-PAGE was obtained after SP-sepharose chromatography (Figure 2), indicating that a two-step protocol is sufficient to purify plantaricin IIA-1A5, a protein with an molecular weight of approximately 6.4 kDa (Figure 2). This is similar to plantaricin LR that has a molecular weight of 6.2 kDa (Tiwari and Srivastava, 2008), whereas MALDI-TOF MS analysis of plantaricin ASM1 gave a molecular mass of 5.046 kDa (Hata et al., 2010). Isolation of plantaricin 423 with Amberlite XAD-1180 and separation on a tricine-SDS-PAGE gel yielded an active peptide band with apparent molecular weight of 3.5 kDa (Van-Reenen et al., 1998). These results indicate that different strains of L. plantarum may produce plantaricins with different characteristics. Based on our results of the plantaricin genes (Figure 1) and molecular weight (Figure 2), plantaricin IIA1A5 has been included in the class II bacteriocins.

Many reports have attempted to purify plantaricin with at least four-step purifications methods. In this work, we employed a two-step protocol: ammonium sulphate precipitation followed by cation exchange column chromatography for purification of plantaricin IIA-1A5 from culture medium.

Given an average molecular weight of 110 Da for each of the amino acid, 6.4 kDa of plantaricin IIA-1A5 corresponds to a peptide composed of 58-59 amino acids. The gene size for expression of a peptide composed of 58-59 amino acids should be around 174-177 bp. This calculated size (174-177 bp) is remarkably lower to the apparent sizes (amplicon size

Given that plnJ encodes a plantaricin precursor, the absence of plnJ at least eliminates the possibility that the precursor for plantaricin IIA-1A5 is encoded by plnJ. In this respect, the plantaricin IIA-1A5 precursor might be encoded by plnA, plnEF, or plnW genes. If the plnJKLR operon is really absent in the chromosomal DNA of L. plantarum IIA1A5, the gene responsible for immunity response of L. plantarum IIA-1A5 towards its bacteriocin is likely located in different operons.

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kDa

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100

30 25 20 15

experiment also cover partial sequences of another gene in the operon. The primers used in this experiments were designed based on previous reports (Ben-Omar et al., 2006) which are used solely for identification of the presence of the genes in bacteriocin operons. Altogether, it is not feasible to identify the gene responsible for production of plantaricin IIA-1A5 solely based on the amplicon size. Further confirmation is necessary through analysis of the complete gene sequence and N-terminal sequence of the expressed protein band.

Amino acid composition of plantaricin IIA-1A5 10

5

Figure 2. SDS-PAGE (15%) and Coomassie Blue staining of purified plantaricin IIA-1A5 (~5 mg/ml) obtained after SPsepharose chromatography. The band corresponds to the migration of plantaricin IIA-1A5 is indicated by the arrow. Lines representing low molecular weight proteins markers in kDa show the apparent size of plantaricin IIA-1A5.

in the gel) of plnA or plnEF (about 450 bp), but comparable to the apparent size of the plnW gene, which is about 200 bp (Arief et al., 2013). However, the apparent size of plnA, plnEF, and plnW are believed to be different to their real sizes because the primers used in our experiment might not cover the start- and stop-codons of each gene. The real size of all these genes should be larger than 450 bp (plnA and plnEF) or 200 bp (plnW). Therefore, despite that the apparent size of plnW (~200 bp) is close to the calculated gene size for the plantaricin IIA-1A5 peptide (174-177 bp), it does not necessarily imply that plantaricin IIA-1A5 is encoded by plnW. It should be noted that bacteriocin precursors have frequently been reported to include N-terminal leader sequences (Maldonado et al., 2003; Diep et al., 1996), although leaderless bacteriocins have also been found (Masuda et al., 2012). Assuming plantaricin IIA-A5 has a N-terminal leader sequence, the real size of precursor of plantaricin IIA-1A5 is possibly larger than 6.4 kDa (>5859 amino acids). Based on the amplicon size, 450 bp of plnA and plnEF genes will be translated into a peptide with minimum size of 16.5 kDa, while ~200 bp of plnW gene yields at least a 7.5 kDa peptide. 16.5 kDa is considerably larger compared to the common size of bacteriocin, which is less than 10 kDa, with the exception of with the exception of the 122 kDa of bacteriocin from L. plantarum ATCC 8014 (Lash et al., 2005). It is suggested that plantaricin IIA1A5 might have a N-terminal leader sequence of unknown size. There is a possibility that the primers used in this 608

PCR analysis revealed that at least three genes encoding precursor plantaricins are present in the chromosomal DNA of L. plantarum IIA-1A5. In addition, mature plantaricin IIA-1A5 has been purified to give a single band in SDS-PAGE. However, neither the genes nor the mature plantaricin IIA-1A5 have been completely sequenced. In the absence of the information of its gene and N-terminal amino acid sequences, we have determined the amino acid composition of plantaricin IIA-1A5 by using HPLC. The amino acids found were proline, leucine, tyrosine, cysteine, glutamic acid, and valine at 127.16, 28.77, 161.51, 743.20, 903.38, and 206.18 mg/kg, respectively. The high glutamic acid-content suggests plantaricin IIA-1A5 to be a cationic peptide, typical for class II bacteriocins. In addition, the high cysteine-content allows plantaricin IIA1A5 to form disulphide bonds among the cysteine residues. The presence of disulphide bonds is known to be unique amongst the ‘pediocin’-subclass of class II bacteriocins, and is responsible for the heat stability of class II bacteriocins.

Antimicrobial activity of plantaricin IIA-1A5 against Staphylococcus aureus Antimicrobial activity of plantaricin IIA-1A5 against S. aureus was examined by zone inhibition test (Table 2). Plantaricin IIA-1A5 displayed antibacterial activity against S. aureus although this was lower than that of commercially available antibiotic amoxillin. Savadogo et al. (2006) stated that, generally, bacteriocins are cationic and amphifilic peptides, and attracted to the negatively charged bacterial membrane. Several different plantaricins that have been characterised were shown to inhibit a wide range of Gram-positive bacteria (Diep et al.,2009; Gong et al., 2010; Todorov et al.,2014). Therefore, we also tested the adsorption of plantaricin IIA-1A5 onto S. aureus cells surface.

Effects of detergents, organics solvents on adsorption of plantaricin IIA-1A5 onto Staphylococcus aureus Bacteriocin adsorption is a critical step in the initiation of lethal interaction between the peptides and sensitive cells. Following adsorption of bacteriocin molecules Beneficial Microbes 6(4)

Bactericidal activity of plantaricin IIA-1A5 against Staphylococcus aureus



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Table 2. Diameter of inhibition zone of plantaricin IIA-1A5 against Staphylococcus aureus.1 Treatments

Diameter of inhibition zone (mm)2

Control Plantaricin IIA-1A5 Amoxillin

0.00±0.00a 10.39±1.80b 26.43±3.50c

1

Value represent mean ± standard deviation (n=9).

2 Means in column with different superscript differ significantly (P

Plantaricin IIA-1A5 from Lactobacillus plantarum IIA-1A5 displays bactericidal activity against Staphylococcus aureus.

Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5 isolated from Indonesian beef. This research aimed to identify the ge...
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