Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821.
Purification and characterization of a novel bacteriocin produced by strain Enterococcus faecium D081821.
Enterococcus faecium D081821, isolated from the traditional Taiwanese fermented food dochi (fermented black beans), was previously found to produce a bacteriocin against Listeria monocytogenes and some Gram-positive bacteria. This bacteriocin, termed enterocin TW21, was purified from culture supernatant by ammonium sulfate precipitation, Sep-Pak C18 cartridge, ion-exchange and gel filtration chromatography. Mass spectrometry analysis showed the mass of the peptide to be approximately 5300·6 Da. The N-terminal amino acid sequencing yielded a partial sequence NH2-ATYYGNGVYxNTQK by Edman degradation, and it contains the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The open reading frame (ORF) encoding the bacteriocin was identified from the draft genome sequence of Enterococcus faecium D081821, and sequence analysis of this peptide indicated that enterocin TW21 is a novel bacteriocin.
Enterococcus faecium D081821 produced a bacteriocin named enterocin TW21, the molecular weight and amino acid sequence both revealed it to be a novel bacteriocin.
A new member of class IIa bacteriocin was identified. This bacteriocin shows great inhibitory ability against L. monocytogenes and could be applied as a natural food preservative.
Bacteriocins are ribosomally synthesized peptides produced by bacteria that are active against the growth of related bacteria (Jack et al. 1995). Bacteriocins produced by lactic acid bacteria (LAB) have attracted special interest as potential safe, alternative food preservatives (Cleveland et al. 2001; Nishie et al. 2012). Numerous bacteriocin-like inhibitory substances (BLISs) have been reported. To confirm a novel bacteriocin, the amino acid sequence and the gene encoding the bacteriocin are required to facilitate the classification. Many of these BLISs have been characterized extensively using biochemical procedures and genetic analyses (Fujita et al. 2007; Nissen-Meyer et al. 2009; Himeno et al. 2012; Iwatani et al. 2012; Knerr and van der Donk 2012).
Bacteriocins produced by enterococci have frequently been reported, and there are numerous researches to investigate these peptides and their potential applications (Foulquié Moreno et al. 2003; García et al. 2004; Yanagida et al. 2005; Molinos et al. 2010; Salvucci et al. 2012). Several attempts have been made to classify LAB bacteriocins. Class I bacteriocins, also called lantibiotics, are produced as precursor peptides which undergo extensive post-translational modifications. Class II bacteriocins are defined as nonlanthionine-containing bacteriocins and are further subdivided into four groups (classes IIa, IIb, IIc and IId) (Eijsink et al. 2002; Nishie et al. 2012). Class IIa bacteriocins are pediocin-like peptides which are characterized by the occurrence of a YYGNGVxC sequence motif in their N-terminal region. Another common characteristic of Class IIa bacteriocins is their strong inhibitory ability against the food pathogen Listeria monocytogenes (Papagianni and Anastasiadou 2009).
In the previous studies, we reported that Enterococcus faecium D081821 and D081833, isolated from the Taiwanese fermented black beans dochi, are capable of producing BLISs (Chen et al. 2006). In addition, inhibitory activities of these BLISs have also been discussed (Chen et al. 2007). Similar to other bacteriocins from enterococci, BLISs from E. faecium D081821 and D081833 also showed great inhibitory activities against Listeria monocytogenes (Lauková et al. 2001; García et al. 2004; Molinos et al. 2008, 2010). However, the amino acid sequences and molecular weight of these BLISs remained unclear in the previous study. The current study describes the purification and analysis of the bacteriocin from strain E. faecium D081821 and discusses its characteristics comparing to other known bacteriocins.
Enterococcus faecium D081821 was grown in a modified GYP medium (containing 1·1% dextrose, 1·5% polypeptone, 0·5% yeast extract, 0·5% sodium acetate and 0·3% (v/v) salt solution, pH 6·8. The salt solution contained 4% MgSO4･7H2O, 0·16% MnSO4･4H2O, 0·2% FeSO4･7H2O and 0·2% NaCl) at 37°C under aerobic condition without shaking (Chen et al. 2007). Listeria monocytogenes ATCC 19111 was used as the indicator strain for inhibitory activity assay and was grown in BHI medium (BD, MD, USA) at 37°C. Inhibitory activity was determined using the agar-well diffusion assay (Srionnual et al. 2007).
Enterococcus faecium D081821 was grown in 1·6 l modified GYP medium at 37°C for 16 h. Cells were removed by centrifugation at 6100 g for 10 min, and the supernatant was collected and then filtered through a 0·45-μm pore-size membrane (Advantec, Tokyo, Japan). Proteins were precipitated with 50% saturated ammonium sulfate under continuous stirring at 4°C for 8 h. The pellets were harvested by centrifugation at 6100 g for 20 min and resuspended in 20% acetonitrile solution. The crude bacteriocin solution was desalted using C18 cartridges (Sep-Pak C18, Waters; prewashed with acetonitrile containing 0·1% trifluoroacetic acid (TFA), conditioned with 0·1% TFA) and eluted in 50% acetonitrile with 0·1% TFA (Yanagida et al. 2005). Eluted bacteriocin fractions were freeze-dried and stored at −20°C for further study.
The freeze-dried crude bacteriocin sample was resuspended in 50 mmol l−1 sodium phosphate buffer (buffer A, pH5·7). The supernatant was filtered (0·22 μm filter, Advantec, Tokyo, Japan) and loaded onto a cation-exchange column at 2 ml/min (SP-Sepharose Fast Flow cation-exchange Column; GE Healthcare Life Sciences, Piscataway, NJ, USA) that was preconditioned with buffer A (pH5·7). The column was washed with 5 column volumes of buffer A after supernatant was loaded onto the column. Bacteriocin was eluted in a gradient of 0·8 mol l−1 NaCl in the same buffer (buffer B). Eluted fractions were collected and tested for activity against L. monocytogenes ATCC 19111. Active fractions were collected, desalted and concentrated using C18 cartridges (Sep-Pak C18, Waters, Milford, MA, USA). Bacteriocin fractions were eluted in 50% acetonitrile with 0·1% TFA, freeze-dried and stored at −20°C. Active peptides were subsequently placed on a size exclusion column (5 μm, 300 × 8 mm, ReproSil 50 SEC, Dr. Maisch, Germany) integrated into a high-performance liquid chromatography (HPLC) system (L-2130, L-2450, Hitachi, Tokyo, Japan). Detection of peptides was monitored by a UV detector at 200 nm. Antimicrobial activity of detected peaks was tested using agar-spot diffusion assay.
The molecular size of the purified bacteriocin was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), following the method described by Srionnual et al. (2007). A 4–20% Mini-Protean TGX precast gel (Bio-Rad Laboratories, Hercules, CA, USA) was used, and BLIS molecular size was estimated using Precision Plus Protein Dual Xtea Standards (Bio-Rad).
The molecular mass of the purified bacteriocin was determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) using a mass spectrometer (SCIEX QSTAR Elite; Applied Biosystems, Foster City, CA, USA).
The active peak obtained from the HPLC analysis was further subjected to N-terminal amino acid sequencing using an ABI Procise 494 protein sequencer (Applied Biosystems).
Sequence comparison was carried out using the N-terminal sequence of enterocin TW21 against the draft genome of Enterococcus faecium D081821 (unpublished) to reveal the open reading frame (ORF) encoding the putative structural gene. Homology search was performed using NCBI BLAST (http://blast.ncbi.nlm.nih.gov/). The nucleotide sequence is available in GenBank under the accession number JX880073.
In the purification step of cation-exchange chromatography, compounds with bacteriocin activity were eluted between 0·48 and 0·62 mol l−1 of NaCl. In this step, 15 active fractions were collected. After desalting using the C18 cartridges (Sep-Pak C18; Waters), these active fractions were further purified using the size exclusion column. A separated peak with bacteriocin activity was observed at 12·8 min. Fraction of this peak was collected and used for further study (Fig. 1).
The molecular size of purified bacteriocin from E. faecium D081821 was analysed by SDS-PAGE, and the specific band associated with the antibacterial activity was approximately 5–10 kDa (Fig. 2). The purified bacteriocin was further analysed by MALDI-TOF-MS, and the result showed a major peak at 5300·6 Da (Fig. 3). N-terminal amino acid analysis of the purified bacteriocin revealed the following partial sequence: NH2-ATYYGNGVYxNTQK.
Using the N-terminal sequence of the purified bacteriocin to search against the draft genome of Enterococcus faecium D081821 (unpublished), we are able to reveal the nucleotide sequences encoding the structural gene for this bacteriocin and its flanking region. The structural gene is preceded by a promoter region, including the putative -10 box (TATAAT) and -35 box (TTGTAA) and ribosomal binding site (GGAGG). The sequence presented in this article has been submitted to GenBank and assigned the accession number JX880073 (Fig. 4).
The deduced bacteriocin comprised 76 amino acid residues in the full-length precursor peptide and 48 residues in the mature peptide. The molecular weight of the 48 amino acids was calculated using the tool Compute pl/Mw in the ExPASy Proteomics Server (http://web.expasy.org/compute_pi/), and a result of 5302·98 Da was obtained.
A corresponding sequence (Accession No.: ZP_03980284) was found in the NCBI database (http://blast.ncbi.nlm.nih.gov/). The sequence was generated as a part of the whole-genome sequence project of Enterococcus faecium TX1330, isolated from the healthy human stool (Qin et al. 2012). However, it was marked as a putative bacteriocin-related but function unknown protein. In addition, bacteriocin from E. faecium D081821 showed similarity to several known bacteriocins which included enterocin SE-K4 (Eguchi et al. 2001), durancin GL (Du et al. 2012) and hiracin JM79 (Sánchez et al. 2007) (Fig. 5). When comparing the bacteriocin sequences, three different amino acid residues were observed between the bacteriocin from E. faecium D081821 and enterocin SE-K4 and at least 20 amino acid substitutions were observed in durancin GL and hiracin JM79 (Fig. 5). Based on the results described above, we suggest that E. faecium D081821 produces a novel bacteriocin and have termed it enterocin TW21.
The data obtained in the current study indicated that enterocin TW21 includes a conserved YYGNGVxC motif in its N-terminal region. Additionally, another feature of Class IIa bacteriocins is their strong inhibitory ability against the food pathogen L. monocytogenes. It has been previously reported that strain E. faecium D081821 showed strong inhibitory activity against L. monocytogenes, Clostridium perfringens and Sporolactobacillus kofuensis (Chen et al. 2007). Therefore, enterocin TW21 is classified as a typical Class IIa bacteriocin.
Genetic analysis suggests that a 76 residues precursor peptide is post-translational modified into a 48 residues mature peptide. The results of SDS-PAGE analysis indicated that the specific band associated with the antibacterial activity was approximately 5–10 kDa, which corresponded to those reported previously (Chen et al. 2007). The calculated molecular weight of the 48 amino acids was 5302·98 Da, which corresponded to the observed molecular weight of 5300·6 Da by MALDI-TOF MS. However, the details of the precursor peptide processed into the mature bacteriocin are still unknown.
BLAST searches of NCBI databases for enterocin TW21 showed diversity to other known LAB bacteriocins. Comparing the mature peptides after the post-translational modification, enterocin TW21 is most closely related to enterocin SE-K4, with one threonine to lysine and two valine to isoleucine substitutions. The sequence of enterocin TW21 displays some similarity to durancin GL and hiracin JM79, especially near the N-terminal of the peptides (Fig. 5).
In conclusion, the evidence presented in this report indicates that the dochi-isolated E. faecium D081821 produces a novel bacteriocin, which we have named enterocin TW21. Enterocin TW21 has a narrow inhibitory spectrum against other LAB but shows great inhibitory ability against L. monocytogenes (Chen et al. 2007). The application of specific bacteriocin, or the producer, could result in improvement of food preservation. Future work in our laboratory will focus on the possibility of applying it as a biopreservative.
The authors would like to thank Mr. Jang-Hau Lian for his technical assistance during the amino acid sequence analyses and Yourgene Bio Science for the assistance in Next Generation Sequencing.