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Lactococcin Q, a Novel Two-Peptide Bacteriocin Produced by Lactococcus lactis QU 4

Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture,¹ Department of Functional Metabolic Design, Bio-Architecture Center, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan,² Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan³

Received 8 September 2005/ Accepted 28 February 2006

	ABSTRACT

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A bacteriocin-producing strain, Lactococcus lactis QU 4, was isolated from corn. The bacteriocin, termed lactococcin Q, showed antibacterial activity only against L. lactis strains among a wide range of gram-positive indicator strains tested. Lactococcin Q was purified by acetone precipitation, cation exchange chromatography, and reverse-phase chromatography. Lactococcin Q consisted of two peptides, and ß, whose molecular masses were determined to be 4,260.43 Da and 4,018.36 Da, respectively. Amino acid and DNA sequencing analyses revealed that lactococcin Q was a novel two-peptide bacteriocin, homologous to lactococcin G. Comparative study using chemically synthesized lactococcin Q (Q plus Qß) and lactococcin G (G plus Gß) clarified that hybrid combinations (Q plus Gß and G plus Qß) as well as original combinations showed antibacterial activity, although each single peptide showed no significant activity. These four pairs of lactococcin peptides acted synergistically at a 1:1 molar ratio and exhibited identical antibacterial spectra but differed in MIC. The MIC of Q plus Gß was 32 times higher than that of Q plus Qß, suggesting that the difference in ß peptides was important for the intensity of antibacterial activity.

	INTRODUCTION

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Some strains of lactic acid bacteria (LAB) produce ribosomally synthesized antimicrobial peptides or proteins, termed bacteriocins, which generally inhibit the growth of closely related strains. Because of their potential use as food preservatives and pharmaceuticals, LAB bacteriocins have been widely explored and studied in recent years (3).

On the basis of their structures, LAB bacteriocins are classified mainly into two groups: lantibiotic (class I) and nonlantibiotic (class II) (13). Class I bacteriocins contain posttranslationally modified unusual amino acids, like lanthionine and dehydrated amino acids. In contrast, class II bacteriocins comprise only unmodified amino acid residues. Class II bacteriocins are further classified into three subclasses, IIa, IIb, and IIc, by their detailed characteristics (21, 22).

Most LAB bacteriocins were found as single active peptides, but some of them were found as two-peptide systems (9). Each two peptides are encoded by adjacent open reading frames (ORFs) in the same operon, transcribed, and synthesized together. They are fully active generally in consequence of the synergistic action of the two peptides, whereas each peptide alone has no activity or less activity. In all cases studied so far, the optimal combination ratio of the peptides is approximately 1:1. Two-peptide bacteriocins are found in both lantibiotics and unmodified bacteriocins. Unmodified two-peptide bacteriocins are classified into class IIb, although two-peptide lantibiotics have not been categorized into a defined class. Recently many class IIb bacteriocins have been discovered from LAB: e.g., ABP-118 (6), plantaricin NC8 (14), and enterocin 1071 (2, 7).

The first reported one was lactococcin G produced by Lactococcus lactis subsp. lactis LMG 2081 (23). Its activity, structure, and mode of action have been examined extensively. It was reported that lactococcin G was active against various LAB and clostridia and that the constituents, and ß peptides, acted synergistically at a 1:1 molar ratio. In addition, it was suggested that both peptides of lactococcin G formed amphiphilic -helices and that their complex showed bactericidal activity by forming a transmembrane pore that conducted potassium ions (10, 17, 18). However, the recognition of target cells and partner peptides is still unclear.

Until now, only one homolog of lactococcin G, namely enterocin 1071, produced by Enterococcus faecalis BFE 1071 and E. faecalis FAIR-E 309, has been reported (2, 7). The primary structure of enterocin 1071, consisting of enterocin 1071A and 1071B, was reported firstly by Balla et al. (2) and corrected by Franz et al. (7). Enterocin 1071 inhibited the growth of the wide range of gram-positive bacteria, especially enterococcal strains. In addition, the peptides were presumed to have amphiphilic -helices in their structures as in the case of lactococcin G.

We have screened a variety of plant sources in Japan and isolated various bacteriocin-producing LAB, including 11 strains from corn. Among the new corn isolates, five strains, including Lactococcus lactis QU 4, produced a bacteriocin with a specific antibacterial spectrum. In this study, we described purification, structural analysis, and characterization of a novel two-peptide bacteriocin, named lactococcin Q, produced by L. lactis QU 4.

	MATERIALS AND METHODS

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Bacterial strains and media.
The bacteriocin-producing strain, L. lactis QU 4 was isolated from fresh corn grown in Aso, Kumamoto, Japan. L. lactis QU 4 and the indicator strain, L. lactis subsp. lactis ATCC 19435^T, were stored at –80°C in MRS medium (Oxoid, Basingstoke, United Kingdom) with 15% glycerol and propagated in MRS medium at 30°C for 18 h before use. The other indicator strains for determination of antibacterial spectra were propagated at appropriate temperatures (30 or 37°C) recommended by culture collections for 18 h before use. MRS medium was used to culture LAB strains, except Streptococcus sp. strains. Tryptic soy broth (Difco, Sparks, Md.) supplemented with 0.6% yeast extract (Nacalai Tesque, Kyoto, Japan) was used to culture Streptococcus strains and the other gram-positive indicator strains. Escherichia coli JM109 was grown in LB medium (1) at 37°C. Transformants of E. coli JM109 were selected on LB agar plates containing 50 mg liter^–1 of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside, 40 mg liter^–1 of isopropyl-ß-D-thiogalactopyranoside, and 20 mg liter^–1 of ampicillin.

Identification of strain QU 4.
The sugar fermentation pattern of strain QU 4 was tested by the API 50 CHL system (bioMérieux, Marcy l'Etoile, France). The obtained pattern was analyzed by using the APILAB Plus software (bioMérieux). A partial 16S rRNA gene region of strain QU 4, corresponding to positions 8 to 519 of E. coli 16S rRNA gene, was analyzed as described previously (29).

Determination of bacteriocin activity.
Bacteriocin activity was determined by the spot-on-lawn method as described previously (5, 16) using L. lactis subsp. lactis ATCC 19435^T as an indicator strain unless otherwise mentioned. Briefly, 10 microliters of a bacteriocin preparation were spotted onto a double-layered agar plate comprised of 5 ml of Lactobacilli Agar AOAC (Difco) inoculated with an overnight culture of an indicator strain as an upper layer and 10 ml of MRS medium with 1.2% agar as a lower layer. After overnight incubation at appropriate temperatures for the indicator strains, bacterial lawns were checked for inhibition zones.

Purification of lactococcin Q.
L. lactis QU 4 was grown at 30°C for 12 h in 250 ml of MRS medium, and the culture supernatant was obtained by centrifugation. Proteins including lactococcin Q in the supernatant were precipitated with 750 ml of acetone at –30°C overnight. The pellet obtained by centrifugation at 4°C was dried under vacuum and dissolved in 50 ml of 20 mM sodium phosphate buffer, pH 5.7 (buffer A). This solution was applied at a flow rate of 2 ml min^–1 to a SP-Sepharose cation-exchange column (100-mm length, 15-mm internal diameter; Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with buffer A. After a subsequent washing with 20 ml of buffer A, the active fraction containing lactococcin Q was eluted with a stepwise gradient consisting of 20 ml each of 0.5, 0.75, and 1 M NaCl in buffer A. The active fraction obtained was then applied to a reverse-phase column (Resource RPC 3-ml; Amersham Pharmacia Biotech) incorporated in LC-10A high-performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) and equilibrated with 20% acetonitrile containing 0.1% trifluoroacetic acid (TFA). Lactococcin Q was eluted with a linear gradient ranging from 20 to 50% acetonitrile containing 0.1% TFA for 30 min at a flow rate of 1 ml min^–1. Purified lactococcin Q was stored at –30°C. The antibacterial activity of each fraction in the purification steps was determined as described above.

Mass spectrometry and amino acid sequence analysis of lactococcin Q.
The molecular masses of the purified lactococcin Q peptides were analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) using a Voyager DE-STR mass spectrometer (Applied Biosystems, Foster City, Calif.) in the linear positive-ion mode and -cyano-4-hydroxycinnamic acid (Sigma, St. Louis, Mo.) as a matrix. The N-terminal amino acid sequences of the peptides were determined by Edman degradation using a 476A gas-phase automatic sequencer (Applied Biosystems).

Analysis of the gene encoding lactococcin Q.
DNA polymerases, restriction enzymes, and other DNA modifying enzymes were used as recommended by the manufacturers. The total genomic DNA of L. lactis QU 4 was extracted from the cells treated with lysozyme (Seikagaku, Tokyo, Japan) and labiase (Seikagaku) as described previously (1) and used as a template for PCR. All primers used to analyze the gene encoding lactococcin Q are listed in Table 1. Two degenerate primers, lcnQ1 and lcnQ2, designed from the obtained amino acid sequences of lactococcin Q and Qß, respectively, were used to amplify a fragment corresponding to a part of the gene encoding lactococcin Q. Degenerate PCR was performed using Taq DNA polymerase (Promega, Madison, Wis.) under the condition consisting of denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 51°C for 30 s, and polymerization at 72°C for 1 min. The PCR fragment was cloned using pGEM-T vector systems (Promega) into E. coli JM109 and sequenced with an ALF Express DNA sequencer (Amersham Pharmacia Biotech), according to the manufacturer's instructions. To obtain the region downstream of the lactococcin Q gene, cassette PCR was applied. Two primers, S-1 and S-2, were designed from the DNA sequence obtained by degenerate PCR and used for cassette PCR with a HindIII cassette (Takara Bio, Otsu, Japan) (Table 1) and primers C-1 and C-2 corresponding to the sequence of the HindIII cassette. The total genomic DNA of strain QU 4 was completely digested with HindIII (Fermentas, Hanover, Md.), and the generated DNA fragments were ligated to the HindIII cassette. They were used as templates for PCR (first PCR) using primers S-1 and C-1. PCR was performed with LA Taq DNA polymerase (Takara Bio) under the condition consisting of denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 2 min, and polymerization at 72°C for 1 min. The products of first PCR were then used as templates for nested PCR using primers S-2 and C-2 under the same conditions as were employed for the first PCR. The product of the nested PCR was cloned and sequenced as described above. To obtain the region upstream of the lactococcin Q gene, inverse PCR was applied. The total genomic DNA of strain QU 4 was completely digested with MboI (Fermentas) and self-ligated for use as the template. Inverse PCR, using primers S-2 and lcnQ3, designed from the sequence obtained by the degenerate PCR, was performed with KOD Dash DNA polymerase (Toyobo, Osaka, Japan) under the condition including denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and polymerization at 74°C for 3.5 min. The product obtained by inverse PCR was cloned and sequenced as described above. Based on the sequences obtained from the PCR products, new specific primers were synthesized and used to confirm the obtained DNA sequence, according to the procedure described above.

Peptide synthesis of lactococcins Q and G.
Lactococcin peptides, Q, Qß, G, and Gß, were synthesized by a PSSM-8 automated peptide synthesizer (Shimadzu) as described previously (12). All the synthesized peptides were purified by reverse-phase HPLC, and their molecular masses were confirmed by MALDI-TOF MS as described above. Concentrations of the peptides were determined spectrophotometrically using molar adsorption coefficients at 280 nm (₂₈₀) deduced from amino acid composition of the peptides.

Evaluation of antibacterial activity of synthetic lactococcin peptides.
Synthetic lactococcin peptides were dissolved in 0.05% TFA and diluted by 0.1% Tween 80 solution to appropriate concentrations. Single peptides and mixtures of and ß peptides at various molar ratios were tested for antibacterial activity as described above, and MIC was determined. For analysis of antibacterial spectra, preparations containing 500 nM (each) of synthetic and ß peptides were assayed. The supernatant of an overnight culture of L. lactis QU 4, adjusted to pH 6.0 with 5 M NaOH and filter sterilized, was also assayed as a crude lactococcin Q.

Nucleotide sequence accession number.
The DNA sequence presented in this article has been deposited in the DDBJ database with accession no. AB182406.

	RESULTS

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Identification of strain QU 4.
By the sugar fermentation pattern test using the API 50 CHL system, strain QU 4 was identified as Lactococcus lactis subsp. lactis. The 16S rRNA gene sequence of strain QU 4 showed 99% identity to those of the reference strains of L. lactis. Other characteristics of strain QU 4, such as being a gram positive diplococcus, being catalase negative, having growth at 10°C and no growth at 45°C, and producing homo L-lactic acid from glucose, also agreed well with the criteria of L. lactis. Considering these results, we concluded that strain QU 4 belonged to L. lactis.

Purification of lactococcin Q.
Lactococcin Q was purified by a three-step procedure including acetone precipitation, cation exchange, and reverse-phase HPLC. Most of the activity in the culture supernatant was recovered by acetone precipitation. In cation exchange chromatography, the bacteriocin activity was mainly recovered in a 0.75 M NaCl fraction. This fraction was applied subsequently to a reverse-phase HPLC, in which two peaks with antibacterial activity were obtained (Fig. 1). These antibacterial substances, termed peptides and ß, were eluted at retention times of 21.9 and 22.7 min, respectively. This implied that lactococcin Q consists of two peptides.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Reverse-phase chromatography of lactococcin Q. The bacteriocin activity was detected in the fractions containing the peaks indicated by and ß. The peaks and ß correspond to peptides and ß of lactococcin Q, respectively.

Analysis of the gene encoding lactococcin Q.
The structural gene sequence of lactococcin Q was obtained by degenerate PCR and subsequent cassette and inverse PCR. Sequencing analysis revealed two ORFs, termed laqA and laqB, encoding lactococcins Q and Qß, respectively (Fig. 2). Putative promoter and terminator sequences and ribosome binding sites were identified (Fig. 2). As in the cases of lactococcin G (20) and enterocin 1071 (7), the ORF for peptide ß was encoded adjacent to and downstream of that for peptide , and the two ORFs constituted an operon. The two ORFs, laqA and laqB, encoded prepeptides consisting of 54 and 61 amino acids, respectively. The deduced prepeptides possessed double-glycine sites for the cleavage of the leader peptides. The deduced amino acid sequences of mature peptides were identical to those of lactococcins Q and Qß obtained by Edman degradation.

View larger version (31K): [in this window] [in a new window]   FIG. 2. Nucleotide sequence of the region encoding lactococcin Q and lactococcin Qß of L. lactis QU 4 and the deduced amino acid sequences. The putative –35 and –10 promoter regions and ribosome binding sites (RBS) are underlined. The vertical arrows indicate the cleavage sites of the peptides. The reversed horizontal arrows indicate the putative transcriptional terminator.

View larger version (33K): [in this window] [in a new window]   FIG. 3. Alignment of leader peptides (a) and mature peptides (b) of lactococcin Q, lactococcin G, and enterocin 1071. Identical amino acid residues are boxed.

View larger version (14K): [in this window] [in a new window]   FIG. 4. Synergistic activities of and ß peptides of lactococcin Q. Various molar ratios of mixtures of and ß peptides were assayed. Minimum concentrations of the peptides in the mixtures exhibiting antibacterial activity were plotted. Even when or ß peptide existed in excess, intensities of antibacterial activity depended on the other peptide at a lower ratio. This indicated that synthetic lactococcin Q and Qß acted at a 1:1 molar ratio.

	DISCUSSION

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We isolated 11 strains of bacteriocin-producing LAB from a corn sample. Whereas five strains of the isolates showed specific antibacterial spectra similar to that of lactococcin Q, the other isolates showed broader antibacterial spectra, one of which was a nisin Z producer. Lactococcin Q and nisin Z were active against each other's producer, implying that they compete in corn microbiota. The remaining isolates showed unique antibacterial spectra, and further characterization is under investigation.

Amino acid and DNA sequence analyses revealed that lactococcin Q consisted of two peptides, termed lactococcins Q and Qß. Lactococcins Q and Qß contained 39 and 35 amino acids, respectively, but neither lanthionines nor dehydrated amino acids. MALDI-TOF MS analysis showed that each purified peptide was cross-contaminated in very small amounts (data not shown). Although amino acid sequence analysis was not affected, this indicated that further characterization of lactococcin Q and Qß using peptides purified through the procedures described above might lead to errors. An antibacterial activity assay using chemically synthesized lactococcin Q and Qß showed that two peptides exhibited full activity synergistically at a molar ratio of 1:1, whereas each peptide alone showed no activity. These characteristics are in accordance with those of class IIb bacteriocins of LAB, suggesting that lactococcin Q belongs to class IIb bacteriocin.

A database search showed that lactococcin Q was a novel class IIb bacteriocin with high homology to lactococcin G (Fig. 3). Based on the amino acid sequences, amphiphilic -helical structures of lactococcin G and enterocin 1071 were predicted by Edmundson -helical wheel representation (2, 23, 26). Amphiphilic -helical structures are often found in pore-forming antimicrobial peptides (24). The analysis applied to lactococcin Q is shown in Fig. 5. It was presumed that each peptide of lactococcin Q formed an amphiphilic -helix in the same positions as lactococcin G (23). In the amphiphilic region of peptide, four residues were different between lactococcins Q and G, and in that of ß peptide, three residues were different. In all the substitutions, the polarities of residues were not changed; hydrophobic and hydrophilic residues were changed to the corresponding ones. This supported the idea that lactococcins Q and G possessed similar secondary structures and therefore similar modes of action.

View larger version (27K): [in this window] [in a new window]   FIG. 5. Edmundson -helical wheel representation of the amphiphilic regions in lactococcin Q (a) and lactococcin Qß (b). For lactococcin Q, the amphiphilic region starts with residue 3 and ends with residue 27, and for lactococcin Qß, it starts with residue 8 and ends with residue 25, as in the cases of lactococcin G and lactococcin Gß, respectively. The shaded areas indicate nonpolar residues, and the nonshaded areas indicate polar residues. The residues different from those of lactococcin G are underlined.

MICs of lactococcin peptides were considerably different by combinations of and ß peptides (Table 2). These differences were probably caused by ß peptides. The antibacterial activity of Q plus Qß was 32 times as high as that of Q plus Gß, whereas those of Q plus Qß and G plus Qß were comparable. This suggested that the contribution of lactococcin Qß for antibacterial activity was greater than that of lactococcin Gß. Three amino acid residues that are different between lactococcins Qß and Gß might play important roles for the intensity of antibacterial activity.

Lactococcin Q showed antibacterial activity only against L. lactis strains (Table 3). Among LAB bacteriocins so far reported, lactococcin A (11), lactococcin B (19, 27, 28), lactococcin M (19, 27, 28), lactococcin 972 (15), LsbA (8), and LsbB (8) showed narrow and specific antibacterial spectra similar to lactococcin Q. They inhibited only the growth of lactococci and very few additional strains belonging to other genera. However, there is no structural homology between lactococcin Q and each of them.

Antibacterial spectra of lactococcin G and hybrid pairs were identical to lactococcin Q (Table 3). Their antibacterial activities were limited to the species of L. lactis. This suggested that different amino acid residues between lactococcins Q and G were not determinants of specificity of antibacterial activity. Another homologue, enterocin 1071, showed the activity against strains of various genera of gram-positive bacteria, including enterococci, but not against L. lactis (2). Amino acid residues that differ between lactococcins and enterocin 1071 might be involved in the specific recognition of target cells.

Recognition of target cells and partner peptides in a two-peptide bacteriocin system is still unknown. Further comparative studies on two-peptide bacteriocins are needed to solve the question of these types of recognition. This study is a good foothold to determine which parts of the peptides play important roles for the recognition. In addition, the results in this study implied that properties of two-peptide bacteriocins could be changed by substitution of partner peptides as well as amino acid residues. Other combinations of the peptides are worth further trying and consideration. Changing partner peptides of two-peptide bacteriocins may be an advantage over single-peptide bacteriocins for improvement of the properties.

	ACKNOWLEDGMENTS

 
We thank T. Horii and H. Nagasawa, Graduate School of Agricultural and Life Sciences, the University of Tokyo, for amino acid sequence analysis.

This work was partially supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, an AIST research grant, the Urakami Foundation for Food and Food Culture, the Hokuto Foundation for Bioscience, and the Iijima Memorial Foundation for the Promotion of Food Science and Technology.

	FOOTNOTES

 
* Corresponding author. Mailing address: Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Phone: (81) 92 642 3019. Fax: (81) 92 642 3019. E-mail: sonomoto{at}agr.kyushu-u.ac.jp.

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