Vol. 58, No. 5
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1992, P. 1417-1422
0099-2240/92/051417-06$02.00/0 Copyright X 1992, American Society for Microbiology
Purification and Characterization of a New Bacteriocin Isolated from a Camobacterium sp. G.
STOFFELS,' J. NISSEN-MEYER,2 A. GUDMUNDSDOTTIR,l H. HOLO,2
K. SLETlEN,3
I. F. NES2*
AND
Science Institute, University of Iceland, 107 ReykjaviK Iceland,1 and Laboratory of Microbial Gene Technology, NLVF, N-1432,4s, 2 and Department of Biochemistry, University of Oslo, 0367 Oslo 3,3 Norway Received 2 December 1991/Accepted 12 February 1992
A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin U149 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin U149 belongs to the class of bacteriocins termed lantibiotics.
was
Antagonistic compounds produced by lactic acid bacteria (LAB) have been known of for years. They have been the subject of much research because of their potential use in fermented food and feed. Of the numerous antagonistic compounds produced by LAB, the bacteriocins have been the focus of attention for the past few years (18, 19). This has resulted in important biochemical and genetic information on bacteriocins produced by LAB (4, 7, 11, 13, 15, 16, 22, 24, 32, 33). The search for new bacteriocins is of great significance in the development of future good starter cultures. Since bacteriocin-producing LAB are frequently isolated from fermented food and feed, bacteriocins are certainly already "in use" (1, 2, 3, 9, 21, 25) and should therefore be acceptable per se as food and feed additives. To exploit the potential of bacteriocin-producing starter cultures, more research is required on both new and established bacteriocin activities. It is of particular importance to investigate their structure, mode of action, and genetics. Among the bacteriocins isolated from LAB, nisin is by far the most studied (4, 7, 14, 16). Nisin is produced by some strains of Lactococcus lactis subsp. lactis. It belongs to a group of small ribosomally synthesized polypeptides containing modified amino acids such as lanthionine, 3-methyllanthionine, and their precursors, dehydroalanine and dehydrobutyrine (11). It has been suggested that antagonistic compounds which belong to this group should be termed lantibiotics to distinguish them from other bacteriocins which do not contain these modified residues (26). To our knowledge, only one additional lantibiotic produced by LAB has so far been isolated and characterized, that is, lactocin S from Lactobacillus sake L45 (22, 23). This investigation was initiated to purify and characterize new bacteriocins from LAB from fish. One LAB isolate was *
found to produce a bacteriocinlike substance. This isolate was identified as belonging to the group of nonaciduric LAB
termed Carnobacterium sp. (29). The genus Carnobacterium
was proposed by Collins et al. (5) to accommodate species of
nonaciduric Lactobacillus piscicola and Lactobacillus divergens in addition to some other nonaciduric lactobacilli isolated from poultry and vacuum-packed meat (5, 27, 31). This work presents the purification and characterization of a new bacteriocin, which has been termed carnocin U149.
MATERIALS AND METHODS Bacterial cultures and media. Carnobacterium sp. strain UI49 isolated from fish was propagated at 30°C in MRS broth (Oxoid Ltd., Basingstoke, England). The isolate was characterized as a Carnobacterium sp. by carbohydrate utilization pattern, lactic acid production and tolerance, percent guanine-plus-cytosine content in DNA, and sequencing of part of its 16S RNA gene (29). L. lactis SIK-83 (2), used as the indicator strain, was propagated in GM17 broth (M17 [Oxoid] medium with 0.5% glucose) at 30°C. Bacteriocin assay. To determine the bacteriocin concentration, a microtiter plate assay was used as described earlier (13). Twofold dilutions (100 ,ul) of bacteriocin extracts in MRS broth were prepared in microtiter plates (96 wells; Costar, Cambridge, Mass.). Twenty microliters of fresh indicator culture of exponentially growing bacteria (L. lactis SIK-83 at anA6. of 0.1 to 0.3) was added to each well. The wells were filled to 200 ,ul by adding 80 RI of GM17 medium. After 3 h of incubation at 30°C, the growth inhibition was evaluated by spectrophotometrically measuring (at 600 nm) the growth by using an MR 700 Microplate Reader (Dynatech Laboratory, Channel Islands, Great Britain). One bacteriocin unit (BU) was defined as the amount of bacteriocin which inhibited growth of the indicator organism by 50% (50% of the turbidity of the control culture without bacteriocin) under standard assay conditions.
Corresponding author. 1417
1418
APPL. ENVIRON. MICROBIOL.
STOFFELS ET AL. TABLE 1. Purification scheme for carnocin UI49
Fraction
Fraction
no.
I II III IV V
Culture supernatant Ammonium sulfate precipitation Cation-exchange chromatography Phenyl-Superose chromatography C2-C18 reverse-phase chromatography
Vol
Optical density
Total activity
Sp act
Purification
Yield
(ml)
(28)
(BU)
(BU/ml/A280)
(%)
2,000 40 20 4 1.5
25 86 0.7 0.2 0.1
(fold) 1 17 1,300
Effects of pH, heat, and storage on bacteriocin. Partially purified bacteriocin (fractions I and III) was diluted with 50 mM sodium citrate (pH 2 to 5) or 50 mM sodium phosphate (pH 5 to 13) to 1,000 BU/ml and 60,000 BU/ml, respectively. The bacteriocin solutions were exposed to the various pHs for 24 h at 4°C. Aliquots of the bacteriocin (fractions I and III) were subjected to various heat treatments and conditions of storage as described in Results. The activities were determined by use of the microtiter assay. Mode of action. Various concentrations of bacteriocin were added to the indicator organism (107 bacteria per ml) in 50 mM sodium phosphate buffer (pH 6.5) and incubated at 22°C. At appropriate time intervals, the number of viable bacteria was determined by dilution and plating on GM17 agar plates. Amino acid composition and sequencing. The purified fraction IV of the bacteriocin was hydrolyzed and analyzed on an amino acid analyzer setup as described earlier (8). The half-cystine content was determined as cysteic acid by the method of Hirs (12). The amino acid sequence was determined by Edman degradation by using an Applied Biosystems 477A automatic sequence analyzer with an on-line 120A phenylthiohydantoin amino acid analyzer (6). SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by using Phast Gel high-density strips, Phast Gel SDS buffer strips, and the Phast System (Pharmacia-LKB Biotechnology, Uppsala, Sweden). Molecular weight standards were a mixture of seven peptides spanning molecular weights of 2,510 to 17,950 (Sigma Chemical Co., St. Louis, Mo.). The molecular weight of the bacteriocin was estimated from the calibration curve of the standard proteins as suggested by the producer (28). Mass spectroscopy analysis. Mass analyses of peptide fractions were performed by using the Biolon Mass Analyser (Applied Biosystem, Kebolab, Denmark) as described earlier (30). Peptide fractions were dissolved in 50 to 100 ,ul of trifluoroacetic acid containing 20% acetonitrile. From each fraction, 5 ,ul was loaded onto a target and data were accumulated for 10 min at 16 kV. Purification. (i) Ammonium sulfate precipitation. All of the purification steps were performed at room temperature. The chromatographic equipment and material were obtained from Pharmacia-LKB. Camobactenum sp. strain UI49 was grown in 2,000 ml of MRS broth to an optical density at 600 nm of 0.4. After 24 to 48 h, the culture contained a bacteriocin titer of 500 to 1,000 BU/ml. The cells were removed by centrifugation at 10,000 x g for 15 min. Ammonium sulfate was added to a final concentration of 40% (wt/vol) and left on ice for at least 30 min. The bacteriocin was pelleted by centrifugation at 10,000 x g for 30 min, and the pellet and floating solid material were combined and dissolved in 40 ml of buffer A (20 mM glycine-NaOH buffer, pH 9.2) (fraction
II).
1.1 1.3 4.0 1.2 1
x 106 x 106 x 105 x
22 378 28 x 103 156 x 103 67 x 103
105
x 104
100 118 36 11 1
7,100 3,050
(ii) Cation-exchange chromatography. Fraction II was desalted by being passed through Sephadex G-25 PD-10 gel filtration columns equilibrated with buffer A. The material was applied to an S-Sepharose cation-exchange column (30 ml) which had been equilibrated with buffer A. After the column was washed in buffer A, the bacteriocin activity was eluted with 0.2 M NaCl in buffer A. The activity was recovered in a 20-ml fraction (fraction III). (iii) Hydrophobic interaction chromatography. Ammonium sulfate (8 g/100 ml of solution) was added to Fraction III, which subsequently was applied to a Phenyl-Superose HR 5/5 column equilibrated with 8% (wtlvol) ammonium sulfate in buffer B (20 mM sodium phosphate, pH 7.5). The bacteriocin was eluted with a linear decreasing gradient of ammonium sulfate at a flow rate of 1 ml/min and recovered in a 4-ml fraction (fraction IV). (iv) Reverse-phase chromatography. Fraction IV was subjected to reverse-phase chromatography. A C2-C18 reversephase column (PepRPC HR5/5), equilibrated with buffer B, was used. The bacteriocin was eluted with a linear gradient, starting with buffer B and ending with a solvent containing 90% (vol/vol) methanol and 10% (vol/vol) isopropanol (flow rate, 1 ml/min). The bacteriocin (fraction V) was stored at -20°C in 80% methanol-8% isopropanol. RESULTS
Purification of the bacteriocin. Results from the purification of the bacteriocin are summarized in Table 1. The Phenyl-Superose and the C2-C18 reverse-phase chromatographic elution profiles of the bacteriocin are shown in Fig. 1 and 2, respectively. Upon chromatography on the Phenyl-
0.35-
40X103
8
0.25-3T
0.20
-
0
W^ 6 \ 025 ]
c'j 0.15 -
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1992, P. 1417-1422
0099-2240/92/051417-06$02.00/0 Copyright X 1992, American Society for Microbiology
Purification and Characterization of a New Bacteriocin Isolated from a Camobacterium sp. G.
STOFFELS,' J. NISSEN-MEYER,2 A. GUDMUNDSDOTTIR,l H. HOLO,2
K. SLETlEN,3
I. F. NES2*
AND
Science Institute, University of Iceland, 107 ReykjaviK Iceland,1 and Laboratory of Microbial Gene Technology, NLVF, N-1432,4s, 2 and Department of Biochemistry, University of Oslo, 0367 Oslo 3,3 Norway Received 2 December 1991/Accepted 12 February 1992
A bacteriocin-producing Carnobacterium sp. was isolated from fish. The bacteriocin, termed carnocin UI49, purified to homogeneity by a four-step purification procedure, including hydrophobic interaction chromatography and reverse-phase chromatography. Carnocin U149 has a bactericidal mode of action. It was shown to be heat tolerant and stable between pH 2 and 8. At pH above 8, carnocin UI49 was rapidly inactivated. Amino acid analysis revealed a composition of about 35 to 37 amino acids in addition to an unidentified peak which migrates at the position of lanthionine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis suggests a molecular weight of about 4,500 to 5,000. Mass spectrometry gave a molecular weight of 4,635, which is about 1,000 larger than that calculated from the amino acid analysis data. Performic acid oxidation of carnocin UI49, followed by amino acid hydrolysis, revealed the presence of cysteic acid. The sequence of the first seven amino acid residues was determined to be N-Gly-Ser-Glu-Ile-Gln-Pro-Arg. After the seventh amino acid, carnocin UI49 was not available for further Edman degradation. The results suggest that carnocin U149 belongs to the class of bacteriocins termed lantibiotics.
was
Antagonistic compounds produced by lactic acid bacteria (LAB) have been known of for years. They have been the subject of much research because of their potential use in fermented food and feed. Of the numerous antagonistic compounds produced by LAB, the bacteriocins have been the focus of attention for the past few years (18, 19). This has resulted in important biochemical and genetic information on bacteriocins produced by LAB (4, 7, 11, 13, 15, 16, 22, 24, 32, 33). The search for new bacteriocins is of great significance in the development of future good starter cultures. Since bacteriocin-producing LAB are frequently isolated from fermented food and feed, bacteriocins are certainly already "in use" (1, 2, 3, 9, 21, 25) and should therefore be acceptable per se as food and feed additives. To exploit the potential of bacteriocin-producing starter cultures, more research is required on both new and established bacteriocin activities. It is of particular importance to investigate their structure, mode of action, and genetics. Among the bacteriocins isolated from LAB, nisin is by far the most studied (4, 7, 14, 16). Nisin is produced by some strains of Lactococcus lactis subsp. lactis. It belongs to a group of small ribosomally synthesized polypeptides containing modified amino acids such as lanthionine, 3-methyllanthionine, and their precursors, dehydroalanine and dehydrobutyrine (11). It has been suggested that antagonistic compounds which belong to this group should be termed lantibiotics to distinguish them from other bacteriocins which do not contain these modified residues (26). To our knowledge, only one additional lantibiotic produced by LAB has so far been isolated and characterized, that is, lactocin S from Lactobacillus sake L45 (22, 23). This investigation was initiated to purify and characterize new bacteriocins from LAB from fish. One LAB isolate was *
found to produce a bacteriocinlike substance. This isolate was identified as belonging to the group of nonaciduric LAB
termed Carnobacterium sp. (29). The genus Carnobacterium
was proposed by Collins et al. (5) to accommodate species of
nonaciduric Lactobacillus piscicola and Lactobacillus divergens in addition to some other nonaciduric lactobacilli isolated from poultry and vacuum-packed meat (5, 27, 31). This work presents the purification and characterization of a new bacteriocin, which has been termed carnocin U149.
MATERIALS AND METHODS Bacterial cultures and media. Carnobacterium sp. strain UI49 isolated from fish was propagated at 30°C in MRS broth (Oxoid Ltd., Basingstoke, England). The isolate was characterized as a Carnobacterium sp. by carbohydrate utilization pattern, lactic acid production and tolerance, percent guanine-plus-cytosine content in DNA, and sequencing of part of its 16S RNA gene (29). L. lactis SIK-83 (2), used as the indicator strain, was propagated in GM17 broth (M17 [Oxoid] medium with 0.5% glucose) at 30°C. Bacteriocin assay. To determine the bacteriocin concentration, a microtiter plate assay was used as described earlier (13). Twofold dilutions (100 ,ul) of bacteriocin extracts in MRS broth were prepared in microtiter plates (96 wells; Costar, Cambridge, Mass.). Twenty microliters of fresh indicator culture of exponentially growing bacteria (L. lactis SIK-83 at anA6. of 0.1 to 0.3) was added to each well. The wells were filled to 200 ,ul by adding 80 RI of GM17 medium. After 3 h of incubation at 30°C, the growth inhibition was evaluated by spectrophotometrically measuring (at 600 nm) the growth by using an MR 700 Microplate Reader (Dynatech Laboratory, Channel Islands, Great Britain). One bacteriocin unit (BU) was defined as the amount of bacteriocin which inhibited growth of the indicator organism by 50% (50% of the turbidity of the control culture without bacteriocin) under standard assay conditions.
Corresponding author. 1417
1418
APPL. ENVIRON. MICROBIOL.
STOFFELS ET AL. TABLE 1. Purification scheme for carnocin UI49
Fraction
Fraction
no.
I II III IV V
Culture supernatant Ammonium sulfate precipitation Cation-exchange chromatography Phenyl-Superose chromatography C2-C18 reverse-phase chromatography
Vol
Optical density
Total activity
Sp act
Purification
Yield
(ml)
(28)
(BU)
(BU/ml/A280)
(%)
2,000 40 20 4 1.5
25 86 0.7 0.2 0.1
(fold) 1 17 1,300
Effects of pH, heat, and storage on bacteriocin. Partially purified bacteriocin (fractions I and III) was diluted with 50 mM sodium citrate (pH 2 to 5) or 50 mM sodium phosphate (pH 5 to 13) to 1,000 BU/ml and 60,000 BU/ml, respectively. The bacteriocin solutions were exposed to the various pHs for 24 h at 4°C. Aliquots of the bacteriocin (fractions I and III) were subjected to various heat treatments and conditions of storage as described in Results. The activities were determined by use of the microtiter assay. Mode of action. Various concentrations of bacteriocin were added to the indicator organism (107 bacteria per ml) in 50 mM sodium phosphate buffer (pH 6.5) and incubated at 22°C. At appropriate time intervals, the number of viable bacteria was determined by dilution and plating on GM17 agar plates. Amino acid composition and sequencing. The purified fraction IV of the bacteriocin was hydrolyzed and analyzed on an amino acid analyzer setup as described earlier (8). The half-cystine content was determined as cysteic acid by the method of Hirs (12). The amino acid sequence was determined by Edman degradation by using an Applied Biosystems 477A automatic sequence analyzer with an on-line 120A phenylthiohydantoin amino acid analyzer (6). SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out by using Phast Gel high-density strips, Phast Gel SDS buffer strips, and the Phast System (Pharmacia-LKB Biotechnology, Uppsala, Sweden). Molecular weight standards were a mixture of seven peptides spanning molecular weights of 2,510 to 17,950 (Sigma Chemical Co., St. Louis, Mo.). The molecular weight of the bacteriocin was estimated from the calibration curve of the standard proteins as suggested by the producer (28). Mass spectroscopy analysis. Mass analyses of peptide fractions were performed by using the Biolon Mass Analyser (Applied Biosystem, Kebolab, Denmark) as described earlier (30). Peptide fractions were dissolved in 50 to 100 ,ul of trifluoroacetic acid containing 20% acetonitrile. From each fraction, 5 ,ul was loaded onto a target and data were accumulated for 10 min at 16 kV. Purification. (i) Ammonium sulfate precipitation. All of the purification steps were performed at room temperature. The chromatographic equipment and material were obtained from Pharmacia-LKB. Camobactenum sp. strain UI49 was grown in 2,000 ml of MRS broth to an optical density at 600 nm of 0.4. After 24 to 48 h, the culture contained a bacteriocin titer of 500 to 1,000 BU/ml. The cells were removed by centrifugation at 10,000 x g for 15 min. Ammonium sulfate was added to a final concentration of 40% (wt/vol) and left on ice for at least 30 min. The bacteriocin was pelleted by centrifugation at 10,000 x g for 30 min, and the pellet and floating solid material were combined and dissolved in 40 ml of buffer A (20 mM glycine-NaOH buffer, pH 9.2) (fraction
II).
1.1 1.3 4.0 1.2 1
x 106 x 106 x 105 x
22 378 28 x 103 156 x 103 67 x 103
105
x 104
100 118 36 11 1
7,100 3,050
(ii) Cation-exchange chromatography. Fraction II was desalted by being passed through Sephadex G-25 PD-10 gel filtration columns equilibrated with buffer A. The material was applied to an S-Sepharose cation-exchange column (30 ml) which had been equilibrated with buffer A. After the column was washed in buffer A, the bacteriocin activity was eluted with 0.2 M NaCl in buffer A. The activity was recovered in a 20-ml fraction (fraction III). (iii) Hydrophobic interaction chromatography. Ammonium sulfate (8 g/100 ml of solution) was added to Fraction III, which subsequently was applied to a Phenyl-Superose HR 5/5 column equilibrated with 8% (wtlvol) ammonium sulfate in buffer B (20 mM sodium phosphate, pH 7.5). The bacteriocin was eluted with a linear decreasing gradient of ammonium sulfate at a flow rate of 1 ml/min and recovered in a 4-ml fraction (fraction IV). (iv) Reverse-phase chromatography. Fraction IV was subjected to reverse-phase chromatography. A C2-C18 reversephase column (PepRPC HR5/5), equilibrated with buffer B, was used. The bacteriocin was eluted with a linear gradient, starting with buffer B and ending with a solvent containing 90% (vol/vol) methanol and 10% (vol/vol) isopropanol (flow rate, 1 ml/min). The bacteriocin (fraction V) was stored at -20°C in 80% methanol-8% isopropanol. RESULTS
Purification of the bacteriocin. Results from the purification of the bacteriocin are summarized in Table 1. The Phenyl-Superose and the C2-C18 reverse-phase chromatographic elution profiles of the bacteriocin are shown in Fig. 1 and 2, respectively. Upon chromatography on the Phenyl-
0.35-
40X103
8
0.25-3T
0.20
-
0
W^ 6 \ 025 ]
c'j 0.15 -
