This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nilsson, L.
Right arrow Articles by Gram, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nilsson, L.
Right arrow Articles by Gram, L.
Agricola
Right arrow Articles by Nilsson, L.
Right arrow Articles by Gram, L.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, May 2002, p. 2251-2260, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2251-2260.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Role of Acetate in Production of an Autoinducible Class IIa Bacteriocin in Carnobacterium piscicola A9b

Lilian Nilsson,* Michael K. Nielsen, Yin Ng, and Lone Gram

Danish Institute for Fisheries Research, Department of Seafood Research, c/o Technical University of Denmark, DK-2800 Lyngby, Denmark

Received 20 September 2001/ Accepted 14 February 2002


arrow
ABSTRACT
 
Carnobacterium piscicola strain A9b isolated from cold smoked salmon inhibits growth of the food-borne pathogen Listeria monocytogenes partly due to the production of a proteinaceous compound (L. Nilsson, L. Gram, and H. H. Huss. J. Food Prot. 62:336-342, 1999). The purpose of the present study was to purify the compound and describe factors affecting its production, with particular emphasis on food-relevant factors. Amino acid sequencing showed that the compound is a class IIa bacteriocin with an N-terminal amino acid sequence identical to that of carnobacteriocin B2. The production of the bacteriocin was autoinducible, and the threshold level for induction was 9.6 x 10-10 M. We also report, for the first time, that acetate acts as an induction factor, with a threshold concentration of 0.3 to 12 mM. Acetate could not act as an inducer during the late exponential phase of C. piscicola A9b. The induction of bacteriocin production showed a dose-dependent relationship at acetate concentrations of up to 10 to 20 mM (depending on the growth medium) and at a concentration of 1.9 x 10-8 M for the bacteriocin itself; a saturation level of bacteriocin specific activity was reached at these concentrations of induction factors. The combined use of both inducers did not enhance the saturation level of bacteriocin production compared to that seen with the use of each inducer alone. Increasing NaCl and glucose concentrations negatively influenced the efficiency of acetate as an induction factor. Based on the results, carnobacteriocin B2 was used as an induction factor to manipulate the production of bacteriocin in cold smoked salmon juice and thus improve the ability to inhibit L. monocytogenes.


arrow
INTRODUCTION
 
The ubiquitous nature of Listeria monocytogenes, its ability to survive and grow in a wide range of food products, and the severity of the disease it causes (20 to 30% fatality) have heightened the awareness of this pathogen as a public health problem over the past decade (13). Research on the control of this pathogen has expanded significantly. In addition to traditional preservation techniques, several new preservation strategies have been developed to inhibit its growth in food products, thus preventing future outbreaks. The use of bacteriocinogenic lactic acid bacteria (LAB) and/or their isolated bacteriocins may offer a promising solution (8, 10, 27).

A number of studies have been conducted on class IIa bacteriocins, which are highly active against Listeria species. Class IIa bacteriocins, also known as pediocin-like bacteriocins, are small, heat-stable, non-lanthionine-containing peptides that contain at least two cysteines with disulfide bridges. The primary structure of class IIa bacteriocins is characterized by a conserved N-terminal hydrophilic domain which has the consensus sequence YGNGV(Xaa)C(Xaa)4V-(Xaa)4A (where Xaa is any amino acid) and a highly variable C-terminal hydrophobic region (12). Class IIa bacteriocins do not undergo posttranslational modification, except for cleavage of the leader peptides across the cytoplasmic membrane and the formation of disulfide bridges. These leader peptides contain a conserved double-glycine motif in the C terminus, and this motif is believed to serve as a signal peptide for the processing and secretion of bacteriocins by an independent ATB-binding cassette transporter (24, 44).

The production of most class IIa bacteriocins is regulated by a three-component system which includes a histidine protein kinase, a response regulator, and an induction factor. Some class IIa bacteriocins are autoregulated by a two-component signal transduction system (32), which is a well-known phenomenon in lantibiotics (37). A threshold concentration of the bacteriocin, which functions as a signal molecule accumulating during growth, triggers the transcription of the genes coding for bacteriocin production, suggesting a self-inducing cell density (quorum-sensing)-regulated system (32, 37).

Bacteriocin production is influenced by several environmental factors, such as pH (1, 25), temperature (9), and NaCl (20, 43) and ethanol (23) concentrations. These environmental factors may influence growth negatively and thereby the secretion of the induction factor (9). Further, it has been suggested that some environmental factors reduce the binding of the induction factor to its receptor (25). Understanding the influence of food-related environmental factors on the induction of bacteriocins is essential for the effective commercial application of bacteriocin-producing LAB in the preservation of foods.

Several species within the genus Carnobacterium, which forms a distinct phylogenetic clade within the LAB, produce class IIa bacteriocins (carnobacteriocins BM1 and B2 and piscicolin 126). Carnobacteria are associated with chilled vacuum-packed meat, poultry, and fish products (2, 7, 16). In a previous study, Carnobacterium piscicola strain A9b isolated from cold smoked salmon was used for the biopreservation of vacuum-packed cold smoked salmon (27). C. piscicola A9 was able to inhibit L. monocytogenes both in model experiments with cold smoked salmon juice and in vacuum-packed cold smoked salmon (27). The competitive ability of C. piscicola strain A9b was attributed to rapid growth in the food system and the production of antilisterial substances. Further development of this strain for the biopreservation of food products requires an understanding of the mechanisms of action of the antilisterial activity and of the influence of food-related parameters on the inhibitory action. Here we report on the isolation and characterization of a class IIa bacteriocin produced by C. piscicola A9b. We show that acetate and the bacteriocin itself function as dose-dependent inducers of bacteriocin production both in laboratory media and in cold smoked salmon juice.


arrow
MATERIALS AND METHODS
 
Bacterial strains, culture conditions, and media.
The bacteriocin producer C. piscicola strain A9b was isolated from vacuum-packed cold smoked salmon stored at 5°C (29). A bacteriocin-negative (Bac-) mutant of C. piscicola A9b was created by exposure to 5, 10, 15, and 20 µg of acriflavin/ml (26). L. monocytogenes strain O57 (3) and C. piscicola DSM 20730 (obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany) were used as indicator organisms for bacteriocin quantification in a well diffusion assay. C. piscicola DSM 20730 was used for measuring bacteriocin production in salmon juice experiments when only small amounts of bacteriocin were produced. Bacteriocin-producing LAB strains used in induction experiments with acetate included Leuconostoc gelium UAL187-22 (14), Leuconostoc mesenteroides TA33a (30), Pediococcus acidilactici PA-2 (21), Lactobacillus sake MI401 (18) (reclassified from Lactobacillus bavaricus), and Enterococcus faecium TSB6 (4).

All strains were propagated twice in broth before use. C. piscicola and L. monocytogenes strains were grown in brain heart infusion (BHI) broth (Oxoid Ltd., Hampshire, England; product code CM225) at 25°C for 18 h. Leuconostoc spp., P. acidilactici, and L. sake were grown in deMan-Rogosa-Sharpe (MRS) broth (CM359; Oxoid) at 30°C for 18 h before use. E. faecium was grown in BHI broth at 30°C. Working cultures were maintained at 5°C on BHI agar (BHI broth containing 1.2% agar; Bie & Berntsen A/S, Højbjerg, Denmark; code BBB10030) or MRS agar (Oxoid; code CM361), and frozen stock cultures were stored at -80°C.

For bacteriocin production studies, a 1.0 or 0.001% stationary-phase culture of C. piscicola A9b (incubation at 25°C for 18 h) was inoculated into MRS7-GCA, MRS7-CA, or MRS7-AG broth (Oxoid; see below) or all-purpose Tween (APT) broth (Difco; 0655-17-9). MRS7-GCA broth was prepared as described previously (15) by assembling the individual components of MRS broth in accordance with the manufacturer's formula but with the exclusion of glucose, triammonium citrate, and sodium acetate from the medium. The components were dissolved in distilled water and the pH was adjusted to 7.0 (MRS7 broth) before autoclaving. Tween 80, dipotassium hydrogen phosphate, magnesium sulfate, and manganese sulfate were added as sterile filtered solutions after autoclaving. In MRS7-CA broth and MRS7-AG broth, glucose and citrate were added, respectively.

Cold smoked salmon juice was made as described by Nilsson et al. (27). Salt analysis was performed as described previously (1a), and NaCl was added to a final concentration of 4.0% (wt/vol). The juice was heat treated for 30 min at 100°C and stored in the dark at 5°C for a maximum of 3 days before use.

Assays for bacteriocin and induction activity.
The bacteriocin concentration was determined in an agar diffusion assay (28) by using 106 CFU of the indicator organism/ml in 48°C BHI agar (BHI broth containing 0.8% agar) supplemented with 0.1% Tween 80 (Merck; code 822187) and poured into 14-cm petri dishes (Sterilin, Bibby, Sterilin Ltd., Stone, Staffs, United Kingdom). Supernatant fluid from the producer strain, C. piscicola A9b, was adjusted to pH 6.5 with 2 N NaOH and filter sterilized (0.2-µm-pore-size nylon membrane filter; Sartorius; 16534K). Samples (50 µl) were pipetted into 7-mm wells cut into the agar. The plates were preincubated at 5°C for 24 h followed by 18 h at 25°C. Twofold dilution series of supernatant fluid were analyzed (28); the reciprocal of the highest dilution causing a zone of inhibition of the indicator organism was expressed as the number of bacteriocin units (BU) per 50 µl, and BU per milliliter were calculated.

The concentration of the proteinaceous extracellular induction factor produced by C. piscicola A9b was determined by preparing twofold dilutions of a sterile filtered sample (or of fractions from the bacteriocin purification) in MRS7-CA broth inoculated with C. piscicola A9b (0.001% inoculum from a stationary-phase culture). The cultures were incubated at 25°C for 22 h, and sterile, cell-free supernatants were obtained by centrifugation (10,000 x g, 5 min, 5°C) and heating (100°C, 10 min). The concentration of bacteriocin in each tube was determined as described above. Induction activity was expressed as induction units (IU) per milliliter, and 1 IU was arbitrarily defined as the minimum concentration of induction factor resulting in detectable bacteriocin production.

Stability of bacteriocin and an extracellular induction factor produced by C. piscicola A9b
Proteins from a cell-free, filter-sterilized supernatant of an overnight culture of C. piscicola A9b were precipitated and desalted as described below. Inactivation by enzymes (proteases I, IV, and XIV; proteinase K; pepsin; lipase VII; phospholipase C; trypsin IX; and {alpha}-amylase) and changes in pHs and temperatures was studied. After each treatment, samples were assayed for residual bacteriocin activity and induction activity. Precipitated proteins were mixed with each enzyme at a final concentration of 1 mg/ml in selected buffers recommended by the enzyme supplier (Sigma-Aldrich, Vallensbaek Strand, Denmark). Precipitated proteins were exposed to various pHs (2.0 to 11.0) for 24 h at 5°C. Protein solutions were diluted 1:1 with 0.05 M glycine hydrochloride buffer (pH 2.0), 0.05 M citrate buffer (pHs 3 to 6), 0.05 M hydrochloride (pHs 7 to 9), or 0.05 M carbonate-bicarbonate buffer (pHs 10 to 11). All buffers were purchased from Sigma. The temperature stability of the bacteriocin was examined for 1 month at -20, 0, 5, and 25°C.

Production, purification, and amino acid sequencing of bacteriocin and an extracellular induction factor produced by C. piscicola A9b
Bacteriocin produced by C. piscicola A9b was purified from BHI broth, although optimal bacteriocin production was obtained in MRS7 broth (15). However, it was observed that MRS7 broth interferes with the purification of bacteriocin due to the presence of the detergent Tween (5). C. piscicola A9b was grown in BHI broth to the stationary phase (24 h at 25°C), corresponding to an optical density (OD) of 1.3 to 1.4. Ammonium sulfate (Merck; code 1.01217) was added to a cell-free supernatant (40% [wt/vol]), and the mixture was stirred for 20 h at 2°C. The bacteriocin precipitate was harvested (10,000 x g for 30 min), resuspended in 50 mM sodium-acetate buffer (pH 4.5), and desalted by dialysis against the same phosphate buffer (cutoff, 1,000; Spectrum Laboratories, Inc., Rancho Dominguez, Calif.). The bacteriocin was purified by cation-exchange chromatography with a HiTrap SP column (Amersham Pharmacia Biotech., H/orsholm, Denmark) equilibrated with 50 mM acetate buffer (pH 4.5). The bacteriocin was eluted at a flow rate of 5.0 ml/min with a linearly increasing gradient (starting with 50 mM sodium-acetate buffer [pH 4.5] and ending with 50 mM sodium-acetate buffer [pH 4.5] containing 1 M NaCl). The active fraction was applied to a hydrophobic interaction column (HiLoad 16/10 Phenyl-Sepharose; Amersham) and eluted with a linear gradient of 8% ammonium sulfate. This active fraction was used for final purification by reverse-phase high-pressure liquid chromatography (HPLC) with a C2/C18 Nucleosil column (250 by 4.6 mm) and a linear gradient from 100 0.1% trifluoroacetic acid to 90% acetonitrile in 0.1% trifluoroacetic acid. Chromatographic purification was performed at room temperature by fast protein liquid chromatography (Bio-Rad, Copenhagen, Denmark) with a SMART system (Amersham).

Protein content, estimated by measuring the A280, bacteriocin activity (L. monocytogenes O57 was used as the target strain), and induction activity were determined at each step of the purification process.

The purity of the bacteriocin produced by C. piscicola A9b was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) with 12% NuPAGE Bis-Tris gels (Novex, San Diego, Calif.). SDS-PAGE was carried out by using a Mini-Cell system (Novex) according to the recommendations of the supplier. A molecular mass marker (range, 2.5 to 200 kDa) kit (Novex) was used for protein size estimation. After electrophoresis, the gel was silver stained and the position of the bacteriocin activity band was confirmed in a biological assay with an identical but unstained SDS gel. The gel was fixed and washed in deionized water (three times for 15 min each time) prior to overlay with agar that had been seeded with the indicator strain, C. piscicola DSM 20730.

The amino acid sequence of the HPLC-purified bacteriocin was determined by N-terminal Edman degradation with an automatic sequencer (Procise; Applied Biosystems, Perkin-Elmer).

Induction studies.
All induction experiments described below were done at 25°C. Cell density was determined by measurement of the OD at 600 nm (OD600) (Novaspec II; Amersham). Bacteriocin activity was determined when cultures had reached the stationary phase of growth (22 to 48 h).

The influence of carbon sources (acetate, citrate, and glucose), extracellular compounds produced by C. piscicola A9b, and inoculum size on both cell density and the production of bacteriocin by C. piscicola A9b was investigated. Acetate (Sigma; S7670), citrate (Sigma; A1332), and glucose (Sigma; G7528) were added to the basal medium (MRS7-GCA broth) at final concentrations of 36.74, 8.22, and 111.01 mM, respectively, corresponding to the concentrations in manufactured MRS broth. In some combinations, sterile filtered supernatant from the bacteriocin-producing strain (Bac+) or the non-bacteriocin-producing mutant (Bac-) of C. piscicola A9b was added at a concentration of 1.0%. A stationary-phase culture of C. piscicola A9b (incubation at 25°C for 18 h) was used as an inoculum for each medium at a 0.001% (vol/vol) or a 1.0% (vol/vol) final concentration.

The induction capacity of acetate and an extracellular proteinaceous factor produced by C. piscicola A9b Bac+ was investigated with MRS7-CA, MRS7-C (MRS7 broth without the addition of citrate), or APT broth. Acetate was added to the broth at increasing concentrations ranging from 0 to 90 mM, and the proteinaceous induction factor, obtained from a sterile, cell-free culture supernatant of C. piscicola A9b Bac+ (see the decription of the induction assay), was added to the broth at increasing concentrations ranging from 0 to 4,096 IU ml-1. A stationary-phase culture of C. piscicola A9b was used as an inoculum at 0.001% (vol/vol). Induction by the extracellular proteinaceous factor was examined in duplicate. Induction by acetate was examined in a single experiment; however, representative points were examined in an independent experiment, and results from the first experiment were confirmed.

We investigated if the induction capacity of acetate was influenced by the time at which acetate was added to the growth medium of C. piscicola A9b. Acetate corresponding to a final concentration of 36.74 mM was added to MRS7-AC broth every second hour (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 h) after inoculation with C. piscicola A9b (0.001% inoculum from a stationary-phase culture).

The effects of glucose and NaCl on the induction capacity of acetate and the proteinaceous induction factor produced by C. piscicola A9b were investigated as follows. (i) Acetate and glucose were added to MRS7-CA broth at different concentrations (from 0 to 36.74 mM and from 0 to 111.11 mM, respectively). (ii) Acetate and the extracellular induction factor were added to MRS7-CA broth containing increasing concentrations of NaCl (from 0 to 7.0%). (iii) Acetate and the extracellular induction factor were added to salmon juice at different concentrations (from 0 to 36.74 mM and from 0 to 111.11 mM, respectively). We examined if acetate could induce bacteriocin production in other LAB: L. gelium UAL187-22, L. mesenteroides TA33a, P. acidilactici PA-2, L. sake MI401, and E. faecium TSB6.

Influence of metabolic conversion of glucose and production of acetate on C. piscicola A9b bacteriocin production
The production of acetate and the consumption of glucose by C. piscicola A9b in MRS7-GCA broth, MRS7-CA broth, or salmon juice were determined at 25°C. When cultures reached the stationary phase, the OD600, bacteriocin activity, pH, and acetate and glucose concentrations were determined. Concentrations of acetate and glucose were determined with enzymatic kits (R-Biopharm; Boehringer Mannheim GmbH, Darmstadt, Germany).

Inhibition of L. monocytogenes O57 in salmon juice by C. piscicola A9b in the presence of induction factors
C. piscicola A9b was preincubated at 5°C for 5 days in BHI broth (1% preculture) supplemented with 4.0% (wt/vol) NaCl (27). L. monocytogenes O57 was grown at 15°C for 24 h in BHI broth (1% inoculum from a preculture) supplemented with 3.0% (wt/vol) NaCl (27) and diluted in 0.1% peptone water to appropriate cell numbers. Cold smoked salmon juice with 4.0% (wt/vol) NaCl was inoculated with C. piscicola A9b corresponding to inoculum levels of 1.0 and 0.001%. L. monocytogenes O57 was added at a final concentration of 103 CFU/ml. A sterile filtered supernatant from bacteriocin-producing strain A9b was added (1.0%) to some of the cultures. The flasks were incubated for 27 days at 5°C in the dark. Samples were removed once or twice a week for the determination of colony counts and bacteriocin production.

L. monocytogenes was enumerated by direct plating of 0.1 ml of appropriate dilutions onto listeria selective agar base (Oxoid; code CM856) to which listeria selective supplement (Oxoid; code SR140) had been added. The plates were incubated at 25°C for 48 h. Cell numbers of C. piscicola were estimated by direct plating on nitrate-polymyxin agar made from APT agar (Difco; 0654-17) (pH 6.7). The plates were incubated at 25°C for 48 h.


arrow
RESULTS
 
Stability of the bacteriocin from C. piscicola A9b
The inhibitory compound produced by C. piscicola A9b was inactivated by the proteolytic enzymes proteases I, IV, and XIV, proteinase K, and trypsin IX, suggesting a proteinaceous nature of the compound (bacteriocin). No significant decrease in activity was found after treatment with pepsin, lipase VII, phospholipase C, or {alpha}-amylase. The bacteriocin activity was stable at -20, 0, and 5°C for 1 month and during heat treatment at 100°C for 10 min. However, the bacteriocin lost 50% of the initial activity (10,240 BU ml-1) after 1 month of storage at 25°C. The bacteriocin activity was stable at pHs of between 2 and 5, but activity was lost at increasing pHs of 6 to 11.

Bacteriocin production, purification, and amino acid sequence.
The bacteriocin produced by C. piscicola A9b was purified by ammonium sulfate precipitation, cation-exchange chromatography, hydrophobic interaction chromatography (HIC), and reverse-phase chromatography. The HIC step resulted in a single A280 peak, and SDS-PAGE analysis showed an electrophoretically pure peptide. However, since a desalting procedure (ultrafiltration) resulted in only 10% recovery of the active material (data not shown), a final separation by reverse-phase HPLC was performed. Reverse-phase HPLC yielded one absorbance peak coincident with the activity peak (data not shown), and the entire purification process resulted in an approximately 1.4 x 106-fold increase in specific activity (data not shown). SDS-PAGE analysis of the peptide obtained from cation-exchange chromatography showed a molecular mass of approximately 4.5 kDa and inhibitory activity against L. monocytogenes O57 (data not shown).

Amino-terminal sequence analysis of the pure bacteriocin obtained from reverse-phase HPLC showed that 48 amino acid residues had a sequence identical to the N-terminal sequence of carnobacteriocin B2 produced by C. piscicola LV17B (33). The molecular mass calculated on the basis of the amino acid sequence was 4,969.5 Da. The bacteriocin produced by C. piscicola A9b is called carnobacteriocin B2 from here on.

Influence of inoculum size and carbon sources on specific bacteriocin production.
Inoculum size had a large effect on bacteriocin production in media without acetate, with the exception of MRS7-GCA broth (Table 1). In these media, specific bacteriocin production was low or undetectable with an inoculum size of 0.001% (from 0 to 160 BU/ml) compared to an inoculum size of 1.0% (from 450 to 1,100 BU/ml). However, bacteriocin production was restored by the addition of a culture supernatant from the wild-type bacteriocin-producing culture (Bac+) of strain A9b corresponding to an inoculum of 1.0%. A supernatant from the non-bacteriocin-producing mutant of A9b (Bac-) did not influence the production of bacteriocin, a result which indicates that A9b Bac+ produces an extracellular induction factor (Table 1).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Influence of inoculum size and addition of carbohydrate sources on bacteriocin production by C. piscicola A9b in MRS7-GCA broth at 25°Ca

The bacteriocin specific activity was doubled in the presence of acetate (increased from 600 to 1,000 BU/OD600 unit) and suppressed in the presence of glucose (decreased from 600 to 0 to 20 BU/OD600 unit) and citrate (decreased from 600 to 160 BU/OD600 unit). However, the suppressing effect of glucose and citrate on bacteriocin production was reversed by the addition of acetate or a filter-sterilized supernatant from the bacteriocin-producing culture (Bac+) corresponding to an inoculum of 1.0% (Table 1). The level of production obtained in the presence of acetate and/or the supernatant from the Bac+ culture was similar to that obtained when a 1.0% inoculum was used.

Characterization of the extracellular induction factor produced by C. piscicola A9b
The pattern of protease sensitivity of the extracellular induction factor produced by C. piscicola A9b was similar to that obtained for the bacteriocin, indicating that the induction factor could be the bacteriocin itself. The induction activity was recorded at each purification step of the bacteriocin except for reverse-phase HPLC. The induction factor was present in all fractions, (i) supernatant, (ii) ammonium sulfate precipitation, (iii) cation exchange, and (iv) HIC, corresponding to total induction activities of 3 x 107, 6.8 x 107, 3.3 x 106, and 2.1 x 106 IU ml-1, respectively (data not shown). These data strongly indicate that carnobacteriocin B2 produced by C. piscicola A9b functions as an inducer of its own synthesis.

The induction activity was dose dependent up to a level of 20 IU of the induction factor ml-1 in MRS7-CA broth (Fig. 1A), corresponding to a concentration of 1.9 x 10-8 M (calculated from a protein concentration of 8.92 x 10-8 mg ml-1 and a size of 4,634 g mol-1). At higher levels of the induction factor, the specific activity reached a plateau of approximately 800 BU/OD600 unit. The kinetics of the relationship in MRS7-CA broth could be described by a Michaelis-Menten equation. The critical concentration of carnobacteriocin B2 required to induce its own biosynthesis was 9.6 x 10-10 M (calculated from a protein concentration of 4.46 x 10-9 mg ml-1 and a size of 4,634 g mol-1), a value which corresponded to an approximate antilisterial activity of 0.007 BU.



View larger version (17K):
[in this window]
[in a new window]
  		FIG. 1. Dose-dependent induction of bacteriocin production in C. piscicola A9b by increasing concentrations of carnobacteriocin B2 and acetate as induction factors. (A) Carnobacteriocin B2 was added to MRS7-CA broth ({blacktriangleup}) and MRS7-C broth ({square}) at 25°C. One IU milliliter-1 equals 9.6 x 10-10 M induction factor. Data are from duplicate determinations. (B) Acetate was added to MRS7-CA broth ({blacktriangleup}) and APT broth ({blacksquare}) at 25°C. Data are from a single experiment.

The combination of acetate and increasing concentrations of carnobacteriocin B2 as an induction factor did not affect the saturation level of bacteriocin production (800 BU/OD600 unit) (Fig. 1A). However, the kinetics of the dose-dependent relationship in MRS7-C broth differed from those in MRS7-CA broth, since the saturation level for bacteriocin production in MRS7-C broth was reached at all levels of the induction factor carnobacteriocin B2 (from 0 to 100 IU ml-1) (Fig. 1A).

Dose-dependent induction of bacteriocin production by acetate.
The induction of bacteriocin production showed a dose-dependent relationship at acetate concentrations of up to 10 and 20 mM in MRS7-CA broth and APT broth, respectively (Fig. 1B). At higher acetate concentrations, the specific activity reached plateaus of 900 to 1,200 BU/OD600 unit and 120 BU/OD600 unit in MRS7-CA broth and APT broth, respectively. In MRS7-CA broth, the plateau was unaffected by the addition of higher acetate concentrations of up to 90 mM, but the induction capacity of acetate was lost at approximately 70 mM acetate in APT broth, probably due to a low biomass. The kinetics of the relationship in MRS7-CA broth could be described by a Michaelis-Menten equation. The critical concentrations of acetate required to induce bacteriocin production were in the ranges of 0.3 to 1 mM and 12 mM in MRS7-CA broth and APT broth, respectively.

Influence of acetate on the induction of bacteriocin production.
The production of bacteriocin by strain A9b in MRS7-C broth was detectable from an OD600 of approximately 0.2 (Fig. 2). We hypothesized that bacteriocin production had to be induced before the culture reached this stage of growth (OD600, ~0.2). Acetate was added to the culture of A9b Bac+ at different stages in the growth phase (Fig. 2). Bacteriocin production reached a maximum level of 1,200 BU/ml when acetate was added within 8 h from the start of the experiment, corresponding to a cell density of less than approximately 5 x 106 CFU/ml (Fig. 2). A minor reduction in production was observed when acetate was added after 10 to 12 h of incubation, and only small amounts of bacteriocin were detected when acetate was added after 14 h of incubation, corresponding to an OD600 of approximately 0.2. At OD600s of greater than 0.2 to 0.5, the addition of acetate did not result in detectable bacteriocin production (Fig. 2).



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 2. Influence of acetate on the induction of bacteriocin production in C. piscicola A9b. Acetate was added to a growing culture of strain A9b Bac+ at different stages in the growth phase, and bacteriocin production was detected when the culture reached the stationary phase of growth after 24 h of incubation at 25°C (white bars). When acetate was added at time zero, bacteriocin production ({blacksquare}) was determined during growth ({square}). The error bars indicate the means and standard deviations of duplicate determinations.

The induction capacity of acetate was investigated with several LAB strains. Acetate did not influence the induction of bacteriocin production in any of the other LAB strains investigated (data not shown).

Influence of glucose on the efficiency of acetate as an induction factor.
The acetate concentration was determined during growth in MRS7-GCA broth and MRS7-CA broth to determine if glucose suppressed the production of acetate (Fig. 3). Bacteriocin production in MRS7-GCA broth reached a maximum concentration of 360 BU/ml in the stationary phase, whereas no bacteriocin was produced when glucose was added (in MRS7-CA broth) (Fig. 3A and B). Within the first 16 h of incubation, no significant differences in acetate concentration (Fig. 3D) or growth rate (Fig. 3A) were observed in comparison to the results obtained with MRS7-GCA broth. Although the concentration of glucose decreased by 15 mM (from 142.2 to 127.4 mM) within the first 16 h of incubation in MRS7-CA broth compared to only 0.1 mM (from 0.12 to 0.017 mM) in MRS7-GCA broth (Fig. 3C), no significant difference in pH was observed (Fig. 3E). These data indicate that the suppressing effect of glucose on the induction of bacteriocin production is not due to a decrease in the production of acetate or a decrease in the pH as a result of the metabolic conversion of glucose. However, from the present study, it is not clear whether the decrease in the pH in MRS7-CA broth (from 6.5 to 5.0) after the induction of bacteriocin production reduces the production of bacteriocin to low or undetectable levels.



View larger version (19K):
[in this window]
[in a new window]
  		FIG. 3. Growth of C. piscicola A9b Bac+ in MRS7-GCA broth ({square}) and MRS7-CA broth ({blacksquare}) at 25°C. OD (A), bacteriocin production (B), glucose concentration (C), acetate concentration (D), and pH (E) were determined. The error bars indicate means and standards deviations of duplicate determinations.

We hypothesized that glucose may interfere with the response to acetate as an induction factor. To test this hypothesis, acetate and glucose were added to MRS7-GCA broth at different concentrations (Fig. 4). The induction efficiency of acetate decreased with increasing concentrations of glucose. However, glucose had no apparent effect on the induction efficiency of acetate at a high concentration (36.74 mM). In media with low acetate concentrations (1.8 and 5.5 mM), the addition of even small amounts of glucose (from 13.9 mM) reduced the induction efficiency of acetate dramatically (from approximately 350 BU/ml to 0 to 45 BU/ml).



View larger version (64K):
[in this window]
[in a new window]
  		FIG. 4. Influence of the concentrations (conc.) of glucose and acetate on specific bacteriocin production by C. piscicola in MRS7-GCA broth at 25°C. Data represent means of duplicates.

Influence of food-relevant environmental factors on the induction capacities of acetate and carnobacteriocin B2.
The growth of C. piscicola in cold smoked salmon juice caused an increase in the acetate concentration from 3 to 5 mM to 9 mM in 32 days at 5°C and a decrease in the glucose concentration from 4 mM to 0.5 mM in 20 days. Since the concentration of acetate was above the threshold level for induction in MRS7-CA broth (0.3 to 1 mM) and the concentration of glucose was low, we hypothesized that acetate could act as a natural induction factor in cold smoked salmon juice. To test this hypothesis, acetate was added to cold smoked salmon juice (with 4 and 5% [wt/vol] NaCl) at increasing concentrations ranging from 0 to 36.74 mM. No bacteriocin production was found in any of the samples (data not shown), even in the presence of a high acetate concentration (Table 2). These data indicate that acetate could not act as an inducer for bacteriocin production in cold smoked salmon juice.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Influence of induction factor and NaCl on growth and production of bacteriocin by C. piscocola A9b inoculated at 0.001% into MRS7-CA broth and salmon juice at 5 or 25°Ca

The induction capacity of acetate decreased dramatically with increasing concentrations of salt in MRS7-CA broth, and no induction effect was observed in the presence of 1.5% NaCl (Table 2). The induction capacity of carnobacteriocin B2 was not influenced by the presence of 0 to 2.5% NaCl in MRS7-CA broth but was decreased in the presence of 3.0% NaCl (reduced from 640 to 320 BU ml-1) (Table 2) to 7.0% NaCl (reduced from 640 to 20 BU ml-1) (Table 2).

The induction capacity of carnobacteriocin B2 in cold smoked salmon juice in the presence of 4% NaCl was investigated. Bacteriocin was induced by carnobacteriocin B2, and bacteriocin activity was similar to that obtained in MRS7-CA broth with 4% NaCl. However, the critical level of carnobacteriocin B2 needed to induce its own biosynthesis at 25°C was approximately 450 times higher in salmon juice with 4% NaCl than in MRS7-CA broth without NaCl (increased from 9.6 x 10-10 to 4.3 x 10-7 M) (data not shown).

We tested the antilisterial effect of C. piscicola A9b in a coculture with L. monocytogenes in cold smoked salmon juice in the absence and presence of the induction factor carnobacteriocin B2 (Fig. 5). The number of L. monocytogenes cells as a pure culture increased from 103 to 108 CFU/ml after 22 days of storage at 5°C. The growth of L. monocytogenes was unaffected in the mixed culture with C. piscicola A9b (initial level of 103 CFU/ml) when the induction factor was not added. In the absence of the induction factor, no bacteriocin was produced during storage. However, in the presence of the induction factor (114 IU ml-1), the growth of L. monocytogenes was suppressed after 17 days of incubation and the maximum cell number was reduced by 2.5 log units. The inhibition of L. monocytogenes coincided with the detection of produced bacteriocin. The growth of L. monocytogenes was strongly reduced in the presence of a high initial cell number of C. piscicola (106 CFU/ml) and in the presence of the induction factor carnobacteriocin B2 (114 IU ml-1). Bacteriocin was detected after 6 days of incubation, and the level increased from 80 BU ml-1 initially to 320 BU ml-1 at the end of storage. A higher concentration of added inducer (457 IU ml-1) resulted in the production of bacteriocin after 2 days of incubation and an enhanced inhibition of L. monocytogenes (data not shown).



View larger version (21K):
[in this window]
[in a new window]
  		FIG. 5. Growth of L. monocytogenes O57 (A) and C. piscicola A9b (B) as monocultures and cocultures in salmon juice at 5°C. Symbols: {square}, L. monocytogenes O57 alone; {triangledown}, L. monocytogenes as a coculture with C. piscicola (0.001% inoculum); , L. monocytogenes as a coculture with C. piscicola (0.001% inoculum) and the addition of carnobacteriocin (114 IU ml-1) at time zero; {triangleup}, L. monocytogenes as a coculture with C. piscicola (1.0% inoculum, which results in an induction factor concentration of 114 IU ml-1). The error bars indicate means and standard deviations of duplicate determinations. The arrow indicates the onset of bacteriocin production.


arrow
DISCUSSION
 
C. piscicola strain A9b was originally isolated as part of the dominant flora of cold smoked salmon (29) and used as a bioprotective culture for inhibiting L. monocytogenes in cold smoked salmon (27). In this study, we demonstrated that C. piscicola A9b produces an autoinducible antilisterial bacteriocin that is induced by acetate. To our knowledge, this is the first time that acetate has been described as an induction factor for bacteriocin production.

The N-terminal amino acid sequence of the bacteriocin produced by C. piscicola A9b was identical to that of carnobacteriocin B2 from C. piscicola LV17B (32, 33), which also produces carnobacteriocins A and BM1. The bacteriocin produced by C. piscicola A9b is therefore referred to here as carnobacteriocin B2. Carnobacteriocin B2 from LV17B is encoded on a 61-kb plasmid, and BM1 is encoded from a genetic fragment (1, 33). C. piscicola A9b produces only carnobacteriocin B2, and the genetic background differs from that of C. piscicola LV17B. It frequently happens that the same bacteriocin is isolated from several strains of the same species, e.g., lactobin A and amylovorin L471 (6), carnobacteriocin BM1 and piscicocin V1b (5), piscicocin V1a and piscicolin 126 (12), and mesenterocin 52A and mesentericin Y105 (36).

Our purification procedure consisted of four steps; in contrast, Quadri et al. (33) purified bacteriocins B2 and BM1 by a three-step procedure. Our last step, reverse-phase chromatography, was used only for desalting purpose but resulted in a 35-fold increase in specific activity.

Bacteriocin production in LAB can be influenced by environmental parameters, which may act on the bacteriocin regulatory system (9, 12) or affect the binding of the induction factor to its receptor (25). In this study, we have demonstrated that in MRS broth with glucose as a C source, acetate is required to induce bacteriocin production in C. piscicola A9b. In other studies, growth and bacteriocin production of C. piscicola strains were obtained in MRS broth without acetate (39, 40). A large inoculum (0.5%) may explain why bacteriocins were produced in these studies. No similar effect of acetate was observed in any other examined bacteriocin-producing LAB. Bacteriocin production is stimulated by less favorable growth conditions, such as low temperature (23), a competitive flora (41), high osmolarity (NaCl) (43), ethanol (23), or mitomycin (19). It has been suggested that bacteriocin production could be the result of the transcription of genes involved in stress responses (9). The fact that Carnobacterium does not grow well in media containing acetate (7, 22) could explain the induction capacity of acetate. Ecologically, the effect of unfavorable growth conditions on bacteriocin production is an advantage, as antagonism due to, e.g., organic acid is not sufficient for successful competition by other microorganisms (9).

Acetate induction has several features in common with peptide-based induction of bacteriocin production in two- or three-component signal transduction systems. Acetate must be present during the early exponential growth phase for induction to occur, as is the case for most peptide-based inducers regulating bacteriocin production, such as those in C. piscicola LV17B (38) and E. faecium CTC492 (25). The acetate induction of bacteriocin was dose dependent, and increasing NaCl or glucose concentrations reduced the efficiency of acetate as an inducer. These relationships are similar to those for the peptide-based induction factor in E. faecium CTC492 (25), where it has been suggested that NaCl negatively influences the binding of the induction factor to its receptor (25).

We found that glucose repressed the induction efficiency of acetate for carnobacteriocin production (Fig. 4). This result could have been caused by a direct effect of glucose on bacteriocin production or indirectly through changes in pH. Glucose can negatively affect response regulators; e.g., in Staphylococcus aureus, glucose reduces the expression of the accessory gene regulator (agr), which regulates the expression of numerous exoproteins in two-component signal transduction systems (35). Further, pH is also known to influence the regulation of signal transduction systems (25, 34). The inducing effect of acetate occurred before changes in pH or acetate concentrations could be measured. We therefore suggest that glucose by itself repressed the induction efficiency of acetate.

We also demonstrated that the biosynthesis of carnobacteriocin B2 in C. piscicola strain A9b is an autoinduced process similar to B2 and BM1 production in C. piscicola LV17B (32, 38) and to the production of some lantibiotics, e.g., nisin (17). The autoinduction is dose dependent, like that for bacteriocin production in E. faecium CTC492 (25).

A threshold concentration of carnobacteriocin B2 from A9b of approximately 10-9 M was required to trigger its own biosynthesis. This level is similar to the threshold level of about 10-10 M previously reported for nisin (17), sakacin P (11), and carnobacteriocins B2 and BM1 (32) but significantly higher than the level of 10-17 M found for induction factor EntF in E. faecium (25). The highest yield (800 to 1,200 BU ml-1) was unaffected by the type of induction factor (acetate or bacteriocin) present, and Nilsen et al. (25) similarly showed that bacteriocin production reaches a saturation level. The production of carnobacteriocin B2 from strain A9b was not enhanced by the combined presence of both induction factors and, to our knowledge, this is the first time that the combined action of two induction factors on bacteriocin production has been examined.

Acetate is present in freshly produced dry salted cold smoked salmon at levels of 2 to 3 µmol g-1 (42), and levels increase to approximately 23 µmol g-1 after 35 days of storage at 5°C as a result of microbiological growth (42). However, in the present study, acetate did not induce bacteriocin production in cold smoked salmon juice due to the presence of moderate NaCl levels (3 to 5% water-phase salt). The induction capacity of acetate was lost at NaCl concentrations above 1.5% in MRS7-CA broth. Increasing NaCl concentrations also reduced the efficiency of carnobacteriocin B2 from strain A9b as an induction factor, but induction did occur, albeit at a lower efficiency, even at an NaCl concentration of 7%. Nilsen et al. (25) also showed that NaCl negatively influenced the induction efficiency of EntF; at 6.5% NaCl, 300 times more EntF was needed to induce detectable bacteriocin production (25). In cold smoked salmon juice, however, adding carnobacteriocin B2 as an induction factor restored bacteriocin production, and detection coincided with the inhibition of L. monocytogenes. It was suggested that NaCl negatively influences the binding of the induction factor to its receptor (25).

In conclusion, in this study we have demonstrated that although both acetate and carnobacteriocin induce the production of carnobacteriocin B2 in strain A9b, only the bacteriocin itself can be used to manipulate the production of bacteriocin in cold smoked salmon juice and thereby improve the ability of C. piscicola strain A9b to inhibit L. monocytogenes. Further, high initial levels (e.g., 106 CFU/g) of the bacteriocinogenic culture must be used when bacteriocin production does not occur before the middle to late exponential phase of growth, as occurs in C. piscicola.


arrow
ACKNOWLEDGMENTS
 
This work was funded by the Danish Ministry of Food, Agriculture and Fisheries (biotechnology program).

We are grateful to Brian Himelbloom, University of Alaska, Fairbanks, for proving valuable input to the experiments. We thank Anne Louise Petersen and Karen Reimers for skillful assistance and Henrik Hauch Nielsen and Flemming Jessen for suggestions about peptide purification. Further, we thank Vibeke Barkholt and Anne Blicker, Department of Biochemistry and Nutrition, Technical University of Denmark, for performing the amino acid sequencing and Thomas Rand and Ole C. Hansen, Biotechnological Institute, Hørsholm, Denmark, for helping with reverse-phase HPLC purification.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Danish Institute for Fisheries Research, Department of Seafood Research, c/o Technical University of Denmark, Building 221, DK-2800 Lyngby, Denmark. Phone: 45 45 25 49 21. Fax: 45 45 88 47 74. E-mail: lni{at}dfu.min.dk. Back


arrow
REFERENCES
 
    1
  1. Ahn, C., and M. E. Stiles. 1990. Plasmid-associated bacteriocin production by a strain of Carnobacterium piscicola from meat. Appl. Environ. Microbiol. 56:2503-2510.[Abstract/Free Full Text]
    2. 1
  2. Association of Official Analytical Chemists. 1975. Official methods of analysis, 12th ed. Association of Official Analytical Chemists, Washington, D.C.
    3. 2
  3. Baya, A. M., A. E. Toranzo, B. Lupiani, T. Li, B. S. Roberson, and F. M. Hetrick. 1991. Biochemical and serological characterization of Carnobacterium spp. isolated from farmed and natural populations of striped bass and catfish. Appl. Environ. Microbiol. 57:3114-3120.[Abstract/Free Full Text]
    4. 3
  4. Ben Embarek, P. K., and H. H. Huss. 1993. Heat resistance of Listeria monocytogenes in vacuum packaged pasteurized fish fillets. Int. J. Food Microbiol. 20:85-95.[CrossRef][Medline]
    5. 4
  5. Ben Embarek, P. K., V. F. Jeppesen, and H. H. Huss. 1994. Antibacterial potential of Enterococcus faecium strains isolated from sous-vide cooked fish fillets. Food Microbiol. 2:525-536.
    6. 5
  6. Bhugaloo-Vial, P., X. Dousset, A. Metivier, O. Sorokine, P. Anglade, P. Boyaval, and D. Marion. 1996. Purification and amino acid sequences of piscicocins V1a and V1b, two class IIa bacteriocins secreted by Carnobacterium piscicola V1 that display significantly different levels of specific inhibitory activity. Appl. Environ. Microbiol. 62:4410-4416.[Abstract]
    7. 6
  7. Callewaert, R., H. Holo, B. Devreese, J. van Beeumen, I. Nes, and L. de Vuyst. 1999. Characterization and production of amylovorin L471, a bacteriocin purified from Lactobacillus amylovorus DCE 471 by a novel three-step method. Microbiology 145:2559-2568.[Abstract/Free Full Text]
    8. 7
  8. Collins, M. D., C. Ash, J. A. E. Ferrow, B. A. Phillips, S. Ferusus, and D. Jones. 1987. Classification of Lactobacillus divergens, Lactobacillus piscicola, and some catalase-negative, asporogenous, rod-shaped bacteria from poultry in a new genus, Carnobacterium. Int. J. Syst. Bacteriol. 37:310-317.[Abstract/Free Full Text]
    9. 8
  9. Degnan, A. J., A. E. Yousef, and J. B. Luchansky. 1992. Use of Pediococcus acidilactici to control Listeria monocytogenes in temperature-abused vacuum packed wieners. J. Food Prot. 55:98-103.
    10. 9
  10. De Vuyst, L., R. Callewaert, and K. Crabbé. 1996. Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiology 142:817-827.
    11. 10
  11. Duffes, F., C. Corre, F. Leroi, X. Dousset, and P. Boyaval. 1999. Inhibition of Listeria monocytogenes by in situ produced and semipurified bacteriocins of Carnobacterium spp. On vacuum-packed, refrigerated cold-smoked salmon. J. Food Prot. 62:1394-1403.[Medline]
    12. 11
  12. Eijsink, V. G. H., M. B. Brurberg, P. H. Middelhoven, and I. F. Nes. 1996. Induction of bacteriocin production in Lactobacillus sake by secreted peptide. J. Bacteriol. 178:2232-2237.[Abstract/Free Full Text]
    13. 12
  13. Ennahar, S., T. Sashihara, K. Sonomoto, and A. Ishizaki. 2000. Class IIa bacteriocins: biosynthesis, structure and activity. FEMS Microbiol. Rev. 24:85-106.[CrossRef][Medline]
    14. 13
  14. Farber, J. M., and P. I. Peterkin. 1991. Listeria monocytogenes, a food-borne pathogen. Microbiol. Rev. 55:476-511.[Abstract/Free Full Text]
    15. 14
  15. Hastings, J. W., M. Sailer, K. Johnson, K. L. Roy, J. C. Vederas, and M. E. Stiles. 1991. Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelium. J. Bacteriol. 173:7491-7500.[Abstract/Free Full Text]
    16. 15
  16. Himelbloom, B., L. Nilsson, and L. Gram. 2001. Factors affecting production of an antilisterial bacteriocin by Carnobacterium piscicola strain A9b in laboratory media and model fish systems. J. Appl. Microbiol. 91:506-513.[CrossRef][Medline]
    17. 16
  17. Hiu, S. F., R. A. Holt, N. Sriranganathan, R. J. Seidler, and J. L. Fryer. 1984. Lactobacillus piscicola, a new species from salmonid fish. Int. J. Syst. Bacteriol. 34:393-400.
    18. 17
  18. Kuipers, O. P., M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos. 1995. Autoregulation of nisin biosynthesis in Lactococcus lactis by signal transduction. J. Biol. Chem. 270:27299-27304.[Abstract/Free Full Text]
    19. 18
  19. Larsen, A. G., F. K. Vogensen, and J. Josephsen. 1993. Antimicrobial activity of lactic acid bacteria isolated from sour doughs: purification and characterization of bavaricin A, a bacteriocin produced by Lactobacillus bavaricus MI401. J. Appl. Bacteriol. 75:113-122.[Medline]
    20. 19
  20. Lazdunski, C. J., D. baty, V. Geli, D. Gavard, J. Morlon, R. Lloubes, S. P. Howard, M. Knibiehler, M. Chartier, S. Varenne, M. Frenette, J. L. Dasseux, and F. Pattus. 1988. The membrane channel-forming colicin A:synthesis, secretion, structure and immunity. Biochim. Biophys. Acta 947:445-464.[Medline]
    21. 20
  21. Leroy, F., and L. de Vuyst. 1999. The presence of salt and a curing agent reduces bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation. Appl. Environ. Microbiol. 65:5350-5356.[Abstract/Free Full Text]
    22. 21
  22. Lozano, J. C. N., J. Nissen-Meyer, K. Sletten, C. Peláz, and I. F. Nes. 1992. Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici. J. Gen. Microbiol. 138:1985-1990.[Medline]
    23. 22
  23. McMullen, L. M., and M. E. Stiles. 1993. Microbial ecology of fresh pork stored under modified atmosphere at -1, 4.4 and 10°C. Int. J. Food Microbiol. 18:1-14.[CrossRef][Medline]
    24. 23
  24. Mørtvedt-Abildgaard, C. I., J. Nissen-Meyer, B. Jelle, B. Grenov, M. Skaugen, and I. F. Nes. 1995. Production and pH-dependent bactericidal activity of lactocin S, a lantibiotic from Lactobacillus sake L45. Appl. Environ. Microbiol. 61:175-179.[Abstract]
    25. 24
  25. Nes, I. F., D. B. Diep, L. S. Håvarstein, M. B. Brurberg, V. Eijsink, and H. Holo. 1996. Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Leeuwenhoek Int. J. Gen. Mol. Microbiol. 70:113-128.
    26. 25
  26. Nilsen, T., I. F. Nes, and H. Holo. 1998. An exported inducer peptide regulates bacteriocin production in Enterococcus faecium CTC492. J. Bacteriol. 180:1848-1854.[Abstract/Free Full Text]
    27. 26
  27. Nilsson, L. 1999. Control of Listeria monocytogenes in cold-smoked salmon by biopreservation. Ph.D. thesis. Danish Institute for Fisheries Research, Department of Seafood Research, Lyngby, Denmark.
    28. 27
  28. Nilsson, L., L. Gram, and H. H. Huss. 1999. Growth control of Listeria monocytogenes on cold-smoked salmon using a competitive lactic acid bacteria flora. J. Food Prot. 62:336-342.[Medline]
    29. 28
  29. Nilsson, L., H. H. Huss, and L. Gram. 1997. Inhibition of Listeria monocytogenes on cold-smoked salmon by nisin and carbon dioxide atmosphere. Int. J. Food Microbiol. 38:217-227.[CrossRef][Medline]
    30. 29
  30. Paludan-Müller, C., P. Dalgaard, H. H. Huss, and L. Gram. 1998. Evaluation of the role of Carnobacterium piscicola in spoilage of vacuum- and modified-packed cold-smoked salmon stored at 5°C. Int. J. Food Microbiol. 39:155-166.[CrossRef][Medline]
    31. 30
  31. Papathanasopoulos, M. A., F. Krier, A.-M. Revol-Junelles, G. Lefebvre, J.-P. Le Caer, A. von Holy, and J. W. Hastings. 1997. Multiple bacteriocin production by Leuconostoc mesenteroides TA33a and other Leuconostoc/Weissella strains. Curr. Microbiol. 35:331-335.[CrossRef][Medline]
    32. 31
  32. Piard, J. C., and M. Desmazeaud. 1992. Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antimicrobial substances. Lait 72:113-142.
    33. 32
  33. Quadri, L. E. N., M. Kleerebezem, O. P. Kuipers, W. M. de Vos, K. L. Roy, J. C. Vederas, and M. E. Stiles. 1997. Characterization of a locus from Carnobacterium piscicola LV17B involved in bacteriocin production and immunity: evidence for global inducer-mediated transcriptional regulation. J. Bacteriol. 179:6163-6171.[Abstract/Free Full Text]
    34. 33
  34. Quadri, L. E. N., M. Sailer, K. L. Roy, J. C. Vederas, and M. E. Stiles. 1994. Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B. J. Biol. Chem. 269:12204-12211.[Abstract/Free Full Text]
    35. 34
  35. Regassa, L. B., and M. J. Betley. 1992. Alkaline pH decreases expression of the accessory gene regulator (arg) in Staphylococcus aureus. J. Bacteriol. 174:5095-5100.[Abstract/Free Full Text]
    36. 35
  36. Regassa, L. B., R. P. Novick, and M. J. Betley. 1992. Glucose and nonmaintained pH decrease expression of the accessory gene regulator (arg) in Staphylococcus aureus. Infect. Immun. 60:3381-3388.[Abstract/Free Full Text]
    37. 36
  37. Revol-Junelles, A.-M., R. Mathis, F. Krier, Y. Fleury, A. Delfour, and G. Lefebvre. 1996. Leuconostoc mesenteroides subsp. mesenteroides FR52 synthesized two distinct bacteriocins. Lett. Appl. Microbiol. 23:120-124.[Medline]
    38. 37
  38. Sahl, H.-G., and G. Bierbaum. 1998. Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from Gram-positive bacteria. Annu. Rev. Microbiol. 52:41-79.[CrossRef][Medline]
    39. 38
  39. Saucier, L., A. Poon, and M. E. Stiles. 1995. Induction of bacteriocin in Carnobacterium piscicola LV17. J. Appl. Bacteriol. 78:684-690.
    40. 39
  40. Schillinger, U., and W. H. Holzapfel. 1990. Antibacterial activity of carnobacteria. Food Microbiol. 7:305-310.
    41. 40
  41. Schillinger, U., M. E. Stiles, and W. H. Holzapfel. 1993. Bacteriocin production by Carnobacterium piscicola LV 61. Int. J. Food Microbiol. 20:131-147.[CrossRef][Medline]
    42. 41
  42. Sip, A., W. Grajek, and P. Boyaval. 1998. Enhancement of bacteriocin production by Carnobacterium divergens AS7 in the presence of a bacteriocin-sensitive strain Carnobacterium piscicola. Int. J. Food Microbiol. 42:63-69.[CrossRef][Medline]
    43. 42
  43. Truelstrup Hansen, L., T. Gill, S. Drewes Røntved, and H. H. Huss. 1996. Importance of autolysis and microbiological activity on quality of cold-smoked salmon. Food Res. Int. 29:181-188.[CrossRef]
    44. 43
  44. Uguen, P., J. Hamelin, J.-P. Le Pennec, and C. Blanco. 1999. Influence of osmolarity and the presence of an osmoprotectant on Lactococcus lactis growth and bacteriocin production. Appl. Environ. Microbial. 65:291-293.[Abstract/Free Full Text]
    45. 44
  45. Van Belkum, M. J., R. W. Worobo, and M. E. Stiles. 1997. Double-glycine-type leader peptides direct secretion of bacteriocins by ABC-transporters: colicin V in Lactococcus lactis. Mol. Microbiol. 23:1293-1301.[CrossRef][Medline]

Applied and Environmental Microbiology, May 2002, p. 2251-2260, Vol. 68, No. 5
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.5.2251-2260.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Verluyten, J., Messens, W., De Vuyst, L. (2004). Sodium Chloride Reduces Production of Curvacin A, a Bacteriocin Produced by Lactobacillus curvatus Strain LTH 1174, Originating from Fermented Sausage. Appl. Environ. Microbiol. 70: 2271-2278 [Abstract] [Full Text]  
  • Gravesen, A., Kallipolitis, B., Holmstrom, K., Hoiby, P. E., Ramnath, M., Knochel, S. (2004). pbp2229-Mediated Nisin Resistance Mechanism in Listeria monocytogenes Confers Cross-Protection to Class IIa Bacteriocins and Affects Virulence Gene Expression. Appl. Environ. Microbiol. 70: 1669-1679 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Nilsson, L.
Right arrow Articles by Gram, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Nilsson, L.
Right arrow Articles by Gram, L.
Agricola
Right arrow Articles by Nilsson, L.
Right arrow Articles by Gram, L.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»