Research Group of Industrial Microbiology, Fermentation Technology and Downstream Processing, Department of Applied Biological Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium1
Laboratory of Microbial Gene Technology, Department of Biotechnological Sciences, Agricultural University of Norway, Post Box 5051, N-1432 s, Norway2
Laboratory of Protein Biochemistry and Protein Engineering, Faculty of Sciences, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium3
Author for correspondence: Luc De Vuyst. Tel: +32 2 629 32 45. Fax: +32 2 629 27 20. e-mail: ldvuyst{at}vub.ac.be
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: lactic acid bacteria, Lactobacillus amylovorus, amylovorin L471, bacteriocin purification, bacteriocin production and adsorption
Abbreviations: RP, reversed-phase; TFA, trifluoroacetic acid
The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is P81927.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several protocols and chromatographic methods have been proposed for the analytical purification to homogeneity of lactic acid bacteria bacteriocins. Chromatography is usually applied after a first concentration step by salt precipitation (Muriana & Klaenhammer, 1991 ) or acid extraction (Yang et al., 1992
). The procedure further includes cationic exchange, hydrophobic interaction, gel filtration and/or reversed-phase chromatography (Hastings et al., 1991
; Nissen-Meyer et al., 1992
). However, only a few authors have reported simple procedures for bacteriocin purification. Methods described include batch adsorption to an appropriate adsorbent based on hydrophobic or electrostatic interactions (Stoffels et al., 1993
), acid extraction and reversed-phase (RP)-HPLC (Daba et al., 1994
), ammonium sulphate precipitation and reversed-phase chromatography (Joosten et al., 1996
), pH-mediated cell adsorptiondesorption and semi-preparative RP-HPLC (Elegado et al., 1997
), and ethanol precipitation, preparative isoelectric focusing and ultrafiltration (Venema et al., 1997
).
Among the large number of bacteriocins from Lactobacillus species, amylovorin L471 was the first bacteriocin to be characterized from Lactobacillus amylovorus (De Vuyst et al., 1996a ). Amylovorin L471 is a small, heat-stable and extremely hydrophobic peptide, produced by L. amylovorus DCE 471 and displaying antagonistic activity against closely related strains such as Lactobacillus delbrueckii subsp. bulgaricus LMG 6901T (the most sensitive strain), L. delbrueckii subsp. delbrueckii ATCC 6949 and Lactobacillus helveticus ATCC 15009. Under native conditions, the bacteriocin forms aggregates, the molecular mass of which exceeds 30 kDa. Under reduced conditions, two peptide bands can be visualized by Tricine/SDS-PAGE, both with a molecular mass of less than 6000 Da. Production of amylovorin L471 in batch fermentation is maximal at a controlled pH of 5·0. Production starts early in the growth cycle and displays primary metabolite kinetics (De Vuyst et al., 1996b
).
In this paper we describe the purification to homogeneity of amylovorin L471 using a novel, rapid and simple isolation and purification protocol involving only one chromatographic step. We further determine the N-terminal amino acid sequence of amylovorin L471, confirm its narrow antibacterial spectrum and show its bactericidal action and constitutive expression. Finally, it is shown that adsorption of the bacteriocin molecules to the producing cells, which is responsible for the apparent loss of bacteriocin activity upon prolonged fermentation, is prevented by ethanol.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Bacteriocin purification to homogeneity and characterization of the purified peptide.
Amylovorin L471 was produced and isolated as described previously (De Vuyst et al., 1996a ). Briefly, cells were harvested from 2 l cultures (Biostat B fermenter, B. Braun Biotech International) by centrifugation (5500 g, 20 min, 4 °C) after 10 h of fermentation at 37 °C when the pH had dropped to approximately 4·2. The supernatant fraction was adjusted to pH 6·5 (with 10 M NaOH). This material was referred to as crude supernatant. Crude cell-free culture supernatant (pH 6·5) was made up to 40% ammonium sulphate and held overnight at 4 °C with stirring. The sample was centrifuged (5500 g, 30 min, 4 °C) and the surface pellicle (containing the bacteriocin) was recovered and suspended in 50 mM sodium phosphate buffer (pH 6·5). This material was considered as crude bacteriocin. Crude bacteriocin was further treated with at least 15 vols of a mixture of chloroform and methanol (2:1, v/v) for 1 h at 4 °C. The resulting fine-grained white precipitate (containing the bacteriocin) was collected by centrifugation for 30 min at 5500 g (4 °C), air-dried and dissolved in a minimal amount of 10% 2-propanol/0·1% trifluoroacetic acid (TFA). It was considered as partially purified bacteriocin and could be stored at -20 °C. Lactobin A (used in the antimicrobial spectrum assays) was similarly isolated and partially purified.
For final purification to homogeneity, an FPLC system equipped with a 1 ml C2/C18 PepRPC HR 5/5 column (Pharmacia) was used. The mobile phases A and B were 10 % 2-propanol/0·1% TFA and 100% 2-propanol/0·1% TFA, respectively. Before applying the sample to the column, two vols phase B were added to the partially purified bacteriocin, as well as HCl to a final concentration of 30 mM, in order to increase its solubility. After micro-centrifugation (13000 g, 15 min) of the partially purified bacteriocin, 500 µl of the supernatant was applied to the column. A flow rate of 0·5 ml min-1 and a stepwise gradient from 15 to 100% of phase B was applied. Fractions of 0·5 ml were collected. Absorbance was monitored both at 210 and 280 nm (VWM 2141 monitor, Pharmacia). All fractions were assayed for bacteriocin activity as described below. The active FPLC fractions (1·0 ml) were ultrafiltered through a membrane with a molecular mass cut-off of 3000 Da. The active retentate was readjusted to the original volume and was stored at -20 °C.
The molecular mass of purified amylovorin L471 was determined via electrospray mass spectrometry. The analysis was carried out on a BIO-Q triple quadrupole atmospheric pressure mass spectrometer, equipped with an electrospray ionization source (Micromass). Ten microlitres of a sample solution in acetonitrile/1% formic acid (1:1, v/v) was injected via a Rheodyne 5717 injector and pumped to the source using acetonitrile/1% formic acid (1:1, v/v) at a flow rate of 4 µl min-1, delivered by a syringe pump (Havard Instruments). Scans were registered for 2·5 min. Calibrations for precise mass measurements were performed with 100 pmol horse myoglobin (16915·5 Da). Masses of the reference protein and the purified peptide were calculated taking the mean of the residual mass of each amino acid into account (Fenn et al., 1990 ).
The N-terminal amino acid sequencing was performed by automated Edman degradation with a model 477A sequencer/model 120A phenylthiohydantoin analyser (Applied Biosystems). For homology analyses of the amino acid sequences, the clustal w program was used (Thompson et al., 1994 ).
Assay of bacteriocin activity, inhibitory spectrum and mode of action.
Bacteriocin activity was measured by an agar spot test as described previously (De Vuyst et al., 1996a ). Briefly, twofold dilutions of cell-free culture supernatant containing bacteriocin (10 µl) were spotted onto fresh indicator lawns of L. delbrueckii subsp. bulgaricus LMG 6901T. These lawns were prepared by propagating fresh cultures to an OD600 of 0·45 and adding 100 µl of the cell suspension to 3·5 ml of overlay agar. Overlaid agar plates were incubated for at least 24 h at 37 °C. Activity was defined as the reciprocal of the highest dilution demonstrating complete inhibition of the indicator lawn and was expressed in activity units (AU) (ml culture medium)-1.
To assay their spectrum of activity, the antimicrobial activity of concentrated preparations of partially purified amylovorin L471, purified amylovorin L471 and partially purified lactobin A was examined against a wide range of target bacteria in at least two separate tests. All bacteriocin preparations used had an activity of 6400 AU ml-1 against L. delbrueckii subsp. bulgaricus LMG 6901T.
To investigate its mode of action, filter-sterilized, partially purified amylovorin L471 (3·5 ml of a bacteriocin solution containing 12800 AU ml-1) was added to 10 ml of an exponentially growing culture of L. delbrueckii subsp. bulgaricus LMG 6901T (the most sensitive strain), Listeria innocua RZS 01 (an insensitive strain) and L. amylovorus DCE 471 (the producer strain). Growth was followed by measurement of OD600 and by plate counting on MRS (lactobacilli) or LM (Listeria) agar.
Bacteriocin production.
To investigate whether amylovorin L471 production was constitutive or induced, samples were taken at different times during the growth cycle of L. amylovorus DCE 471. From each sample a tenfold dilution series was made, resulting in a much lower density than that from which bacteriocin production can be induced (Diep et al., 1995 ; Saucier et al., 1995
; Nilsen et al., 1998
), and each tube was further incubated until fully grown. The OD600 and the bacteriocin titre were then determined. In addition, L. amylovorus DCE 471 cells were treated with proteinase K and
-chymotrypsin for 2 h at 37 °C in 50 mM sodium phosphate buffer at pH 7·5. Both proteases are able to inactivate amylovorin L471 (De Vuyst et al., 1996a
). Before and after protease treatment cells were washed in 0·85% NaCl. Again, tenfold dilution series of the treated cells were made. Bacteriocin activity of the subsequent full-grown cultures was checked.
To study the influence of ethanol on L. amylovorus DCE 471 bacteriocin production and adsorption, a 15 l stainless steel Biostat C fermenter (B. Braun Biotech International) with temperature and pH control (Micro-MFCS for Windows NT) was used for batch fermentation experiments. The fermenter was operated at a temperature of 37 °C and a controlled pH of 5·0, and was inoculated with 1·0% (v/v) of an exponentially growing cell culture of L. amylovorus DCE 471. This culture was obtained by propagating the bacterium in MRS broth at 37 °C for 12 h; the transfer inoculum was 1·0% (v/v). Slow agitation of 50 r.p.m. was applied to keep the broth homogeneous. The fermenter was filled with 10 l modified MRS medium containing (l-1) 40 g glucose and 88 g of a complex nitrogen source [40 g bacteriological peptone (Oxoid), 32 g meat extract (Lab-Lemco, Oxoid), 16 g yeast extract (Merck)]. The medium was sterilized in situ at 121 °C for 20 min. The energy source was sterilized separately and aseptically added to the fermenter. Ethanol was added to a batch fermentation in order to obtain a gradual increase of the ethanol concentration. Samples were withdrawn aseptically at regular time intervals to determine the cell dry mass (g l-1), the bacteriocin activity (AU ml-1), and the residual glucose and ethanol concentrations (both g l-1). Cell dry mass and bacteriocin activity analyses were performed as described previously (De Vuyst et al., 1996b ). Glucose and ethanol were determined with a HPLC system (Waters). Samples were pretreated with 20% (w/v) TCA to precipitate proteins. A pre-packed column RT 300-7,8 Polyspher OA KC (Merck) and a differential refraction detector (Waters) were used. As the mobile phase, a 2·5 mM H2SO4 solution was used at a fixed flow rate of 0·4 ml min-1.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
Addition of amylovorin L471 (3·5 ml of a bacteriocin solution containing 12800 AU ml-1) to 10 ml of an exponentially growing L. delbrueckii subsp. bulgaricus LMG 6901T culture resulted in an immediate loss of viable cells. The number of colony forming units was already zero within 1 min (not shown in Fig. 4a). This indicates that amylovorin L471 possesses a uniquely high potency, although it displays a narrow inhibitory spectrum. A decrease in OD600 indicated cell lysis (Fig. 4a
). Even by reducing the bacteriocin concentration (addition of 2·0 ml of a bacteriocin solution containing 12800 AU ml-1), a similar decrease in cell number occurred albeit without cell lysis (results not shown). In contrast to L. delbrueckii subsp. bulgaricus LMG 6901T, growth of Listeria innocua RZS 01 was not influenced upon addition of amylovorin L471 to the growing culture, confirming its inability to kill Listeria spp. (Fig. 4b
). Addition of 3·5 ml of amylovorin L471 (12800 AU ml-1) to the amylovorin L471-producing strain did not affect bacterial growth (Fig. 4c
). After addition of amylovorin L471, however, no increase in bacteriocin activity could be detected in the medium, indicating an immediate adsorption of amylovorin L471 to the producing cells. An increased amylovorin L471 production by the cells did not occur either, indicating that amylovorin L471 may be produced constitutively (see below).
|
Since killing of the cells during the stationary phase may go hand-in-hand with adsorption of the bacteriocin to the cell surface of the producing cells, the influence of ethanol has been studied with respect to its capacity to prevent aggregation and adsorption of bacteriocin molecules. Bacterial growth and bacteriocin production were therefore followed during batch fermentation with or without gradual addition of ethanol (Fig. 5). During the fermentation without ethanol addition, glucose was completely consumed after approximately 15 h of incubation. This was followed by a drastic decrease in amylovorin L471 activity (Fig. 5a
). This phenomenon coincided with a drop in c.f.u. as described by De Vuyst et al. (1996a
, b). Gradual addition of ethanol during bacterial growth resulted in a similar biomass production profile (Fig. 5b
) and a higher viability of bacteriocin-producing cells. Maximal bacteriocin activity at the end of the growth phase was higher than the maximal activity obtained during fermentation without addition of ethanol (Fig. 5a
, b
). In addition, no decrease in bacteriocin activity was observed, indicating that no adsorption of the amylovorin L471 molecules to the surface of the producer cells occurred. Bacteriocin adsorption could not be observed during the growth and bacteriocin production phase either (results not shown; De Vuyst et al., 1996b
). However, adsorbed bacteriocin molecules could be recovered from the cells by both acid extraction (De Vuyst et al., 1996b
) and addition of ethanol to fermentation medium containing the cells (results not shown). The ethanol effect described here could also be obtained by co-cultivation of L. amylovorus DCE 471 and a strain of Saccharomyces cerevisiae (unpublished results).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The primary structure of the sequenced N-terminal part of amylovorin L471 is identical to that of the bacteriocin lactobin A, produced by another L. amylovorus strain (Fig. 3). Lactobin A is the only other L. amylovorus bacteriocin whose sequence has so far been determined (Contreras et al., 1997
). We do not know at present how closely these bacteriocins are related at the genetic level. The lactobin-A-producing strain and L. amylovorus DCE 471 were both isolated from a starch-containing substrate, but differ in their total cell protein profile (L. De Vuyst & B. Pot, unpublished results) and in the fact that L. amylovorus DCE 471 cells do not aggregate whereas L. amylovorus LMG P-13139 cells do so within a few minutes after mixing a test tube containing a high density of bacterial cells. The two bacteriocin producers thus behave differently phenotypically. It frequently happens that the same bacteriocin is isolated from several strains of the same species as is the case here, or from several species of the same genus. This often becomes clear only after having determined the amino acid or gene sequence. For instance, sakacin A from Lactobacillus sake (now Lactobacillus sakei) Lb706 (Holck et al., 1992
) and curvacin A from Lactobacillus curvatus LTH1174 (Tichaczek et al., 1992
) are similar. However, it is also found that an IS element has been introduced into the sakacin A gene cluster which is not found in the L. curvatus strain (unpublished results). Although supposed to be species-specific (De Vuyst & Vandamme, 1994
), one bacteriocin (pediocin AcH) has been reported to be synthesized by different strains, namely Pediococcus acidilactici and Lactobacillus plantarum (Ennahar et al., 1996
).
The N-terminal sequence of 35 amino acids of amylovorin L471 reported in this paper shows strong similarity with lactacin X (LafX), a bacteriocinogenic peptide from Lactobacillus johnsonii VPI 11088. The latter peptide is produced simultaneously with lactacin F (LafA). Both LafA and LafX peptides constitute the lactacin F complex, a two-peptide bacteriocin (Frémaux et al., 1993 ). The considerable similarity may indicate that amylovorin L471 is likewise part of a two-peptide bacteriocin, although amylovorin L471 has a very strong activity on its own. The hydropathy profiles of amylovorin L471 and lactobin A show that the whole molecule is strongly hydrophobic, with the most hydrophobic part near the C-terminal end of the currently available sequence. Lactacin X displays a similar profile.
The antimicrobial spectrum of both partially purified and purified amylovorin L471 resembled that of crude cell-free culture supernatants, which may indicate that this bacteriocin is the only one that is expressed by L. amylovorus DCE 471. However, it should be mentioned that the presence of multiple bacteriocin genes is common for a single lactic acid bacterium strain (Casaus et al., 1997 ; Anderssen et al., 1998
; Papathanasopoulos et al., 1998
). Also, the lactobin A producer, L. amylovorus P-13139, produces at least two bacteriocins, namely lactobin A and lactobin B (B. Contreras, E. Sablon & L. De Vuyst, unpublished results). Nevertheless, only lactobin A could be isolated and purified from active culture broths (Contreras et al., 1997
). Amylovorin L471 displays a very narrow inhibitory spectrum, being active only against some lactobacilli, without any significant effect against food spoilage and food-borne pathogenic bacteria. Similar narrow inhibitory spectra were observed for lactacin F (Muriana & Klaenhammer, 1987
) and lactobin A (Contreras et al., 1997
). For instance, lactacin F is bactericidal to the closely related species L. delbrueckii and L. helveticus, and to Lactobacillus fermentum and Enterococcus faecalis. The LafA peptide alone is active against only L. helveticus (Muriana & Klaenhammer, 1991
). Such killing of closely related strains may reflect a sophisticated mode of competition among related species sharing the same ecological niche. The apparent discrepancies between the inhibitory spectra of amylovorin L471 and lactobin A are difficult to explain; they may be due to the absence (lactobin A) or presence (amylovorin L471) of a disulphide bridge, which possibly also explains the small difference in molecular mass between the two peptides. The diversity of bacteriocins and their various spectra of activity may reflect the broad habitats in which lactic acid bacteria compete, ranging from fermenting food substrates to the intestinal tracts of man and animals. Why L. amylovorus DCE 471 (amylovorin L471 producer) is resistant to the bacteriocin produced by L. amylovorus LMG P-13139 (lactobin A and B producer), but not vice versa, is difficult to explain. Felix et al. (1994
) suggested that the leader peptide of the prebacteriocin may play an additional role in producer immunity, since the only differences detected between leucocin B-Ta11a and leucocin A-UAL 187 were in the N-terminal extensions; the leucocin A-UAL 187-producing strain inhibits the leucocin B-Ta11a producer Lactobacillus carnosum Ta11a, but not vice versa. We do not know at present whether amylovorin L471 and lactobin A differ in their N-terminal extensions.
Amylovorin L471 displays a bactericidal activity. The mode of action starts with an immediate and unique, rapid adsorption of the bacteriocin molecules to both sensitive (L. delbrueckii subsp. bulgaricus) and insensitive (Listeria innocua; L. amylovorus) cells. Depending on the amount of bacteriocin molecules added to the cells, a bactericidal or bacteriolytic activity is observed with L. delbrueckii subsp. bulgaricus. Listeria innocua RZS 01 is not killed by the adsorbed bacteriocin molecules, while L. amylovorus DCE 471 is immune to its own bacteriocin. A concentration-dependent bactericidal or bacteriolytic activity has been reported for other bacteriocins previously (Gálvez et al., 1998 ). Also, adsorption to both sensitive and resistant cells is a well-known phenomenon (Bhunia et al., 1991
; Atrih et al., 1993
; Rekhif et al., 1994
; Manca de Nadra et al., 1998
).
It appears that amylovorin L471 production is not dependent upon induction. Indeed, no bacteriocin non-producing culture could be obtained after extensive dilution of active cells as was observed for some other bacteriocin-producing strains (Diep et al., 1995 ; Saucier et al., 1995
). In addition, no improved bacteriocin production could be observed upon addition of amylovorin L471 to producer cells, also suggesting that amylovorin L471 is produced constitutively. Furthermore, higher amounts of carbon and nitrogen as well as unfavourable growth conditions could stimulate specific amylovorin L471 production (De Vuyst et al., 1996b
). Other authors have reported on the stimulation of bacteriocin production by external influences such as mitomycin C (Rammelsberg et al., 1990
; Frémaux et al., 1993
) and foreign proteins (Barefoot et al., 1994
). However, recently it has been shown that the biosynthesis of several bacteriocins is induced via transcriptional activation through a three-component signal transduction pathway (Nes et al., 1996
). Hence, the possibility that the induction factor adheres to the cells very strongly cannot be excluded. For instance, sakacin A production is turned on continuously, although it has a three-component induction system (unpublished results).
We have already reported that loss of bacteriocin activity upon prolonged batch fermentation is due to adsorption of the bacteriocin molecules to the producing cells, which in turn prevents further cell growth and hence bacteriocin production (De Vuyst et al., 1996b ). This paper shows that ethanol clearly affects bacteriocin adsorption to the producer cells upon prolonged fermentation. Inhibition of adsorption of the bacteriocin molecules to the cell surface by ethanol can prevent subsequent cell death due to a limited immunity of bacteriocin producer cells. The higher viability of bacteriocin-producing cells at the end of the fermentation in the presence of ethanol confirms this hypothesis. An increased bacteriocin production upon addition of 1·0% (v/v) ethanol was also observed for lactocin S (Mørtvedt-Abildgaard et al., 1995
). As for amylovorin L471, no positive effect of ethanol on the activity of the bacteriocin in solution was found. Since ethanol had to be present during bacterial growth and lactocin S production, an enhancing role of ethanol on bacteriocin gene expression was suggested in this case. It has further been postulated that the presence of ethanol disturbs the binding of the induction fator to its receptor (the putative protein kinase) during bacteriocin production in E. faecium CTC 492 (Nilsen et al., 1998
). No further studies were performed concerning the ethanol effect on bacteriocin production.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderssen, E. L., Diep, D. B., Nes, I. F., Eijsink, V. G. H. & Nissen-Meyer, J. (1998). Antagonistic activity of Lactobacillus plantarum C11: two new two-peptide bacteriocins, plantaricin EF and JK, and the induction factor plantaricin A. Appl Environ Microbiol 64, 2269-2272.
Atrih, A., Rekhif, N., Milliere, J. B. & Lefebvre, G. (1993). Detection and characterization of a bacteriocin produced by Lactobacillus plantarum C19. Can J Microbiol 39, 1173-1179.
Barefoot, S. F., Chen, Y.-R., Hughes, T. A., Bodine, A. B., Shearer, M. Y. & Hughes, M. D. (1994). Identification and purification of a protein that induces production of the Lactobacillus acidophilus bacteriocin lactacin B. Appl Environ Microbiol 60, 3522-3528.[Abstract]
Bhunia, A. K., Johnson, M. C., Ray, B. & Kalchayanand, N. (1991). Mode of action of pediocin AcH from Pediococcus acidilactici H on sensitive bacterial strains. J Appl Bacteriol 70, 25-30.
Casaus, P., Nilsen, T., Cintas, L. M., Nes, I. F., Hernández, P. E. & Holo, H. (1997). Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology 143, 2287-2294.[Abstract]
Contreras, B. G. L., De Vuyst, L., Devreese, B., Busanyova, K., Raymaeckers, J., Bosman, F., Sablon, E. & Vandamme, E. J. (1997). Isolation, purification, and amino acid sequence of lactobin A, one of the two bacteriocins produced by Lactobacillus amylovorus LMG P-13139. Appl Environ Microbiol 63, 13-20.[Abstract]
Daba, H., Lacroix, C., Huang, R. E. & Lemieux, L. (1994). Simple method of purification and sequencing of a bacteriocin produced by Pediococcus acidilactici UL5. J Appl Bacteriol 77, 682-688.[Medline]
De Vos, W. M., Kuipers, O. M., van der Meer, J. R. & Siezen, R. J. (1995). Maturation pathway of nisin and other lantibiotics: posttranslationally modified antimicrobial peptides exported by Gram-positive bacteria. Mol Microbiol 17, 427-437.[Medline]
De Vuyst, L. & Callewaert, R. (1997). Purification and large-scale isolation of amylovorin L471 and other class II bacteriocins. 8ème Colloque du Club des Bactéries Lactiques, ENSBANA, Université de Bourgogne, Dijon, France, aff. B3.
De Vuyst, L. & Vandamme, E. J. (1994). Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics and Applications. London: Blackie Academic & Professional.
De Vuyst, L., Callewaert, R. & Pot, B. (1996a). Characterization of the antagonistic activity of Lactobacillus amylovorus DCE 471 and large scale isolation of its bacteriocin amylovorin L471. Syst Appl Microbiol 19, 9-20.
De Vuyst, L., Callewaert, R. & Crabbé, K. (1996b). Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiology 142, 817-827.
Diep, D. B., Hvarstein, L. S. & Nes, I. F. (1995). A bacteriocin-like peptide induces bacteriocin synthesis in Lactobacillus plantarum C11. Mol Microbiol 18, 631-639.[Medline]
Elegado, F. B., Kim, W. J. & Kwon, D. Y. (1997). Rapid purification, partial characterization, and antimicrobial spectrum of the bacteriocin, pediocin AcM, from Pediococcus acidilactici M. Int J Food Microbiol 37, 1-11.[Medline]
Ennahar, S., Aoude-Werner, D., Sorokine, O., van Dorsselaer, A., Bringel, F., Hubert, J. C. & Hasselmann, C. (1996). Production of pediocin AcH by Lactobacillus plantarum WHE 92 isolated from cheese. Appl Environ Microbiol 62, 4381-4387.[Abstract]
Felix, J. V., Papathanasopoulos, M. A., Smith, A. A., von Holy, A. & Hastings, J. W. (1994). Characterization of leucocin B-Ta11a: a bacteriocin from Leuconostoc carnosum Ta11a isolated from meat. Curr Microbiol 29, 207-212.[Medline]
Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. & Whitehouse, C. M. (1990). Electrospray ionization principles and practice. Mass Spectrom Rev 9, 37-70.
Frémaux, C., Ahn, C. & Klaenhammer, T. R. (1993). Molecular analysis of the lactacin F operon. Appl Environ Microbiol 59, 3906-3915.[Abstract]
Gálvez, A., Valdivia, E., Abriouel, H., Camafeita, E., Mendez, E., Martínez-Bueno, M. & Maqueda, M. (1998). Isolation and characterisation of enterocin EJ97, a bacteriocin produced by Enterococcus faecalis EJ97. Arch Microbiol 171, 59-65.[Medline]
Hastings, J. M., Sailer, M., Johnson, K. L., Ray, K. L., Vederas, J. C. & Stiles, M. E. (1991). Characterization of leucocin A-UAL 187 and cloning of the bacteriocin gene from Leuconostoc gelidum. J Bacteriol 173, 7491-7500.[Medline]
Holck, A., Axelsson, L., Birkeland, S.-E., Aukrust, T. & Blom, H. (1992). Purification and amino acid sequence of sakacin A, a bacteriocin from Lactobacillus sake Lb706. J Gen Microbiol 138, 2715-2720.[Medline]
Joerger, M. C. & Klaenhammer, T. R. (1986). Characterization and purification of helveticin J and evidence for a chromosomally determined bacteriocin produced by Lactobacillus helveticus 481. J Bacteriol 167, 439-446.[Medline]
Joosten, H. M., Nunez, M., Devreese, B., Van Beeumen, J. & Marugg, J. D. (1996). Purification and characterization of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4. Appl Environ Microbiol 62, 4220-4223.[Abstract]
Klaenhammer, T. R. (1988). Bacteriocins of lactic acid bacteria. Biochimie 70, 337-349.[Medline]
Klaenhammer, T. R. (1993). Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol Rev 12, 39-85.[Medline]
Kyte, J. & Doolittle, R. F. (1982). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105-132.[Medline]
Manca de Nadra, M. C., Sendino de Lamelas, D. & Strasser de Saad, A. M. (1998). Pediocin N5p from Pediococcus pentosaceus: adsorption on bacterial strains. Int J Food Microbiol 39, 79-85.[Medline]
Mørtvedt-Abildgaard, C. I., Nissen-Meyer, J., Jelle, B., Grenov, B., Skaugen, M. & Nes, I. F. (1995). Production and pH-dependent bactericidal activity of lactocin S, a lantibiotic from Lactobacillus sake L45. Appl Environ Microbiol 61, 175-179.[Abstract]
Motlagh, A. M., Bhunia, A. K., Szostek, F., Hansen, T. R., Johnson, M. G. & Ray, B. (1992). Nucleotide and amino acid sequence of pap-gene (pediocin AcH production) in Pediococcus acidilactici H. Lett Appl Microbiol 15, 45-48.[Medline]
Muriana, P. M. & Klaenhammer, T. R. (1987). Conjugal transfer of plasmid-encoded determinants for bacteriocin production and immunity in Lactobacillus acidophilus 88. Appl Environ Microbiol 53, 553-560.
Muriana, P. M. & Klaenhammer, T. R. (1991). Purification and partial characterisation of lactacin F, a bacteriocin produced by Lactobacillus acidophilus 11088. Appl Environ Microbiol 57, 114-121.[Medline]
Nes, I. F., Diep, D. B., Hvarstein, I. S., Brurberg, M. B., Eijsink, V. & Holo, H. (1996). Biosynthesis of bacteriocins in lactic acid bacteria. Antonie Leeuwenhoek 70, 113-128.
Nieto-Lozano, J. C., Meyer, J. N., Sletten, K., Peláz, C. & Nes, I. F. (1992). Purification and amino acid sequence of a bacteriocin produced by Pediococcus acidilactici. J Gen Microbiol 138, 1985-1990.[Medline]
Nilsen, T., Nes, I. F. & Holo, H. (1998). An exported inducer peptide regulates bacteriocin production in Enterococcus faecium CTC492. J Bacteriol 180, 1848-1854.
Nissen-Meyer, J., Holo, H., Hvarstein, L. S., Sletten, K. N. & Nes, I. F. (1992). A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides. J Bacteriol 174, 5686-5692.[Abstract]
Papathanasopoulos, M. A., Dykes, G. A., Revol-Junelles, A.-M., Delfour, A., von Holy, A. & Hastings, J. W. (1998). Sequence and structural relationships of leucocins A-, B- and C-TA33a from Leuconostoc mesenteroides TA33a. Microbiology 144, 1343-1348.[Abstract]
Rammelsberg, M., Muller, E. & Radler, F. (1990). Caseicin 80: purification and characterization of a new bacteriocin from Lactobacillus casei. Arch Microbiol 154, 249-252.
Rekhif, N., Atrih, A. & Lefebvre, G. (1994). Characterisation and partial purification of plantaricin LC74, a bacteriocin produced by Lactobacillus plantarum LC74. Biotechnol Lett 16, 771-776.
Saucier, L., Poon, A. & Stiles, M. E. (1995). Induction of bacteriocin in Carnobacterium piscicola LV17. J Appl Bacteriol 78, 684-690.
Stoffels, G., Sahl, H.-G. & Gudmundsdóttir, Á. (1993). Carnocin UI49, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various Gram-positive bacteria including Listeria sp. Int J Food Microbiol 20, 199-210.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]
Thompson, J. K., Collins, M. A. & Mercer, W. D. (1996). Characterization of a proteinaceous antimicrobial produced by Lactobacillus helveticus CNRZ450. J Appl Bacteriol 80, 338-348.[Medline]
Tichaczek, P. S., Nissen-Meyer, J., Nes, I. F., Vogel, R. F. & Hammes, W. P. (1992). Characterization of the bacteriocins curvacin A from Lactobacillus curvatus LTH1174 and sakacin P from Lactobacillus sake LTH673. Syst Appl Microbiol 15, 460-468.
Vaughan, E. E., Daly, C. & Fitzgerald, G. F. (1992). Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829. J Appl Bacteriol 73, 299-308.[Medline]
Venema, K., Chikindas, M. L., Seegers, J. F. M. L., Haandrikman, A. J., Leenhouts, K. J., Venema, G. & Kok, J. (1997). Rapid and efficient purification method for small, hydrophobic, cationic bacteriocins: purification of lactococcin B and pediocin PA-1. Appl Environ Microbiol 63, 305-309.[Abstract]
Worobo, R. W., van Belkum, M. J., Sailer, M., Roy, K. L., Vederas, J. C. & Stiles, M. E. (1995). A signal peptide secretion-dependent bacteriocin from Carnobacterium divergens. J Bacteriol 177, 3143-3149.[Abstract]
Yang, R., Johnson, M. C. & Ray, B. (1992). Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl Environ Microbiol 58, 3355-3359.[Abstract]
Received 22 February 1999;
revised 7 May 1999;
accepted 20 May 1999.