Section of Microbiology1 and Department of Food Science and Technology (NYSAES)2, Cornell University, Ithaca, NY 14853, USA
Agricultural Research Service, USDA, Ithaca, NY 14853, USA3
Author for correspondence: James B. Russell. Tel: +1 607 255 4508. Fax: +1 607 255 3904. e-mail: jbr8{at}cornell.edu
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ABSTRACT |
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Keywords: rumen, lactic acid bacteria, purification, N-terminal amino acids
Abbreviations: MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; TFA, trifluoroacetic acid
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INTRODUCTION |
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Nisin is too expensive to be used as a feed additive and experiments with the ruminal bacterium Streptococcus bovis indicated that resistance developed quickly (Mantovani & Russell, 2001 ). Whitford et al. (2001)
screened several ruminal streptococci for their ability to produce bacteriocins, and they purified and sequenced a peptide from Streptococcus gallolyticus LRC0255 (bovicin 255). Bovicin 255 inhibited some freshly isolated strains of S. bovis, but many isolates were not inhibited and adaptation greatly decreased its potential activity (Mantovani et al., 2001
). Because nisin-resistant S. bovis JB1 could not be inhibited by bovicin 255, it appeared that there was a common mechanism of resistance.
Ruminal isolations yielded a lactic-acid-producing bacterium (HC5) that was identified by 16S rDNA to be S. bovis (Mantovani et al., 2001 ). S. bovis HC5 had a broader antibacterial spectrum than S. gallolyticus LRC0255 and freshly isolated strains of S. bovis did not adapt to S. bovis HC5 (Mantovani et al., 2001
). The antibacterial activity of S. bovis HC5 appeared to be a bacteriocin, but further work was needed to demonstrate that it was indeed a pore-forming peptide. The following experiments describe the purification and characterization of the S. bovis HC5 bacteriocin.
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METHODS |
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Spectrum of activity.
S. bovis HC5 was spotted onto basal medium plates and these plates were incubated anaerobically for 24 h at 39 °C. Molten agar (basal medium, 4 mg glucose ml-1) inoculated with target bacteria (approx. 106 viable cells ml-1) was poured over agar plates that already had a S. bovis HC5 colony (deferred antagonism assay). The agar overlays were incubated anaerobically for 4 h at 25 °C (a temperature that does not allow growth of most ruminal bacteria). The agar overlays were then incubated anaerobically at 39 °C for 48 h and zones of clearing were measured.
Crude extracts of the S. bovis HC5 bacteriocin.
Stationary-phase S. bovis HC5 cells were harvested by centrifugation (20 min, 8200 g, 4 °C). The cell-free supernatant was treated (4 °C, 1 h) in stepwise fashion with increasing amounts of (NH4)2SO4, and precipitated materials were harvested by centrifugation (30 min, 8200 g, 4 °C). The 4060% (NH4)2SO4 fraction was resuspended in K2HPO4 buffer (6·6 ml, 100 mM, pH 6·0) and dialysed (3500 Mr cut-off; Pierce Chemicals) against potassium phosphate buffer. Crude extracts were in some cases treated with peptidases and proteinases (4 U Pronase E, 11·3 U trypsin, 12 U proteinase K, 41 U -chymotrypsin ml-1) and activity was assessed by adding these extracts to agar wells that had been cut into agar plates inoculated with S. bovis JB1 (105 cells ml-1). The agar plates were incubated anaerobically at 39 °C and bacteriocin activity was assessed from the size of the zone of clearing.
Intracellular potassium.
Stationary phase S. bovis JB1 cells (10 ml, approximately 160 µg protein ml-1) were harvested by centrifugation (4000 g, 15 min, 22 °C), washed anaerobically in basal medium lacking ammonia, and resuspended in 10 ml of the same medium. The washed cell suspensions were energized with glucose (22 mmol ml-1) and some suspensions were treated with either nisin (1 µM) or partially purified S. bovis HC5 bacteriocin (amount equivalent to a culture density of 160 µg protein ml-1). Samples (1 ml) were centrifuged (13000 g, 5 min, 22 °C) through 0·3 ml silicon oil (1:1 ratio, Dow-Corning 550 and 556). The microcentrifuge tubes were frozen (-20 °C) and the bottom of the tubes containing the cell pellets were removed with a pair of dog nail clippers. Cell pellets were digested (22 °C, 24 h, 3 M HNO3) and insoluble cell debris was removed (13000 g, 5 min, 22 °C). Potassium was determined with a flame photometer (model 2655-00 digital flame analyser; Cole-Parmer Instruments).
Purification of S. bovis HC5 bacteriocin.
Stationary-phase S. bovis cultures were harvested by centrifugation and the cells were washed in sodium phosphate buffer (5 mM, pH 6·7). The cell pellets were resuspended in acidic sodium chloride (100 mM, pH 2·0, 2 h, 4 °C). The cell suspensions were recentrifuged to remove cells and the cell-free supernatant was lyophilized. The lyophilized material was resuspended in sterile water. The bacteriocin extract was then applied to an SP Sepharose column (1·0x10 cm; Amersham Pharmacia) washed with 3 vols water followed by 0·2 M and 0·4 M NaCl. Multiple 100 µl injections of the active fractions were applied to a Discover BIO wide pore C-18 column [4·6x150 mm, 5 µm Supelco, 1 ml 0·1% trifluoroacetic acid (TFA) in water min-1, 2% acetonitrile gradient min-1, 22 °C]. Antibacterial activity of the eluted fractions was assayed with Bacillus subtilis ATCC 6537, an aerobic bacterium that was also sensitive. The final purification of the active peptide was completed by reinjecting the active fractions onto a Discovery RP amide C16 column using ethanol as a carrier solvent (1% min-1, 4·6 mmx25 cm, 5 µm Supelco). The active fractions were then collected and lyophilized.
The purified peptide was separated by Tris/Tricine SDS-PAGE (16·5% acrylamide) (Ausubel et al., 1997 ). One half of the gel was washed in water (10 min), and fixed with glutaraldehyde (5%, 1 h; Sigma), prior to staining with Coomassie brilliant blue R-250 (0·025%, 1 h; Sigma). Gels were destained overnight in acetic acid (10%, v/v). The other half of the gel was fixed (10% acetate/20% 2-propanol, v/v, 30 min) and washed with MilliQ-H2O (1 h). The gel was then covered with moist Kimwipes (Kimberly Clark), overlaid with soft agar (10 ml) containing 106 cells B. subtilis ATCC 6537 and incubated overnight at 37 °C.
Determination of N-terminal amino acid sequence of bovicin HC5.
Purified bacteriocin from S. bovis HC5 was subjected to Edman degradation analysis on a PE/ABD Procise 494 cLC Protein Sequencing System (Harvard Microchemistry Facility, Cambridge, MA).
Mass spectrometry of bovicin HC5.
The S. bovis HC5 bacteriocin obtained by HPLC purification was added to a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) sample plate and the sample was supplemented with 1 ml of an -cyano-4-hydroxy-trans-cinnamic acid solution (10 mg in 50% acetonitrile and 0·3% TFA). The mixture was mixed and allowed to dry at room temperature prior to mass spectrometry. The mass spectrometry data were acquired on a Voyager DE-STR MALDI-TOF MS system (Perspective Biosystems) with delayed extraction in the reflectron mode.
Other analysis.
Cell protein was determined by the Lowry method, using serum albumin as a standard.
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RESULTS |
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When the acidic NaCl extract was applied to an SP Sepharose column, the antibacterial activity could be eluted by 0·4 M NaCl. HPLC (C18 column) indicated that this fraction had a variety of peaks, but only one peak had activity. Rechromatography of the active fraction (RP amide, C16 column) yielded a single purified peptide that had activity (Fig. 3). Tris/Tricine PAGE supported the idea that bacteriocin had indeed been purified (Fig. 4
). When the purified bacteriocin was lyophilized and subjected to Edman degradation, the N-terminal amino acid sequence could be determined (VGXRYASXPGXSWKYVXF), but there were four residues (indicated by an X) that did not correspond to any of the 20 amino acids commonly found in proteins. Mass spectrometry based on MALDI-TOF indicated that the purified bacteriocin had a molecular mass of approximately 2440 Da (Fig. 5
).
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DISCUSSION |
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Some bacteriocins are highly specific and can only inhibit closely related strains (Jack et al., 1995 ), but S. bovis HC5 was able to inhibit a variety of Gram-positive ruminal bacteria as well as Gram-positive species from other habitats (Table 1
). Sel. ruminantium and M. elsdenii are closely related to Gram-positive bacteria, but these species have outer membranes (Stackebrandt et al., 1985
) and were resistant to the antibacterial activity of S. bovis HC5. S. bovis HC5 did not inhibit E. coli or A. fermentans, but a small zone of clearing was observed with Prevotella bryantii, a Gram-negative ruminal bacterium that has been used as a model of monensin resistance (Callaway & Russell, 2000
). Based on these results, it appeared that S. bovis HC5 has approximately the same spectrum of activity as monensin, a commonly used antibiotic in cattle rations (Russell & Strobel, 1989
).
The idea that the antibacterial activity of S. bovis HC5 was a bacteriocin was supported by the observation that it could be precipitated by ammonium sulfate and inactivated by Pronase E, a mixture of proteinases and peptidases. The crude extracts were resistant to -chymotrypsin, proteinase K and heat, and these properties could be advantageous for commercial applications. Many bacteriocins are peptides that insert into cell membranes to form pores, but some bacteriocins are thought to inhibit peptidoglycan synthesis and DNA replication (Jack et al., 1995
; Sablon et al., 2000
). Because the crude extract catalysed potassium efflux from S. bovis JB1, it appeared to contain a pore-forming peptide.
Bacteriocins are frequently cell associated and detergents are often added to the culture media to promote bacteriocin release (Parente & Ricciardi, 1999 ; Nel et al., 2001
). The bacteriocin activity of S. bovis HC5 cell-free supernatant was greatly enhanced by Tween 80. Bacteriocin activity could be precipitated from the cell-free supernatant by ammonium sulfate, but it had an abundance of contaminating peptides. Some of these peptides could be removed by dialysis, but even this latter treatment did not remove all of the contamination.
Yang et al. (1992) noted that bacteriocins of some lactic acid bacteria could be dislodged from the cell surface by acidic NaCl, and this treatment liberated S. bovis HC5 bacteriocin without causing detectable cell lysis. Because the cells could be washed prior to the acidic NaCl treatment, contaminating peptides from the basal medium were largely eliminated. HPLC indicated that the acidic NaCl extracts had some inactive peptides, but the active peptide could be purified and concentrated by rechromatography. The active peptide could not be silver stained, and other workers have noted a similar phenomenon (Carolissen-Mackay et al., 1997
; Pattnaik et al., 2001
; Villani et al., 1995
). However, it reacted with Coomassie stain after the gel had been fixed with glutaraldehyde (Fig. 4
).
Edman degradation analysis indicated that the N-terminal amino acid sequence had 4 amino acid residues that did not correspond to any of the 20 amino acids commonly found in proteins. Lantibiotics have rings that are created from the condensation of cysteine and dehydro-amino acids (e.g. dehydroalanine) (DeVos et al., 1995 ; Guder et al., 2000
). The unnamed amino acids had approximately the same position as the N-terminal dehydroalanines of nisin, subtilin and epidermin, but samples that were reduced and alkylated prior to Edman degradation did not have cysteine residues. Because cysteine residues were not detected under these conditions, it appeared that the unnamed residues were modified or other unusual amino acids.
A BLAST search of GenBank sequences indicated that our N-terminal amino acid sequence was unique. The only other bacteriocin that had significant similarity was the lantibiotic precursor of S. pyogenes SF370 (Ferreti et al., 2001 ), but the identity was only 55% (Fig. 6
). Based on these results, the bacteriocin of S. bovis HC5 appears to be novel and we now designate it as bovicin HC5. Further work will be needed to locate and sequence the bovicin HC5 gene.
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ACKNOWLEDGEMENTS |
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Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.
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REFERENCES |
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Callaway, T. R. & Russell, J. B. (2000). Variations in the ability of ruminal gram-negative Prevotella species to resist monensin. Curr Microbiol 40, 185-190.[Medline]
Callaway, T. R., Carneiro De Melo, A. M. S. & Russell, J. B. (1997). The effect of nisin and monensin on ruminal fermentations in vitro. Curr Microbiol 35, 90-96.[Medline]
Carolissen-Mackay, V., Arendse, G. & Hastings, J. W. (1997). Purification of bacteriocins of lactic acid bacteria: problems and pointers. Int J Food Microbiol 34, 1-16.[Medline]
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De Vos, W. M., Kuipers, O. P., van der Meer, J. R. & Siezen, R. J. (1995). Maturation pathway of nisin and other lantibiotics: post-translationally modified antimicrobial peptides exported by Gram-positive bacteria. Mol Microbiol 17, 427-437.[Medline]
Ferretti, J. J., McShan, W. M., Ajdic, D. & 20 other authors (2001). Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 98, 46584663.
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Nel, H. A., Bauer, R., Vandamme, E. J. & Dicks, L. M. T. (2001). Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1. J Appl Microbiol 91, 1131-1138.[Medline]
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Sablon, E., Contreras, B. & Vandamme, E. (2000). Antimicrobial peptides of lactic acid bacteria: mode of action, genetics and biosynthesis. Adv Biochem Eng 68, 21-60.
Stackebrandt, E., Pohla, H., Kroppenstedt, R., Hippe, H. & Woese, C. R. (1985). 16S rRNA analysis of Sporomusa, Selenomonas, and Megasphaera: on the phylogenetic origin of Gram-positive eubacteria. Arch Microbiol 143, 270-276.
Villani, F., Pepe, O., Mauriello, G., Salzano, G., Moschetti, G. & Coppola, S. (1995). Antilisterial activity of thermophilin 347, a bacteriocin produced by Streptococcus thermophilus. Int J Food Microbiol 25, 179-190.[Medline]
Whitford, M. F., McPherson, M. A., Forster, R. J. & Teather, R. M. (2001). Identification of bacteriocin-like inhibitors from rumen Streptococcus spp. and isolation and characterization of bovicin 255. Appl Environ Microbiol 67, 569-574.
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Received 2 April 2002;
revised 25 June 2002;
accepted 15 July 2002.