APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1992, p. 2393-2396
Vol. 58, No. 8
0099-2240/92/082393-04$02.00/0 Copyright X3 1992, American Society for Microbiology
Characterization of Bacteriocins from Two Strains of Bacillus thermoleovorans, a Thermophilic Hydrocarbon-Utilizing Species JAMES F. NOVOTNY, JR., AND JEROME J. PERRY* Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695 Received 18 February 1992/Accepted 19 May 1992
Bacillus thermoleovorans S-II and B. thermokovorans NR-9 produce bacteriocins, and these bacteriocins are designated thermoleovorin-S2 and thermoleovorin-N9, respectively. The bacteriocins are effective against all but the producing strain of B. thermoleovorans, as well as being effective against Salmonella typhimurium, Branhamella catarrhalis, Streptococcus faecalis, and Thermus aquaticus. Thermoleovorins are produced during log-phase growth and are inhibitory to actively growing cells. The bacteriocins are proteinaceous in nature, being sensitive to selected proteases (protease type XI and pepsin). They are stable at pHs of 3 to 10. Thermoleovorin-S2 was more thermostable than thermoleovorin-N9 at 70 and 80°C. Thermoleovorins-S2 and -N9 apparently act by binding to the susceptible organisms, resulting in lysis of the cell. Thermoleovorin-S2 has an estimated Mr of 42,000, while thermoleovorin-N9 has a Mr of 36,000. The discovery and characterization of antimicrobial compounds produced by organisms isolated from extreme environments are of interest and potentially important to industry. Owing to the unique nature of the compounds produced by these organisms, they might provide new or moreefficient means for the inhibition of selected microorganisms. One such area of interest is the use of bacteriocins in eliminating organisms that are responsible for food spoilage or food-related pathogenicity (3, 5, 10, 13, 16). Although bacteriocin activity is considered to be species specific, bacteriocins produced by gram-positive organisms have proven to have a greater spectra of activity and thus possibly have a broader industrial application (16). We report here the characterization of two thermostable bacteriocins from Bacillus thennoleovorans S-II and NR-9. B. thermoleovorans strains are aerobic, endospore-forming, obligate thermophiles that utilize a wide array of substrates, including C13 to C20 n-alkanes. B. thermoleovorans strains were isolated by enrichment from various mud and water samples obtained from across the United States. The enrichment substrate was n-heptadecane (20). These organisms have some relatedness to the moderate thermophile Bacillus stearothennophilus (60% DNA-DNA hybridization) but no relatedness to the mesophilic Bacillus species (20). The bacteriocins, designated thermoleovorin-S2 and thermoleovorin-N9, exhibit the classical characteristics of most bacteriocins but also affect several pathogenic organisms that are not within the expected spectrum of activity. The potential use of the thermoleovorins and the characterization of their properties are described here.
(L-salts) (11) supplemented with 0.1% (wt/vol) tryptone and 0.1% (wt/vol) yeast extract (TY). Salmonella typhimurium, Shigella flexneri, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Staphylococcus epidermidis, Proteus mirabilis, Escherichia coli, Streptococcus pyogenes, Streptococcus faecalis, Klebsiella pneumoniae, Vibrio cholera, Branhamella catarrhalis, Corynebacterium xerosis, and Mycobacterium smegmatis were donated by James F. Novotny, Sr. (University of New England College of Osteopathic Medicine, Biddeford, Maine) and were maintained on brain-heart infusion broth (Difco, Detroit, Mich.) at 35°C. Lactobacillus acidophilus, Lactobacillus salivarius, and Lactobacillus reuteri were obtained from Walter J. Dobrogosz (North Carolina State University, Raleigh, N.C.) and grown on Lactobacilli MRS broth (Difco) anaerobically at 370C. Bacteriocin detection and activity assay. Bacteriocin activity was determined by an agar-well diffusion method (18) using soft TYA medium plates (TY medium containing 0.75% Bacto Agar). Bacteriocin was added to precut wells and allowed to diffuse for 2 h. A lawn of the indicator strain was then replica plated onto the well plate and incubated at the appropriate temperature. One arbitrary unit (AU) per milliliter of bacteriocin was defined as the reciprocal of the greatest dilution that demonstrated a zone of inhibition. Thermoleovorins were shown not to be viruses by the use of a plaque assay. Soft TYA medium was cooled to 40°C and inoculated with a 1% inoculum of strain LEH-1. Fifteenmilliliter aliquots were added to increasing dilutions of bacteriocin (0 to 250 AU) and poured onto an existing TYA medium plate. Plates were allowed to solidify and were incubated at 550C overnight. Partial purification. B. thennoleovorans S-IT and NR-9 were grown on TY overnight at 55°C. Cells were removed by centrifugation at 8,200 x g for 10 min at 4'C, and the bacteriocins were precipitated from the supernatant with ammonium sulfate (65% saturation). The resulting precipitate was centrifuged at 13,700 x g for 20 min, suspended in 2.0 ml of 0.1 M sodium phosphate buffer (pH 7.0), and dialyzed against the phosphate buffer at 4°C for 24 h in a
MATERIALS AND METHODS Bacterial strains and growth conditions. B. thermoleovorans strains and Thennus aquaticus were grown overnight at 55°C, while Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, and Micrococcus luteus were grown overnight at 30'C in a mineral salts medium *
Corresponding author. 2393
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6,000- to 8,000-Mr cutoff membrane. The dialysate was concentrated by using an Amicon stirred cell (Amicon, Danvers, Mass.) and then passed through a 0.45 ,u-m-poresize filter. Mr determination. Estimation of molecular weight was accomplished on a Sephadex G-200 column (Pharmacia) (0.5 by 45.0 cm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.0) by using molecular weight markers (Pharmacia) as standards. Susceptibility. The ability of thermoleovorin SII and N9 to inhibit various indicator strains was determined by two methods as follows. B. thermoleovorans strains, T. aquaticus, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, and Micrococcus luteus were assayed by the agar-well diffusion method (18). Salmonella typhimurium, Shigella fle-xneri, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Staphylococcus epidermidis, Proteus mirabilis, Escherichia coli, Streptococcus pyogenes, Streptococcus faecalis, Klebsiella pneumoniae, Vibrio cholera, Branhamella catarrhalis, Corynebacterium xerosis, and Mycobacterium smegmatis were assayed by a spot-on-lawn method as follows. Cultures were streaked onto plates of Mueller-Hinton agar (Difco), and bacteriocin was spotted on each plate, which was incubated at 35°C for 1 h right side up and then inverted. Lactobacillus cultures were assayed by the spot-on-lawn method using lactobacilli MRS agar plates. Sensitivity to heat, pH, and enzyme activity. Thermostability of the thermoleovorins was determined at 60, 70, and 80°C. A 200-,ul aliquot of each bacteriocin was overlaid with paraffin oil to prevent evaporation and incubated at the desired temperature. At intervals, 50 AU was removed and assayed by the agar-well diffusion method using B. thermoleovorans LEH-1 as the indicator strain. Effects of pH upon bacteriocin activity were determined by adjusting the pH of the thermoleovorin sample (50 AU in 45 ,ul of phosphate buffer) with dilute NaOH or HCl. Samples were incubated for 1 h, readjusted to pH 7.0, and assayed as described above. Inhibition of activity by selected enzymes at 1 mg/ml was done in the buffer mixture recommended by the supplier (Sigma, St. Louis, Mo.). Fifty arbitrary units of bacteriocin was incubated at 25 or 37°C for 1 h, followed by the standard assay. Enzymes used were protease type VI, type X, and type XI; pepsin; papain; ot-chymotrypsin; trypsin type III and type IX; lysozyme; lipase type II; phospholipase A2 and C; RNase A and T1; and DNase I. The protease inhibitors pepstatin A, phenylmethylsulfonyl fluoride, and leupeptin were tested at 1 mg/ml. Bacteriocin production. Synthesis of bacteriocin was monitored during the growth cycle of the organism. Each hour, the optical density at 600 nm (OD600) was recorded, a 1.0-ml sample of the culture was centrifuged (10,000 x g for 3 min), and a 40-plI aliquot of the resulting supernatant was assayed by the agar-well diffusion method. Production of bacteriocin by using various growth substrates was investigated by assaying the supernatant as described above. C13, C16, and C20 n-alkanes were added to L-salts at 0.1% (vol/vol). All other growth substrates were added at concentrations of 0.2%. Binding and lysis. Binding of thermoleovorins to susceptible and nonsusceptible cells was investigated as follows. Cultures of B. thermoleovorans LEH-1, S-II, and NR-9 and of B. subtilis were grown for 7 and 18 h, respectively, at the appropriate temperature in 500 ml of TY. The cells were centrifuged at 8,200 x g for 10 min, washed in 0.1 M sodium
V
0
C
C
go
V
C.
0
'U
Time (hrs)
FIG. 1. Production of thermoleovorin-S2 during the growth cycle of B. thermoleovorans S-II. At different intervals, samples were taken and the OD6. (-) and bacteriocin production (0) were determined.
phosphate buffer (pH 7.0), and centrifuged again, (8,200 x g for 10 min). The cell pellet was resuspended in 5.0 ml of phosphate buffer (pH 7.0) and divided into five portions of equal volume. Bacteriocin (10 AU) was added to each tube and incubated at the test organism's growth temperature. At intervals, cells were removed by centrifugation (10,000 x g for 3 min) and 25 pul of supernatant was assayed by the agar-well diffusion method. The effect of thermoleovorins on B. thermoleovorans LEH-1 at various stages of growth was investigated. Bacteriocin was added at selected time points to lawns that had been started simultaneously. After 24 h of incubation, zone sizes were recorded and correlated with the LEH-1 growth curve. Lysis of strain LEH-1 was investigated by growing 50 ml of TY-grown cultures to a selected phase of growth (4, 7, 10, and 24 h) and centrifugation at 4,200 x g, followed by resuspension of the cells to an OD600 of 0.5 in the growth medium supernatant. Excess bacteriocin (200 AU) was added, and the cells were incubated at 55°C. At 15-min intervals, the optical density of the sample was recorded. RESULTS
Production of bacteriocins. Two strains of B. thermoleovorans produce an antimicrobial substance that is a bacteriocin. Strains S-II and NR-9 were the producing organisms, and the bacteriocins were designated thermoleovorin-S2 and thermoleovorin-N9, respectively. Figure 1 illustrates the production of thermoleovorin-S2 during log-phase growth of the organism. The two thermoleovorins were synthesized by the organisms regardless of the substrate. These substrates include C13, C16, and C20 n-alkanes, acetate, pyruvate, galactose, maltose, mannose, ribose, sucrose, trehalose, mannitol, casein, nutrient broth, peptone, and TY. The thermoleovorins were produced and released into the media. Fractionation of the cultures into supernatant, cell extract, and membrane fractions demonstrated that bacteriocin activity was present in the supernatant and was not in the cytoplasm or associated with the cell membrane. The bacteriocins were differentiated from viruses by a plaque assay procedure. In this test, plaques were not formed as the bacteriocin titer increased.
BACTERIOCINS FROM BACILLUS THERMOLEOVORANS
VOL. 58, 1992 Thermoleovorin-N9
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TABLE 1. Activity spectrum of thermoleovorins
Thermoleovorin-S2
Inhibition by: Indicator organism
100
Xg0 t80 70
40
30 20
10 0
15
30
45
60
75
Time (miin)
90 105 120
0
15
30
45
60
75
90 105 120
Time (min)
FIG. 2. Thermostability of thermoleovorin-S2 and -N9 when incubated at 60°C (X), 70°C (C1), and 80°C (A).
Molecular weight. The Mr of the bacteriocins was determined by gel filtration through a Sephadex G-200 column. Thermoleovorin-S2 had a Mr of 42,000, and thermoleovorin-N9 had a Mr of 36,000. Enzyme and pH sensitivity. The effects of various proteases, lipases, nucleases, and lysozyme upon bacteriocin activity were investigated. The thermoleovorins were inhibited by protease type XI (proteinase K) and pepsin. Protease type XI had a greater effect on thermoleovorin-N9, whereas pepsin inhibited thermoleovorin-S2 to a greater extent. The protease inhibitors pepstatin A, phenylmethylsulfonyl fluoride, and leupeptin were not effective against the thermoleovorins. pH did not affect thermoleovorin activity over the pH range of 3 to 10. Thermostability. Results in Fig. 2 illustrate the thermostability of the two bacteriocins at 60, 70, and 80°C. Thermoleovorin-S2 was considerably more stable at 70 and 80°C than thermoleovorin-N9, and although it initially lost about 25% of its activity at 80°C, it retained this level of activity over the 2-h incubation period. Inhibitory spectrum. Results in Table 1 show the effect of the thermoleovorins upon various gram-positive and gramnegative organisms. Thermoleovorin-S2 and -N9 inhibited all strains of B. thermoleovorans except the producing strain but did not inhibit mesophilic bacilli. Other than members of the genus Bacillus, thermoleovorin-S2 inhibited the thermophile T. aquaticus and the mesophiles Salmonella typhimurium, Streptococcus faecalis, and Branhamella catarrhalis. The latter three organisms were also inhibited by thermoleovorin-N9. Binding and lysis. The binding of bacteriocins to susceptible and nonsusceptible cells at various growth points was investigated by incubating an excess of cells with the minimum detectable amount of bacteriocin. When bacteriocin was incubated with the susceptible strain LEH-1, no zone of inhibition was seen. This indicates binding of bacteriocin by the organism. Cells that were previously determined to not be susceptible to thermoleovorin inhibition (Bacillus subtilis and the producing strain) did not bind the bacteriocin, as determined on the basis of the identical sizes of the zone of inhibition over the test time. These tests were done by using 7- (log phase) and 18-h-old (stationary phase) cultures. Irrespective of the age of the cultures, equivalent results were obtained. The lytic effects of the thermoleovorins upon B. thermoleovorans LEH-1 were investigated. Figure 3 illustrates a decrease in bacteriocin effect (inhibitory zone diameter) over the course of bacterial growth, indicating that the thermole-
Thermoleovorin-S2
Thermoleovorin-N9
+
+
B. thermoleovorans S-II B. thernoleovorans NR-9 B. thermoleovorans CC-6 B. thermoleovorans LEH-1 B. thernoleovorans TX-1 B. thermoleovorans TX2 B. thermoleovorans Op B. thermoleovorans Wh B. thermoleovorans BI-1 B. thermoleovorans S-1V Bacillus subtilis Bacillus megaterium Bacillus cereus Lactobacillus acidophilus Lactobacillus salivarius Lactobacillus reuteri Staphylococcus aureus Staphylococcus epidermidis Streptococcus pyogenes Streptococcus faecalis Shigella flexneri Salmonella typhimurium Proteus mirabilis Escherichia coli Klebsiella pneumoniae Vibrio cholera Branhamella catarrhalis Corynebacterium xerosis Mycobacterium smegmatis Micrococcus luteus Thermus aquaticus
++ + + + + + + + + + + + +
+
+
+
+
+
+
ovorins affect actively growing cells. This result was supported upon examination of the lysis of LEH-1 at 4 h (early log phase), 7 h (late log), 10 h (stationary), and 24 h (late stationary). Results in Fig. 4 indicate that lysis of LEH-1 (decrease in OD600) occurs more rapidly when the cells are in the log phase of growth. The effects of thermoleovorin-N9 indicate that early log phase or rapidly dividing cells are lysed, whereas thermoleovorin-S2 will lyse cells over a greater portion of their growth cycle but with the greatest activity being during the log phase of growth.
Thermoleovorin-N9
Thermoleovorin-S2
I
I 0.6-
0
I
E s 10
a
1
0.4-
5
-a
8
m
-a
0
i 0.2
00
a
0
m 0
2
4
6
Time (hrs)
8
10
0
2
4
6
a
10
Time (hrs)
FIG. 3. Effect of thermoleovorin-S2 and -N9 during the growth cycle of B. thermoleovorans LEH-1. *, OD600 of LEH-1; 0, zone of inhibition diameter of the bacteriocin.
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Thermoleovorin-N9
Thermoleovorin-S2
ability of thermoleovorins to inhibit Salmonella typhimu-
num, Branhamella catarrhalis, and Streptococcus faecalis was an unexpected finding. The antimicrobial effect on
Salmonella typhimurium warrants further investigation and may provide a use for these bacteriocins either in the food industry or as a feed additive for poultry. The ability of the thermoleovorins to withstand a wide range of temperatures and pH, as well as their spectrum of activity, might make them useful agents against Salmonella typhimurium.
0
15
30
45
nJ FIG. 4. Lysis of B. thermoleovorans LEH-1 by thermoleovorin-S2 and -N9. Cultures of LEH-1 were grown for certain times (, 4 h; C1, 7 h; *, 10 h; 0, 24 h) and incubated in the presence of thermoleovorin-S2 or -N9. At 15-min intervals, the OD6. was recorded and the percent unlysed cells was calculated by designating the time zero reading as 100%. Time (min)
[vn;
DISCUSSION Bacteriocins are produced by a wide variety of grampositive organisms (16). Within the genus Bacillus, bacteriocins or bacteriocinlike substances have been reported from several species. These include B. thuringiensis (4, 6, 19), B. stearothennophilus (15), B. licheniformis (1), and B. megaterium (7, 8). There are two strains of B. thermoleovorans, a thermophilic species capable of growth on hydrocarbons, that produce bacteriocins. The thermoleovorins meet the criteria that differentiate bacteriocins from antibiotics. Those criteria are a narrow spectrum of activity centered around its species, an essential protein moiety, a bactericidal mode of action, and attachment to cell receptors on susceptible cells (16). A fifth requirement, plasmid-encoded function, has yet to be proven, although preliminary results indicate that B. therinoleovorans S-Il has a 72.2-kb plasmid of unknown function (unpublished results). The thermoleovorins are quite similar to bacteriocins produced by other gram-positive organisms. Production during the log phase of growth (14), bactericidal action against actively growing cells (9, 12, 17), insensitivity to pH (2), and Mr (16) were similar to those of other described bacteriocins. The ability to inhibit certain organisms outside of their genus is also typical of gram-positive bacteriocins. The thermoleovorins S2 and N9 differ in several aspects. Thermoleovorin-S2 inhibits T. aquaticus, is more thermostable (Fig. 2), appears to have a greater lytic effect on cells over their life cycle (Fig. 4), and is inhibited by pepsin. Thermoleovorin-N9 has a lower Mr and is inhibited by protease type XI. Interest in the characterization of bacteriocins for commercial use in the food industry has increased research and development in this area (3, 5, 13). Bacteriocins from the lactobacilli have been the more prominent ones, as these organisms are involved in the production of foods (10). The
REFERENCES 1. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 2. Cheol, A., and M. E. Stiles. 1990. Plasmid-associated bacteriocin production by a strain of Camobacterium piscicola from meat. Appl. Environ. Microbiol. 56:2503-2510. 3. Daeshel, M. A. 1989. Antimicrobial substances from lactic acid bacteria for use as food preservatives. Food Technol. 43:164167. 4. de Borjac, H., and J. Lajudie. 1974. Mise en evidence de facteurs antagonistes du type des bacteriocines chez Bacillus thuringiensis. Ann. Microbiol. (Paris) 125B:529-537. 5. Delves-Broughton, J. 1990. Nisin and its uses as a food preservative. Food Technol. 44:100-113. 6. Goze, A. 1972. Thuricines et cerecines moleculaires. C. R. Seances Soc. Biol. Fil. 166:200-204. 7. Ivanovics, G. 1962. Bacteriocins and bacteriocin-like substances. Bacteriol. Rev. 26:108-118. 8. Ivanovics, G. 1965. Megacins and megacin-like substances. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 196:318-329. 9. Jetten, A. M., and G. D. Vogels. 1973. Effects of colicin A and staphylococcin 1580 on amino acid uptake into membrane vesicles of Escherichia coli and Staphylococcus aureus. Biochim. Biophys. Acta 311:483-495. 10. Klaenhammer, T. R. 1988. Bacteriocins of lactic acid bacteria. Biochimie 70:337-349. 11. Leadbetter, E. R., and J. W. Foster. 1958. Studies on some methane utilizing bacteria. Arch. Mikrobiol. 30:91-118. 12. Mahony, D. E., and M. E. Butler. 1971. Bacteriocins of Clostridiumperfringens. 1. Isolation and preliminary studies. Can. J. Microbiol. 17:1-6. 13. Radler, F. 1990. Possible use of nisin in wine making. I. Action of nisin against lactic acid bacteria and wine yeast in solid and liquid media. Am. J. Enol. Vitic. 41:1-16. 14. Schlegel, R., and H. D. Slade. 1973. Properties of a Streptococcus sanguis (Group H) bacteriocin and its separation from the competence factor of transformation. J. Bacteriol. 115:655-661. 15. Shafia, F. 1966. Thermocins of Bacillus stearothermophilus. J. Bacteriol. 92:524-525. 16. Tagg, T. R., A. S. Dajani, and L. W. Wannamaker. 1976. Bacteriocins of gram-positive bacteria. Bacteriol. Rev. 40:722756. 17. Tagg, J. R., A. S. Dajani, L. W. Wannamaker, and E. D. Gray. 1973. Group A streptococcal bacteriocin. Production, purification and mode of action. J. Exp. Med. 138:1168-1183. 18. Tagg, J. R., and A. R. McGiven. 1971. Assay system for bacteriocin. Appl. Microbiol. 21:943. 19. Vankova, J. 1957. Study of the effect of Bacillus thuringiensis on insects. Folia Biol. (Prague) 3:175-181. 20. Zarilla, K. A., and J. J. Perry. 1987. Bacillus thermoleovorans, sp. nov., a species of obligately thermophilic hydrocarbon utilizing endospore-forming bacteria. Syst. Appl. Microbiol. 9:258-264.
Vol. 58, No. 8
0099-2240/92/082393-04$02.00/0 Copyright X3 1992, American Society for Microbiology
Characterization of Bacteriocins from Two Strains of Bacillus thermoleovorans, a Thermophilic Hydrocarbon-Utilizing Species JAMES F. NOVOTNY, JR., AND JEROME J. PERRY* Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695 Received 18 February 1992/Accepted 19 May 1992
Bacillus thermoleovorans S-II and B. thermokovorans NR-9 produce bacteriocins, and these bacteriocins are designated thermoleovorin-S2 and thermoleovorin-N9, respectively. The bacteriocins are effective against all but the producing strain of B. thermoleovorans, as well as being effective against Salmonella typhimurium, Branhamella catarrhalis, Streptococcus faecalis, and Thermus aquaticus. Thermoleovorins are produced during log-phase growth and are inhibitory to actively growing cells. The bacteriocins are proteinaceous in nature, being sensitive to selected proteases (protease type XI and pepsin). They are stable at pHs of 3 to 10. Thermoleovorin-S2 was more thermostable than thermoleovorin-N9 at 70 and 80°C. Thermoleovorins-S2 and -N9 apparently act by binding to the susceptible organisms, resulting in lysis of the cell. Thermoleovorin-S2 has an estimated Mr of 42,000, while thermoleovorin-N9 has a Mr of 36,000. The discovery and characterization of antimicrobial compounds produced by organisms isolated from extreme environments are of interest and potentially important to industry. Owing to the unique nature of the compounds produced by these organisms, they might provide new or moreefficient means for the inhibition of selected microorganisms. One such area of interest is the use of bacteriocins in eliminating organisms that are responsible for food spoilage or food-related pathogenicity (3, 5, 10, 13, 16). Although bacteriocin activity is considered to be species specific, bacteriocins produced by gram-positive organisms have proven to have a greater spectra of activity and thus possibly have a broader industrial application (16). We report here the characterization of two thermostable bacteriocins from Bacillus thennoleovorans S-II and NR-9. B. thermoleovorans strains are aerobic, endospore-forming, obligate thermophiles that utilize a wide array of substrates, including C13 to C20 n-alkanes. B. thermoleovorans strains were isolated by enrichment from various mud and water samples obtained from across the United States. The enrichment substrate was n-heptadecane (20). These organisms have some relatedness to the moderate thermophile Bacillus stearothennophilus (60% DNA-DNA hybridization) but no relatedness to the mesophilic Bacillus species (20). The bacteriocins, designated thermoleovorin-S2 and thermoleovorin-N9, exhibit the classical characteristics of most bacteriocins but also affect several pathogenic organisms that are not within the expected spectrum of activity. The potential use of the thermoleovorins and the characterization of their properties are described here.
(L-salts) (11) supplemented with 0.1% (wt/vol) tryptone and 0.1% (wt/vol) yeast extract (TY). Salmonella typhimurium, Shigella flexneri, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Staphylococcus epidermidis, Proteus mirabilis, Escherichia coli, Streptococcus pyogenes, Streptococcus faecalis, Klebsiella pneumoniae, Vibrio cholera, Branhamella catarrhalis, Corynebacterium xerosis, and Mycobacterium smegmatis were donated by James F. Novotny, Sr. (University of New England College of Osteopathic Medicine, Biddeford, Maine) and were maintained on brain-heart infusion broth (Difco, Detroit, Mich.) at 35°C. Lactobacillus acidophilus, Lactobacillus salivarius, and Lactobacillus reuteri were obtained from Walter J. Dobrogosz (North Carolina State University, Raleigh, N.C.) and grown on Lactobacilli MRS broth (Difco) anaerobically at 370C. Bacteriocin detection and activity assay. Bacteriocin activity was determined by an agar-well diffusion method (18) using soft TYA medium plates (TY medium containing 0.75% Bacto Agar). Bacteriocin was added to precut wells and allowed to diffuse for 2 h. A lawn of the indicator strain was then replica plated onto the well plate and incubated at the appropriate temperature. One arbitrary unit (AU) per milliliter of bacteriocin was defined as the reciprocal of the greatest dilution that demonstrated a zone of inhibition. Thermoleovorins were shown not to be viruses by the use of a plaque assay. Soft TYA medium was cooled to 40°C and inoculated with a 1% inoculum of strain LEH-1. Fifteenmilliliter aliquots were added to increasing dilutions of bacteriocin (0 to 250 AU) and poured onto an existing TYA medium plate. Plates were allowed to solidify and were incubated at 550C overnight. Partial purification. B. thennoleovorans S-IT and NR-9 were grown on TY overnight at 55°C. Cells were removed by centrifugation at 8,200 x g for 10 min at 4'C, and the bacteriocins were precipitated from the supernatant with ammonium sulfate (65% saturation). The resulting precipitate was centrifuged at 13,700 x g for 20 min, suspended in 2.0 ml of 0.1 M sodium phosphate buffer (pH 7.0), and dialyzed against the phosphate buffer at 4°C for 24 h in a
MATERIALS AND METHODS Bacterial strains and growth conditions. B. thermoleovorans strains and Thennus aquaticus were grown overnight at 55°C, while Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, and Micrococcus luteus were grown overnight at 30'C in a mineral salts medium *
Corresponding author. 2393
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6,000- to 8,000-Mr cutoff membrane. The dialysate was concentrated by using an Amicon stirred cell (Amicon, Danvers, Mass.) and then passed through a 0.45 ,u-m-poresize filter. Mr determination. Estimation of molecular weight was accomplished on a Sephadex G-200 column (Pharmacia) (0.5 by 45.0 cm) equilibrated with 0.1 M sodium phosphate buffer (pH 7.0) by using molecular weight markers (Pharmacia) as standards. Susceptibility. The ability of thermoleovorin SII and N9 to inhibit various indicator strains was determined by two methods as follows. B. thermoleovorans strains, T. aquaticus, Bacillus subtilis, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, and Micrococcus luteus were assayed by the agar-well diffusion method (18). Salmonella typhimurium, Shigella fle-xneri, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, Staphylococcus epidermidis, Proteus mirabilis, Escherichia coli, Streptococcus pyogenes, Streptococcus faecalis, Klebsiella pneumoniae, Vibrio cholera, Branhamella catarrhalis, Corynebacterium xerosis, and Mycobacterium smegmatis were assayed by a spot-on-lawn method as follows. Cultures were streaked onto plates of Mueller-Hinton agar (Difco), and bacteriocin was spotted on each plate, which was incubated at 35°C for 1 h right side up and then inverted. Lactobacillus cultures were assayed by the spot-on-lawn method using lactobacilli MRS agar plates. Sensitivity to heat, pH, and enzyme activity. Thermostability of the thermoleovorins was determined at 60, 70, and 80°C. A 200-,ul aliquot of each bacteriocin was overlaid with paraffin oil to prevent evaporation and incubated at the desired temperature. At intervals, 50 AU was removed and assayed by the agar-well diffusion method using B. thermoleovorans LEH-1 as the indicator strain. Effects of pH upon bacteriocin activity were determined by adjusting the pH of the thermoleovorin sample (50 AU in 45 ,ul of phosphate buffer) with dilute NaOH or HCl. Samples were incubated for 1 h, readjusted to pH 7.0, and assayed as described above. Inhibition of activity by selected enzymes at 1 mg/ml was done in the buffer mixture recommended by the supplier (Sigma, St. Louis, Mo.). Fifty arbitrary units of bacteriocin was incubated at 25 or 37°C for 1 h, followed by the standard assay. Enzymes used were protease type VI, type X, and type XI; pepsin; papain; ot-chymotrypsin; trypsin type III and type IX; lysozyme; lipase type II; phospholipase A2 and C; RNase A and T1; and DNase I. The protease inhibitors pepstatin A, phenylmethylsulfonyl fluoride, and leupeptin were tested at 1 mg/ml. Bacteriocin production. Synthesis of bacteriocin was monitored during the growth cycle of the organism. Each hour, the optical density at 600 nm (OD600) was recorded, a 1.0-ml sample of the culture was centrifuged (10,000 x g for 3 min), and a 40-plI aliquot of the resulting supernatant was assayed by the agar-well diffusion method. Production of bacteriocin by using various growth substrates was investigated by assaying the supernatant as described above. C13, C16, and C20 n-alkanes were added to L-salts at 0.1% (vol/vol). All other growth substrates were added at concentrations of 0.2%. Binding and lysis. Binding of thermoleovorins to susceptible and nonsusceptible cells was investigated as follows. Cultures of B. thermoleovorans LEH-1, S-II, and NR-9 and of B. subtilis were grown for 7 and 18 h, respectively, at the appropriate temperature in 500 ml of TY. The cells were centrifuged at 8,200 x g for 10 min, washed in 0.1 M sodium
V
0
C
C
go
V
C.
0
'U
Time (hrs)
FIG. 1. Production of thermoleovorin-S2 during the growth cycle of B. thermoleovorans S-II. At different intervals, samples were taken and the OD6. (-) and bacteriocin production (0) were determined.
phosphate buffer (pH 7.0), and centrifuged again, (8,200 x g for 10 min). The cell pellet was resuspended in 5.0 ml of phosphate buffer (pH 7.0) and divided into five portions of equal volume. Bacteriocin (10 AU) was added to each tube and incubated at the test organism's growth temperature. At intervals, cells were removed by centrifugation (10,000 x g for 3 min) and 25 pul of supernatant was assayed by the agar-well diffusion method. The effect of thermoleovorins on B. thermoleovorans LEH-1 at various stages of growth was investigated. Bacteriocin was added at selected time points to lawns that had been started simultaneously. After 24 h of incubation, zone sizes were recorded and correlated with the LEH-1 growth curve. Lysis of strain LEH-1 was investigated by growing 50 ml of TY-grown cultures to a selected phase of growth (4, 7, 10, and 24 h) and centrifugation at 4,200 x g, followed by resuspension of the cells to an OD600 of 0.5 in the growth medium supernatant. Excess bacteriocin (200 AU) was added, and the cells were incubated at 55°C. At 15-min intervals, the optical density of the sample was recorded. RESULTS
Production of bacteriocins. Two strains of B. thermoleovorans produce an antimicrobial substance that is a bacteriocin. Strains S-II and NR-9 were the producing organisms, and the bacteriocins were designated thermoleovorin-S2 and thermoleovorin-N9, respectively. Figure 1 illustrates the production of thermoleovorin-S2 during log-phase growth of the organism. The two thermoleovorins were synthesized by the organisms regardless of the substrate. These substrates include C13, C16, and C20 n-alkanes, acetate, pyruvate, galactose, maltose, mannose, ribose, sucrose, trehalose, mannitol, casein, nutrient broth, peptone, and TY. The thermoleovorins were produced and released into the media. Fractionation of the cultures into supernatant, cell extract, and membrane fractions demonstrated that bacteriocin activity was present in the supernatant and was not in the cytoplasm or associated with the cell membrane. The bacteriocins were differentiated from viruses by a plaque assay procedure. In this test, plaques were not formed as the bacteriocin titer increased.
BACTERIOCINS FROM BACILLUS THERMOLEOVORANS
VOL. 58, 1992 Thermoleovorin-N9
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TABLE 1. Activity spectrum of thermoleovorins
Thermoleovorin-S2
Inhibition by: Indicator organism
100
Xg0 t80 70
40
30 20
10 0
15
30
45
60
75
Time (miin)
90 105 120
0
15
30
45
60
75
90 105 120
Time (min)
FIG. 2. Thermostability of thermoleovorin-S2 and -N9 when incubated at 60°C (X), 70°C (C1), and 80°C (A).
Molecular weight. The Mr of the bacteriocins was determined by gel filtration through a Sephadex G-200 column. Thermoleovorin-S2 had a Mr of 42,000, and thermoleovorin-N9 had a Mr of 36,000. Enzyme and pH sensitivity. The effects of various proteases, lipases, nucleases, and lysozyme upon bacteriocin activity were investigated. The thermoleovorins were inhibited by protease type XI (proteinase K) and pepsin. Protease type XI had a greater effect on thermoleovorin-N9, whereas pepsin inhibited thermoleovorin-S2 to a greater extent. The protease inhibitors pepstatin A, phenylmethylsulfonyl fluoride, and leupeptin were not effective against the thermoleovorins. pH did not affect thermoleovorin activity over the pH range of 3 to 10. Thermostability. Results in Fig. 2 illustrate the thermostability of the two bacteriocins at 60, 70, and 80°C. Thermoleovorin-S2 was considerably more stable at 70 and 80°C than thermoleovorin-N9, and although it initially lost about 25% of its activity at 80°C, it retained this level of activity over the 2-h incubation period. Inhibitory spectrum. Results in Table 1 show the effect of the thermoleovorins upon various gram-positive and gramnegative organisms. Thermoleovorin-S2 and -N9 inhibited all strains of B. thermoleovorans except the producing strain but did not inhibit mesophilic bacilli. Other than members of the genus Bacillus, thermoleovorin-S2 inhibited the thermophile T. aquaticus and the mesophiles Salmonella typhimurium, Streptococcus faecalis, and Branhamella catarrhalis. The latter three organisms were also inhibited by thermoleovorin-N9. Binding and lysis. The binding of bacteriocins to susceptible and nonsusceptible cells at various growth points was investigated by incubating an excess of cells with the minimum detectable amount of bacteriocin. When bacteriocin was incubated with the susceptible strain LEH-1, no zone of inhibition was seen. This indicates binding of bacteriocin by the organism. Cells that were previously determined to not be susceptible to thermoleovorin inhibition (Bacillus subtilis and the producing strain) did not bind the bacteriocin, as determined on the basis of the identical sizes of the zone of inhibition over the test time. These tests were done by using 7- (log phase) and 18-h-old (stationary phase) cultures. Irrespective of the age of the cultures, equivalent results were obtained. The lytic effects of the thermoleovorins upon B. thermoleovorans LEH-1 were investigated. Figure 3 illustrates a decrease in bacteriocin effect (inhibitory zone diameter) over the course of bacterial growth, indicating that the thermole-
Thermoleovorin-S2
Thermoleovorin-N9
+
+
B. thermoleovorans S-II B. thernoleovorans NR-9 B. thermoleovorans CC-6 B. thermoleovorans LEH-1 B. thernoleovorans TX-1 B. thermoleovorans TX2 B. thermoleovorans Op B. thermoleovorans Wh B. thermoleovorans BI-1 B. thermoleovorans S-1V Bacillus subtilis Bacillus megaterium Bacillus cereus Lactobacillus acidophilus Lactobacillus salivarius Lactobacillus reuteri Staphylococcus aureus Staphylococcus epidermidis Streptococcus pyogenes Streptococcus faecalis Shigella flexneri Salmonella typhimurium Proteus mirabilis Escherichia coli Klebsiella pneumoniae Vibrio cholera Branhamella catarrhalis Corynebacterium xerosis Mycobacterium smegmatis Micrococcus luteus Thermus aquaticus
++ + + + + + + + + + + + +
+
+
+
+
+
+
ovorins affect actively growing cells. This result was supported upon examination of the lysis of LEH-1 at 4 h (early log phase), 7 h (late log), 10 h (stationary), and 24 h (late stationary). Results in Fig. 4 indicate that lysis of LEH-1 (decrease in OD600) occurs more rapidly when the cells are in the log phase of growth. The effects of thermoleovorin-N9 indicate that early log phase or rapidly dividing cells are lysed, whereas thermoleovorin-S2 will lyse cells over a greater portion of their growth cycle but with the greatest activity being during the log phase of growth.
Thermoleovorin-N9
Thermoleovorin-S2
I
I 0.6-
0
I
E s 10
a
1
0.4-
5
-a
8
m
-a
0
i 0.2
00
a
0
m 0
2
4
6
Time (hrs)
8
10
0
2
4
6
a
10
Time (hrs)
FIG. 3. Effect of thermoleovorin-S2 and -N9 during the growth cycle of B. thermoleovorans LEH-1. *, OD600 of LEH-1; 0, zone of inhibition diameter of the bacteriocin.
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Thermoleovorin-S2
ability of thermoleovorins to inhibit Salmonella typhimu-
num, Branhamella catarrhalis, and Streptococcus faecalis was an unexpected finding. The antimicrobial effect on
Salmonella typhimurium warrants further investigation and may provide a use for these bacteriocins either in the food industry or as a feed additive for poultry. The ability of the thermoleovorins to withstand a wide range of temperatures and pH, as well as their spectrum of activity, might make them useful agents against Salmonella typhimurium.
0
15
30
45
nJ FIG. 4. Lysis of B. thermoleovorans LEH-1 by thermoleovorin-S2 and -N9. Cultures of LEH-1 were grown for certain times (, 4 h; C1, 7 h; *, 10 h; 0, 24 h) and incubated in the presence of thermoleovorin-S2 or -N9. At 15-min intervals, the OD6. was recorded and the percent unlysed cells was calculated by designating the time zero reading as 100%. Time (min)
[vn;
DISCUSSION Bacteriocins are produced by a wide variety of grampositive organisms (16). Within the genus Bacillus, bacteriocins or bacteriocinlike substances have been reported from several species. These include B. thuringiensis (4, 6, 19), B. stearothennophilus (15), B. licheniformis (1), and B. megaterium (7, 8). There are two strains of B. thermoleovorans, a thermophilic species capable of growth on hydrocarbons, that produce bacteriocins. The thermoleovorins meet the criteria that differentiate bacteriocins from antibiotics. Those criteria are a narrow spectrum of activity centered around its species, an essential protein moiety, a bactericidal mode of action, and attachment to cell receptors on susceptible cells (16). A fifth requirement, plasmid-encoded function, has yet to be proven, although preliminary results indicate that B. therinoleovorans S-Il has a 72.2-kb plasmid of unknown function (unpublished results). The thermoleovorins are quite similar to bacteriocins produced by other gram-positive organisms. Production during the log phase of growth (14), bactericidal action against actively growing cells (9, 12, 17), insensitivity to pH (2), and Mr (16) were similar to those of other described bacteriocins. The ability to inhibit certain organisms outside of their genus is also typical of gram-positive bacteriocins. The thermoleovorins S2 and N9 differ in several aspects. Thermoleovorin-S2 inhibits T. aquaticus, is more thermostable (Fig. 2), appears to have a greater lytic effect on cells over their life cycle (Fig. 4), and is inhibited by pepsin. Thermoleovorin-N9 has a lower Mr and is inhibited by protease type XI. Interest in the characterization of bacteriocins for commercial use in the food industry has increased research and development in this area (3, 5, 13). Bacteriocins from the lactobacilli have been the more prominent ones, as these organisms are involved in the production of foods (10). The
REFERENCES 1. Bradley, D. E. 1967. Ultrastructure of bacteriophages and bacteriocins. Bacteriol. Rev. 31:230-314. 2. Cheol, A., and M. E. Stiles. 1990. Plasmid-associated bacteriocin production by a strain of Camobacterium piscicola from meat. Appl. Environ. Microbiol. 56:2503-2510. 3. Daeshel, M. A. 1989. Antimicrobial substances from lactic acid bacteria for use as food preservatives. Food Technol. 43:164167. 4. de Borjac, H., and J. Lajudie. 1974. Mise en evidence de facteurs antagonistes du type des bacteriocines chez Bacillus thuringiensis. Ann. Microbiol. (Paris) 125B:529-537. 5. Delves-Broughton, J. 1990. Nisin and its uses as a food preservative. Food Technol. 44:100-113. 6. Goze, A. 1972. Thuricines et cerecines moleculaires. C. R. Seances Soc. Biol. Fil. 166:200-204. 7. Ivanovics, G. 1962. Bacteriocins and bacteriocin-like substances. Bacteriol. Rev. 26:108-118. 8. Ivanovics, G. 1965. Megacins and megacin-like substances. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 196:318-329. 9. Jetten, A. M., and G. D. Vogels. 1973. Effects of colicin A and staphylococcin 1580 on amino acid uptake into membrane vesicles of Escherichia coli and Staphylococcus aureus. Biochim. Biophys. Acta 311:483-495. 10. Klaenhammer, T. R. 1988. Bacteriocins of lactic acid bacteria. Biochimie 70:337-349. 11. Leadbetter, E. R., and J. W. Foster. 1958. Studies on some methane utilizing bacteria. Arch. Mikrobiol. 30:91-118. 12. Mahony, D. E., and M. E. Butler. 1971. Bacteriocins of Clostridiumperfringens. 1. Isolation and preliminary studies. Can. J. Microbiol. 17:1-6. 13. Radler, F. 1990. Possible use of nisin in wine making. I. Action of nisin against lactic acid bacteria and wine yeast in solid and liquid media. Am. J. Enol. Vitic. 41:1-16. 14. Schlegel, R., and H. D. Slade. 1973. Properties of a Streptococcus sanguis (Group H) bacteriocin and its separation from the competence factor of transformation. J. Bacteriol. 115:655-661. 15. Shafia, F. 1966. Thermocins of Bacillus stearothermophilus. J. Bacteriol. 92:524-525. 16. Tagg, T. R., A. S. Dajani, and L. W. Wannamaker. 1976. Bacteriocins of gram-positive bacteria. Bacteriol. Rev. 40:722756. 17. Tagg, J. R., A. S. Dajani, L. W. Wannamaker, and E. D. Gray. 1973. Group A streptococcal bacteriocin. Production, purification and mode of action. J. Exp. Med. 138:1168-1183. 18. Tagg, J. R., and A. R. McGiven. 1971. Assay system for bacteriocin. Appl. Microbiol. 21:943. 19. Vankova, J. 1957. Study of the effect of Bacillus thuringiensis on insects. Folia Biol. (Prague) 3:175-181. 20. Zarilla, K. A., and J. J. Perry. 1987. Bacillus thermoleovorans, sp. nov., a species of obligately thermophilic hydrocarbon utilizing endospore-forming bacteria. Syst. Appl. Microbiol. 9:258-264.
