- Email: [email protected]
Isolation of enterocin SE-K4-encoding plasmid and a high enterocin SE-K4 producing strain of Enterococcus faecalis K-4
JOURNAL OF BIOSCIENCEAND BIOENGINEERING Vol. 93, No. 4, 434-436. 2002
Isolation of Enterocin SE-K4-Encoding Plasmid and a High Enterocin SE-K4 Producing Strain of Enterococcus faecalis K-4 K A T S U M I D O I ] * T O M O K O E G U C H I ] S E O N G - H Y U N CHOI, l A T S U S H I IWATAKE,1 S A D A H I R O O H M O M O , 2 AND SEIYA O G A T A 1 Laboratory of Applied Microbial Genetics, Faculty of Agriculture, Graduate Sehools, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan ~ and Japan International Research Centerfor Agricultural Sciences, 1-10hwashi, Tsukuba, Ibaraki 305-8686, Japan2 Received 18 October2001/Accepted12 January2002
Enterococcusfaecalis K-4, whieh produees a elass IIa bacteriocin, enteroein SE-K4, earries two plasmids, pEK4S (approximately 60 kb) and pEK4L (approximately 75 kb). Piasmid-curing experiments showed that pEK4S was involved in the production of and immunity to enterocin SEK4 in strain K-4. A derivative strain, M6, with pEK4S produced a higher amount of enterocin SE-K4 than the parental strain K-4, although its growth rate was lower than that of parental strain K-4. Phenotypic changes in strain Mó are attributed to an increase in plasmid copy number. [Key words: plasmid-encoded bacteriocin, enterocin-encoding plasmid, Enterococcusfaecalis] lated on MRS agar plates. After 48-h incubation at 37°C, the colonies grown were examined for the existence ofplasmids. Three derivätives were obtained: M6 and M10 that lack plasmid pEK4L, and K-4N that lacks both pEK4L and pEK4S (Fig. 1). The bacteriocin production of strain K-4 and its three isolate derivatives was examined using a paper disk method (7) with E. faecium IFO 13712 as the indicator strain. Derivatives M6 and M10 harboring pEK4S produced enterocin SE-K4 and exhibited immunity to high concentrations of enterocin SE-K4. Strain M6 exhibited immunity to the highest concentration of enterocin SE-K4 (over 135,000 AU/ml), while strains K-4 and M10 exhibited resistance against 8500 AU/ml enterocin SE-K4. The derivative K-4N harboring no plasmid lost the potential to produce enterocin SE-K4 (see Fig. 3B), and became sensitive to its own bacteriocin (data not shown). These results indicate that the genes encoding enterocin SE-K4 and immunity protein are located in pEK4S. The relationship between the growth and bacteriocin production of the four strains was examined. All strains were cultivated aerobically at 37 and 43°C in MRS medium, then centrifuged at 11,000 x g for 15 min at 4°C. Bacteriocin activity was determined by agar well diffusion (8), using the indicator strain mentioned above. A 30-gl portion of the supernatant of each culture was placed in a well (10 mm in diameter) in MRS agar plates seeded with the indicator strain (ca. 10 6 CFU/ml). The plates were left for 2 h at 25°C until the bacteriocin diffused, and then incubated ovemight at 37°C. After incubation, the clear zone around the agar well was measured and bacteriocin activity calculated (3). Strain K-4 showed a much higher enterocin SE-K4 productivity in MRS medium than in TGE medium (3): the
Bacteriocins produced by enterococci have gained interest because bacteriocin-produeing strains can readily be isolated from fermented foods and silages (1, 2) and many of them are active against food-bome pathogens. We isolated Enterococcus faecalis K-4, which produces enteroein SE-K4, a small, heat-stable and antilisterial bacteriocin, from grass silage in Thailand (3). Seven bacteriocins of E. faecalis are biochemically and genetically separated into four types (4). Genes required for production of these seven baeteriocins are encoded by plasmids. We isolated a plasmid pEK4S from E. faecalis K-4 which apparently encodes the genes for production of enterocin SE-K4. E. faecalis K-4 was grown aerobically at 37°C in Bacto Lactobacilli MRS Broth (Becton, Dickinson and Campany, USA). Plasmid DNA from strain K-4 was isolated using the method of Anderson and Mckay (5), and agarose gel electrophoresis was carried out for 3.5 h at 50 V with 0.3% agarose in TAE buffer. The electrophoresis revealed two plasmids, pEK4L (approximately 75 kb) and pEK4S (approximately 60 kb) (Fig. 1). The sizes of the plasmids were estimated by restriction analysis using various restriction enzymes, and a restriction map of pEK4S was constructed (Fig. 2). To determine if production of and immunity to enterocin SE-K4 in strain K-4 were related to the plasmids, plasmidcuring experiments were carried out, as described by RuizBarba et al. (6). Strain K-4 was incubated at various concentrations of novobiocin (0.1-0.7 gg/tal) for 72 h at 37°C. Cultures that grew at the highest concentration of the curing reagent were serially diluted with MRS broth and inocu* Correspondingauthor, e-mail: [email protected] phone/fax: +81-(0)92-642-3059 434
VOL. 93, 2002
NOTES
"I
1
2
3
EcoRI
4
KpnI'~
A
SalI FIG. 1. Agarose gel electrophoresis of plasmid DNAs of E. faecalis K-4 and its derivatives. Lane 1, Strain K-4; lane 2, strain M10 (harboring pEK4S); lane 3, strain M6 (harboring pEK4S); lane 4, strain K-4N (harboring no plasmid).
0,[, .~ ~~
~°~t // 0
3
6
~~"~Sall KpnI
3.0[ B
i
",
%
j j ;~-~:--~ 9
-Sall
FIG. 2. Restriction map of plasmid pEK4S. Enterocin SE-K4-encoding fragment (bold line) was detected by Southern hybridization, using an oligonueleotide probe designed from the amino acid sequence of enterocin SE-K4 in Ref. 3.
1
o,/.<--
435
12 15 18 21 24 Time (h)
0
_. 3
_. 6
_. 9
_. _. ~ 12 15 18
.....
,
_~ 21 24
Time (h)
FIG. 3. Growth curves (A) and antimicrobiol activities of Enterocin SE-K4 (B) of E. faecalis strain K-4 and its derivatives. All strains were cultivated in MRS broth at 37°C (indicated by a straight line) or 43°C (indicated by a dotted line). Symbols indicate growth and antimicrobiol activity of enterocin SE-K4 of E. faecalis K-4 (open circle), M I0 (open triangle), M6 (closed circle) and K-4N (closed triangle). The titer of bacteriocin in arbitrary units (AU/ml -~) was expressed as a reciprocal of the highest dilution showing a definite zone (> 11 mm diameter) of inhibition. amounts were 540 AU/ml and 133 AU/ml, respectively. The growth rates o f K-4 and M10 were the same at 37 and 43°C, hut the levels o f enterocin SE-K4 production by these both strains at 43°C were more stable than those at 37°C through out the time period examined using MRS medium. Such higher and stable level o f the bacteriocin production might be related to the MRS medium used. The growth rate o f M6 was lower than those o f strains K-4, M6 and K-4N. However, strain M6 produced the highest amount o f SE-K4 among the strains used at 43°C. The amount (2080 AU/ml) exceeded more than 3.5-fold those produced by strains K-4 and M10. Fitzwater et al. (9) reported that the increase in plasmid copy number led to repression o f growth o f host cells by plasmid ColE1. On the other hand, using Bio-profile V. 6.0 and BIO-1D (Vilber Lourmat, France), the methods o f Projan et al. (10), the copy numbers o f pEK4S in both K-4 and M10 were calculated to be one or two. Apparently, the band corresponding to pEK4S in M6 was more distinct than those in K-4 and M10 (Fig. 1). The copy number o f pEK4S in M6 became twofold or more than those in strains K4 and M10. This means that the increase in plasmid copy number
in M6 might increase its production level and its immunity to enterocin SE-K4, and decrease its growth. However, the copy number o f pEKS4 did not increase with an increase in temperature. To clarify temperature-dependent production o f enterocin SE-K4, detailed analysis o f the bacteriocin productivity in strain M6 is under way. We thank Drs. J. Shima and S. Kawamoto of National Food Research Institute for their invaluable adviee in analysis of bacteriocin activity.
REFERENCES 1. Cintas, L.M., Casaus, P., Hävarstein, L.S., Hernändez, P.E., and Nes, I.F.: Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P 13 with a broad antimierobial spectrum. Appl. Environ. Microbiol., 63, 4321-4330 (1997).
2. Ohmomo, S., Murata, S., Katayama, N., Nitisinprasart, S., Kobayashi, M., Nakajima, T., Yajima, M., and Nakanishi, K.: Purification and some characteristics of enterocin ON157, a bacteriocin produced by Enterococcus faecium N[AI 157. J. Appl. Microbiol., 88, 81-89 (2000).
436
DOI ET AL.
3. Eguchi, T., Kaminaka, K., Shima, J., Kawamoto, S., Mori, K., Choi, S.-H., Doi, K., Ohmomo, S., and Ogata, S.: Isolation and characterization of enterocin SE-K4 produced by thermophilic Enterococci, Enterococcusfaecalis K-4. Biosci. Biotechnol. Biochem., 65, 247-253 (2001). 4. Balla, E., Dicks, L. M., Du Toit, M., van der Merwe, M. J., and Holzapfel, W.H.: Characterization and cloning of the genes encoding enterocin 107lA and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071. Appl. Environ. Microbiol., 66, 1298-1304 (2000). 5. Anderson, D.G. and Mckay, L.L.: Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol., 46, 549-552 (1983). 6. Ruiz-Barba, J.L., Piard, J.C., and Jiménez-Diaz, R.: Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations. J. Appl. Bacteriol., 71, 417~t21 (1991).
J. BIoscl. BIOENG., 7. Hoover, D. G. and Harlander, S. K.: Screening methods for detection of bacteriocin activity, p. 23-39. In Hoover, D.G. and Steenson, L. R. (ed.), Bacteriocins of lactic acid bacteria. Academic Press Inc., San Diego, CA (1993). 8. Floriano, B., Ruiz-Barba, J. L., and Jiménez-Diaz, R.: Purification and genetic characterization of enterocin I from Enterococcus faecalis 6Tla, a novel antilisterial plasmidencoded bacteriocin which does not belong to the pediocin family of bacteriocins. Appl. Environ. Microbiol., 64, 48834890 (1998). 9. Fitzwater, T., Zhang, X.Y., Elble, R., and Polisky, B.: Conditional high copy nurnber ColE1 mutants: resistance to RNA1 inhibition in vivo and in vitro. EMBO J., 7, 3289-3297 (1988). 10. Projan, S.J., Carleton, S., and Novick, R.P.: Determination of plasmid copy number by fluorescence densitometry. Plasmid, 9, 182-190 (1983).
Isolation of Enterocin SE-K4-Encoding Plasmid and a High Enterocin SE-K4 Producing Strain of Enterococcus faecalis K-4 K A T S U M I D O I ] * T O M O K O E G U C H I ] S E O N G - H Y U N CHOI, l A T S U S H I IWATAKE,1 S A D A H I R O O H M O M O , 2 AND SEIYA O G A T A 1 Laboratory of Applied Microbial Genetics, Faculty of Agriculture, Graduate Sehools, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan ~ and Japan International Research Centerfor Agricultural Sciences, 1-10hwashi, Tsukuba, Ibaraki 305-8686, Japan2 Received 18 October2001/Accepted12 January2002
Enterococcusfaecalis K-4, whieh produees a elass IIa bacteriocin, enteroein SE-K4, earries two plasmids, pEK4S (approximately 60 kb) and pEK4L (approximately 75 kb). Piasmid-curing experiments showed that pEK4S was involved in the production of and immunity to enterocin SEK4 in strain K-4. A derivative strain, M6, with pEK4S produced a higher amount of enterocin SE-K4 than the parental strain K-4, although its growth rate was lower than that of parental strain K-4. Phenotypic changes in strain Mó are attributed to an increase in plasmid copy number. [Key words: plasmid-encoded bacteriocin, enterocin-encoding plasmid, Enterococcusfaecalis] lated on MRS agar plates. After 48-h incubation at 37°C, the colonies grown were examined for the existence ofplasmids. Three derivätives were obtained: M6 and M10 that lack plasmid pEK4L, and K-4N that lacks both pEK4L and pEK4S (Fig. 1). The bacteriocin production of strain K-4 and its three isolate derivatives was examined using a paper disk method (7) with E. faecium IFO 13712 as the indicator strain. Derivatives M6 and M10 harboring pEK4S produced enterocin SE-K4 and exhibited immunity to high concentrations of enterocin SE-K4. Strain M6 exhibited immunity to the highest concentration of enterocin SE-K4 (over 135,000 AU/ml), while strains K-4 and M10 exhibited resistance against 8500 AU/ml enterocin SE-K4. The derivative K-4N harboring no plasmid lost the potential to produce enterocin SE-K4 (see Fig. 3B), and became sensitive to its own bacteriocin (data not shown). These results indicate that the genes encoding enterocin SE-K4 and immunity protein are located in pEK4S. The relationship between the growth and bacteriocin production of the four strains was examined. All strains were cultivated aerobically at 37 and 43°C in MRS medium, then centrifuged at 11,000 x g for 15 min at 4°C. Bacteriocin activity was determined by agar well diffusion (8), using the indicator strain mentioned above. A 30-gl portion of the supernatant of each culture was placed in a well (10 mm in diameter) in MRS agar plates seeded with the indicator strain (ca. 10 6 CFU/ml). The plates were left for 2 h at 25°C until the bacteriocin diffused, and then incubated ovemight at 37°C. After incubation, the clear zone around the agar well was measured and bacteriocin activity calculated (3). Strain K-4 showed a much higher enterocin SE-K4 productivity in MRS medium than in TGE medium (3): the
Bacteriocins produced by enterococci have gained interest because bacteriocin-produeing strains can readily be isolated from fermented foods and silages (1, 2) and many of them are active against food-bome pathogens. We isolated Enterococcus faecalis K-4, which produces enteroein SE-K4, a small, heat-stable and antilisterial bacteriocin, from grass silage in Thailand (3). Seven bacteriocins of E. faecalis are biochemically and genetically separated into four types (4). Genes required for production of these seven baeteriocins are encoded by plasmids. We isolated a plasmid pEK4S from E. faecalis K-4 which apparently encodes the genes for production of enterocin SE-K4. E. faecalis K-4 was grown aerobically at 37°C in Bacto Lactobacilli MRS Broth (Becton, Dickinson and Campany, USA). Plasmid DNA from strain K-4 was isolated using the method of Anderson and Mckay (5), and agarose gel electrophoresis was carried out for 3.5 h at 50 V with 0.3% agarose in TAE buffer. The electrophoresis revealed two plasmids, pEK4L (approximately 75 kb) and pEK4S (approximately 60 kb) (Fig. 1). The sizes of the plasmids were estimated by restriction analysis using various restriction enzymes, and a restriction map of pEK4S was constructed (Fig. 2). To determine if production of and immunity to enterocin SE-K4 in strain K-4 were related to the plasmids, plasmidcuring experiments were carried out, as described by RuizBarba et al. (6). Strain K-4 was incubated at various concentrations of novobiocin (0.1-0.7 gg/tal) for 72 h at 37°C. Cultures that grew at the highest concentration of the curing reagent were serially diluted with MRS broth and inocu* Correspondingauthor, e-mail: [email protected] phone/fax: +81-(0)92-642-3059 434
VOL. 93, 2002
NOTES
"I
1
2
3
EcoRI
4
KpnI'~
A
SalI FIG. 1. Agarose gel electrophoresis of plasmid DNAs of E. faecalis K-4 and its derivatives. Lane 1, Strain K-4; lane 2, strain M10 (harboring pEK4S); lane 3, strain M6 (harboring pEK4S); lane 4, strain K-4N (harboring no plasmid).
0,[, .~ ~~
~°~t // 0
3
6
~~"~Sall KpnI
3.0[ B
i
",
%
j j ;~-~:--~ 9
-Sall
FIG. 2. Restriction map of plasmid pEK4S. Enterocin SE-K4-encoding fragment (bold line) was detected by Southern hybridization, using an oligonueleotide probe designed from the amino acid sequence of enterocin SE-K4 in Ref. 3.
1
o,/.<--
435
12 15 18 21 24 Time (h)
0
_. 3
_. 6
_. 9
_. _. ~ 12 15 18
.....
,
_~ 21 24
Time (h)
FIG. 3. Growth curves (A) and antimicrobiol activities of Enterocin SE-K4 (B) of E. faecalis strain K-4 and its derivatives. All strains were cultivated in MRS broth at 37°C (indicated by a straight line) or 43°C (indicated by a dotted line). Symbols indicate growth and antimicrobiol activity of enterocin SE-K4 of E. faecalis K-4 (open circle), M I0 (open triangle), M6 (closed circle) and K-4N (closed triangle). The titer of bacteriocin in arbitrary units (AU/ml -~) was expressed as a reciprocal of the highest dilution showing a definite zone (> 11 mm diameter) of inhibition. amounts were 540 AU/ml and 133 AU/ml, respectively. The growth rates o f K-4 and M10 were the same at 37 and 43°C, hut the levels o f enterocin SE-K4 production by these both strains at 43°C were more stable than those at 37°C through out the time period examined using MRS medium. Such higher and stable level o f the bacteriocin production might be related to the MRS medium used. The growth rate o f M6 was lower than those o f strains K-4, M6 and K-4N. However, strain M6 produced the highest amount o f SE-K4 among the strains used at 43°C. The amount (2080 AU/ml) exceeded more than 3.5-fold those produced by strains K-4 and M10. Fitzwater et al. (9) reported that the increase in plasmid copy number led to repression o f growth o f host cells by plasmid ColE1. On the other hand, using Bio-profile V. 6.0 and BIO-1D (Vilber Lourmat, France), the methods o f Projan et al. (10), the copy numbers o f pEK4S in both K-4 and M10 were calculated to be one or two. Apparently, the band corresponding to pEK4S in M6 was more distinct than those in K-4 and M10 (Fig. 1). The copy number o f pEK4S in M6 became twofold or more than those in strains K4 and M10. This means that the increase in plasmid copy number
in M6 might increase its production level and its immunity to enterocin SE-K4, and decrease its growth. However, the copy number o f pEKS4 did not increase with an increase in temperature. To clarify temperature-dependent production o f enterocin SE-K4, detailed analysis o f the bacteriocin productivity in strain M6 is under way. We thank Drs. J. Shima and S. Kawamoto of National Food Research Institute for their invaluable adviee in analysis of bacteriocin activity.
REFERENCES 1. Cintas, L.M., Casaus, P., Hävarstein, L.S., Hernändez, P.E., and Nes, I.F.: Biochemical and genetic characterization of enterocin P, a novel sec-dependent bacteriocin from Enterococcus faecium P 13 with a broad antimierobial spectrum. Appl. Environ. Microbiol., 63, 4321-4330 (1997).
2. Ohmomo, S., Murata, S., Katayama, N., Nitisinprasart, S., Kobayashi, M., Nakajima, T., Yajima, M., and Nakanishi, K.: Purification and some characteristics of enterocin ON157, a bacteriocin produced by Enterococcus faecium N[AI 157. J. Appl. Microbiol., 88, 81-89 (2000).
436
DOI ET AL.
3. Eguchi, T., Kaminaka, K., Shima, J., Kawamoto, S., Mori, K., Choi, S.-H., Doi, K., Ohmomo, S., and Ogata, S.: Isolation and characterization of enterocin SE-K4 produced by thermophilic Enterococci, Enterococcusfaecalis K-4. Biosci. Biotechnol. Biochem., 65, 247-253 (2001). 4. Balla, E., Dicks, L. M., Du Toit, M., van der Merwe, M. J., and Holzapfel, W.H.: Characterization and cloning of the genes encoding enterocin 107lA and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071. Appl. Environ. Microbiol., 66, 1298-1304 (2000). 5. Anderson, D.G. and Mckay, L.L.: Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Appl. Environ. Microbiol., 46, 549-552 (1983). 6. Ruiz-Barba, J.L., Piard, J.C., and Jiménez-Diaz, R.: Plasmid profiles and curing of plasmids in Lactobacillus plantarum strains isolated from green olive fermentations. J. Appl. Bacteriol., 71, 417~t21 (1991).
J. BIoscl. BIOENG., 7. Hoover, D. G. and Harlander, S. K.: Screening methods for detection of bacteriocin activity, p. 23-39. In Hoover, D.G. and Steenson, L. R. (ed.), Bacteriocins of lactic acid bacteria. Academic Press Inc., San Diego, CA (1993). 8. Floriano, B., Ruiz-Barba, J. L., and Jiménez-Diaz, R.: Purification and genetic characterization of enterocin I from Enterococcus faecalis 6Tla, a novel antilisterial plasmidencoded bacteriocin which does not belong to the pediocin family of bacteriocins. Appl. Environ. Microbiol., 64, 48834890 (1998). 9. Fitzwater, T., Zhang, X.Y., Elble, R., and Polisky, B.: Conditional high copy nurnber ColE1 mutants: resistance to RNA1 inhibition in vivo and in vitro. EMBO J., 7, 3289-3297 (1988). 10. Projan, S.J., Carleton, S., and Novick, R.P.: Determination of plasmid copy number by fluorescence densitometry. Plasmid, 9, 182-190 (1983).