Letters in Applied Microbiology 1992, 14,12-76
Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobacillus casei W . G A I E RR~ .,F . V O G E L *& W . P . H A M M Elnstitut S f u r Lebensmitteltechnologie, Universitat Hohenheim, GarbenstraDe 25,0-7000 Stuttgart 70, FRG FLDI3: received 8 October 1991 and accepted 24 October 1991 G A I E RW., , VOGEL,R . F . & H A M M E SW.P. , 1992. Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobacillus casei. Letters in Applied Microbiology 14, 72-76.
The lysostaphin structural gene was cloned in Bacillus subtilis DSM402 and in Lactobacillus casei 102s. The gene was expressed in both organisms and active lysostaphin was released into the medium. Lysostaphin produced by these organisms induced lysis of growing and heat inactivated staphylococci. Expression in a protective starter organism is a prerequisite to produce lysostaphin in situ in fermenting foods and hence, to reduce the hygienical risk of staphylococcal food poisoning.
Staphylococcus aureus is a major food poisoning organism whose growth in food causes enterotoxicosis in man. The murein of staphylococci is sensitive to hydrolysis by lysostaphin, which selectively cleaves the pentaglycyl interpeptide bridges (Schindler & Schuhardt 1964). Lysostaphin is released by S. staphylolyticus together with two additional lytic activities, a hexaminidase and an amidase (Iversen & Grov 1973). S . staphylolyticus is now classified as a strain of S. simulans. The cloning in Escherichia coli and the sequence of the lysostaphin gene were reported by Recsei et al. (1987) and Heinrich et al. (1987). The sequence revealed that the glycinase lysostaphin is synthesized as a preproenzyme, which is released into the medium followed by extracellular processing to form the mature enzyme consisting of 296 amino acids. In this communication the potential was investigated of a protective starter organism to form the active enzyme as a prerequisite to produce lysostaphin in situ.
Materials and Methods BACTERIAL STRAINS A N D G R O W T H CONDITIONS
The bacterial strains and plasmids are shown in
* Corresponding author.
t Present address: NESTEC S.A., Centre de Recherche, Vers-chez-le-blanc,CH-1000 Lausanne 26.
Table 1. S . carnosus and E . coli cells were grown in LB-medium (Sambrook et al. 1989) and lactobacilli in MRS-medium (De Man et al. 1960). Transformants were selected in medium containing erythromycin (10 pg/ml). Selective growth of staphylococci was performed on SK-medium (Schleifer & Kramer 1980). Lysostaphin-producing transformants were detected on MRS-lyso-agar. This medium was basically MRS-medium with the acetate omitted, to allow growth of Bacillus subtilis and E . coli. A suspension of heat inactivated cells of S. carnosus cells (80"C, 10 min) was prepared, washed twice with peptone solution containing NaCl (8.5 g/l) and peptone (0.1 g/l) and resuspended in one volume peptone solution. This suspension was added to the medium at 30-60 ml/l to obtain a turbid medium. CLONING TECHNIQUES
Plasmid DNA was isolated from Lactobacillus casei by the method of Anderson & McKay (1983). Plasmids from B. subtilis and E . coli were isolated according to Birnboim & Doly (1979) with the modification that the lysozyme treatment of B. subtilis was changed to 37°C for 30 min. Other techniques like CsC1-gradients, restriction cutting and ligations were performed according to Sambrook et al. (1989). For the transformation of E . coli by electroporation
Expression of lysostaphin
73
Table 1. Bacterial strains and plasmids auulred
Strain/plasmid
Description
Referenceisource
BaciMus subfilis DSM402 Escherichia coli BHB2600 Lactohacillus casei 102s
Cloning host Cloning host Plasmid cured derivative of L. casei ATCC393 Plasmid-free variant of L . curvatus LTH683 Lysostaphin indicator strain pUC19 containing the lysostaphin gene E . coli-Lactococcus shuttle-vector pGKV210 containing the lysostaphin gene on a HindIII/BamHI fragment pGKV210 containing the lysostaphin gene on a HindIII fragment
Spizizen (1958)
Lactohacillus curcatus LTH 1432 Staphylococcus carnosus TM300 pUls23
pGKV?IO pWGlOO pWG200
(Dower et al. 1988), ligation mixes were precipitated with ethanol and dissolved in 5 p1 aqua bidest. Competent cells of B. subtilis were prepared and transformed as described by Spizizen (1958) and cells of L. casei and L. curuatus were transformed by electroporation as described by Chassy & Flickinger (1987) and Gaier et a / . (1990), respectively. D E T E R M I N A T I O N O F LYSOSTAPHIN
ACTIVITY
For the determination of the staphylolytic activity plate and photometric assays were employed. On MRS-lyso-agar lysostaphinproducing colonies were detected by formation of a clear halo. Quantitative determination of lysostaphin activity was performed in a photometric assay. Na-phosphate buffer (20 mM, pH 7.0) was adjusted with a suspension of inactivated S. carnosus cells to an optical density (578 nm) of 0.8. The suspension was mixed with an equal volume of culture supernatant in a cuvette. The cuvette was held at 37'C and the decrease in optical density was followed. The lysostaphin activity was determined on growing S. carnosus cells during co-incubation with a producer-strain. LB-medium ( 5 ml) was inoculated with 50 , ~ t lof an overnight culture of each S. carnosus and B. subtilis and shaken at 37°C. The cell count of staphylococci was determined on SK-agar plates. L. casei strains were
Hohn (1979) Chassy & Flickinger (1987) Heidel (1989) Gotz, Universitat
Tiibingen, FRG Heinrich et al. (1987) van der Vossen et ul. (1985) Present study Present study
co-cultivated at 30°C with S. carnosus in 5 ml MRS-medium without acetate after inoculation with 100 p1 of an overnight culture of each strain.
Results and Discussion C L O N I N G OF THE LYSOSTAPHIN G E N E
For the expression of lysostaphin in B. subtilis and L. casei the lysostaphin gene present on pUls23 was recloned into the E . coliLactococcus shuttle-vector pGKV210. The resulting plasmids pWG100 and pWG200 are depicted in Fig. 1. For the construction of pWGlOO or pWG2OO the 1.8 k b insert of pUls23 was cloned into pGKV210 as a HindIIIl BarnHI or a HindIII fragment, respectively. The plasmid pWGl00 differs from pWG200 only by the lack of the small HindIII/BumHI fragment from pGKV210. The direction of the lysostaphin gene in pWG200 was determined by restriction enzyme analysis with BarnHI, yielding fragments of 5.5 and 0.7 kb. No clones were detected with the 1.8 kb HindIII fragment inserted in the inverse direction. Both pWGlOO and pWG200 were isolated from E . coli and transferred to B. subtilis, L. cnsei and L. curuatus. Erythromycin-resistant transformants were obtained with all hosts. Analysis of the plasmid patterns of the transformants revealed that B. subtilis transformants contained pWGl00 and pWG200 of the
W . Gaier et al.
74 Bum HI
SmuI I
pWGlOO
Fig. 1. Restriction map of the plasmids pWGl00 and pWG200 containing the lysostaphin gene from Staphylococcus staphylolyticus. DNA fragments of pUls23 (m) and pGKV2lO (0) are shown. Location and direction of structural genes are indicated by arrows. Ery, erythromycin resistance; (cat86), part(s) of the promoterless chloramphenicol-acetyltransferase gene on pGKV210; Lys; lysostaphin gene.
unchanged size, whereas transformants of L. casei contained plasmids of various sizes, which were slightly smaller than the original plasmids. After transformation of L. curvatus only two erythromycin-resistant strains were obtained, both of which contained a plasmid of approx. 3.2 kb size. The small plasmids in L. casei and L. curvatus are probably the result of deletions occurring during the replication of these plasmids. Such deletions in plasmids like pGKV210 replicating via a rolling circle mechanism are not unusual, as can be seen from studies with B.
subtilis plasmids by Gruss & Ehrlich (1989) and Janniere et al. (1990). E X P R E S S I O N OF T H E L Y S O S T A P H I N GENE
The lysostaphin activity of E. coli, B . subtilis and L. casei transformants was determined initially by the plate assay method and is shown in Table 2 together with the data obtained in the photometric assay. No lysostaphin activity was detected with transformants of L. curvatus. The
Table 2. Lysostaphin activity of Bacillus subtilis, Escherichia coli and Lactococcus casei transformants
Strain
Plasmid
B. subtilis DSM402 B. subtilis DSM402 B. subtilis DSM402 E. coli BHB2600 E.coliBHB2600 E. coli BHB2600 L. casei 102s L. casei 102s L. casei 102s
None pWGl00 pWG200 None pWG100 pWG2OO None pWGl00 pWG2OO
*
+
Plate* assay
Photometric assay (% decrease in OD,,, x min-')
-
-
+
0.67 1.15
+
-
+ + +I ~
+$
~
0.25 0.36 ~
0.48 0.20
Co-cultivation assay (cfu/ml)t 6h
24 h
2.6 x 107 3.4 x 103 2.1 x 104 ND ND ND 3.8 107 1.7 x 107 4.3 x 107
ND ND ND ND ND ND 1.8 x 105 2.6 x 103 1.2 105
= formation of a clear halo around the colonies. $ Cell count of staphylococci on SK-medium. $ Not all plasmid containing transformants exhibited lysostaphin activity.
Expression of lysostaphin lysostaphin gene is probably deleted in the small plasmids harboured by these strains. Transformants were subsequently subjected to quantitative activity determination in the photometric assay. As an example, the time course of the decrease in OD,,, during the photometric assay performed with L . casei transformants is depicted in Fig. 2. For comparison of the lysostaphin activity of B. subtilis, E. coli and L. casei transformants, the slope was determined of the initial linear part of the lysis curve. The activity was calculated as the decrease in the percentage of the optical density x min-' (Table 2). Activities of different L. casei transformants ranged from zero up to the values shown in Table 2. These various activities may be explained by deletion of pWGlOO and pWG200 in the host L. casei. In addition to the lysis assays with heat-inactivated Staphylococcus cells, the staphylolytic activity on cells of B. subtilis and L. casei transformants was determined with growing staphylococci. The results shown in Table 2 are qualitatively consistent with those obtained in the plate and photometric assays
40 50
1
0
l
-
/
II 0 l /
0
/
/
# I00
Transformants of L. cusei exhibiting the highest lysostaphin activity had significantly reduced growth rates. This may be explained by a membrane-destabilizing effect of the lysostaphin signal peptide occurring during the transport of lysostaphin through the Lactobacillus cell membrane. Reduced growth or cell death due to lysostaphin transport might be the reason that no lysostaphin-positive L. curuatus transformants were obtained. On the other hand, lysostaphin is expressed in B. subtilis and L. casei, released into the medium and processed into an active form. The use in food production of lactic acid bacteria, which produce lysostaphin. could possibly reduce the risk of staphylococcal food poisoning, by using genetically engineered protective starter cultures. Previously, a Penicillium nalgiovense strain producing lysostaphin was constructed by Geisen et a/. (1990). As this mould grows on the surface of fermenting sausages, a possible inhibition of staphylococcal growth can occur in the outer region of the sausage only. The production of lysostaphin by lactobacilli could provide its homogeneous distribution and uniform inhibition of staphylococcal growth in fermenting foods. However, for this purpose, the gene has to be maintained stably by the host and antibiotic resistances have to be replaced by food grade markers. The authors are indebted to F. Gotz, Universitat Tubingen, FRG and J. K o k , University of Groningen, NL for kindly providing the plasmids pUls23 and pCKVZI0, respectively. This work was supported by the Bundesministerium Technologie grant fur Forschung und 0319280A. The authors are responsible for the content of this publication.
0
E
75
200
References
300
Time (min!
Fig. 2. Time course of the lysis of Staphylococcus carnosus cells by lysostaphin in the culture supernatant of Lactobacillus case; 102s transformants. Decrease in ODs7* of a suspension of S . carnosus TM300 cells during incubation with culture supernatant of L. casei 102s harboring no plasmid (m), pWGl00 ( 0 )or pWG200 (A).
ANDERSOF;. D.G. & M c K A ~ .L.L. IY83 Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Applied and Environmental Microhioloqx 46,549-552. BIRNBOIM,H.C. & DOLY.J. 1Y79 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nuclei
Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobacillus casei W . G A I E RR~ .,F . V O G E L *& W . P . H A M M Elnstitut S f u r Lebensmitteltechnologie, Universitat Hohenheim, GarbenstraDe 25,0-7000 Stuttgart 70, FRG FLDI3: received 8 October 1991 and accepted 24 October 1991 G A I E RW., , VOGEL,R . F . & H A M M E SW.P. , 1992. Cloning and expression of the lysostaphin gene in Bacillus subtilis and Lactobacillus casei. Letters in Applied Microbiology 14, 72-76.
The lysostaphin structural gene was cloned in Bacillus subtilis DSM402 and in Lactobacillus casei 102s. The gene was expressed in both organisms and active lysostaphin was released into the medium. Lysostaphin produced by these organisms induced lysis of growing and heat inactivated staphylococci. Expression in a protective starter organism is a prerequisite to produce lysostaphin in situ in fermenting foods and hence, to reduce the hygienical risk of staphylococcal food poisoning.
Staphylococcus aureus is a major food poisoning organism whose growth in food causes enterotoxicosis in man. The murein of staphylococci is sensitive to hydrolysis by lysostaphin, which selectively cleaves the pentaglycyl interpeptide bridges (Schindler & Schuhardt 1964). Lysostaphin is released by S. staphylolyticus together with two additional lytic activities, a hexaminidase and an amidase (Iversen & Grov 1973). S . staphylolyticus is now classified as a strain of S. simulans. The cloning in Escherichia coli and the sequence of the lysostaphin gene were reported by Recsei et al. (1987) and Heinrich et al. (1987). The sequence revealed that the glycinase lysostaphin is synthesized as a preproenzyme, which is released into the medium followed by extracellular processing to form the mature enzyme consisting of 296 amino acids. In this communication the potential was investigated of a protective starter organism to form the active enzyme as a prerequisite to produce lysostaphin in situ.
Materials and Methods BACTERIAL STRAINS A N D G R O W T H CONDITIONS
The bacterial strains and plasmids are shown in
* Corresponding author.
t Present address: NESTEC S.A., Centre de Recherche, Vers-chez-le-blanc,CH-1000 Lausanne 26.
Table 1. S . carnosus and E . coli cells were grown in LB-medium (Sambrook et al. 1989) and lactobacilli in MRS-medium (De Man et al. 1960). Transformants were selected in medium containing erythromycin (10 pg/ml). Selective growth of staphylococci was performed on SK-medium (Schleifer & Kramer 1980). Lysostaphin-producing transformants were detected on MRS-lyso-agar. This medium was basically MRS-medium with the acetate omitted, to allow growth of Bacillus subtilis and E . coli. A suspension of heat inactivated cells of S. carnosus cells (80"C, 10 min) was prepared, washed twice with peptone solution containing NaCl (8.5 g/l) and peptone (0.1 g/l) and resuspended in one volume peptone solution. This suspension was added to the medium at 30-60 ml/l to obtain a turbid medium. CLONING TECHNIQUES
Plasmid DNA was isolated from Lactobacillus casei by the method of Anderson & McKay (1983). Plasmids from B. subtilis and E . coli were isolated according to Birnboim & Doly (1979) with the modification that the lysozyme treatment of B. subtilis was changed to 37°C for 30 min. Other techniques like CsC1-gradients, restriction cutting and ligations were performed according to Sambrook et al. (1989). For the transformation of E . coli by electroporation
Expression of lysostaphin
73
Table 1. Bacterial strains and plasmids auulred
Strain/plasmid
Description
Referenceisource
BaciMus subfilis DSM402 Escherichia coli BHB2600 Lactohacillus casei 102s
Cloning host Cloning host Plasmid cured derivative of L. casei ATCC393 Plasmid-free variant of L . curvatus LTH683 Lysostaphin indicator strain pUC19 containing the lysostaphin gene E . coli-Lactococcus shuttle-vector pGKV210 containing the lysostaphin gene on a HindIII/BamHI fragment pGKV210 containing the lysostaphin gene on a HindIII fragment
Spizizen (1958)
Lactohacillus curcatus LTH 1432 Staphylococcus carnosus TM300 pUls23
pGKV?IO pWGlOO pWG200
(Dower et al. 1988), ligation mixes were precipitated with ethanol and dissolved in 5 p1 aqua bidest. Competent cells of B. subtilis were prepared and transformed as described by Spizizen (1958) and cells of L. casei and L. curuatus were transformed by electroporation as described by Chassy & Flickinger (1987) and Gaier et a / . (1990), respectively. D E T E R M I N A T I O N O F LYSOSTAPHIN
ACTIVITY
For the determination of the staphylolytic activity plate and photometric assays were employed. On MRS-lyso-agar lysostaphinproducing colonies were detected by formation of a clear halo. Quantitative determination of lysostaphin activity was performed in a photometric assay. Na-phosphate buffer (20 mM, pH 7.0) was adjusted with a suspension of inactivated S. carnosus cells to an optical density (578 nm) of 0.8. The suspension was mixed with an equal volume of culture supernatant in a cuvette. The cuvette was held at 37'C and the decrease in optical density was followed. The lysostaphin activity was determined on growing S. carnosus cells during co-incubation with a producer-strain. LB-medium ( 5 ml) was inoculated with 50 , ~ t lof an overnight culture of each S. carnosus and B. subtilis and shaken at 37°C. The cell count of staphylococci was determined on SK-agar plates. L. casei strains were
Hohn (1979) Chassy & Flickinger (1987) Heidel (1989) Gotz, Universitat
Tiibingen, FRG Heinrich et al. (1987) van der Vossen et ul. (1985) Present study Present study
co-cultivated at 30°C with S. carnosus in 5 ml MRS-medium without acetate after inoculation with 100 p1 of an overnight culture of each strain.
Results and Discussion C L O N I N G OF THE LYSOSTAPHIN G E N E
For the expression of lysostaphin in B. subtilis and L. casei the lysostaphin gene present on pUls23 was recloned into the E . coliLactococcus shuttle-vector pGKV210. The resulting plasmids pWG100 and pWG200 are depicted in Fig. 1. For the construction of pWGlOO or pWG2OO the 1.8 k b insert of pUls23 was cloned into pGKV210 as a HindIIIl BarnHI or a HindIII fragment, respectively. The plasmid pWGl00 differs from pWG200 only by the lack of the small HindIII/BumHI fragment from pGKV210. The direction of the lysostaphin gene in pWG200 was determined by restriction enzyme analysis with BarnHI, yielding fragments of 5.5 and 0.7 kb. No clones were detected with the 1.8 kb HindIII fragment inserted in the inverse direction. Both pWGlOO and pWG200 were isolated from E . coli and transferred to B. subtilis, L. cnsei and L. curuatus. Erythromycin-resistant transformants were obtained with all hosts. Analysis of the plasmid patterns of the transformants revealed that B. subtilis transformants contained pWGl00 and pWG200 of the
W . Gaier et al.
74 Bum HI
SmuI I
pWGlOO
Fig. 1. Restriction map of the plasmids pWGl00 and pWG200 containing the lysostaphin gene from Staphylococcus staphylolyticus. DNA fragments of pUls23 (m) and pGKV2lO (0) are shown. Location and direction of structural genes are indicated by arrows. Ery, erythromycin resistance; (cat86), part(s) of the promoterless chloramphenicol-acetyltransferase gene on pGKV210; Lys; lysostaphin gene.
unchanged size, whereas transformants of L. casei contained plasmids of various sizes, which were slightly smaller than the original plasmids. After transformation of L. curvatus only two erythromycin-resistant strains were obtained, both of which contained a plasmid of approx. 3.2 kb size. The small plasmids in L. casei and L. curvatus are probably the result of deletions occurring during the replication of these plasmids. Such deletions in plasmids like pGKV210 replicating via a rolling circle mechanism are not unusual, as can be seen from studies with B.
subtilis plasmids by Gruss & Ehrlich (1989) and Janniere et al. (1990). E X P R E S S I O N OF T H E L Y S O S T A P H I N GENE
The lysostaphin activity of E. coli, B . subtilis and L. casei transformants was determined initially by the plate assay method and is shown in Table 2 together with the data obtained in the photometric assay. No lysostaphin activity was detected with transformants of L. curvatus. The
Table 2. Lysostaphin activity of Bacillus subtilis, Escherichia coli and Lactococcus casei transformants
Strain
Plasmid
B. subtilis DSM402 B. subtilis DSM402 B. subtilis DSM402 E. coli BHB2600 E.coliBHB2600 E. coli BHB2600 L. casei 102s L. casei 102s L. casei 102s
None pWGl00 pWG200 None pWG100 pWG2OO None pWGl00 pWG2OO
*
+
Plate* assay
Photometric assay (% decrease in OD,,, x min-')
-
-
+
0.67 1.15
+
-
+ + +I ~
+$
~
0.25 0.36 ~
0.48 0.20
Co-cultivation assay (cfu/ml)t 6h
24 h
2.6 x 107 3.4 x 103 2.1 x 104 ND ND ND 3.8 107 1.7 x 107 4.3 x 107
ND ND ND ND ND ND 1.8 x 105 2.6 x 103 1.2 105
= formation of a clear halo around the colonies. $ Cell count of staphylococci on SK-medium. $ Not all plasmid containing transformants exhibited lysostaphin activity.
Expression of lysostaphin lysostaphin gene is probably deleted in the small plasmids harboured by these strains. Transformants were subsequently subjected to quantitative activity determination in the photometric assay. As an example, the time course of the decrease in OD,,, during the photometric assay performed with L . casei transformants is depicted in Fig. 2. For comparison of the lysostaphin activity of B. subtilis, E. coli and L. casei transformants, the slope was determined of the initial linear part of the lysis curve. The activity was calculated as the decrease in the percentage of the optical density x min-' (Table 2). Activities of different L. casei transformants ranged from zero up to the values shown in Table 2. These various activities may be explained by deletion of pWGlOO and pWG200 in the host L. casei. In addition to the lysis assays with heat-inactivated Staphylococcus cells, the staphylolytic activity on cells of B. subtilis and L. casei transformants was determined with growing staphylococci. The results shown in Table 2 are qualitatively consistent with those obtained in the plate and photometric assays
40 50
1
0
l
-
/
II 0 l /
0
/
/
# I00
Transformants of L. cusei exhibiting the highest lysostaphin activity had significantly reduced growth rates. This may be explained by a membrane-destabilizing effect of the lysostaphin signal peptide occurring during the transport of lysostaphin through the Lactobacillus cell membrane. Reduced growth or cell death due to lysostaphin transport might be the reason that no lysostaphin-positive L. curuatus transformants were obtained. On the other hand, lysostaphin is expressed in B. subtilis and L. casei, released into the medium and processed into an active form. The use in food production of lactic acid bacteria, which produce lysostaphin. could possibly reduce the risk of staphylococcal food poisoning, by using genetically engineered protective starter cultures. Previously, a Penicillium nalgiovense strain producing lysostaphin was constructed by Geisen et a/. (1990). As this mould grows on the surface of fermenting sausages, a possible inhibition of staphylococcal growth can occur in the outer region of the sausage only. The production of lysostaphin by lactobacilli could provide its homogeneous distribution and uniform inhibition of staphylococcal growth in fermenting foods. However, for this purpose, the gene has to be maintained stably by the host and antibiotic resistances have to be replaced by food grade markers. The authors are indebted to F. Gotz, Universitat Tubingen, FRG and J. K o k , University of Groningen, NL for kindly providing the plasmids pUls23 and pCKVZI0, respectively. This work was supported by the Bundesministerium Technologie grant fur Forschung und 0319280A. The authors are responsible for the content of this publication.
0
E
75
200
References
300
Time (min!
Fig. 2. Time course of the lysis of Staphylococcus carnosus cells by lysostaphin in the culture supernatant of Lactobacillus case; 102s transformants. Decrease in ODs7* of a suspension of S . carnosus TM300 cells during incubation with culture supernatant of L. casei 102s harboring no plasmid (m), pWGl00 ( 0 )or pWG200 (A).
ANDERSOF;. D.G. & M c K A ~ .L.L. IY83 Simple and rapid method for isolating large plasmid DNA from lactic streptococci. Applied and Environmental Microhioloqx 46,549-552. BIRNBOIM,H.C. & DOLY.J. 1Y79 A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nuclei
