JOURNAL OF BACMRmol,oGY, Feb. 1978, p. 614-620 0021-9193/78/0133-0614$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 133, No. 2 Printed in U.S.A.
Effects of Streptomycin and Novobiocin on Staphylococcus aureus Gene Expression KRISTINA NORDSTROM' AND MARTIN LINDBERG2* Department of Clinical Bacteriology' and Department of Microbiology,' The Biomedical Center, University of Uppsala, Uppsala, Sweden Received for publication 19 July 1977
Streptomycin and novobiocin induced production of protein A and inhibited production of alpha- and beta-hemolysins in mutants of Staphylococcus aureus strains RN450 and RN1 resistant to these antibiotics. Streptomycin, but not novobiocin, also inhibited propagation of bacteriophages of serological group B, whereas phages of group A were unaffected. Streptomycin had to be present at adsorption of the phage, and 10 mM CaCl2 reversed the inhibitory effect. Lysogenization and competence induction occurred in the presence of streptomycin, suggesting that some early phage genes were expressed.
The localization of genes for extracellular protein virulence factors and surface antigens in Staphylococcus aureus is of considerable interest. Protein A is a cell wall protein covalently linked to the peptidoglycan with a molecular weight of 42,000 (29). It reacts with the Fc region of immunoglobulin G (IgG) from many mammalian species (28). Most strains of S. aureus produce cell-bound as well as extracellular protein A (6). Strain RN450 synthesizes only minute amounts of protein A, and it may therefore be used as recipient strain to study cotransformation of resiance markers and the gene(s) for protein A. In the experiments reported here we find that streptomycin and novobiocin induce production of protein A in reistant mutants of strain RN450. The influence of these antibiotics on the production of other extracellular proteins and toxins was also examined. The production of alpha- and beta-hemolysins was inhibited when the antibiotics were added to the growth media. Since protein A inhibits adsorption of some phages (21), the influence of the two drugs on the plating efficiency of different phages was also examined.
minimal inhibitory concentration for novobiocin-resistant mutants was 20 jLg per ml. Cell density was estimated by measuring the absorbancy at 530 nm. An absorbancy of 0.2 corresponds to approximately 10' colony-forming units (CFU) per ml. Quantitation of protein A. Protein A was determined from concentrated cultures containing 109 CFU/ml. It was extracted from the cells by the method of Jensen (7) or in some experiments by lysostaphin treatment (20). Affinity chromatography on IgGSepharose 4B was used for purification of protein A (14). The content of the protein was estimated by the hemagglutination technique (30) or by radial immunodiffusion in gels (18). The latter contained equal volumes of 3% agarose and 10% normal dog serum in 0.04 M barbiturate buffer at pH 8.9. For radial immunodiffusion, the extracts were concentrated 100 times by lyophilization. Purified protein A from strain S. aureus Cowan I was used as a standard in all assays. All assays were standardized to give the protein A content per milliliter in a broth culture containing 109 CFU/ml. Sodium dodecyl sulfate-polyacrylamide electrophoresis was carried out in 11% polyacrylamide gels in 5 mM tris(hydroxymethyl)aminomethane-glycine buffer at pH 8.3 (19). Assays of hemolysins. The assay for alpha- and beta-hemolysin has been described (17). Cell-free supernatants were concentrated in an ultrafiltration cell (Diaflo m50, Amicon Corp.). Titers were expressed MATERIALS AND METHODS relative to a culture containing 10' CFU/ml. Bacterial sri and cultivation technique. Sera. Antiserum against protein A was obtained The S. aureus strains are listed in Table 1. The bac- from rabbits immunized with highly purified protein teria were grown in Trypticase soy broth (TSB) or A from strain Cowan I (8). on Trypticase soy agar (TSA). For cultivation of thyPhages. Several staphylococcal phages of serologmine-requiring mutants, thymine was added at 20 ical groups A and B (2, 25) were used in this study ug/ml. Spontaneous streptomycin- and novobiocin-re- (see Table 3). All phages except 011 and js14 were sistant mutants were isolated after incubation at 37°C from the standard typing set obtained from the Nafor 48 h on TSA containing 500 ,ug of streptomycin tional Bacteriological Laboratory, Stockholm, Sweper ml or 10 ytg of novobiocin per ml. The minimal den. Phage ll was originally obtained from R. P. inhibitory concentration for streptomycin-resistant Novick (23). The mutant phage 4,llvir (31) and phage mutants was more than 2,000 ug per ml, and the 4,14 (26) were isolated in this laboratory. Phage 414vir 614
S. AUREUS GENE EXPRESSION
VOL. 133, 1978
615
TABLE 1. Strains of S. aureus Strain no. RN1 U100 RN450
U102 U84 U32 U97 U98 U99 U1o0
Characteristcs and derivation Strain 8325, lysogenic for phages 411, 412, and 4)13 Novobiocin-resistant mutant of strain 8325 Derivative of strain 8325 cured for phages 411, 412, and 413 = strain 8325-4 Novobiocin-resistant mutant of strain 8325-4 Streptomycin-resistant mutant of strain 8325-4 Thymine-requiring mutant of strain 83254 Streptomycin-resistant mutant of strain 8325-4 thy Strain 8325-4 lysogenized with phage 414 Streptomycin-resistant mutant of strain 8325-4 (4)14) Novobiocin-resistant mutant of strain 8325-4 thy
isolated at a frequency of approximately 10-i by plating phage 414 on a lawn of strain 8325-4 (4)14). The mutant phage was purified by single-plaque propagation on strain 8325-4. Phage stocks were prepared in TSB medium (for propagation of phage 414 it is necessary to add 5 mM CaCl2) or by the soft-agar overlay technique. TSA and TSB with 0.5% agar were used as bottom and soft agar, respectively, for phage titration. Induction of lysogenic strains was performed by ultraviolet irradiation as described earlier (26). Transduction procedure. Recipient cells grown in TSB medium to late logarithmic phase (about 2 x i09 CFU/ml) were centifuged and resuspended in phage lysate. The tramnduction mixture was incubated at 370C for 15 min and plated. Transformation procedure. The method for tranormation has been decribed (27). Competent cells were obtained by lytic infection with competenceinducing phages (see Table 4). Tris(hydroxymethyl)aminomethane-hydrochloride buffer, 0.1 M at pH 7.0, containing 1 M NH4Cl was used as transformation medium. Screening methods for transductants and tranformants. CY agar (22) or CY agar plus streptomycin (500 jg/ml) was used for selection of Thy' transductants and transformants. Tetracycline-resistant btaductants and novobiocin-resistant transformants were scored after 2 h of phenotypic expression on TSA or TSA plus streptomycin (500 pg/ml) at 37°C by adding soft agar with antibiotics to give the foilowing concentrations in the agar medium: tetracycline, 5 #g/ml; novobiocin, 10 pug/ml; and streptomycin, 500 pg/ml. Antibiotics. The foflowing antibiotics were used: novobiocin (sodium novobiocin), BDH Chemical, England; streptomycin, Glaxo, England; terramycin (oxitetracycline), Pfizer Inc., New York, N.Y. was
RESULTS Induction of protein A production by streptomycin and novobiocin. S. aureus strain RN450 produces minute amounts of protein A. Spontaneous streptomycin-resistant mutants of strain RN450 are also low producers of protein A. Figure 1 illustrates the production of cell-bound protein A during sequential cultivations of strain U84 in TSB medium supple-
Source and reference R. Novick (23) Our laboratory R. Novick (23) Our laboratory Our laboratory J. E. Sjostrom (32) Our laboratory L. Rudin (26) Our laboratory Our laboratory
mented with 500 ug of streptomycin per ml. In the first passage the protein A titer increased from % to /63 as determined by the hemagglutination technique. After the second passage the titer reached V28s, and it increased after the third passage to %ou. Additional passages did not increase production further. Thus a 500-fold increase in protein A titer was observed after three sequential passages in streptomycin-containing medium. The maximum effect was obtained at 500 jig of streptomycin per ml, but there was already a measurable increase in protein A titer at 10 pug of streptomycin per ml. Control experiments showed that streptomycin itself does not interfere in the hemagglutination reaction. When cells from the third passage in streptomycin-containing medium were inoculated into antibiotic-free medium, the production of protein A ceased, and after two serial subcultivations, the protein A titer had diminished to the
original level (Fig. 2). Streptomycin, therefore, does not select protein A-producing mutants, but causes altered gene expression. In spontaneous novobiocin-resistant mutants of strains RN450 and RN1, novobiocin had the same effect as streptomycin on the production of protein A (Table 2). Maximum production was obtained after three passages in TSB medium containing 10 pg of novobiocin per ml. After subcultivation of the culture in antibioticfree medium, the production of protein A decreased to the original level after two passages (not shown). Purification of protein A by affinity chromatography on IgG-Sepharose. Strain U84 was grown in 750 ml of TSB medium with and without streptomycin. The cells were collected by centrifugation, and cell-bound protein A was released by lysostaphin (20). Cell-bound and extracellular protein A was purified by affinity chromatography on IgG-Sepharose (14). In radial immunodiffusion tests no protein A was detected from the culture grown in TSB medium, whereas the streptomycin-grown culture gave approximately 14 mg of cell-bound protein
616
J. BACTERIOL.
NORDSTROM AND LINDBERG
of strain U84 in TSB medium supplemented with 500 ,ug of streptomycin per ml. The production of both hemolysins decreased in-
passages
0o
w
,
0
mediately after addition of streptomycin, and
the production was almost undetectable after the second passage. The maximum effect was obtained at 500 to 1,000 ,ig of streptomycin per ml, but there was already a measurable decrease in hemolysin titers after one passage in 50 ,tg of streptomycin per ml. Control experiments showed that the antibiotic itself did not interfere with the hemolysin assays. O1100 A
1.0
E
E
B
10
ISO
(
CD)
t
49~ ~ 70-
U,
4 4
uiw
90 54
~~LkiZim
determined (. 30a a reIn X~~~~~~~~4 C. TSB0 meimwr ncusrpoyi-otiing 20
z TIME
TIME (MINUTES)
m
z
m
d-0 t Repression nevls aplswr 37C foro A wihrw ofprotein TFIG 2. synthesis in streph temnto fcl-on cells afterpotenAxelsfo inoculation to antibiotictomyci-induced free TSB medium. Cells cultivated three times in
0.
streptomycin-containing TSB medium
0
(n
m 4
(MINUTES)
filrs
lated into
antibiotic-fr-ee
were
inocu-
medium and incubated at
samples were withdrawn for deof cell- bound protein A. Cells fr-om the stationary phase of the first passage (A) were subculTSB medium, and protein A content was tured at 530 nm (0); bars, sty re-determnined (B). Absorbancy titer of cell-bound proreciprocal hemagglutination gep 370C. At intervals,
termination
e.IC1
Cen
~
TIME (MINUTES ) FIG. 1. Induction of protein A synthesis by tomycin in strain U84 during sequential passai Cells grown overnight in TSB medium were inc lated into fresh medium and incubated at 37°C. In the beginning of the exponential phase in the first passage (A), streptomycin was added to a final c centration of 500 pg/ml (indicated by the arrow). At intervals, samples were withdrawn for determinai tion of cell-bound protein A. Cells in the stationary phiase (800 min) from the first passage (A) were subcultu red for two additional sequential passages (B and ' C), and protein A was determined. Absorbancy at 530 nm (0); bars, reciprocal hemagglutination titerr of cell-bound protein A corresponding to 109 CFU, /ml. Note the difference in scale between (A) and (B) on the one hand and (C) on the other. )cu-
tein A
corresponding
to
iO1 CFU/ml.
*.
'on-
of protein A and hemolysins during growth with different concentrations of
TABLE 2. Production
novobiocina
Detenrination Protein Ah Passage 1 Passage 2 Passage 3
Novobiocin (jug/ml) 0
7.5
10
4 4 4
64 128 512
128 256 1,024
64 64 64
2
Vol. 133, No. 2 Printed in U.S.A.
Effects of Streptomycin and Novobiocin on Staphylococcus aureus Gene Expression KRISTINA NORDSTROM' AND MARTIN LINDBERG2* Department of Clinical Bacteriology' and Department of Microbiology,' The Biomedical Center, University of Uppsala, Uppsala, Sweden Received for publication 19 July 1977
Streptomycin and novobiocin induced production of protein A and inhibited production of alpha- and beta-hemolysins in mutants of Staphylococcus aureus strains RN450 and RN1 resistant to these antibiotics. Streptomycin, but not novobiocin, also inhibited propagation of bacteriophages of serological group B, whereas phages of group A were unaffected. Streptomycin had to be present at adsorption of the phage, and 10 mM CaCl2 reversed the inhibitory effect. Lysogenization and competence induction occurred in the presence of streptomycin, suggesting that some early phage genes were expressed.
The localization of genes for extracellular protein virulence factors and surface antigens in Staphylococcus aureus is of considerable interest. Protein A is a cell wall protein covalently linked to the peptidoglycan with a molecular weight of 42,000 (29). It reacts with the Fc region of immunoglobulin G (IgG) from many mammalian species (28). Most strains of S. aureus produce cell-bound as well as extracellular protein A (6). Strain RN450 synthesizes only minute amounts of protein A, and it may therefore be used as recipient strain to study cotransformation of resiance markers and the gene(s) for protein A. In the experiments reported here we find that streptomycin and novobiocin induce production of protein A in reistant mutants of strain RN450. The influence of these antibiotics on the production of other extracellular proteins and toxins was also examined. The production of alpha- and beta-hemolysins was inhibited when the antibiotics were added to the growth media. Since protein A inhibits adsorption of some phages (21), the influence of the two drugs on the plating efficiency of different phages was also examined.
minimal inhibitory concentration for novobiocin-resistant mutants was 20 jLg per ml. Cell density was estimated by measuring the absorbancy at 530 nm. An absorbancy of 0.2 corresponds to approximately 10' colony-forming units (CFU) per ml. Quantitation of protein A. Protein A was determined from concentrated cultures containing 109 CFU/ml. It was extracted from the cells by the method of Jensen (7) or in some experiments by lysostaphin treatment (20). Affinity chromatography on IgGSepharose 4B was used for purification of protein A (14). The content of the protein was estimated by the hemagglutination technique (30) or by radial immunodiffusion in gels (18). The latter contained equal volumes of 3% agarose and 10% normal dog serum in 0.04 M barbiturate buffer at pH 8.9. For radial immunodiffusion, the extracts were concentrated 100 times by lyophilization. Purified protein A from strain S. aureus Cowan I was used as a standard in all assays. All assays were standardized to give the protein A content per milliliter in a broth culture containing 109 CFU/ml. Sodium dodecyl sulfate-polyacrylamide electrophoresis was carried out in 11% polyacrylamide gels in 5 mM tris(hydroxymethyl)aminomethane-glycine buffer at pH 8.3 (19). Assays of hemolysins. The assay for alpha- and beta-hemolysin has been described (17). Cell-free supernatants were concentrated in an ultrafiltration cell (Diaflo m50, Amicon Corp.). Titers were expressed MATERIALS AND METHODS relative to a culture containing 10' CFU/ml. Bacterial sri and cultivation technique. Sera. Antiserum against protein A was obtained The S. aureus strains are listed in Table 1. The bac- from rabbits immunized with highly purified protein teria were grown in Trypticase soy broth (TSB) or A from strain Cowan I (8). on Trypticase soy agar (TSA). For cultivation of thyPhages. Several staphylococcal phages of serologmine-requiring mutants, thymine was added at 20 ical groups A and B (2, 25) were used in this study ug/ml. Spontaneous streptomycin- and novobiocin-re- (see Table 3). All phages except 011 and js14 were sistant mutants were isolated after incubation at 37°C from the standard typing set obtained from the Nafor 48 h on TSA containing 500 ,ug of streptomycin tional Bacteriological Laboratory, Stockholm, Sweper ml or 10 ytg of novobiocin per ml. The minimal den. Phage ll was originally obtained from R. P. inhibitory concentration for streptomycin-resistant Novick (23). The mutant phage 4,llvir (31) and phage mutants was more than 2,000 ug per ml, and the 4,14 (26) were isolated in this laboratory. Phage 414vir 614
S. AUREUS GENE EXPRESSION
VOL. 133, 1978
615
TABLE 1. Strains of S. aureus Strain no. RN1 U100 RN450
U102 U84 U32 U97 U98 U99 U1o0
Characteristcs and derivation Strain 8325, lysogenic for phages 411, 412, and 4)13 Novobiocin-resistant mutant of strain 8325 Derivative of strain 8325 cured for phages 411, 412, and 413 = strain 8325-4 Novobiocin-resistant mutant of strain 8325-4 Streptomycin-resistant mutant of strain 8325-4 Thymine-requiring mutant of strain 83254 Streptomycin-resistant mutant of strain 8325-4 thy Strain 8325-4 lysogenized with phage 414 Streptomycin-resistant mutant of strain 8325-4 (4)14) Novobiocin-resistant mutant of strain 8325-4 thy
isolated at a frequency of approximately 10-i by plating phage 414 on a lawn of strain 8325-4 (4)14). The mutant phage was purified by single-plaque propagation on strain 8325-4. Phage stocks were prepared in TSB medium (for propagation of phage 414 it is necessary to add 5 mM CaCl2) or by the soft-agar overlay technique. TSA and TSB with 0.5% agar were used as bottom and soft agar, respectively, for phage titration. Induction of lysogenic strains was performed by ultraviolet irradiation as described earlier (26). Transduction procedure. Recipient cells grown in TSB medium to late logarithmic phase (about 2 x i09 CFU/ml) were centifuged and resuspended in phage lysate. The tramnduction mixture was incubated at 370C for 15 min and plated. Transformation procedure. The method for tranormation has been decribed (27). Competent cells were obtained by lytic infection with competenceinducing phages (see Table 4). Tris(hydroxymethyl)aminomethane-hydrochloride buffer, 0.1 M at pH 7.0, containing 1 M NH4Cl was used as transformation medium. Screening methods for transductants and tranformants. CY agar (22) or CY agar plus streptomycin (500 jg/ml) was used for selection of Thy' transductants and transformants. Tetracycline-resistant btaductants and novobiocin-resistant transformants were scored after 2 h of phenotypic expression on TSA or TSA plus streptomycin (500 pg/ml) at 37°C by adding soft agar with antibiotics to give the foilowing concentrations in the agar medium: tetracycline, 5 #g/ml; novobiocin, 10 pug/ml; and streptomycin, 500 pg/ml. Antibiotics. The foflowing antibiotics were used: novobiocin (sodium novobiocin), BDH Chemical, England; streptomycin, Glaxo, England; terramycin (oxitetracycline), Pfizer Inc., New York, N.Y. was
RESULTS Induction of protein A production by streptomycin and novobiocin. S. aureus strain RN450 produces minute amounts of protein A. Spontaneous streptomycin-resistant mutants of strain RN450 are also low producers of protein A. Figure 1 illustrates the production of cell-bound protein A during sequential cultivations of strain U84 in TSB medium supple-
Source and reference R. Novick (23) Our laboratory R. Novick (23) Our laboratory Our laboratory J. E. Sjostrom (32) Our laboratory L. Rudin (26) Our laboratory Our laboratory
mented with 500 ug of streptomycin per ml. In the first passage the protein A titer increased from % to /63 as determined by the hemagglutination technique. After the second passage the titer reached V28s, and it increased after the third passage to %ou. Additional passages did not increase production further. Thus a 500-fold increase in protein A titer was observed after three sequential passages in streptomycin-containing medium. The maximum effect was obtained at 500 jig of streptomycin per ml, but there was already a measurable increase in protein A titer at 10 pug of streptomycin per ml. Control experiments showed that streptomycin itself does not interfere in the hemagglutination reaction. When cells from the third passage in streptomycin-containing medium were inoculated into antibiotic-free medium, the production of protein A ceased, and after two serial subcultivations, the protein A titer had diminished to the
original level (Fig. 2). Streptomycin, therefore, does not select protein A-producing mutants, but causes altered gene expression. In spontaneous novobiocin-resistant mutants of strains RN450 and RN1, novobiocin had the same effect as streptomycin on the production of protein A (Table 2). Maximum production was obtained after three passages in TSB medium containing 10 pg of novobiocin per ml. After subcultivation of the culture in antibioticfree medium, the production of protein A decreased to the original level after two passages (not shown). Purification of protein A by affinity chromatography on IgG-Sepharose. Strain U84 was grown in 750 ml of TSB medium with and without streptomycin. The cells were collected by centrifugation, and cell-bound protein A was released by lysostaphin (20). Cell-bound and extracellular protein A was purified by affinity chromatography on IgG-Sepharose (14). In radial immunodiffusion tests no protein A was detected from the culture grown in TSB medium, whereas the streptomycin-grown culture gave approximately 14 mg of cell-bound protein
616
J. BACTERIOL.
NORDSTROM AND LINDBERG
of strain U84 in TSB medium supplemented with 500 ,ug of streptomycin per ml. The production of both hemolysins decreased in-
passages
0o
w
,
0
mediately after addition of streptomycin, and
the production was almost undetectable after the second passage. The maximum effect was obtained at 500 to 1,000 ,ig of streptomycin per ml, but there was already a measurable decrease in hemolysin titers after one passage in 50 ,tg of streptomycin per ml. Control experiments showed that the antibiotic itself did not interfere with the hemolysin assays. O1100 A
1.0
E
E
B
10
ISO
(
CD)
t
49~ ~ 70-
U,
4 4
uiw
90 54
~~LkiZim
determined (. 30a a reIn X~~~~~~~~4 C. TSB0 meimwr ncusrpoyi-otiing 20
z TIME
TIME (MINUTES)
m
z
m
d-0 t Repression nevls aplswr 37C foro A wihrw ofprotein TFIG 2. synthesis in streph temnto fcl-on cells afterpotenAxelsfo inoculation to antibiotictomyci-induced free TSB medium. Cells cultivated three times in
0.
streptomycin-containing TSB medium
0
(n
m 4
(MINUTES)
filrs
lated into
antibiotic-fr-ee
were
inocu-
medium and incubated at
samples were withdrawn for deof cell- bound protein A. Cells fr-om the stationary phase of the first passage (A) were subculTSB medium, and protein A content was tured at 530 nm (0); bars, sty re-determnined (B). Absorbancy titer of cell-bound proreciprocal hemagglutination gep 370C. At intervals,
termination
e.IC1
Cen
~
TIME (MINUTES ) FIG. 1. Induction of protein A synthesis by tomycin in strain U84 during sequential passai Cells grown overnight in TSB medium were inc lated into fresh medium and incubated at 37°C. In the beginning of the exponential phase in the first passage (A), streptomycin was added to a final c centration of 500 pg/ml (indicated by the arrow). At intervals, samples were withdrawn for determinai tion of cell-bound protein A. Cells in the stationary phiase (800 min) from the first passage (A) were subcultu red for two additional sequential passages (B and ' C), and protein A was determined. Absorbancy at 530 nm (0); bars, reciprocal hemagglutination titerr of cell-bound protein A corresponding to 109 CFU, /ml. Note the difference in scale between (A) and (B) on the one hand and (C) on the other. )cu-
tein A
corresponding
to
iO1 CFU/ml.
*.
'on-
of protein A and hemolysins during growth with different concentrations of
TABLE 2. Production
novobiocina
Detenrination Protein Ah Passage 1 Passage 2 Passage 3
Novobiocin (jug/ml) 0
7.5
10
4 4 4
64 128 512
128 256 1,024
64 64 64
2
