VIROLOGY
72, 322-329(1976)
Recombination
between Phage Sl and the TC-Resistant Staphylococcus aureus Plasmid
MATSUHISA
Gene on
INOUE’ AND SUSUMU MITSUHASHI
Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan Accepted February 24,1976 When bacteriophage Sl is grown in a Staphylococcus aureus strain carrying the nonconjugative plasmid rmsi’ that encodes tetracycline (TC) resistance, a phage lysate capable of transducing TC resistance at an extremely high frequency was obtained. A linear relationship was found between transduction frequency and multiplicity of phage infection and a single phage particle can form a plaque containing TC lysogenic cells in its center. Treatment with anti-S1 phage serum and heating at 63” eliminated both transducing and plaque-forming activities of the lysate. These results indicated that a single recombinant of Sl particle (called Slptet) carries the tet gene(s) of the rmd plasmid and is responsible for the transduction. INTRODUCTION
In the transduction of an R factor with bacteriophage Pl, bacteriophage Plcml was obtained which carried all pertinent phage activities of Pl, as well as the chloramphenicol resistance (cml) character from the R plasmid (Kondo et al., 1964; Scott, 1972; Mise, personal communication). Similarly, the same type of converting bacteriophage with kanamycin resistance in Proteus (Coetzee, 1974, 1975), ampicillin resistance in E. coli (Smith, 19721, and tetracycline resistance in Enterobacteriaceae (Kameda et al., 1965) was reported. In the transduction of tetracycline (TC) resistance from Staphylococcus aureus carrying a nonconjugative plasmid with TC resistance, S. aureus MS3878 111157,a phage capable of transducing the TC resistance character at an extremely high frequency was obtained. This paper deals with the genetic and molecular properties of the phage particle responsible for the transduction. MATERIALS
AND METHODS
Bacterial strains. Staphylococcus aureus MS353 and MS3878 were isolated
from clinical specimens and stocked in this laboratory. S. aureus MS353 strains carrying various nonconjugative plasmid (r) encoding drug resistance were obtained by transduction from MS3878 rms7 (Inoue et al., 1970; Mitsuhashi et al., 1973). Table 1 lists resistances of these strains and plasmids . Media. Nutrient broth and nutrient agar (Difco) were used for routine cultivation. Selection for drug resistance in transduction experiments was made on heart infusion (HI) agar. HI agar was also used for the determination of drug resistance. Chemicals. Tetracycline (TC) hydrochloride was donated by the Taito-Pfizer Co. Other chemicals were from commercial sources. Transduction experiments. Transduction was performed by the method described previously (Inoue et al., 1970). An overnight culture of the recipient bacteria grown on HI agar was collected and suspended in nutrient broth at 30”. After 30 min of incubation, 10e3M CaCl, was added to the culture. The recipient bacteria were mixed with phage lysates at multiplicities of infection of lo+ to 10’ and the mixture was incubated for 45 min at 30”. The mixed culture was sedimented by centrifugation and the precipitated bacteria were washed
1 Author to whom reprint requests should be addressed. 322 Copyright 6 19’76by Academic Press, Inc. All rights of reproduction in any form reserved.
ACTIVE
TRANSDUCING
PHAGE
TABLE
323
Slptel
1
BACTERIAL STRAINS AND PLASMIDS” Strain MS3878
MS353 MS15001
MS15002
phenotype
Phage and plasmid
Resistant to TC and lysogenized with Sl and S2 Drug sensitive TC-resistant transductant of MS353
Sl and S2 phage rms7
Sl
rms7 and Sl
Relevant
lysogen MS15001
of
Source Clinical
Clinical isolate Obtained by transduction of TC resistance from MS3878 to MS353 with temperate phage Obtained by lysogenization of MS15001 with Sl
rms7
n Abbreviations: TC, tetracycline; rms7, nonconjugative temperate phage isolated from MS3878.
with nutrient broth. The sedimented cells were resuspended in 1.0 ml of nutrient broth and spread on HI agar plate containing 12.5 pg/ml of TC. Colonies formed after 24 or 48 hr of incubation on selective plates were picked, and their drug resistance and immunity to phage Sl and S2 were examined. In each transduction experiment, two control tests were set up. The phage lysate was plated on nutrient broth to test sterility and the noninfected culture was tested for spontaneous mutation. Transduction frequency was expressed as the number of transductants divided by the plaque-forming units. Phage purification. The Sl phage was purified by the following methods. One milliliter of phage lysate from MS3876 was mixed with 1.0 ml of nutrient broth containing 0.1 ml of antiserum of serotype B phage and the mixture was incubated at 37” without aeration. After 20 min of incubation, 0.1 ml was diluted with nutrient broth, mixed with soft agar containing MS353 and plated on a nutrient agar plate. After 24 hr of incubation at 37”, each plaque was picked with sterilized capillary glass and suspended in nutrient broth. The phage was subjected to three cycles of propagation on MS353 and the serotype of the resulting phage was determined. Lysogenization with Sl phage. MS15001 was spread on HI agar plates and spotted with appropriate dilutions of Sl phage. After 24 hr of incubation at 37”, the colo-
plasmid
isolate
encoding
TC resistance;
Sl and S2,
nies in the center of phage plaques were picked, purified three times on HI agar, and tested for lysogeny by Sl phage. The MS15062 strain lysogenized with phage Sl were used for further experiments. Plaque assay after inactivation. After appropriate times of treatment with either anti-S1 phage serum or heat, a O.l-ml amount of the sample was taken, diluted with nutrient broth, mixed with soft agar containing MS353, and plated on nutrient agar . Plaque center test. When the plaques were directly picked to TC plates, the colonies are too small, and the surrounding of the colonies are lysed by phage. Accordingly, it is difficult to count the relation between phage particles and TC resistance. Therefore, plaque centers were tested by the following method. The center of the plaque was stabbed with a needle and inoculated onto HI agar plate. After 20 hr of incubation at 37”, this was replicaplated on HI agar plate in the presence or absence of TC (12.5 pg/ml). Density-gradient centrifugation. Onetenth-milliliter phage lysate was suspended in 5.0 ml of C&O, (p = 1.365 at 25%0.01 M Tris (hydroxmethyl) aminomethane-O.01 M NaCl (pH 7.6) buffer, and the mixture was centrifuged in the Hitachi RPS65T rotor of a Spinco ultracentrifuge for 20 hr at 55,000 rpm. Fractions were collected from the bottom and assayed for phage and the density of each fraction. Electron microscopy. Thirty milliliters
324
INOUE AND MITSUHASHI TABLE 2
PLAQUE-FORMING TITERS AND TRANSDUCTION FREQUENCIES OF PHAGE LYSATES PREPARED FROM TC-RESISTANT TRANSDUCTANTP
Strain
PlaqueMulti litforming ti- it 0 inter (per ml) YP ection
MS353 TC-12 MS353 TC-13 MS353 TC-22 MS353 TC-23 MS353 TC-32 MS353 TC-33 MS353 TC-42 MS353 TC-43
1.3 2.0 7.0 2.1 1.4 1.6 6.0 1.8
x x x x x x x x
lo9 lo9 lo8 lo9 109 lo9 lOa lo9
3.0 0.6 3.0 0.4 0.03 0.5 0.05 0.5
Transduction frequency per input phage 3.5 1.0 1.3 5.0 3.1 1.0 7.8 9.5
x x x x x x x x
10-a 10-l 10-Z 10-Z 10-I 10-Z 10-Z 10-Z
a Fifty clones of TC-resistant transductants were obtained at a multiplicity of infection of lo-’ in the experiments shown in Fig. 1. Each clone of the transductanta developed on HI agar plates containing TC was inoculated in nutrient broth for 18 hr at 37” and each 1 ml of the cultures was inoculated in 10 ml of fresh BHI broth. After 3 hr of incubation at 37”, the cultures were treated with mitomycin C as described in Methods. The supernatant fluid was used as the source of phage after filtration through a HA (0.45) membrane filter. MA353 was used as the recipient cell. of phage lysate (5 x log/ml) obtained in liquid culture was centrifuged for 20 min at 10,000 rpm in the cold to remove bacterial debris. The supernatant thus obtained was filtered through a millipore filter and suspended in 0.01 A4 Tris-0.01 M NaCl (pH 7.6) buffer. The phage was further purified by centrifugation as described above and the visible band of phage was collected from the bottom. The purified phage sample was diluted with deionized water immediately before electron microscopic observation and applied to a carbon-coated grid. The grid was stained with phosphotungstic acid. RESULTS
Transduction rms7 plasmid.
of MS15002 with mitomycin C. The transduction frequency of tet in this lysate was about 4 x lo-lo per phage, and when transductants were used as donors to MS353,
of TC resistance
FIG. 1. Linear relationship between number of TC-resistant transductants and multiplicity of infection. Phage lysates were prepared by treatment of (a) MS353 TC-11, (b) MS353 TC-21, (c) MS353 TC31, and (d) MS353 TC-41 with mitomycin C. MS353 was used as the recipient cell.
109
108
“? 107 % IO 5
z
lo6
2 $
105
104
103 5
of the
In a phage lysate obtained by treatment of MS3878 rms7(Sl)(S2) with mitomycin C, the transduction frequency for tet ranged from 1 x 10e6to 4 x lo-+ per phage and all transductants were nonlysogenic. Four tet transductants obtained by the above experiments were lysogenized with purified Sl phage. Following phage lysates were made by treatment
10 15 20 25 min
5
10 15 20 25 30 min
FIG. 2. Inactivation of Sl and Slptet phages. One-tenth milliliter of anti-S1 phage serum was added to 6 ml of nutrient broth containing phage, and mixture was incubated at 37”. When the phage lysate was heated at 63”, one milliliter of the phage 1ysat.e was incubated. After appropriate time intervals, the mixture was diluted with nutrient broth and the number of plaques was assayed. (A) Treatment with antiserum; and (B) treatment with heat at 63”, Sl (-0-j and Slptet (-0-4.
ACTIVE
TRANSDUCING
the frequency of tet transduction was increased enormously; the tet transduction frequency now ranged from 1.0 x 10m2to 9.0 x lo+’ per phage. All transductants possessed the same level of TC resistance and could produce plaques on a lawn of MS353, indicating a difference from transductants resulting from Hft tranduction by hdg (Campbell, 1957). Relation between transduction frequency and multiplicities of phage infection (m.0.i.). Four TC-resistant transduc-
tants, MS353-11, -21, -31, and -41, were randomly chosen from the experiments shown in Table 2, and the relation between the number of TC-resistant transductants and m.o.i. was examined. As shown in Fig. 1, there was a linear relationship over a wide range of m.o.i. from 10e7to 1. From eight tet transductants obtained at a m.o.i. 7 x 10m7’,phage lysates were prepared by treatment with mitomytin C. As shown in Table 2, all TC-resistante transductants liberated active plaque-forming phages; their induced lysate had similar titer to that of the lysate as shown in Fig. 1. Thus, the ability to produce Hft lysates is passed on to the tet transductants. From these results, it is concluded that OF Slptet
Phage source” MS353 TC-11
MS353 TC-21
MS353 TC-31
MS353 TC-41
Sl”
BY TREATMENT
TABLE
Antiserum 63” r Antiserum 63” Antiserum 63” Antiserum 63” Antiserum 63”
with
Ab
A
A
A
A
4
LYSOGENIC CONVERSION OF TETRACYCLINE RESISTANCE WITH Slptet PHAGE
Strain”
MS353 MS353 MS353 MS353
Number of pla ues containing % C-resistant cells in the center of plaqueb (%)
Number of plaqueforming units
TC-11 TC-21 TC-31 TC-41
MS15001’
15 18 36 32
15 18 36 32
300
0
(100) (100) (100) (100)
o The strains were obtained in the experiments shown in Fig. 1. ’ A sample obtained from the center of each plaque from the strains n with needle was inoculated on HI agar plate. After 24 hr of incubation at 37”, the plates were replica-plated on HI agar plates containing TC (12.5 pg/ml). After 24 hr of incubation at 37”, number of colonies developed on TC plate was enumerated. The number in parentheses indicates the percentage of the number of plaque-forming units per that of plaques containing TC-resistant cells in the plaque center. ’ MS15001, see Table 1. Phage lysate was obtained by propagation of phage Sl on this strain.
WITH ANTI-Sl
Treatment
325
Slptet
the TC resistant character is transduced by a single particle which possesses a plaque-forming activity in addition to a
TABLE INACTIVATION
PHAGE
3 PHAGE
SERUM
AND WITH HIGH
Plaque-fF;r$ng 3.5 1.9 3.3 4.4 1.2 2.3 1.5 2.2 2.1 1.3 2.7 8.6 8.7 7.1 2.4
x x x x x x x x x x x x x x x
103 105 102 109 105 lo3 109 105 103 109 lo5 103 10y 105 lo3
unit
TEMPERATURE
Number of transductants (ml) 3.7 2.4 1.7 4.1 2.1 1.2 3.7 7.2 4.2 1.5 6.4 8.4 3.0
x x x x x x x x x x x x x 0 0
108 IO4 102 10” 104 103 108 lo4 10’ 10’ lo4 10’ 10”
a MS353 TC-11, -21, -31, and -41 were obtained, respectively, by transduction of TC resistance in independent experiments, and they were lysogenized with phage Sl. b One-tenth milliliter of anti-S1 phage serum was added to 6 ml of nutrient broth containing phage, and the mixture was incubated at 37” for 20 min. ’ Incubated at 63” for 30 min. See Methods for details. d Phage lysate was obtained by propagation of phage Sl on MS15001.
326
INOUE
AND
Density ( g.cmm31 1.3725
1.3652
1.3550
MITSUHASHI Density ( g.cmm31 1.3725 1.3650
1.3550
\ (a)
Fraction Number
Fraction Number
FIG. 3. Preparative density-gradient centrifugation in Cs$O, of Slptet or Sl phage. Solid circles indicate the number of plaque-forming particles and open circles indicate the number of plaque-forming particles and open circles indicate the number of TC-resistant transductants. See the footnote of Table 4 for details. (a) Sl phage; and (bl Slptet phage.
transducing activity. This phage particle is designated “Slptet.” Properties of Slptet. Slptet particles have a number of properties that are also characteristic of phage Sl. (1) Anti-S1 phage serum neutralize both Sl and Sl ptet with a rate constant of lop4 per 20 min. (2) Heat inactivation of Sl and Slptet at 63” for 30 min gives lo+ surviving phage. Slptet lost both transducing and plaqueforming ability upon this treatment (Fig. 2; Table 3). (3) When the transduction with Slptet was carried out in BHI broth without the addition of CaCl,, the transduction frequency was decreased about l/50 that obtained by the addition of CaCl,. (4) The host range of phage Slptet (6/423/ 54/52/80/81/84) is identical to that of phage Sl (6/42E/(54Y52/80/81/84). Plaque center test of Slptet. As shown in Table 4, all TC resistant transductants were lysogenic. Therefore, an investigation was directed toward determining
whether all lysogenized clones were TCresistant. Each phage induced with mitomycin C from MS353 TC-11, -21, -31, and -41 was used to infect S. aureus MS353 at a m.o.i. of 0.01. Among 101 lysogenic clones tested, all were TC-resistant, and of 300 TC-resistant clones tested, all were lysogenie for Sl phages. Next, we examined that a single particle can form a plaque as well as transduce tet. A majority of the plaques contained TCresistant cells at their centers (Table 4). The test gave a similar result when S. aureus MS9403 was used as the recipient. Density-gradient centrifugation of Sl phages. It can be seen that the transducing Slptet phage and the original Sl phage in the lysate are almost the same in buoyant density analysis (p = 1.365). When Sl ptet was subjected to preparative density-gradient centrifugation (Fig. 3), the peak of transducing and plaqueforming activities appeared at the same
ACTIVE
FIG. 4. Electron
microscopy
TRANSDUCING
of the Sl@t
PHAGE
phage. Bar indicates
Slptet
327
0.1 pm. (a) Sl phage; (b) Slptet phage
328
INOUE
AND
position. This confirms the finding that in an Slptet lysate, transducing and plaqueforming ability reside in the same particle. Electron micrographs of Slptet phuge. Figures 4A and B illustrate the shape of phage Slptet and Sl, respectively. The diameter of the hexagonal head of these phages is about 0.39 pm and the length of the tail is about 3.35 pm. DISCUSSION
Converting phage Plcml or Plcry carries a gene for chloramphenicol (cml) resistance (Kondo and Mitsuhashi, 1964; Scott, 1972, 1973) and phage 34kan carries a gene for kanamycin @an) resistance (Coetzee, 1975). Both are also plaque-forming phage and are capable of general transduction. Jessop (1972) also described a nondefective, plaque-forming, specialized transducing phage P22 that was capable of high-frequency transduction of proline genes and general transduction of other markers. The cryptic high frequency transducing phage Pllde was isolated from S. aureeus and analyzed by Novick (1967). From a study of its origin and properties, Novick deduced that Pllde was apparently composed of a segment of the Pll genome stably linked to a segment of a y-plasmid. However, the production of Pllde transducing particles is found to require the activity of helper Pll phage, while the transduction process itself does not. Strains harboring Pllde are somewhat unstable, segregating antibiotic-sensitives at rates in the neighborhood 1% per cell generation. Transduction efficiency of the TC-resistante character with Slptet was not affected by varying the m.o.i. or by the addition of normal Sl phage. The results shown in Fig. 1 may be interpreted to mean that plaque-forming particles have transducing ability, because TC-resistance transductants obtained at a multiplicity of infection of 7 x lop7 liberate active plaqueforming phages with transduction frequencies similar to those of the lysate used in the initial infection. Moreover, plaques, which develop from single infection, contain TC-resistant cells in their center.
MITSUHASHI
Thus, it can be concluded that the transduction of tet character by Slptet phage does not require a helper phage, and that all of the essential phage functions are still retained by the Slptet phage particles. In Plcml, the association of cml gene(s) with Pl genome appears to be less firm (Kondo and Mitsuhashi, 1964). However, the tet gene in a Slptet phage is stably incorporated in the Sl genome since loss of the tet gene in Slptet upon treatment with various conditions was not observed. Accordingly, the acquisition of TC resistance by the recipient cell is always accompanied by the lysogenization of Slptet, and neither the establishment of TC resistance alone without lysogenization nor the formation of Sl-defective TC-transducing particle was observed. Therefore, the TC resistance-conferring ability of Slptet is entirely controlled by the phage itself, and the Sl element in Slptet represents a nonconjugative genome of Sl. The Sl element in Slptet represents a nondefective Sl genome. It possesses at least a genetic region which is responsible for the production of Slptet-transducing phage in stabilizing lysogeny and in synthesizing tail-protein of Sl, as well as in providing all information for replicating itself. The buoyant density of Slptet was not different from that of phage Sl under these conditions. ACKNOWLEDGMENT We should like to thank Mr. Takeshi Saito for his excellent technical assistance with electron microscopy. REFERENCES A. (1957). Transduction and segregation in Escherichia coli K12. Virology 4, 366-384. COETZEE, J. N. (1974). High frequency transduction of kanamycin resistance in Proteus mirabilis. J. Gen. Microbial. 84, 285-296. COETZEE, J. N. (1974). High frequency transduction of resistance to ampicillin and kanamycin in PFOteus mirabilis. J. Gen. Microbial. 87, 173-176. INOUE, M., HASHIMOTO, H., YAMAGISHI, S., and MITSUHASHI, S. (1970). Transduction analysis of the genetic determinants for chloramphenicol resistance in staphylococci. Japan J. Microbial. 14, 261-268. INOUE, M., OKUBO, T., SAITO, T., and MITSUHASHI, CAMPBELL,
ACTIVE
TRANSDUCING
S. (1973). The properties of staphylococcal temperate phages isolated from various strains of Stuphylococcus aureus. Japan. J. Bacterial. 28, 459. JESSOP, A. P. (1972). A specialized transducing phage P22 for which the ability to form plaques is associated with transduction of the proAB region. Molec. Gen. Genet. 114, 214-222. KAMEDA, M., HARADA, K., SUZUKI, M., and MITSUof enterobacHASHI, S. (1965). Drug resistance teria. V. High frequency of transduction of R factors with bacteriophage epsilon. J. Bacterial. 90, 1174-1181. KONDO, E., and MITSUHASHI, S. (1964). Drug resistance of enterobacteria. IV. Active transducing bacteriophage PlCM produced by the recombination of R factor with bacteriophage Pl. J. Bacteriol. 88, 1266-1276. MITSUHASHI, S., INOUE, M., KAWABE, H., OSHIMA,
PHAGE
Slptet
329
H., and OKUBO, T. (1973). In “Stanphylococci and Staphylococcal Infections.” (J. Jeljasaewicz and H. Hrynieewicz, eds.), Vol. 1, p. 144-165, S. Karger, Basel. NOVICK, R. (1967). Properties of a cryptic high frequency transducing phage in Staphylococcus aureus. Virology 33, 155-166. SCOTT, J. R. (1970). A defective Pl prophage with a chromosomal location. Virology 44, 144-151. SCOTT, J. R. (1973). Prophage Pl cryptic. II. Location and regulation of prophage genes. Virology 53, 327-336. SMITH-KEARY, P. F. (1966). Restricted transduction by bacteriophage P22 in Salmonella typhimurium. Genet. Res. 8, 73-82. SMITH, H. W. (1972). Ampicillin resistance in Escherichia coli by phage infection. Nature (London, 238, 205-206.
72, 322-329(1976)
Recombination
between Phage Sl and the TC-Resistant Staphylococcus aureus Plasmid
MATSUHISA
Gene on
INOUE’ AND SUSUMU MITSUHASHI
Department of Microbiology, School of Medicine, Gunma University, Maebashi, Japan Accepted February 24,1976 When bacteriophage Sl is grown in a Staphylococcus aureus strain carrying the nonconjugative plasmid rmsi’ that encodes tetracycline (TC) resistance, a phage lysate capable of transducing TC resistance at an extremely high frequency was obtained. A linear relationship was found between transduction frequency and multiplicity of phage infection and a single phage particle can form a plaque containing TC lysogenic cells in its center. Treatment with anti-S1 phage serum and heating at 63” eliminated both transducing and plaque-forming activities of the lysate. These results indicated that a single recombinant of Sl particle (called Slptet) carries the tet gene(s) of the rmd plasmid and is responsible for the transduction. INTRODUCTION
In the transduction of an R factor with bacteriophage Pl, bacteriophage Plcml was obtained which carried all pertinent phage activities of Pl, as well as the chloramphenicol resistance (cml) character from the R plasmid (Kondo et al., 1964; Scott, 1972; Mise, personal communication). Similarly, the same type of converting bacteriophage with kanamycin resistance in Proteus (Coetzee, 1974, 1975), ampicillin resistance in E. coli (Smith, 19721, and tetracycline resistance in Enterobacteriaceae (Kameda et al., 1965) was reported. In the transduction of tetracycline (TC) resistance from Staphylococcus aureus carrying a nonconjugative plasmid with TC resistance, S. aureus MS3878 111157,a phage capable of transducing the TC resistance character at an extremely high frequency was obtained. This paper deals with the genetic and molecular properties of the phage particle responsible for the transduction. MATERIALS
AND METHODS
Bacterial strains. Staphylococcus aureus MS353 and MS3878 were isolated
from clinical specimens and stocked in this laboratory. S. aureus MS353 strains carrying various nonconjugative plasmid (r) encoding drug resistance were obtained by transduction from MS3878 rms7 (Inoue et al., 1970; Mitsuhashi et al., 1973). Table 1 lists resistances of these strains and plasmids . Media. Nutrient broth and nutrient agar (Difco) were used for routine cultivation. Selection for drug resistance in transduction experiments was made on heart infusion (HI) agar. HI agar was also used for the determination of drug resistance. Chemicals. Tetracycline (TC) hydrochloride was donated by the Taito-Pfizer Co. Other chemicals were from commercial sources. Transduction experiments. Transduction was performed by the method described previously (Inoue et al., 1970). An overnight culture of the recipient bacteria grown on HI agar was collected and suspended in nutrient broth at 30”. After 30 min of incubation, 10e3M CaCl, was added to the culture. The recipient bacteria were mixed with phage lysates at multiplicities of infection of lo+ to 10’ and the mixture was incubated for 45 min at 30”. The mixed culture was sedimented by centrifugation and the precipitated bacteria were washed
1 Author to whom reprint requests should be addressed. 322 Copyright 6 19’76by Academic Press, Inc. All rights of reproduction in any form reserved.
ACTIVE
TRANSDUCING
PHAGE
TABLE
323
Slptel
1
BACTERIAL STRAINS AND PLASMIDS” Strain MS3878
MS353 MS15001
MS15002
phenotype
Phage and plasmid
Resistant to TC and lysogenized with Sl and S2 Drug sensitive TC-resistant transductant of MS353
Sl and S2 phage rms7
Sl
rms7 and Sl
Relevant
lysogen MS15001
of
Source Clinical
Clinical isolate Obtained by transduction of TC resistance from MS3878 to MS353 with temperate phage Obtained by lysogenization of MS15001 with Sl
rms7
n Abbreviations: TC, tetracycline; rms7, nonconjugative temperate phage isolated from MS3878.
with nutrient broth. The sedimented cells were resuspended in 1.0 ml of nutrient broth and spread on HI agar plate containing 12.5 pg/ml of TC. Colonies formed after 24 or 48 hr of incubation on selective plates were picked, and their drug resistance and immunity to phage Sl and S2 were examined. In each transduction experiment, two control tests were set up. The phage lysate was plated on nutrient broth to test sterility and the noninfected culture was tested for spontaneous mutation. Transduction frequency was expressed as the number of transductants divided by the plaque-forming units. Phage purification. The Sl phage was purified by the following methods. One milliliter of phage lysate from MS3876 was mixed with 1.0 ml of nutrient broth containing 0.1 ml of antiserum of serotype B phage and the mixture was incubated at 37” without aeration. After 20 min of incubation, 0.1 ml was diluted with nutrient broth, mixed with soft agar containing MS353 and plated on a nutrient agar plate. After 24 hr of incubation at 37”, each plaque was picked with sterilized capillary glass and suspended in nutrient broth. The phage was subjected to three cycles of propagation on MS353 and the serotype of the resulting phage was determined. Lysogenization with Sl phage. MS15001 was spread on HI agar plates and spotted with appropriate dilutions of Sl phage. After 24 hr of incubation at 37”, the colo-
plasmid
isolate
encoding
TC resistance;
Sl and S2,
nies in the center of phage plaques were picked, purified three times on HI agar, and tested for lysogeny by Sl phage. The MS15062 strain lysogenized with phage Sl were used for further experiments. Plaque assay after inactivation. After appropriate times of treatment with either anti-S1 phage serum or heat, a O.l-ml amount of the sample was taken, diluted with nutrient broth, mixed with soft agar containing MS353, and plated on nutrient agar . Plaque center test. When the plaques were directly picked to TC plates, the colonies are too small, and the surrounding of the colonies are lysed by phage. Accordingly, it is difficult to count the relation between phage particles and TC resistance. Therefore, plaque centers were tested by the following method. The center of the plaque was stabbed with a needle and inoculated onto HI agar plate. After 20 hr of incubation at 37”, this was replicaplated on HI agar plate in the presence or absence of TC (12.5 pg/ml). Density-gradient centrifugation. Onetenth-milliliter phage lysate was suspended in 5.0 ml of C&O, (p = 1.365 at 25%0.01 M Tris (hydroxmethyl) aminomethane-O.01 M NaCl (pH 7.6) buffer, and the mixture was centrifuged in the Hitachi RPS65T rotor of a Spinco ultracentrifuge for 20 hr at 55,000 rpm. Fractions were collected from the bottom and assayed for phage and the density of each fraction. Electron microscopy. Thirty milliliters
324
INOUE AND MITSUHASHI TABLE 2
PLAQUE-FORMING TITERS AND TRANSDUCTION FREQUENCIES OF PHAGE LYSATES PREPARED FROM TC-RESISTANT TRANSDUCTANTP
Strain
PlaqueMulti litforming ti- it 0 inter (per ml) YP ection
MS353 TC-12 MS353 TC-13 MS353 TC-22 MS353 TC-23 MS353 TC-32 MS353 TC-33 MS353 TC-42 MS353 TC-43
1.3 2.0 7.0 2.1 1.4 1.6 6.0 1.8
x x x x x x x x
lo9 lo9 lo8 lo9 109 lo9 lOa lo9
3.0 0.6 3.0 0.4 0.03 0.5 0.05 0.5
Transduction frequency per input phage 3.5 1.0 1.3 5.0 3.1 1.0 7.8 9.5
x x x x x x x x
10-a 10-l 10-Z 10-Z 10-I 10-Z 10-Z 10-Z
a Fifty clones of TC-resistant transductants were obtained at a multiplicity of infection of lo-’ in the experiments shown in Fig. 1. Each clone of the transductanta developed on HI agar plates containing TC was inoculated in nutrient broth for 18 hr at 37” and each 1 ml of the cultures was inoculated in 10 ml of fresh BHI broth. After 3 hr of incubation at 37”, the cultures were treated with mitomycin C as described in Methods. The supernatant fluid was used as the source of phage after filtration through a HA (0.45) membrane filter. MA353 was used as the recipient cell. of phage lysate (5 x log/ml) obtained in liquid culture was centrifuged for 20 min at 10,000 rpm in the cold to remove bacterial debris. The supernatant thus obtained was filtered through a millipore filter and suspended in 0.01 A4 Tris-0.01 M NaCl (pH 7.6) buffer. The phage was further purified by centrifugation as described above and the visible band of phage was collected from the bottom. The purified phage sample was diluted with deionized water immediately before electron microscopic observation and applied to a carbon-coated grid. The grid was stained with phosphotungstic acid. RESULTS
Transduction rms7 plasmid.
of MS15002 with mitomycin C. The transduction frequency of tet in this lysate was about 4 x lo-lo per phage, and when transductants were used as donors to MS353,
of TC resistance
FIG. 1. Linear relationship between number of TC-resistant transductants and multiplicity of infection. Phage lysates were prepared by treatment of (a) MS353 TC-11, (b) MS353 TC-21, (c) MS353 TC31, and (d) MS353 TC-41 with mitomycin C. MS353 was used as the recipient cell.
109
108
“? 107 % IO 5
z
lo6
2 $
105
104
103 5
of the
In a phage lysate obtained by treatment of MS3878 rms7(Sl)(S2) with mitomycin C, the transduction frequency for tet ranged from 1 x 10e6to 4 x lo-+ per phage and all transductants were nonlysogenic. Four tet transductants obtained by the above experiments were lysogenized with purified Sl phage. Following phage lysates were made by treatment
10 15 20 25 min
5
10 15 20 25 30 min
FIG. 2. Inactivation of Sl and Slptet phages. One-tenth milliliter of anti-S1 phage serum was added to 6 ml of nutrient broth containing phage, and mixture was incubated at 37”. When the phage lysate was heated at 63”, one milliliter of the phage 1ysat.e was incubated. After appropriate time intervals, the mixture was diluted with nutrient broth and the number of plaques was assayed. (A) Treatment with antiserum; and (B) treatment with heat at 63”, Sl (-0-j and Slptet (-0-4.
ACTIVE
TRANSDUCING
the frequency of tet transduction was increased enormously; the tet transduction frequency now ranged from 1.0 x 10m2to 9.0 x lo+’ per phage. All transductants possessed the same level of TC resistance and could produce plaques on a lawn of MS353, indicating a difference from transductants resulting from Hft tranduction by hdg (Campbell, 1957). Relation between transduction frequency and multiplicities of phage infection (m.0.i.). Four TC-resistant transduc-
tants, MS353-11, -21, -31, and -41, were randomly chosen from the experiments shown in Table 2, and the relation between the number of TC-resistant transductants and m.o.i. was examined. As shown in Fig. 1, there was a linear relationship over a wide range of m.o.i. from 10e7to 1. From eight tet transductants obtained at a m.o.i. 7 x 10m7’,phage lysates were prepared by treatment with mitomytin C. As shown in Table 2, all TC-resistante transductants liberated active plaque-forming phages; their induced lysate had similar titer to that of the lysate as shown in Fig. 1. Thus, the ability to produce Hft lysates is passed on to the tet transductants. From these results, it is concluded that OF Slptet
Phage source” MS353 TC-11
MS353 TC-21
MS353 TC-31
MS353 TC-41
Sl”
BY TREATMENT
TABLE
Antiserum 63” r Antiserum 63” Antiserum 63” Antiserum 63” Antiserum 63”
with
Ab
A
A
A
A
4
LYSOGENIC CONVERSION OF TETRACYCLINE RESISTANCE WITH Slptet PHAGE
Strain”
MS353 MS353 MS353 MS353
Number of pla ues containing % C-resistant cells in the center of plaqueb (%)
Number of plaqueforming units
TC-11 TC-21 TC-31 TC-41
MS15001’
15 18 36 32
15 18 36 32
300
0
(100) (100) (100) (100)
o The strains were obtained in the experiments shown in Fig. 1. ’ A sample obtained from the center of each plaque from the strains n with needle was inoculated on HI agar plate. After 24 hr of incubation at 37”, the plates were replica-plated on HI agar plates containing TC (12.5 pg/ml). After 24 hr of incubation at 37”, number of colonies developed on TC plate was enumerated. The number in parentheses indicates the percentage of the number of plaque-forming units per that of plaques containing TC-resistant cells in the plaque center. ’ MS15001, see Table 1. Phage lysate was obtained by propagation of phage Sl on this strain.
WITH ANTI-Sl
Treatment
325
Slptet
the TC resistant character is transduced by a single particle which possesses a plaque-forming activity in addition to a
TABLE INACTIVATION
PHAGE
3 PHAGE
SERUM
AND WITH HIGH
Plaque-fF;r$ng 3.5 1.9 3.3 4.4 1.2 2.3 1.5 2.2 2.1 1.3 2.7 8.6 8.7 7.1 2.4
x x x x x x x x x x x x x x x
103 105 102 109 105 lo3 109 105 103 109 lo5 103 10y 105 lo3
unit
TEMPERATURE
Number of transductants (ml) 3.7 2.4 1.7 4.1 2.1 1.2 3.7 7.2 4.2 1.5 6.4 8.4 3.0
x x x x x x x x x x x x x 0 0
108 IO4 102 10” 104 103 108 lo4 10’ 10’ lo4 10’ 10”
a MS353 TC-11, -21, -31, and -41 were obtained, respectively, by transduction of TC resistance in independent experiments, and they were lysogenized with phage Sl. b One-tenth milliliter of anti-S1 phage serum was added to 6 ml of nutrient broth containing phage, and the mixture was incubated at 37” for 20 min. ’ Incubated at 63” for 30 min. See Methods for details. d Phage lysate was obtained by propagation of phage Sl on MS15001.
326
INOUE
AND
Density ( g.cmm31 1.3725
1.3652
1.3550
MITSUHASHI Density ( g.cmm31 1.3725 1.3650
1.3550
\ (a)
Fraction Number
Fraction Number
FIG. 3. Preparative density-gradient centrifugation in Cs$O, of Slptet or Sl phage. Solid circles indicate the number of plaque-forming particles and open circles indicate the number of plaque-forming particles and open circles indicate the number of TC-resistant transductants. See the footnote of Table 4 for details. (a) Sl phage; and (bl Slptet phage.
transducing activity. This phage particle is designated “Slptet.” Properties of Slptet. Slptet particles have a number of properties that are also characteristic of phage Sl. (1) Anti-S1 phage serum neutralize both Sl and Sl ptet with a rate constant of lop4 per 20 min. (2) Heat inactivation of Sl and Slptet at 63” for 30 min gives lo+ surviving phage. Slptet lost both transducing and plaqueforming ability upon this treatment (Fig. 2; Table 3). (3) When the transduction with Slptet was carried out in BHI broth without the addition of CaCl,, the transduction frequency was decreased about l/50 that obtained by the addition of CaCl,. (4) The host range of phage Slptet (6/423/ 54/52/80/81/84) is identical to that of phage Sl (6/42E/(54Y52/80/81/84). Plaque center test of Slptet. As shown in Table 4, all TC resistant transductants were lysogenic. Therefore, an investigation was directed toward determining
whether all lysogenized clones were TCresistant. Each phage induced with mitomycin C from MS353 TC-11, -21, -31, and -41 was used to infect S. aureus MS353 at a m.o.i. of 0.01. Among 101 lysogenic clones tested, all were TC-resistant, and of 300 TC-resistant clones tested, all were lysogenie for Sl phages. Next, we examined that a single particle can form a plaque as well as transduce tet. A majority of the plaques contained TCresistant cells at their centers (Table 4). The test gave a similar result when S. aureus MS9403 was used as the recipient. Density-gradient centrifugation of Sl phages. It can be seen that the transducing Slptet phage and the original Sl phage in the lysate are almost the same in buoyant density analysis (p = 1.365). When Sl ptet was subjected to preparative density-gradient centrifugation (Fig. 3), the peak of transducing and plaqueforming activities appeared at the same
ACTIVE
FIG. 4. Electron
microscopy
TRANSDUCING
of the Sl@t
PHAGE
phage. Bar indicates
Slptet
327
0.1 pm. (a) Sl phage; (b) Slptet phage
328
INOUE
AND
position. This confirms the finding that in an Slptet lysate, transducing and plaqueforming ability reside in the same particle. Electron micrographs of Slptet phuge. Figures 4A and B illustrate the shape of phage Slptet and Sl, respectively. The diameter of the hexagonal head of these phages is about 0.39 pm and the length of the tail is about 3.35 pm. DISCUSSION
Converting phage Plcml or Plcry carries a gene for chloramphenicol (cml) resistance (Kondo and Mitsuhashi, 1964; Scott, 1972, 1973) and phage 34kan carries a gene for kanamycin @an) resistance (Coetzee, 1975). Both are also plaque-forming phage and are capable of general transduction. Jessop (1972) also described a nondefective, plaque-forming, specialized transducing phage P22 that was capable of high-frequency transduction of proline genes and general transduction of other markers. The cryptic high frequency transducing phage Pllde was isolated from S. aureeus and analyzed by Novick (1967). From a study of its origin and properties, Novick deduced that Pllde was apparently composed of a segment of the Pll genome stably linked to a segment of a y-plasmid. However, the production of Pllde transducing particles is found to require the activity of helper Pll phage, while the transduction process itself does not. Strains harboring Pllde are somewhat unstable, segregating antibiotic-sensitives at rates in the neighborhood 1% per cell generation. Transduction efficiency of the TC-resistante character with Slptet was not affected by varying the m.o.i. or by the addition of normal Sl phage. The results shown in Fig. 1 may be interpreted to mean that plaque-forming particles have transducing ability, because TC-resistance transductants obtained at a multiplicity of infection of 7 x lop7 liberate active plaqueforming phages with transduction frequencies similar to those of the lysate used in the initial infection. Moreover, plaques, which develop from single infection, contain TC-resistant cells in their center.
MITSUHASHI
Thus, it can be concluded that the transduction of tet character by Slptet phage does not require a helper phage, and that all of the essential phage functions are still retained by the Slptet phage particles. In Plcml, the association of cml gene(s) with Pl genome appears to be less firm (Kondo and Mitsuhashi, 1964). However, the tet gene in a Slptet phage is stably incorporated in the Sl genome since loss of the tet gene in Slptet upon treatment with various conditions was not observed. Accordingly, the acquisition of TC resistance by the recipient cell is always accompanied by the lysogenization of Slptet, and neither the establishment of TC resistance alone without lysogenization nor the formation of Sl-defective TC-transducing particle was observed. Therefore, the TC resistance-conferring ability of Slptet is entirely controlled by the phage itself, and the Sl element in Slptet represents a nonconjugative genome of Sl. The Sl element in Slptet represents a nondefective Sl genome. It possesses at least a genetic region which is responsible for the production of Slptet-transducing phage in stabilizing lysogeny and in synthesizing tail-protein of Sl, as well as in providing all information for replicating itself. The buoyant density of Slptet was not different from that of phage Sl under these conditions. ACKNOWLEDGMENT We should like to thank Mr. Takeshi Saito for his excellent technical assistance with electron microscopy. REFERENCES A. (1957). Transduction and segregation in Escherichia coli K12. Virology 4, 366-384. COETZEE, J. N. (1974). High frequency transduction of kanamycin resistance in Proteus mirabilis. J. Gen. Microbial. 84, 285-296. COETZEE, J. N. (1974). High frequency transduction of resistance to ampicillin and kanamycin in PFOteus mirabilis. J. Gen. Microbial. 87, 173-176. INOUE, M., HASHIMOTO, H., YAMAGISHI, S., and MITSUHASHI, S. (1970). Transduction analysis of the genetic determinants for chloramphenicol resistance in staphylococci. Japan J. Microbial. 14, 261-268. INOUE, M., OKUBO, T., SAITO, T., and MITSUHASHI, CAMPBELL,
ACTIVE
TRANSDUCING
S. (1973). The properties of staphylococcal temperate phages isolated from various strains of Stuphylococcus aureus. Japan. J. Bacterial. 28, 459. JESSOP, A. P. (1972). A specialized transducing phage P22 for which the ability to form plaques is associated with transduction of the proAB region. Molec. Gen. Genet. 114, 214-222. KAMEDA, M., HARADA, K., SUZUKI, M., and MITSUof enterobacHASHI, S. (1965). Drug resistance teria. V. High frequency of transduction of R factors with bacteriophage epsilon. J. Bacterial. 90, 1174-1181. KONDO, E., and MITSUHASHI, S. (1964). Drug resistance of enterobacteria. IV. Active transducing bacteriophage PlCM produced by the recombination of R factor with bacteriophage Pl. J. Bacteriol. 88, 1266-1276. MITSUHASHI, S., INOUE, M., KAWABE, H., OSHIMA,
PHAGE
Slptet
329
H., and OKUBO, T. (1973). In “Stanphylococci and Staphylococcal Infections.” (J. Jeljasaewicz and H. Hrynieewicz, eds.), Vol. 1, p. 144-165, S. Karger, Basel. NOVICK, R. (1967). Properties of a cryptic high frequency transducing phage in Staphylococcus aureus. Virology 33, 155-166. SCOTT, J. R. (1970). A defective Pl prophage with a chromosomal location. Virology 44, 144-151. SCOTT, J. R. (1973). Prophage Pl cryptic. II. Location and regulation of prophage genes. Virology 53, 327-336. SMITH-KEARY, P. F. (1966). Restricted transduction by bacteriophage P22 in Salmonella typhimurium. Genet. Res. 8, 73-82. SMITH, H. W. (1972). Ampicillin resistance in Escherichia coli by phage infection. Nature (London, 238, 205-206.
