JOURNAL OF VIROLOGY, JUlY 1975, p. 179-183 Copyright O 1975 American Society for Microbiology
Vol. 16, No. 1 Printed in U.S.A.
Bacteriophage-Specific Protein Synthesis During Induction of the Defective Bacillus subtilis Bacteriophage PBSX PHYLLIS THURMI AND ANTHONY J. GARRO* Department of Microbiology, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Received for publication 24 January 1975
Particles of PBSX, a defective, noninfectious phage which is inducible from strains of Bacillus subtilis 168, contain at least seven structural proteins resolvable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Five of these proteins are associated with the phage tail and two with the phage head. An eighth protein, which also may be coded for by the PBSX prophage, has been identified in cells derepressed for PBSX replication.
Many procaryotic and eucaryotic species carry information for the production of defective viruses or virus-like particles as part of their genetic potential (3, 4). Analysis of the relationship between these particles and cellular physiological processes has been hampered both by their noninfectious nature and, in most instances, by the lack of genetic information about the producer cells. One set of defective phages which is amenable to genetic analysis is that of Bacillus subtilis. The different strains of this transformable bacterial species, which has been used extensively for genetic studies, are each inducible for one of three morphologically similar noninfectious phages, PBSX, PBSY, or PBSZ. These phage particles all have small heads, long contractile tails, and package DNA which consists entirely of host chromosomal fragments with a molecular weight of 8.35 x 106 (7, 9). The particles, while noninfectious, exhibit a bacteriocin-like killing activity to which the producer cell is resistant. To characterize and map mutations on the B. subtilis 168 chromosome which alter PBSXspecific gene products, it first was necessary to identify the phage's structural proteins as well as other PBSX-specific proteins which may be synthesized during the course of phage induction. For this purpose we analyzed, by polyacrylamide gel electrophoresis, the protein composition of purified PBSX particles and isolated PBSX tails. We also studied the pattern of protein synthesIs in cells derepressed for PBSX rep ^- ,,
'Presui. .. . ogy, Massachus tts Mass. 02139.
MATERIALS AND METHODS Bacterial strains. The bacterial strain used throughout these studies is BR95 pheA, ilvC, trpC, a derivative of B. subtilis 168 and lysogenic for PBSX. Media. All minimal media contained Spizizen salts (1) plus 0.5% glucose and 25 gg of aspartic and glutamic acids per ml. Required amino acids were added to a final concentration of 50 ug/ml. KS is minimal medium supplemented with 0.1% yeast extract (Difco). NaS contains 6.07 g of NaH2PO4 and 11.36 g of Na3HPO4 per liter in place of the respective potassium salts. VY nutrient broth consists of 2.5% veal infusion plus 0.5% yeast extract in distilled water. Tryptose blood agar base plates (Difco) were used for routine maintenance of cultures and for colony counts. Chemicals and radioactive materials. Mitomycin C (MC) was purchased from Hakko Kogyo Co. Ltd. Stock solutions were prepared every 2 weeks and stored in the dark at 4 C. "4C-labeled amino acid mix (0.1 mCi/ml) and [methyl-3H]thymidine (10 to 15 Ci/mmol) were obtained from New England Nuclear (Boston, Mass.). Preparation of PBSX. Throughout the procedures to be described, cells were grown at 37 C with vigorous aeration, and culture densities were monitored with a Klett-Summerson colorimeter equipped with a no. 66 red filter. One Klett unit is approximately equivalent to 1 x 106 colony-forming units/ml. Overnight cultures of B. subtilis in VY were inoculated into fresh VY to a final cell density of 7 Klett units. When the culture reached early exponential phase (23 to 24 Klett units), MC was added to a final concentration of 0.4 Mg/ml. Thirty minutes later the cells were harvested and resuspended in an equal volume of prewarmed KS medium supplemented with 0.4 Mg/ml of MC and 1.5 ,Ci of "4C-labeled amino acid mix per ml. After 120 min of incubation from the time of resuspension the culture had lysed. The lysate was PI ilis Hammer, Department of Biol- incubated for 30 min at 37 C with 1 Ag of both institute of Technology, Cambridge, pancreatic DNase and pancreatic RNase per ml and made 0.5 M with respect to NaCl before removal of 179
180
THURM AND GARRO
cell debris by low-speed centrifugation (10 min. at 7,000 rpm in a Sorvall SS-34 or GSA rotor). PBSX particles were concentrated by polyethylene glycol precipitation and purified in CsCl step gradients as described by Yamamoto et al. (12). The phage band, identified by its density of 1.375 g/cm3, was dialyzed against TMK buffer (10- 2M Tris-hydrochloride, 5 x 10-a M MgCl2, 0.3 M KCl, pH 7.2). For further purification, the phage was rebanded in a CsCl gradient (1.375 g/cm3 average density), centrifuged for 48 h at 35,000 rpm in a Spinco type 50 rotor, and then dialyzed against TMK. SDS polyacrylamide gel electrophoresis. To label proteins for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, cells were grown in NaS to a culture density of 23 Klett units and induced with 0.4 jig of MC per ml. At various times after induction, 0.2-ml aliquots of the culture were pulse labeled for 3 min with 2.5 gCi of "4C-labeled amino acids. The pulse was terminated by the addition of 2 volumes of cold 0.02 M NaN, and immediate centrifugation (15 min, 5,000 rpm in a Sorvall SS34 rotor). The pelleted cells were lysed by a 30-min incubation in 20 1l of a 0.02 M NaN, solution containing 5 mg of lysozyme per ml, followed by the addition of 20 ul of double strength sample buffer (0.05 M Tris-hydrochloride, pH 6.8, 1% SDS, 1% mercaptoethanol, 0.002 M EDTA, 10% glycerol). The samples then were placed in boiling water for 3 min and cooled before being applied to the gel. Aliquots of each sample were analyzed on SDS slab gels as described by Studier (8) using the discontinuous buffer system of Laemmli (5). Kodak no-screen medical X-ray film was used for autoradiography of dried gels. The pattern of grain densities, converted into absorbance, was recorded on a Canalco microdensitometer. Electron microscopy. Samples for electron microscopy were dialyzed against a solution of 1% ammonium acetate-10-2 M MgCl2. A drop of the solution was placed on carbon-coated formovar-covered grids and negatively stained with 2% uranyl acetate. Specimens were examined on a Hitachi or AEI electron microscope.
RESULTS Structural proteins of PBSX. PBSX structural proteins and their kinetics of synthesis during induction were analyzed using SDS gel electrophoresis. Electropherograms of disrupted purified phage revealed seven distinct bands designated Xl through X7 (Fig. 1). The molecular weights of these proteins together with their relative proportions in the purified particles are presented in Table 1. The kinetics of phage protein synthesis were examined using lysates prepared from cells which had been pulse-labeled with a mixture of "4C-labeled amino acids at various times during induction. A comparison of induced and uninduced cultures demonstrated that many of the normal host proteins were not turned off but
J. VIROL.
continued to be synthesized throughout induction, and some, such as the protein designated H in Fig. 2, were synthesized in relatively large quantities. Synthesis of the more readily identifiable PBSX structural proteins such as Xl, X2, and X4 was first detected at 45 min postinduction, and their rates of synthesis appeared to increase progressively during the latent period. It is noteworthy that in lysates prepared from pulse-labeled cells, labeled material corresponding to protein X5 was never XI X2 X3 X4
0
X5 X6 X7
0
.0 a-
0 co .0
a CP
0
a-
-j
-
-
i
-
direction of migration FIG. 1. Densitometer tracing of an autoradiograph of a 13% SDS polyacrylamide gel on which disrupted, purified PBSX particles had been subjected to electrophoresis. The phage proteins were labeled with "4C-labeled amino acids and the phage was purified on CsCI gradients as described.
TABLE 1. Structural proteins of PBSX PBSX protein
Mol Wta
% Total protein
Xi X2 X3 X4 X5 X6 X7
83,000 71,500 47,200 30,600 17,700 13,700 10,800
3 28
in phageb
2 37 10 15 5
aThe molecular weights of proteins X1, X2, X3, and X4 were determined on 10% SDS polyacrylamide gels using unlabeled serum albumin (68,000 mol wt), ovalbumin (43,000 mol wt) and chymotrypsinogen (25,700 mol wt) as a molecular weight standards (Weber and Osborn [11]). "4C-labeled proteins Xl, X2, X3, and X4 were then used as molecular weight standards on 13% gels to estimate the approximate molecular weights of the remaining proteins. b The amount of protein in each band of radioautographs was determined by the area under each peak measured by automatic integrator function of a Canalco microdensitometer.
PROTEINS OF DEFECTIVE BACILLUS PHAGE PBSX
VOL. 16, 1975
synthesized than are incorporated into mature phage particles. Head and tail proteins of PBSX. It was possible to identify the proteins associated with the head and tail structures using preparations of isolated tails. Tails were isolated by fractionating polyethylene glycol concentrated phage on CsCl density gradients prepared at an average density less than that of intact particles (density of 1.330 U rather than 1.375 U). A fraction consisting almost exclusively of phage tails with some contaminating flagella was isolated (Fig. 4). Electropherograms of this fraction revealed
observed. However, another protein designated X8, which was not observed in preparations of purified phage particles, is synthesized in increasing amounts in induced cells starting at approximately 60 min postinduction. Protein X7 was not resolved from other low-molecularweight peptides on the 10% SDS gel used in this experiment, but it can be seen in the tracing of the 13% gel shown in Fig. 3. In addition to the absence of X5 and the appearance of X8, the labeling pattern of phage proteins in a typical total cell lysate shown in Fig. 3 revealed that relatively greater amounts of Xl and X7 are
o0
15' 30'
A A A
181
45' 60' 75' 90' 105'
A A A _%
-1
+
A
-%
mm t.1
-
0
-Xi15
-X2A
-H-X 3-
i0-X2
_
4
a& 40
w-X I
qu
W
i
-H
-X3 -
;:
-X4-
IV *-X 4
EP
..
-X 8-
-X6-
-X8
a
-X6 ....
FIG. 2. Radioautograph of a 10% SDS gel showing the proteins synthesized in a culture induced for PBSX (+) and a noninduced (-) culture. Xl, X2, X3, X4, and X6 are PBSX structural proteins identified by their similar mobility to reference PBSX proteins present on the same gel; X8 is a protein seen only in the induced culture; H is a protein made by induced and noninduced cells. BR95 was grown in NaS medium, and MC was added to 0.4 sg/ml at 0 min. At the times indicated, 0.2-mI samples were removed and pulsed for 3 min with 2.5 pCi of "4C-labeled amino acids. The pulse was terminated by 0.4 ml of cold 0.02 M NaN,, and the cells were concentrated by centrifugation. The pellets were lysed and processed for electrophoresis as described.
182
THURM AND GARRO
J. VIROL.
the presence of proteins Xl, X2, X3, X5, and for PBSX head assembly (10). The flagella X6 (Fig. 5). The two missing structural pro- protein was identified both by its enrichment in teins, X4 and X7, were assumed to be head other fractions consisting primarily of flagella proteins. Further support for this assumption and by its characteristic molecular weight of 4 was derived from analysis of a mutant defective x 104 (6). XI X2 X3 FX4
XI X2 H X3 X4X8 X5 X6 X7
l
/ 81cell total v erlysate ~
purified PBSX
FIG. 3. Comparison of the proteins found in purified PBSX and those synthesized at 75 min postinduction in BR95 cells. The lysate was prepared as described in the legend to Fig. 2 and subjected to electrophoresis on a 13% SDS-polyacrylamide gel.
X5 X6 X7
PBSX tails
purified PBSX FIG. 5. Electrophoretic pattern of the labeled proteins in the gradient fraction shown in Fig. 4 (13% gels). The samples were prepared by adding an equal volume of double-strength sample buffer to samples previously dialyzed against TMK and then heating the mixture at 100 C for 3 min.
;e "
.1.
--wipp-,
X.
1. t.
X
.1 11
FIG. 4. Electron micrograph of a CsCI density gradient fraction containing PBSX tails. Polyethylene glycol-concentrated PBSX which had been labeled with "4C-laoeled amino acids as described was centrifuged in a Spinco SW27 rotor for 48 h at 35,000 rpm in a CsCI gradient with an average density of 1.330 g/cm3. The gradient was collected dropwise and pooled into seven fractions on the basis of the 14C profile of the gradient and dialyzed against 1% amonium acetate-10-2 M MgCI2. The scale represents 4.5 pm.
PROTEINS OF DEFECTIVE BACILLUS PHAGE PBSX
VOL. 16, 1975
DISCUSSION
PBSX particles contain a minimum of seven structural proteins which can be resolved on SDS polyacrylamide gels. Five of these, Xl, X2, X3, X5, and X6, comprise the phage tail, whereas proteins X4 and X7 are associated with the head structure. An eighth protein which is synthesized in MC-treated cells has also been identified. In the absence of the appropriate mutation it is impossible to unequivocally identify X8 as being a phage-coded protein; however, X8 may be either a nonstructural protein or a precursor for a PBSX structural protein. The fact that X5 could not be detected in pulse-labeled lysates suggested that this structural protein might share a product-precursor relationship with another protein such as X8. Attempts to resolve this question by a pulsechase experiment, however, were inconclusive since the label in two proteins, Xl and X8, appeared to be turning over during the chase (data not shown). There are at least two effects of MC treatment of B. subtilis which have been associated with the induction of PBSX and which may be mediated by PBSX-coded enzymes. These are the fragmentation of the host chromosome (7) and a recently described modification system which increases the plating efficiency of phage SP02 on the restricting strains of B. subtilis 5GR (2). However, with the possible exception of X8, we failed to detect the synthesis of any nonstructural PBSX proteins in induced cells. It probably will be very difficult to identify such proteins, especially if they are made only transiently during the early stages of induction, since they may be masked by the many hostspecific proteins which continue to be synthesized in MC-treated cells.
183
ACKNOWLEDGMENTS We thank V. Honzova for her expert technical assistance and A. Erickson and J. Schwartz for their help with the electron microscopy. This work was supported by research grant no. VC-38 from the American Cancer Society. LITERATURE CITED 1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. 2. Arwert, F., and L. Rutberg. 1974. Restriction and modification in Bacillus subtilis. Induction of a modifying activity in Bacillus subtilis 168. Mol. Gen. Genet. 133: 175-177. 3. Garro, A. J., and J. Marmur. 1970. Defective bacteriophages. J. Cell. Physiol. 76:253-263. 4. Huebner, R. J., and G. J. Todaro. 1969. Oncogenesis of RNA tumor viruses as determinants of cancer. Proc. Natl. Acad. Sci. U.S.A. 64:1087-1094. 5. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 6. Martinez, R. J., D. M. Bown, and A. N. Glaser. 1967. The formation of bacterial flagella. III. Characterization of the subunits of the flagella of Bacillus subtilis and Spirillum serpens. J. Mol. Biol. 28:45-51. 7. Okamoto, K., J. A. Mudd, J. Mangan, W. M. Huang, T. V. Subbaiah, and J. Marmur. 1968. Properties of the defective phage of Bacillus subtilis. J. Mol. Biol. 34:413-428. 8. Studier, F. W. 1973. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79:237-248. 9. Subbaiah, T. V., C. D. Goldthwaite, and J. Marmur. 1965. Nature of bacteriophages induced in Bacillus subtilis, p. 435-446. In V. Bryson and H. J. Vogel (ed.), Evolving genes and proteins. Academic Press Inc., New York. 10. Thurm, P., and A. J. Garro. 1974. Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX. J. Virol. 16:184-191. 11. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl-sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412. 12. Yamamoto, K. R., B. M. Alberts, R. Benzinger, L. Lawhorne, and G. Treiber. 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large scale virus purification. Virology 40:734-744.
Vol. 16, No. 1 Printed in U.S.A.
Bacteriophage-Specific Protein Synthesis During Induction of the Defective Bacillus subtilis Bacteriophage PBSX PHYLLIS THURMI AND ANTHONY J. GARRO* Department of Microbiology, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 Received for publication 24 January 1975
Particles of PBSX, a defective, noninfectious phage which is inducible from strains of Bacillus subtilis 168, contain at least seven structural proteins resolvable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Five of these proteins are associated with the phage tail and two with the phage head. An eighth protein, which also may be coded for by the PBSX prophage, has been identified in cells derepressed for PBSX replication.
Many procaryotic and eucaryotic species carry information for the production of defective viruses or virus-like particles as part of their genetic potential (3, 4). Analysis of the relationship between these particles and cellular physiological processes has been hampered both by their noninfectious nature and, in most instances, by the lack of genetic information about the producer cells. One set of defective phages which is amenable to genetic analysis is that of Bacillus subtilis. The different strains of this transformable bacterial species, which has been used extensively for genetic studies, are each inducible for one of three morphologically similar noninfectious phages, PBSX, PBSY, or PBSZ. These phage particles all have small heads, long contractile tails, and package DNA which consists entirely of host chromosomal fragments with a molecular weight of 8.35 x 106 (7, 9). The particles, while noninfectious, exhibit a bacteriocin-like killing activity to which the producer cell is resistant. To characterize and map mutations on the B. subtilis 168 chromosome which alter PBSXspecific gene products, it first was necessary to identify the phage's structural proteins as well as other PBSX-specific proteins which may be synthesized during the course of phage induction. For this purpose we analyzed, by polyacrylamide gel electrophoresis, the protein composition of purified PBSX particles and isolated PBSX tails. We also studied the pattern of protein synthesIs in cells derepressed for PBSX rep ^- ,,
'Presui. .. . ogy, Massachus tts Mass. 02139.
MATERIALS AND METHODS Bacterial strains. The bacterial strain used throughout these studies is BR95 pheA, ilvC, trpC, a derivative of B. subtilis 168 and lysogenic for PBSX. Media. All minimal media contained Spizizen salts (1) plus 0.5% glucose and 25 gg of aspartic and glutamic acids per ml. Required amino acids were added to a final concentration of 50 ug/ml. KS is minimal medium supplemented with 0.1% yeast extract (Difco). NaS contains 6.07 g of NaH2PO4 and 11.36 g of Na3HPO4 per liter in place of the respective potassium salts. VY nutrient broth consists of 2.5% veal infusion plus 0.5% yeast extract in distilled water. Tryptose blood agar base plates (Difco) were used for routine maintenance of cultures and for colony counts. Chemicals and radioactive materials. Mitomycin C (MC) was purchased from Hakko Kogyo Co. Ltd. Stock solutions were prepared every 2 weeks and stored in the dark at 4 C. "4C-labeled amino acid mix (0.1 mCi/ml) and [methyl-3H]thymidine (10 to 15 Ci/mmol) were obtained from New England Nuclear (Boston, Mass.). Preparation of PBSX. Throughout the procedures to be described, cells were grown at 37 C with vigorous aeration, and culture densities were monitored with a Klett-Summerson colorimeter equipped with a no. 66 red filter. One Klett unit is approximately equivalent to 1 x 106 colony-forming units/ml. Overnight cultures of B. subtilis in VY were inoculated into fresh VY to a final cell density of 7 Klett units. When the culture reached early exponential phase (23 to 24 Klett units), MC was added to a final concentration of 0.4 Mg/ml. Thirty minutes later the cells were harvested and resuspended in an equal volume of prewarmed KS medium supplemented with 0.4 Mg/ml of MC and 1.5 ,Ci of "4C-labeled amino acid mix per ml. After 120 min of incubation from the time of resuspension the culture had lysed. The lysate was PI ilis Hammer, Department of Biol- incubated for 30 min at 37 C with 1 Ag of both institute of Technology, Cambridge, pancreatic DNase and pancreatic RNase per ml and made 0.5 M with respect to NaCl before removal of 179
180
THURM AND GARRO
cell debris by low-speed centrifugation (10 min. at 7,000 rpm in a Sorvall SS-34 or GSA rotor). PBSX particles were concentrated by polyethylene glycol precipitation and purified in CsCl step gradients as described by Yamamoto et al. (12). The phage band, identified by its density of 1.375 g/cm3, was dialyzed against TMK buffer (10- 2M Tris-hydrochloride, 5 x 10-a M MgCl2, 0.3 M KCl, pH 7.2). For further purification, the phage was rebanded in a CsCl gradient (1.375 g/cm3 average density), centrifuged for 48 h at 35,000 rpm in a Spinco type 50 rotor, and then dialyzed against TMK. SDS polyacrylamide gel electrophoresis. To label proteins for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, cells were grown in NaS to a culture density of 23 Klett units and induced with 0.4 jig of MC per ml. At various times after induction, 0.2-ml aliquots of the culture were pulse labeled for 3 min with 2.5 gCi of "4C-labeled amino acids. The pulse was terminated by the addition of 2 volumes of cold 0.02 M NaN, and immediate centrifugation (15 min, 5,000 rpm in a Sorvall SS34 rotor). The pelleted cells were lysed by a 30-min incubation in 20 1l of a 0.02 M NaN, solution containing 5 mg of lysozyme per ml, followed by the addition of 20 ul of double strength sample buffer (0.05 M Tris-hydrochloride, pH 6.8, 1% SDS, 1% mercaptoethanol, 0.002 M EDTA, 10% glycerol). The samples then were placed in boiling water for 3 min and cooled before being applied to the gel. Aliquots of each sample were analyzed on SDS slab gels as described by Studier (8) using the discontinuous buffer system of Laemmli (5). Kodak no-screen medical X-ray film was used for autoradiography of dried gels. The pattern of grain densities, converted into absorbance, was recorded on a Canalco microdensitometer. Electron microscopy. Samples for electron microscopy were dialyzed against a solution of 1% ammonium acetate-10-2 M MgCl2. A drop of the solution was placed on carbon-coated formovar-covered grids and negatively stained with 2% uranyl acetate. Specimens were examined on a Hitachi or AEI electron microscope.
RESULTS Structural proteins of PBSX. PBSX structural proteins and their kinetics of synthesis during induction were analyzed using SDS gel electrophoresis. Electropherograms of disrupted purified phage revealed seven distinct bands designated Xl through X7 (Fig. 1). The molecular weights of these proteins together with their relative proportions in the purified particles are presented in Table 1. The kinetics of phage protein synthesis were examined using lysates prepared from cells which had been pulse-labeled with a mixture of "4C-labeled amino acids at various times during induction. A comparison of induced and uninduced cultures demonstrated that many of the normal host proteins were not turned off but
J. VIROL.
continued to be synthesized throughout induction, and some, such as the protein designated H in Fig. 2, were synthesized in relatively large quantities. Synthesis of the more readily identifiable PBSX structural proteins such as Xl, X2, and X4 was first detected at 45 min postinduction, and their rates of synthesis appeared to increase progressively during the latent period. It is noteworthy that in lysates prepared from pulse-labeled cells, labeled material corresponding to protein X5 was never XI X2 X3 X4
0
X5 X6 X7
0
.0 a-
0 co .0
a CP
0
a-
-j
-
-
i
-
direction of migration FIG. 1. Densitometer tracing of an autoradiograph of a 13% SDS polyacrylamide gel on which disrupted, purified PBSX particles had been subjected to electrophoresis. The phage proteins were labeled with "4C-labeled amino acids and the phage was purified on CsCI gradients as described.
TABLE 1. Structural proteins of PBSX PBSX protein
Mol Wta
% Total protein
Xi X2 X3 X4 X5 X6 X7
83,000 71,500 47,200 30,600 17,700 13,700 10,800
3 28
in phageb
2 37 10 15 5
aThe molecular weights of proteins X1, X2, X3, and X4 were determined on 10% SDS polyacrylamide gels using unlabeled serum albumin (68,000 mol wt), ovalbumin (43,000 mol wt) and chymotrypsinogen (25,700 mol wt) as a molecular weight standards (Weber and Osborn [11]). "4C-labeled proteins Xl, X2, X3, and X4 were then used as molecular weight standards on 13% gels to estimate the approximate molecular weights of the remaining proteins. b The amount of protein in each band of radioautographs was determined by the area under each peak measured by automatic integrator function of a Canalco microdensitometer.
PROTEINS OF DEFECTIVE BACILLUS PHAGE PBSX
VOL. 16, 1975
synthesized than are incorporated into mature phage particles. Head and tail proteins of PBSX. It was possible to identify the proteins associated with the head and tail structures using preparations of isolated tails. Tails were isolated by fractionating polyethylene glycol concentrated phage on CsCl density gradients prepared at an average density less than that of intact particles (density of 1.330 U rather than 1.375 U). A fraction consisting almost exclusively of phage tails with some contaminating flagella was isolated (Fig. 4). Electropherograms of this fraction revealed
observed. However, another protein designated X8, which was not observed in preparations of purified phage particles, is synthesized in increasing amounts in induced cells starting at approximately 60 min postinduction. Protein X7 was not resolved from other low-molecularweight peptides on the 10% SDS gel used in this experiment, but it can be seen in the tracing of the 13% gel shown in Fig. 3. In addition to the absence of X5 and the appearance of X8, the labeling pattern of phage proteins in a typical total cell lysate shown in Fig. 3 revealed that relatively greater amounts of Xl and X7 are
o0
15' 30'
A A A
181
45' 60' 75' 90' 105'
A A A _%
-1
+
A
-%
mm t.1
-
0
-Xi15
-X2A
-H-X 3-
i0-X2
_
4
a& 40
w-X I
qu
W
i
-H
-X3 -
;:
-X4-
IV *-X 4
EP
..
-X 8-
-X6-
-X8
a
-X6 ....
FIG. 2. Radioautograph of a 10% SDS gel showing the proteins synthesized in a culture induced for PBSX (+) and a noninduced (-) culture. Xl, X2, X3, X4, and X6 are PBSX structural proteins identified by their similar mobility to reference PBSX proteins present on the same gel; X8 is a protein seen only in the induced culture; H is a protein made by induced and noninduced cells. BR95 was grown in NaS medium, and MC was added to 0.4 sg/ml at 0 min. At the times indicated, 0.2-mI samples were removed and pulsed for 3 min with 2.5 pCi of "4C-labeled amino acids. The pulse was terminated by 0.4 ml of cold 0.02 M NaN,, and the cells were concentrated by centrifugation. The pellets were lysed and processed for electrophoresis as described.
182
THURM AND GARRO
J. VIROL.
the presence of proteins Xl, X2, X3, X5, and for PBSX head assembly (10). The flagella X6 (Fig. 5). The two missing structural pro- protein was identified both by its enrichment in teins, X4 and X7, were assumed to be head other fractions consisting primarily of flagella proteins. Further support for this assumption and by its characteristic molecular weight of 4 was derived from analysis of a mutant defective x 104 (6). XI X2 X3 FX4
XI X2 H X3 X4X8 X5 X6 X7
l
/ 81cell total v erlysate ~
purified PBSX
FIG. 3. Comparison of the proteins found in purified PBSX and those synthesized at 75 min postinduction in BR95 cells. The lysate was prepared as described in the legend to Fig. 2 and subjected to electrophoresis on a 13% SDS-polyacrylamide gel.
X5 X6 X7
PBSX tails
purified PBSX FIG. 5. Electrophoretic pattern of the labeled proteins in the gradient fraction shown in Fig. 4 (13% gels). The samples were prepared by adding an equal volume of double-strength sample buffer to samples previously dialyzed against TMK and then heating the mixture at 100 C for 3 min.
;e "
.1.
--wipp-,
X.
1. t.
X
.1 11
FIG. 4. Electron micrograph of a CsCI density gradient fraction containing PBSX tails. Polyethylene glycol-concentrated PBSX which had been labeled with "4C-laoeled amino acids as described was centrifuged in a Spinco SW27 rotor for 48 h at 35,000 rpm in a CsCI gradient with an average density of 1.330 g/cm3. The gradient was collected dropwise and pooled into seven fractions on the basis of the 14C profile of the gradient and dialyzed against 1% amonium acetate-10-2 M MgCI2. The scale represents 4.5 pm.
PROTEINS OF DEFECTIVE BACILLUS PHAGE PBSX
VOL. 16, 1975
DISCUSSION
PBSX particles contain a minimum of seven structural proteins which can be resolved on SDS polyacrylamide gels. Five of these, Xl, X2, X3, X5, and X6, comprise the phage tail, whereas proteins X4 and X7 are associated with the head structure. An eighth protein which is synthesized in MC-treated cells has also been identified. In the absence of the appropriate mutation it is impossible to unequivocally identify X8 as being a phage-coded protein; however, X8 may be either a nonstructural protein or a precursor for a PBSX structural protein. The fact that X5 could not be detected in pulse-labeled lysates suggested that this structural protein might share a product-precursor relationship with another protein such as X8. Attempts to resolve this question by a pulsechase experiment, however, were inconclusive since the label in two proteins, Xl and X8, appeared to be turning over during the chase (data not shown). There are at least two effects of MC treatment of B. subtilis which have been associated with the induction of PBSX and which may be mediated by PBSX-coded enzymes. These are the fragmentation of the host chromosome (7) and a recently described modification system which increases the plating efficiency of phage SP02 on the restricting strains of B. subtilis 5GR (2). However, with the possible exception of X8, we failed to detect the synthesis of any nonstructural PBSX proteins in induced cells. It probably will be very difficult to identify such proteins, especially if they are made only transiently during the early stages of induction, since they may be masked by the many hostspecific proteins which continue to be synthesized in MC-treated cells.
183
ACKNOWLEDGMENTS We thank V. Honzova for her expert technical assistance and A. Erickson and J. Schwartz for their help with the electron microscopy. This work was supported by research grant no. VC-38 from the American Cancer Society. LITERATURE CITED 1. Anagnostopoulos, C., and J. Spizizen. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81:741-746. 2. Arwert, F., and L. Rutberg. 1974. Restriction and modification in Bacillus subtilis. Induction of a modifying activity in Bacillus subtilis 168. Mol. Gen. Genet. 133: 175-177. 3. Garro, A. J., and J. Marmur. 1970. Defective bacteriophages. J. Cell. Physiol. 76:253-263. 4. Huebner, R. J., and G. J. Todaro. 1969. Oncogenesis of RNA tumor viruses as determinants of cancer. Proc. Natl. Acad. Sci. U.S.A. 64:1087-1094. 5. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 6. Martinez, R. J., D. M. Bown, and A. N. Glaser. 1967. The formation of bacterial flagella. III. Characterization of the subunits of the flagella of Bacillus subtilis and Spirillum serpens. J. Mol. Biol. 28:45-51. 7. Okamoto, K., J. A. Mudd, J. Mangan, W. M. Huang, T. V. Subbaiah, and J. Marmur. 1968. Properties of the defective phage of Bacillus subtilis. J. Mol. Biol. 34:413-428. 8. Studier, F. W. 1973. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79:237-248. 9. Subbaiah, T. V., C. D. Goldthwaite, and J. Marmur. 1965. Nature of bacteriophages induced in Bacillus subtilis, p. 435-446. In V. Bryson and H. J. Vogel (ed.), Evolving genes and proteins. Academic Press Inc., New York. 10. Thurm, P., and A. J. Garro. 1974. Isolation and characterization of prophage mutants of the defective Bacillus subtilis bacteriophage PBSX. J. Virol. 16:184-191. 11. Weber, K., and M. Osborn. 1969. The reliability of molecular weight determinations by dodecyl-sulfate polyacrylamide gel electrophoresis. J. Biol. Chem. 244:4406-4412. 12. Yamamoto, K. R., B. M. Alberts, R. Benzinger, L. Lawhorne, and G. Treiber. 1970. Rapid bacteriophage sedimentation in the presence of polyethylene glycol and its application to large scale virus purification. Virology 40:734-744.
