JOURNAL OF VIROLOGY, JUlY 1975, p. 184-191 Copyright 0 1975 American Society for Microbiology
Vol. 16, No. 1 Printed in U.S.A.
Isolation and Characterization of Prophage Mutants of the Defective Bacillus subtilis Bacteriophage PBSX PHYLLIS THURM1 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
Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since /105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome.
Three separate sites on the Bacillus subtilis 168 chromosome have been implicated as possible loci for genes coding for the defective phage PBSX (Fig. 1). A site near the origin of chromosomal replication has been suggested on the basis of the multiforked replication of this region which ensues after exposure of cells to inducing agents such as mitomycin C (MC) (7). A second site in the region of the purB marker was suggested by the isolation of a mutant which exhibits a temperature-inducible phenotype for PBSX replication (17). The third site which maps near metC was identified by the xtl-1 mutation which interferes with PBSX tail assembly (6). If, as suggested by these results, the PBSX, PBSY, and PBSZ prophage genes are scattered across their respective cell chromosomes, their defective nature could result from an inability to excise and replicate an intact phage genome during induction. To examine this question more closely we sought to isolate and genetically characterize a number of mutants with impaired PBSX gene functions. Three such mutants are described here; one is defective for
MC-induced PBSX derepression and the other two for PBSX capsid formation.
I Present address: Phyllis Hammer, Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139.
184
MATERIALS AND METHODS Bacterial and phage strains. The bacterial strains used are listed in Table 1. All strains except W23 are B. subtilis 168 derivatives and lysogenic for PBSX. W23 is a PBSZ lysogen and sensitive to PBSX killing. The conditions used for growing bacterial cultures and for monitoring cell densities are given in the accompanying paper (18). Phage 4105 and the clear plaque mutant 0105c4 were obtained from L. Rutberg; SP02 and SPO2c were from J. Marmur. Media and chemicals. The compositions of minimal, NaS, tryptose blood agar base (TBAB), and VY media have been described (18). In CH-min the basic minimal media is supplemented with 0.05% Casamino Acids and 10-5 M MnCl2; in KS, it is supplemented with 0.1% yeast extract (Difco). Selective plates consisted of minimal media plus 50 Ag of required amino acids and 100 ug of purines and pyrimidines per ml and were solidified with 2% agar (Difco). The overlay plates used to assay PBSX-mediated killing of W23 consisted of 2.5 ml of top agar described by Okubo and Romig (13) seeded with a streptomycin-resistant strain of W23 layered on CH-min plates. Both layers contained 3 mg of streptomycin sulfate per ml. Overlay plates of tryptone broth plus 10-2 M MgSO4 were used for 0105 and SP02 plaque assays. Nitrosoguanidine (N-methyl-N'-nitro-N-
GENETICS OF DEFECTIVE BACILLUS PHAGE PBSX
VOL. 16, 1975
185
The rabbits received four toe pad injections once a week for 4 weeks and were bled 1 week after the final inoculations. Sheep antiserum to rabbit gamma globulin was generously donated by B. F. Erlanger. Mutagenesis. BR95 cells were mutagenized by a modification of a procedure described by Adelberg et al. (1). Cells grown in VY to a density of 100 Klett units were resuspended in an equal volume of Spizizen salts adjusted to pH 6.0 and incubated for 10 min at 37 C with 100 Mg of nitrosoguanidine per ml. After thorough washing, the culture was grown in VY to stationary phase to allow cell division and segregation of mutant genotypes before plating on TBAB. Screening of mutants. Single colonies were inocua00 lated into 1 ml of VY and grown overnight to stationary phase. Ten microliters of these cultures was inoculated into 1 ml of VY which contained 250 mg of deoxyadenosine and 8 MCi of [3H Ithymidine per ml to label the bacterial DNA which would be packaged by the phage during induction. The cultures were grown until the cells in each tube reached a visually estimated density of 25 Klett units (approximately 2 to 3 h). MC was added to a final concentration of 0.5 Mg/ml, and incubation was continued for 3 h. Clones potentially defective for PBSX-specific functions were identified by monitoring three phageassociated activities in the MC-induced cultures. Induction defective (Xin-) mutants were detected by their sustained high turbidity at 3 h postinduction.In cultures of MC-treated cells carrying wild-type co. PBSX, turbidity normally increases for 90 min but Q' then falls rapidly as the cells lyse. Mutants with possible tail defects (Xtl -) were identified by the absence of PBSX-mediated killing of B. subtilis W23. FIG. 1. B. subtilis genetic map adapted from J. The screening procedure for head defective (Xhd-) Kejzlarova-Lepesant, N. Harford, J. A. Lepesant, and mutants utilized an assay designed to test the ability R. Dedonder (In P. Gerhardt, H. L. Sadoff, and R. N. of phage heads to protect packaged 3H-labeled DNA Costilow [ed. ], Spores VI, in press). The arrows from degradation by exogenously added nucleases. indicate suggested sites for PBSX prophage genes. Lysates were treated with 100 Mg of lysozyme per ml 1. TABLE Bacterial strains nitrosoguanidine) was purchased from Aldrich Chemicals (Cedar Knolls, N.J.), and spleen phosphodiesterase and micrococcal nuclease were purchased from Worthington Biochemical Corp. (Freehold, N.J.). 14Clabeled amino acid mix (0.1 mCi/ml) and [methyl'H Jthymidine (10 to 15 Ci/mmol) were obtained from New England Nuclear (Boston, Mass.). Antisera. Anti-PBSX antiserum was prepared by immunizing rabbits with CsCl-purified PBSX (500 Ag of phage protein/ml) in complete Freund adjuvant. z
z
Strain
BR95
GB159 GB1323 GB219 44AO BD71 GB26 GB19 GB64 GB75 GB23 GB1001 MU8U5U16 W23
Propertiesa
pheA, ilvC, trpC pheA, ilvC, trpC, xtl-3 pheA, ilvC, trpC, xhd pheA, ilvC, trpC, xin xtl-1 argC, hisA, pyrA trpC, metB, recA-1 xtl-lb, metC, pyrA argC, metC, pyrA xinb, metC, pyrA metC, leuA leuA, metB, tsi-23 purA, leuA, metB Str', PBSX'
Source
L. Rutberg
Mutagenized derivative of BR95 Mutagenized derivative of BR95 Mutagenized derivative of BR95 K. Bott D. Dubnau C. Anagnostopoulos A. Garro A. Garro A. Garro A. Garro E. Siegel N. Sueoka Spontaneous Strr mutant
a xtl, PBSX marker leading to loss of bacteriocidal activity; xin, PBSX marker leading to noninducibility ci PBSX; xhd, PBSX marker leading to formation of defective heads; PBSX, killed by PBSX; Strr, resistant to 5 mg of streptomycin sulfate per ml. °PBSX markers were introduced into strain GB64 by transformation at saturating DNA concentrations selecting argC+ transformants and then assaying for the PBSX mutant phenotype.
186
THURM AND GARRO
for 30 min at 37 C and then adjusted to 0.1 M Tris-hydrochloride-5 x 10- M CaCl2 (pH 7.6) and incubated for 45 min at 37 C with 10 ug of spleen phosphodiesterase and 10 gg of micrococcal nuclease per ml. The percentage of protection was calculated from the ratio of acid-insoluble counts, measured by the method of Li and Felmy (10), before and after nuclease treatment. The Xhd- phenotype was ascribed to lysates which afforded less than 40% protection. This is the average amount of bacterial DNA packaged into PBSX particles. Xtl- cells also exhibited the Xhd- phenotype probably because defective tails allow nuclease to penetrate otherwise normal heads. Immunoprecipitation. Cells were grown in KS and induced with 0.4 Mg of MC/ml when the culture reached a density of 23 Klett units. At 75 min postinduction, a 0.2-ml aliquot was pulsed for 6 to 10 min with 5 MCi of "4C-labeled amino acids. The pulse was terminated by a 30-min incubation in an equal volume of 500 MAg of lysozyme per ml 0.02 M NaN,. The lysate was incubated first with anti-PBSX antiserum for 60 min at 37 C and then with sheep anti-rabbit gamma globulin for 60 min at 37 C. After overnight incubation at 4 C the immunoprecipitates were washed with cold 0.9% saline and dissolved in sample buffer (18). Samples were placed in a boiling water bath for 3 min and cooled before being applied to the sodium dodecyl sulfate (SDS) gels. The procedure used for SDS gel electrophoresis is described in the accompanying paper (18). Only 1 to 3 Ml of the dissolved immunoprecipitate was placed in each slot to avoid distortion of the bands by excessive immunoglobulins. Genetic mapping. Transforming DNA was isolated from exponentially growing cultures using the method of Okamoto et al. (12). Spore DNA was isolated from BR95 spores which had been germinated for 2 h in the presence of 100 Mg of chloramphenicol per ml and then heated for 10 min at 60 C (19). The germinated spores were lysed with 1 mg of lysozyme per ml for 30 min at 37 C followed by the addition of 1% sodium lauryl sulfate. The procedure for cellular DNA was then followed. The preparation of competent cells and transformations were as described by Rudner and Remeza (15). PBS1-transducing lysates (4) were used to mediate three-factor crosses. Since it was not possible to select directly for PBSX markers, primary selection was for the auxotrophic markers argC or metC. Prior to scoring for unselected markers, the prototrophic transductants were purified on appropriately supplemented minimal plates. UV killing assay. Cultures were grown in CH-min media to a cell density of 25 Klett units. Cells, transferred to an equivalent volume of Spizizen salts in a petri dish, were aerated by a magnetic spinbar and irradiated in a darkened room with an 8 W Germicidal lamp (General Electric) at an approximate dose rate of 25 ergs per mm2/S. Samples were taken at intervals during the irradiation and divided into two parts; one was used to determine surviving colony-forming units and the other, diluted 1/5 in VY, was incubated for 120 min to allow production of PBSX.
J. VIROL. Preparation and induction of k105 and SPO2 lysogens. Strains to be lysogenized were used as lawns for plaquing 0105 and SP02. Prospective lysogens, picked from the center of turbid plaques, were streaked on TBAB plates and then tested for immunity by cross-streaking against 0105c4 and SPO2c, respectively. Cultures of /105 and SP02 lysogens, grown in VY to a density of 25 Klett units, were induced with 0.4 and 0.3 gg of MC per ml, respectively.
RESULTS Biochemical characterization of the PBSX mutants. In screening approximately 1,500 mutagenized clones, one was isolated with the phenotype expected for cells producing phage with defective heads (Xhd-) and twelve with that expected for cells producing phage with an altered tail (Xtl-). Since the Xhd- and Xtlphenotypes potentially reflected mutations in PBSX structural components, the patterns of phage protein synthesis in induced cultures of the single Xhd - isolate GB1323 and a randomly chosen Xtl- isolate, GB159, were examined. Both mutants appeared to produce all of the previously identified PBSX proteins (Fig. 2). Since this approach would only detect gross alterations in either the synthesis or the molecular weight of a particular protein, the ability of the proteins synthesized by the mutants to react with an anti-PBSX antiserum was examined. The labeled proteins present in total cell lysates capable of complexing with anti-PBSX antibodies were analyzed by electrophoresis. The results, presented in Fig. 3, show that the immunoprecipitate of the wild-type strain BR95 contains the PBSX structural proteins in approximately the same proportions present in purified phage. However, the immunoprecipitate of the Xhd- mutant GB1323 is conspicuously missing proteins X4 and X7, which were previously identified as head components (18). In the case of the Xtl- mutant GB159 proteins X6 and possibly X5 are not immunoprecipitable; however, a protein which bands at the position of X8 is evident. Electron micrographs were taken of pellets prepared by high-speed centrifugation (43,500 x g for 180 min) of the MC-induced lysates of the two mutants. The pellet from the Xhdmutant contained assembled tails without heads and that from the Xtl - mutant contained head structures without tails (Fig. 4, panels A and B). The inability of the Xtl- mutant GB159 to assemble phage tails also has been observed for two independently isolated mutant strains of B. subtilis, 44A0 and BS10, which carry the xtl-1 and xtl-2 mutations, respectively (6). The screening for PBSX mutants also de-
VOL. 16, 1975
GENETICS OF DEFECTIVE BACILLUS PHAGE PBSX
a
b
C
187
The mutation carried by GB219 is specific for PBSX. SPO2 and 0105 lysogens of this strain are inducible by MC (Table 2); thus the mutation appears only to block MC- or UV-mediated derepression of PBSX. When GB219 was examined for MC induction of PBSX-specific protein synthesis by SDS gel electrophoresis no structural proteins were detected even by immunoprecipitation (data not shown). GB219 is not cured of PBSX. The presence of the wild-type xtl-1 allele, a PBSX genetic function involved with tail assembly and bacteriocidal activity (6), was demonstrated by using GB219 as the donor for PBS1-mediated transduction of an xtl-l- strain to Xtl+ (Table 3). Between 14 and 43% of the metC recombinants were converted to Xtl+. A more precise determination of the cotransduction frequency of xtl-1 with metC could not be made in this cross since 29% of the metC recombinants also become noninducible for PBSX. This high frequency of conversion to Xin- also demonstrates that the mutation responsible for this noninducible phenotype is linked to metC. Genetic mapping of the PBSX mutants. The mutations responsible for the Xhd- phenotype of GB1323 and the Xtl- phenotype of
XIX2 X3X4X8
X5 X6 X7
d FIG. 2. Total cell lysates subjected to electrophoresis on 13% SDS polyacrylamide gels. The lysates were prepared as described after pulse labeling the cells for 3 min at 75 min postinduction. a, XhdGB1323; b, Xtl- GB159; c, BR95; d, purified PBSX.
I
a
tected six clones which failed to lyse or release PBSX particles after exposure to MC. This noninducible phenotype (Xin-) is expected for mutations in PBSX regulatory genes involved -~b in maintenance of repression. However, mutants with altered cellular functions affecting prophage induction such as certain Rec- mutants (16) or mutants with altered permeability to MC also would be expected to exhibit this phenotype. Since Rec- mutants are generally UV sensitive, the six potential Xin- mutants C W-F were tested for their sensitivity to killing by UV I irradiation. Only one of these, GB219, exhibited enhanced resistance to UV. The UV survival curve for this strain together with that of wild-type BR95 and a recA mutant is presented d LI--.Olo in Fig. 5. Also included in this figure is the UV dose dependence of PBSX induction for BR95. FIG. 3. Immunoprecipitated PBSX proteins subGB219 failed to induce PBSX at any of the jected to electrophoresis on 13% SDS polyacrylamide doses tested and was more resistant to UV than gels. a, Xhd-, GB1323; b, Xtl-, GB159; c, BR95; d, the wild-type strain. purified PBSX.
J
J
1%..O-
I
A-..
';4
*n
..
,j,
z
Vol. 16, No. 1 Printed in U.S.A.
Isolation and Characterization of Prophage Mutants of the Defective Bacillus subtilis Bacteriophage PBSX PHYLLIS THURM1 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
Bacillus subtilis mutants with lesions in PBSX prophage genes have been isolated. One of these appears to be a regulatory mutant and is defective for mitomycin C-induced derepression of PBSX; the others are defective for phage capsid formation. All of the PBSX structural proteins are synthesized during induction of the capsid defective mutants; however, several of these proteins exhibit abnormal serological reactivity with anti-PBSX antiserum. The two head proteins X4 and X7 are not immunoprecipitable in a mutant which fails to assemble phage head structures. In the tail mutant, proteins X5 and X6 are not immunoprecipitable, tails are not assembled, and a possible tail protein precursor remains uncleaved. The noninducible mutant does not synthesize any PBSX structural proteins after exposure to mitomycin C. The mutation is specific for PBSX since /105 and SPO2 lysogens of the mutant are inducible. All of the known PBSX-specific mutations were shown to be clustered between argC and metC on the host chromosome. In addition, the metC marker was shown to be present in multiple copies in cells induced for PBSX replication. This suggests that the derepressed prophage replicates while still integrated and that replication extends into the adjacent regions of the host chromosome.
Three separate sites on the Bacillus subtilis 168 chromosome have been implicated as possible loci for genes coding for the defective phage PBSX (Fig. 1). A site near the origin of chromosomal replication has been suggested on the basis of the multiforked replication of this region which ensues after exposure of cells to inducing agents such as mitomycin C (MC) (7). A second site in the region of the purB marker was suggested by the isolation of a mutant which exhibits a temperature-inducible phenotype for PBSX replication (17). The third site which maps near metC was identified by the xtl-1 mutation which interferes with PBSX tail assembly (6). If, as suggested by these results, the PBSX, PBSY, and PBSZ prophage genes are scattered across their respective cell chromosomes, their defective nature could result from an inability to excise and replicate an intact phage genome during induction. To examine this question more closely we sought to isolate and genetically characterize a number of mutants with impaired PBSX gene functions. Three such mutants are described here; one is defective for
MC-induced PBSX derepression and the other two for PBSX capsid formation.
I Present address: Phyllis Hammer, Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139.
184
MATERIALS AND METHODS Bacterial and phage strains. The bacterial strains used are listed in Table 1. All strains except W23 are B. subtilis 168 derivatives and lysogenic for PBSX. W23 is a PBSZ lysogen and sensitive to PBSX killing. The conditions used for growing bacterial cultures and for monitoring cell densities are given in the accompanying paper (18). Phage 4105 and the clear plaque mutant 0105c4 were obtained from L. Rutberg; SP02 and SPO2c were from J. Marmur. Media and chemicals. The compositions of minimal, NaS, tryptose blood agar base (TBAB), and VY media have been described (18). In CH-min the basic minimal media is supplemented with 0.05% Casamino Acids and 10-5 M MnCl2; in KS, it is supplemented with 0.1% yeast extract (Difco). Selective plates consisted of minimal media plus 50 Ag of required amino acids and 100 ug of purines and pyrimidines per ml and were solidified with 2% agar (Difco). The overlay plates used to assay PBSX-mediated killing of W23 consisted of 2.5 ml of top agar described by Okubo and Romig (13) seeded with a streptomycin-resistant strain of W23 layered on CH-min plates. Both layers contained 3 mg of streptomycin sulfate per ml. Overlay plates of tryptone broth plus 10-2 M MgSO4 were used for 0105 and SP02 plaque assays. Nitrosoguanidine (N-methyl-N'-nitro-N-
GENETICS OF DEFECTIVE BACILLUS PHAGE PBSX
VOL. 16, 1975
185
The rabbits received four toe pad injections once a week for 4 weeks and were bled 1 week after the final inoculations. Sheep antiserum to rabbit gamma globulin was generously donated by B. F. Erlanger. Mutagenesis. BR95 cells were mutagenized by a modification of a procedure described by Adelberg et al. (1). Cells grown in VY to a density of 100 Klett units were resuspended in an equal volume of Spizizen salts adjusted to pH 6.0 and incubated for 10 min at 37 C with 100 Mg of nitrosoguanidine per ml. After thorough washing, the culture was grown in VY to stationary phase to allow cell division and segregation of mutant genotypes before plating on TBAB. Screening of mutants. Single colonies were inocua00 lated into 1 ml of VY and grown overnight to stationary phase. Ten microliters of these cultures was inoculated into 1 ml of VY which contained 250 mg of deoxyadenosine and 8 MCi of [3H Ithymidine per ml to label the bacterial DNA which would be packaged by the phage during induction. The cultures were grown until the cells in each tube reached a visually estimated density of 25 Klett units (approximately 2 to 3 h). MC was added to a final concentration of 0.5 Mg/ml, and incubation was continued for 3 h. Clones potentially defective for PBSX-specific functions were identified by monitoring three phageassociated activities in the MC-induced cultures. Induction defective (Xin-) mutants were detected by their sustained high turbidity at 3 h postinduction.In cultures of MC-treated cells carrying wild-type co. PBSX, turbidity normally increases for 90 min but Q' then falls rapidly as the cells lyse. Mutants with possible tail defects (Xtl -) were identified by the absence of PBSX-mediated killing of B. subtilis W23. FIG. 1. B. subtilis genetic map adapted from J. The screening procedure for head defective (Xhd-) Kejzlarova-Lepesant, N. Harford, J. A. Lepesant, and mutants utilized an assay designed to test the ability R. Dedonder (In P. Gerhardt, H. L. Sadoff, and R. N. of phage heads to protect packaged 3H-labeled DNA Costilow [ed. ], Spores VI, in press). The arrows from degradation by exogenously added nucleases. indicate suggested sites for PBSX prophage genes. Lysates were treated with 100 Mg of lysozyme per ml 1. TABLE Bacterial strains nitrosoguanidine) was purchased from Aldrich Chemicals (Cedar Knolls, N.J.), and spleen phosphodiesterase and micrococcal nuclease were purchased from Worthington Biochemical Corp. (Freehold, N.J.). 14Clabeled amino acid mix (0.1 mCi/ml) and [methyl'H Jthymidine (10 to 15 Ci/mmol) were obtained from New England Nuclear (Boston, Mass.). Antisera. Anti-PBSX antiserum was prepared by immunizing rabbits with CsCl-purified PBSX (500 Ag of phage protein/ml) in complete Freund adjuvant. z
z
Strain
BR95
GB159 GB1323 GB219 44AO BD71 GB26 GB19 GB64 GB75 GB23 GB1001 MU8U5U16 W23
Propertiesa
pheA, ilvC, trpC pheA, ilvC, trpC, xtl-3 pheA, ilvC, trpC, xhd pheA, ilvC, trpC, xin xtl-1 argC, hisA, pyrA trpC, metB, recA-1 xtl-lb, metC, pyrA argC, metC, pyrA xinb, metC, pyrA metC, leuA leuA, metB, tsi-23 purA, leuA, metB Str', PBSX'
Source
L. Rutberg
Mutagenized derivative of BR95 Mutagenized derivative of BR95 Mutagenized derivative of BR95 K. Bott D. Dubnau C. Anagnostopoulos A. Garro A. Garro A. Garro A. Garro E. Siegel N. Sueoka Spontaneous Strr mutant
a xtl, PBSX marker leading to loss of bacteriocidal activity; xin, PBSX marker leading to noninducibility ci PBSX; xhd, PBSX marker leading to formation of defective heads; PBSX, killed by PBSX; Strr, resistant to 5 mg of streptomycin sulfate per ml. °PBSX markers were introduced into strain GB64 by transformation at saturating DNA concentrations selecting argC+ transformants and then assaying for the PBSX mutant phenotype.
186
THURM AND GARRO
for 30 min at 37 C and then adjusted to 0.1 M Tris-hydrochloride-5 x 10- M CaCl2 (pH 7.6) and incubated for 45 min at 37 C with 10 ug of spleen phosphodiesterase and 10 gg of micrococcal nuclease per ml. The percentage of protection was calculated from the ratio of acid-insoluble counts, measured by the method of Li and Felmy (10), before and after nuclease treatment. The Xhd- phenotype was ascribed to lysates which afforded less than 40% protection. This is the average amount of bacterial DNA packaged into PBSX particles. Xtl- cells also exhibited the Xhd- phenotype probably because defective tails allow nuclease to penetrate otherwise normal heads. Immunoprecipitation. Cells were grown in KS and induced with 0.4 Mg of MC/ml when the culture reached a density of 23 Klett units. At 75 min postinduction, a 0.2-ml aliquot was pulsed for 6 to 10 min with 5 MCi of "4C-labeled amino acids. The pulse was terminated by a 30-min incubation in an equal volume of 500 MAg of lysozyme per ml 0.02 M NaN,. The lysate was incubated first with anti-PBSX antiserum for 60 min at 37 C and then with sheep anti-rabbit gamma globulin for 60 min at 37 C. After overnight incubation at 4 C the immunoprecipitates were washed with cold 0.9% saline and dissolved in sample buffer (18). Samples were placed in a boiling water bath for 3 min and cooled before being applied to the sodium dodecyl sulfate (SDS) gels. The procedure used for SDS gel electrophoresis is described in the accompanying paper (18). Only 1 to 3 Ml of the dissolved immunoprecipitate was placed in each slot to avoid distortion of the bands by excessive immunoglobulins. Genetic mapping. Transforming DNA was isolated from exponentially growing cultures using the method of Okamoto et al. (12). Spore DNA was isolated from BR95 spores which had been germinated for 2 h in the presence of 100 Mg of chloramphenicol per ml and then heated for 10 min at 60 C (19). The germinated spores were lysed with 1 mg of lysozyme per ml for 30 min at 37 C followed by the addition of 1% sodium lauryl sulfate. The procedure for cellular DNA was then followed. The preparation of competent cells and transformations were as described by Rudner and Remeza (15). PBS1-transducing lysates (4) were used to mediate three-factor crosses. Since it was not possible to select directly for PBSX markers, primary selection was for the auxotrophic markers argC or metC. Prior to scoring for unselected markers, the prototrophic transductants were purified on appropriately supplemented minimal plates. UV killing assay. Cultures were grown in CH-min media to a cell density of 25 Klett units. Cells, transferred to an equivalent volume of Spizizen salts in a petri dish, were aerated by a magnetic spinbar and irradiated in a darkened room with an 8 W Germicidal lamp (General Electric) at an approximate dose rate of 25 ergs per mm2/S. Samples were taken at intervals during the irradiation and divided into two parts; one was used to determine surviving colony-forming units and the other, diluted 1/5 in VY, was incubated for 120 min to allow production of PBSX.
J. VIROL. Preparation and induction of k105 and SPO2 lysogens. Strains to be lysogenized were used as lawns for plaquing 0105 and SP02. Prospective lysogens, picked from the center of turbid plaques, were streaked on TBAB plates and then tested for immunity by cross-streaking against 0105c4 and SPO2c, respectively. Cultures of /105 and SP02 lysogens, grown in VY to a density of 25 Klett units, were induced with 0.4 and 0.3 gg of MC per ml, respectively.
RESULTS Biochemical characterization of the PBSX mutants. In screening approximately 1,500 mutagenized clones, one was isolated with the phenotype expected for cells producing phage with defective heads (Xhd-) and twelve with that expected for cells producing phage with an altered tail (Xtl-). Since the Xhd- and Xtlphenotypes potentially reflected mutations in PBSX structural components, the patterns of phage protein synthesis in induced cultures of the single Xhd - isolate GB1323 and a randomly chosen Xtl- isolate, GB159, were examined. Both mutants appeared to produce all of the previously identified PBSX proteins (Fig. 2). Since this approach would only detect gross alterations in either the synthesis or the molecular weight of a particular protein, the ability of the proteins synthesized by the mutants to react with an anti-PBSX antiserum was examined. The labeled proteins present in total cell lysates capable of complexing with anti-PBSX antibodies were analyzed by electrophoresis. The results, presented in Fig. 3, show that the immunoprecipitate of the wild-type strain BR95 contains the PBSX structural proteins in approximately the same proportions present in purified phage. However, the immunoprecipitate of the Xhd- mutant GB1323 is conspicuously missing proteins X4 and X7, which were previously identified as head components (18). In the case of the Xtl- mutant GB159 proteins X6 and possibly X5 are not immunoprecipitable; however, a protein which bands at the position of X8 is evident. Electron micrographs were taken of pellets prepared by high-speed centrifugation (43,500 x g for 180 min) of the MC-induced lysates of the two mutants. The pellet from the Xhdmutant contained assembled tails without heads and that from the Xtl - mutant contained head structures without tails (Fig. 4, panels A and B). The inability of the Xtl- mutant GB159 to assemble phage tails also has been observed for two independently isolated mutant strains of B. subtilis, 44A0 and BS10, which carry the xtl-1 and xtl-2 mutations, respectively (6). The screening for PBSX mutants also de-
VOL. 16, 1975
GENETICS OF DEFECTIVE BACILLUS PHAGE PBSX
a
b
C
187
The mutation carried by GB219 is specific for PBSX. SPO2 and 0105 lysogens of this strain are inducible by MC (Table 2); thus the mutation appears only to block MC- or UV-mediated derepression of PBSX. When GB219 was examined for MC induction of PBSX-specific protein synthesis by SDS gel electrophoresis no structural proteins were detected even by immunoprecipitation (data not shown). GB219 is not cured of PBSX. The presence of the wild-type xtl-1 allele, a PBSX genetic function involved with tail assembly and bacteriocidal activity (6), was demonstrated by using GB219 as the donor for PBS1-mediated transduction of an xtl-l- strain to Xtl+ (Table 3). Between 14 and 43% of the metC recombinants were converted to Xtl+. A more precise determination of the cotransduction frequency of xtl-1 with metC could not be made in this cross since 29% of the metC recombinants also become noninducible for PBSX. This high frequency of conversion to Xin- also demonstrates that the mutation responsible for this noninducible phenotype is linked to metC. Genetic mapping of the PBSX mutants. The mutations responsible for the Xhd- phenotype of GB1323 and the Xtl- phenotype of
XIX2 X3X4X8
X5 X6 X7
d FIG. 2. Total cell lysates subjected to electrophoresis on 13% SDS polyacrylamide gels. The lysates were prepared as described after pulse labeling the cells for 3 min at 75 min postinduction. a, XhdGB1323; b, Xtl- GB159; c, BR95; d, purified PBSX.
I
a
tected six clones which failed to lyse or release PBSX particles after exposure to MC. This noninducible phenotype (Xin-) is expected for mutations in PBSX regulatory genes involved -~b in maintenance of repression. However, mutants with altered cellular functions affecting prophage induction such as certain Rec- mutants (16) or mutants with altered permeability to MC also would be expected to exhibit this phenotype. Since Rec- mutants are generally UV sensitive, the six potential Xin- mutants C W-F were tested for their sensitivity to killing by UV I irradiation. Only one of these, GB219, exhibited enhanced resistance to UV. The UV survival curve for this strain together with that of wild-type BR95 and a recA mutant is presented d LI--.Olo in Fig. 5. Also included in this figure is the UV dose dependence of PBSX induction for BR95. FIG. 3. Immunoprecipitated PBSX proteins subGB219 failed to induce PBSX at any of the jected to electrophoresis on 13% SDS polyacrylamide doses tested and was more resistant to UV than gels. a, Xhd-, GB1323; b, Xtl-, GB159; c, BR95; d, the wild-type strain. purified PBSX.
J
J
1%..O-
I
A-..
';4
*n
..
,j,
z
