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Studies on Bacillus subtilis bacteriophage φ15
66, 11&122
VIROLOGY
(1973)
Studies JUNETSU Department
on Bacillus
subtilis
MEINKE,
GARY
ITO, WILLIAM of Microbiology,
Scripps
Clinic
Bacteriophage HATHAWilY,’
a.nd Research
Foundation,
415 AND
La Jolla,
JOHN
SPIZIZEN
Calijornia
9%OSY
Accepted August 8, 19YS The growth characteristics and physicochemical properties of Bacillus sztbtilis bacteriophage 415 have been investigated and compared with +29. Differences in growth characteristics of t.hese phages were found in various stages of spore germination and outgrowth which may account for the absence of plaque formation by 615 on wild-type cells. Both 415 and 429 genomes were incorporat.ed into spores when cells were infected at a certain critical time during host cell development.. 415 is morphologically indistinguishable from 429. The DN.4 of $115 is a linear duplex molecule having a mean length of 6.1 pm. The S value of this DNA is 23 & 0.4 S. These values correspond to a molecular weight of approximately 12 X lo6 daltons. At least seven structural proteins of 615 were recognized in SDS-acrylamide gel electrophoresis. The sum of the molecular weights of 615 proteins is approximat,ely 354,000 daltons. This corresponds to about 595 of the coding capacity of doublestranded DNA of 415. 415 DNA was found to hybridize with $29 DNA.
genous mutants (Reilly, 1965; Hoch and Spizizen, 1969; It,o and Spizizen, 1971). In an effort to determine the basis of this selectivit,g, we initiated a study of 415. In this report,, some of the growth characteristics as well as physicochemical properties of 915 and its DNA will be described. A preliminaq report of some of these studies was presented and Spizizen, uto, Bleinke, Hathaway, 1972).
INTRODUCTION
Certain phage-host interactions in Baci1.h have been found to provide useful systems for investigations of cell development. Thus, infection of sporulating cells by some virulent phages results in the incorporation of phage genomes into spores, whereas phagc multiplication takes place in exponentially growing cells. Bacillus subtilis phages /33and 4e (Yehel and Doi, 1967; Sonenshein and Roscoe, 1969) are examples of this relationship. These studies led to the finding that alt,erations in the template specificity of bacterial RNA polymerase occur during sporulation (Losick and Sonenshein, 1969). Similar effects have been observed for the related phages $15 and $29, two of t,he smallest known phages containing doublestranded DNA (Reilly, 1965 ; Anderson, Hickman, and Reilly, 1966; Szybalski, 1968). Phage 915, unlike +Y9, does not produce plaques on wild-type Bacilbus subt~ilis and late-blocked asporogenous mutants, but does plaque on certain early blocked asporo’ Department of Biochemistry, California, Riverside, California.
University
of
MATERIALS
@l 193 by Academic Press. of reproduction in any form
Inc. reserved.
METHODS
Bacteria and pkge. Bacdlus subtilis 168 (trpCd), SR-32 (spoAl2 IrpCd), and Bacillus amyloliquefaciens H were used in this study and described previously (Ito and Spizizen, 1971). Strain SCRlOO (spodlb Zeu- and SCR114 (spoAl2 thyX1 t,hTl2) were con&ructed by PBS-1-transduction (Takahashi, 1963). Recipient cells were BR&% (trpC2 Zeu-) and B. subtil~is 168 th;y-(trpC2 thyX1 thyYl)? which were provided by Drs. Reilly and Anagnostopoulos, respectively. 31edia. Minimal medium was described previously (Spizizen, 1958). Penassay broth [antibiotic medium no. 3 (Difco)] and tryp-
110 Copyright All rights
BND
BACTER.IOPHAGE
tose blood agar base (Difco) were used for t#he propagation of bacterial strains. Plating conditions. Bacteria were plated on TBAB plates and phages were plated on minimal medium agar supplemented with casein acid hydrolysate (0.05 %) and 5 X lo-” di MnC12 with 3 ml of soft agar composed of t,ryptone (Difco), 10 g; NaCl, 8 g; agar (Difco) 0.6 ?J; glucose, 6 g; per liter of dist#illed water. Sporulation medium. Sporulation medium was essentially t,he same as that of Schaeff er’s medium modified by Leighton and Doi (1971). The medium consists of Difco INtrient brot#h (16 mg/ml), 2.5 X lo-* llrl KCl, 2 X lo+’ M CaC12, 1 X lo+’ II1 FeC12, 1 X lo+ Jf RInS04, 1 X 1w4 M lJIgSO4, and 5 X 1O-3M glucose. Preparation. of spores. Bacillus subtilis 168 was grown in sporulation medium at 37 C for 70 hr. Spores were collected by centrifugation, washed once with water, and resuspended in 0.01 M Tris-HCl (pH 7.5) buffer. The spore suspension was then heated for 10 min at 80 C. After cooling to room temperature, spores were treat,ed with lysozyme (20 pg/ml) and deoxyribonuclease I (DNase I) (5 pg/ml) for 20 min at 37 C. Spores were washed again with water and further purified by sedimentation kough Renografin gradients, according to the modified method of Tamir and Gilbarg (1966), (Van Alstyne, Grant, and Simon, 1969). Purified spores were stored in water at 4 C. Preparation and pw$catim of r#d5 aid 429. Unless otherwise st,ated, Bacillus amyloliquefaciens H was used as a host in preparation of lysates of both 415 and #29. Cells were grown in 2.8 1 flasks containing 500 ml Penassay broth supplemented with 0.5 % glucose to a final concentration of 2 X 10’ cells/ml. The cells were pelleted by centrifugat,ion and then resuspended in adsorption medium (0.1 111NaCl, 10-*ill MgS04 in 0.1 ,I;C pot#assium phosphate buffer at pH 7.0). The bacteria were mixed wit,h eit,her 415 or $29 to a multiplicit,y of 5. After 10 min at room temperature the phage-bacteria complex was resuspended in fresh Penassay broth containing 0.5 % glucose to the original volume and shaken at 37 C. Usually after 2 hr the cells were completely lysed. Lysat’es were centrifuged at 80009 for 30 min to remove
415
111
cell debris. The supernatant fluid was treated with DNase (5 pg/ml) and pancreatic RNase (5 pg/ml) at 37 C for 20 min. Phage particles were sediiented by centrifugation at 25,000g for 2 hr at 4 C in a 19 rotor of the Spinco Model L2 preparative ultracentrifuge. Phage pellets were resuspended in phage adsorption medium and centrifuged for 30 min at SOOOgat 4 C. Solid CsCl (the Harshaw Chemical Co., Soton, Ohio) was added to the concent’rated phage suspension until the refractive index reached 1.375. The mixture was then centrifuged for 48 hr at 70,OOOg in the SW5OL rot’or in a Spinco L-2 preparative ultracentrifuge. The peak fractions, with a density of 1.45 g cm+, were pooled and dialyzed against adsorpt.ion medium overnight at 4 C. A rapid purification of phage particles could also be achieved by centrifugation in a three-layered gradient of CsCl as follows. The bottom layer was 1.5 ml of a CsCl solution (1.5 g cme3) ; the middle layer, 1.0 ml (1.3 g cme3) and bhe top layer, 0.5 ml (1.1 g cmd3). Phage suspensions (1 .Oor 1.5 ml) were layered on top and tubes centrifuged in the SW-50L rot,or for 3 hr at 70,OOOg.The phage band was collected t’hrough a hole in the bottom of the tube. Phage particles were diluted with adsorption medium and centrifuged in a 50 Ti rotor at. 80,OOOgfor 30 min at 4 C. The phage pellet was resuspended in phage adsorption medium. Preparation 0j ~raclioadive 415 and +29. Strain SCR 100 (spodld leu-) was grown in minimal casein acid hydrolysate medium consisting of Spizizen’s minimal salt, 0.1 J1 NaCl, lo-* M RInC12 , 10e3 M RIgCl2 ,0.5 ‘% glucose, 20 pg/ml n-leucine, and 0.05 % casein acid hydrolysate (Difco). Cells were grown to about 1 X 10” cells/ml, centrifuged, and resuspended in phage adsorption medium (10 X cone). Phage (either 915 or @29) was added to cell suspensions at a multiplicity of 10 and incubated for 10 min at room t,emperat,ure. The bacterium-phage complex was inoculated into medium consisting of minimal casein acid hydrolysate (above) except that the amount of L-leucine was reduced to 3 pg/ml and [3H]leucine (5 &%/ml, 5 Ci/nid1) or [14C]leucine (1 #Zi/ml, 311 Ci,/mili) added. Infected cells were shaken at 37 C for 2 hr. Radioactive phage
112
ITO ET AL.
particles were isolated and purified as described above. Preparation of 3H-labelecl 415 DNA. Strain SCR114 (spoAl.2 thyX1 thyY1) was grown to 1 X IO8 cells/ml in mmlmal casein acid hydrolysate medium (above) supplemented with 5 pg/ml of [3H]thymidine (15 pCi/mg thymidine), infected with 415 at a multiplicity of 5, and shaken for 3 hr. 3H-415 was purified as described above. DNA was extracted from phage by a phenol method (Mandel and Hershey, 1960). Preparation of 32P-labebed 415 and $829. Bacillus amyloliquefaciens H was grown in low phosphate broth containing 20 PCi of 32Pper ml. Dephosphorylated brot.h was prepared as described previously (Ito and Spizizen, 1971). When the cell density reached 1 X 10” cells/ml cultures were infected with 415 or 429 at a multiplicity of 10, and shaken for 3 hr at 37 C. “P-labeled phage particles were collected and purified as described. [32P]DNA was extracted twice with phenol. The aqueous layer was dialyzed extensively against standard saline citrate (SSC) in 0.01 111Tris-HCl buffer at pH 7.0. Electron microscopy-~15 DNA. Samples of 415 DNA were prepared for electron microscopy as described previously (Rleinke and Goldstein, 1971). Electron micrographs were taken with a Hitachi HU-11 electron microscope. Contour length of 915 DNA molecules were measured w&h a Keuffel and Essen map measurer from tracing of projected negatives. Magnifications were determined by photographing a standard grating replica (Ladd). 415 P&ides. $15 was negatively stained with 1% potassiumphosphotungstate (W,/Vj (Brenner and Horne, 1959). Electron micrographs and magnifications are as described above. Determination of molecular weight by seclimentation velocity. Band sedimentation was performed in 1 M NaC1, pH 7 solvent essentially as described by Studier (1965). Samples of 415 DNA (10 pliters) in 1 X SSC were pipetted into the filling holes of double sector centerpieces (Vinograd et al., 1963) and layered at low speed on the 1 M NaCl solvent. Centrifugation at 88,000 rpm and 20 C was commenced after the disappearance of the initially sharp refractile band.
R.ecordings were made at 4-min int,ervals using the split-beam mode of the phot80elect,ric detector and scanner (Lamers et al., 1963) lvith t,he monochromator set, at 265 nm. Molecular weight was estimated using t#he equations of Freifelder (1970) and t,he sedimentation coefficients calculated from peak positions of the absorption tracings. Sucrose gradient centrifugalion. Linear (520%) sucrose gradients were made by dissolving sucrose in 0.01211 Tris-HCI buffer at pH 7.5 containing 1 M NaCl and 10d3 M EDT-4 (neutral gradients) or by dissolving sucrose in 0.9 II1 NaCl, 0.1 M NaOH, and 10e3 111 EDTA (alkaline gradients). Samples (0.1 ml) were centrifuged in a SW5OL rotor in a Spinco Model L2 ultracentrifuge at 73,000g for 2 hr. To measure the radioactivity in a gradient fraction, seven drops per fraction were collected on 2.0 cm Whatman glass filter paper. Aft,er drying, the filters were treated with 10% cold trichloroacetic acid (TCA) and then with ethanol (95 %). Air dried filters were placed in vials containing 3 ml of toluene-Omnifluor (New England Nuclear) fluid and counted. Equilibrium
Ceretrifugation in CsCl
(a) Phage particles. Solid CsCl was added to phage suspensions and refractive indexes were adjusted to 1.3745. (b) 415 DNA. The refractive index was adjusted to 1.3944 by addition of solid CsCl to DNA solutions containing 5 pg [3”P]915 DNA and 5 pg [3H]BacilZus subtilis DNA (p = 1.7034) as a density reference. Solutions were cent#rifuged for 48 hr at 70,OOOgat 20 C in a SW5OL rotor. Ten drops per fraction were collected into tubes and portions (50 Jiters) assayed for radioactivity and for measurement of densit,ies. Optical melting of 915 DNA. DNA melting profiles were done in a Gilford 2000 continuous recording spectrophotometer equipped with automatic temperature and blank compensation. DNA solutions were pretreated to 50 C to remove dissolved air, then capped with Teflon stoppers before being placed in the sample holder. The melting profile of 25 E.rg/ml of 41.5 DNA was performed in SSC buffer, pH 7.0. The temperature was increased 0.5 C every 7 min.
BACTERIOPHAGE
DNA-DNA hybridization. A modification of the method of Denhardt (1966) was employed as described by Aloni, Winocour, Sachs, and Torten (1969). Polyacrylamide gel electrophoresis of stmctural polypeptides of $15 a&, 429. Purified 415 were suspended in 0.02 M Tris-HCI buffer, pH 7.5 and made 1% (v/v) with sodium dodecyl. sulfate (SDS) and 2-mercaptoethanol. Phage suspensions were heated for 5 min in boiling water and cooled to room t,emperature. SDS-polyacrylamide gel electrophoresis was carried out in 10 % gels according to the methods of Laemmli (1970). Electrophoresis was at room temperature (2 mA per tube) using a buffer containing 0.025 A1 Tris, 0.192 Jr glycine, and 0.1% SDS, pH 8.3. Prior t.o staining, SDS was removed from gels by dialysis overnight against 7 % acet,ic acid. Gels were then immersed for 4 hr in a solution of 0..25 % Coomassie brilliant blue, freshly made in 50 % methanol and 7 ‘% acet#ic acid. Gels were destained overnight against several changes of 30 % met#hanol in 7 % acetic .acid an.d stored in 5 % methanol in 7 4”o acetic acid. Estimation of molecular weigh.ts of polypeptide chaim. The molecular weights of the polypeptide chains were determined by SDS-polyacrylamide gel electrophoresis according to the method of Shapiro, Venuela, and Maize1 (1967). Standard proteins used were muscle phosphorylase a (MW, lOO,OOO), bovine serum albumin (MW, 68,000), ovalbumin (RlW, 43,000), t’rypsin (MW, 23,800), lysozyme (MW, 14,300), cytochrome c (RIW, 11, 700) (Weber, Pringle, and Osborn, 1972). Standard proteins were heated at 100 C for 5 min in 1% SDS and 2-mercaptoethanol and subjected to electrophoresis as above. Chemicals. [h,Iethyl-3H]thymidine (51 Ci/ mM>, and H33”P0, , carrier-free, were obtained from New England Nuclear Corp. RESULTS
Growth characteristics of 415. One-step growth curves of 915 on Bacill,us subtilis 168 and an early blocked asporogenous mutant (spoAl2) are shown in Fig. 1. The latent period of 415 in spoAld strain at 37 C was 45 min and the burst size was about 300. Similar results with a slightly higher burst
113
$15
Time
(mid
FIG. 1. One-step growthof phage+l5 on Bacillus subtilis 168 and spoA12. The bacteria were grown in Penassay broth (Difco) t.o 2 X lo8 cells/ml, centrifuged, and resuspended in an adsorption medium. The bacteria were mixed with 415 at a multiplicity of 0.1 as determined by previous titration of the phage on Bacillus amyloliquefacierls H. After a lo-min incubation period to allow for adsorption, t.he cult,ure was centrifuged and diluted to 10mi into prewarmed brot.h containing 0.5’$Zoglucose and incubated at 37 C wit.h aeration. At indicated times, samples were withdrawn and assayed for the number of infective centers on B. amyloliquejaciens H.
size were obtained when Bacillus amyloliquefaciens H strain was used as host. During this study, it was often observed that some active 415 particles were produced in B. subtilis 168, although plaques were not seen. The burst size of 415 was about 4 (Fig. 1). This value, however, varies from experiment to experiment. In order to study $15 infection of B. subtilis 168 in detail, it was necessary t,o establish a relatively simple but highly reproducible svstem. We have found that infection follo&g spore germination and outgrowth can be pyrformed under reproducible conditions. Spores purified by Renografin gradient centrifugation (Tamir and Gilvarg, 1966) are germinated in sporulation medium and endospores were subsequently formed 8-9 hr after
IT0
114
the inoculation. However, the t.iming of sport formation depends on the size of the inoculum. Figure 2 shows the time course of 41s infection following germination and outgrowth of spores of B. subtilis 168. At. selected times. indicat,ed by the arrows, 415 was added and the optical absorbancy followed. Cells were lysed to a limited extent when infection occurred during the first 120 min of germination, but after 120 min cells were no longer susceptible to lysis. However, 415 adsorbs to these cells and t,hey are capable of forming spores which contain the phage genomc. Similarly, we have found that, qG9 DNA4 is also incorporated into spores when infection oscurs at a lat,er stage of cell development. The phage genome can bc recovered upon germination of such spores. Figure 3 shows grow’th patterns of $15 and +29 whose genomes were incorporated into spores during germination. The latent periods of both phages were 55 min. Sonenshein and Roscor (1969) reported the existence of a crit,ic.al period in host de-
240
300
360
Time (mid
FIG. 2. Infection of 415 on germinat.ed spores of Bacillus subtilis 168. Spores (1 X l@!ml) were inoculated into 20 ml of sporulation medium in 250 ml naphalo culture flasks and shaken in water bath at 3i C. At the times indicated by the arrows, +15 was added (2 X 10g;‘ml) and the optical density was read with a Klett Summerson calorimeter equipped with a 66 filter
h’7’ Al.
Time
(mid
FIG. 3. Recovery of active phages incorpornt,ed into spores upon germination. Spores with trapped +15 or $29 genomes were inoculated into Penassay broth containing 0.5(“. glucose and shaken at 37 C. At indicated times, samples were wit.hdraxn and assayed for t,he number of infective centers on B. ar,~yloliq~cqfaciarrs H.
velopment,al processes during lvhich phage genomts are trapped by spores. We have also determined the optimum t,ime of infect,ion for incorporation of intact 415 DNA into spores by infecting cells at different stages of growt,h. Inf&ed cultures were incubated for 20 hr to allow for completion of spore formaGon. Spores wrs harvested, xvashed, and neutralized with phage antiserum. The percentage of spores n-hich incorporated phage DN.\ was then determined (Fig. 4). The opt,imum time of infect,ion for incorporat8ion of +l5 DNA int,o spores was 3 hr aft.er germination. This time is significantly earlier as compared to t,he +e system which \vas 5-6 hr aft.er t,he end of logarithmic growth (‘7% S.5 hr aft,er inoculation) (Sonenshein and Roscoe, 1969). Compcwison of bwst size of415 and 429. To understand the differences in growth of 015 and 429 and to examine t,he relationship between phage multiplication and incorporation of phage genomes into spores, the avcrage phagc burst size \vas determined in
BACTEKIOPHAGE
0 0
2
4
6
8
10
Time(hours) FIG. 4. Optimum t.ime of infection for incorporation of the +15 genome into spores. Spores were inoculated (1 X 108spores ‘ml) into200 ml of sporulat,ion medium and shaken in 2 lit.er side-armed flasks at 37 C. At the indicated times, 10 ml samples were removed and mixed with $15 (1 X 109 phages’ml). The mixture was shaken at 37 C for 24 hr in 125 ml flask. The spores were harvest,ed, treated with Iysozyme, washed with water, and determined for tot,al spores and infective centers of @15.
single-step growth experiments for cells infected at different stages of growth. In this experiment, fewer spores (5 X lo6 spores/ml) were inoculated into sporulation medium. ,Uhough the appearance of endospores was delayed about 80 min, better synchronization n-as achieved. A high burst size of both phages was obkned in midlogarit~hmic phase cells but t#heyield of 415 then decreased more ra,pidlg when cells at, later stages were infected than that of @29 (Fig. 5). An accurate estimate of burst, size cannot be made because midlogarithmic st#age B. subtilis cells tend to form long chains. Thus, the dat,a indicat,e onlv relative burst size values. The diffrrences’in average burst size of 415 and $29 ma\- account for the fact, t,hat 415 cannot,
115
415
2
4
Time
6
8
10
(hours)
FIG. 5. Average burst size of phage ~15 and $29 for infection at various t.imes during growth of Bacillus subtilis 168. Purified spores were inoculat,ed into 150 ml of sporulat,ion medium containing O.lO;Oglucose (5 X 106,/ml spores). At the indicated times 5 ml samples were removed. -4fter the optical density was read with a Klett Summerson calorimeter equipped wit,h a 66 filter, the culture was used for single-step growth of 615 and $29. Single-step growth experiments were carried out as described in Fig. 1.
form plaques on \vild type or late-blocked asporogenous mutan@ while @29 forms plaques under convrrkional plating conditions. If the decrease in phage burst, size is due to changes in t,he template specificity of bacterial RNA polymerase as demonstrat,ed by Losick and Sonenshein (1969) for de, t#hen we must consider the possibility that host RNA polymerase has different t,emplate specificities for 415 and 629 DNA. Preliminary studies indicate that, host RNA polymerase, isolated at different stages of cell growth, does not distinguish 41.5 DNA from 629 DN-k in llitro. These studies will be presented in a separate publication. These observations
on the
difference
behxveen
415
116
ITO ET -AL.
and @29led us to characterize 415 in greater detail. Morphologica. appea:ralece of 415. Typical electron micrographs of 415 are shon-11in Fig. 6. The phage is morphologically veq similar to $29 (Anderson et ab., 1966) and t#o Nf (Shin~zu et al., 1970). 415 has a hexagonal head with a flattened base and is characterized by head projections. The head is approximately 45 nm long and 33 11111 wide. The tail is 30 nm long and complex. When the tail is dissociated from t,he head it appears to contain two collars at the upper end and a dist’al knob at the lower end. Several tail appendages are seen at#tached near the tail collars. Twelve t,ail appendages have been shown for phages 629 and Nf (Anderson et al., 1966; RKndez et al., 19Tl; Shimizu et al., 1970). The exact number of appendages of 415 are not, known at, present, but there appears to be at least ten. Sedimentat.io?l am1 buoyant density of $115. Purified 415 particles sediment as a single component in sucrose gradients. 415 has a sedimentation coefficient of 260 S, relat,ive
t,o T7 phage which sediments at 453 d (Dubin ct a.l., 1970). A value of 257 S 1la.s been reported for $29 by Undez et al. (1971). The buoyant density of 415 is est,imated as 1.155 g crnm3by CsCl equilibrium centrifugatAion (Fig. 7). Properties of 415 DNA. Purified 415 was made with 1% SDS and the DNA released by heating at 50 C for 10 min, followed by rapid cooling. DNA was treated with DNase free pronase (Ca.lbiochem, 30 pg/ml) for 20 min at 30 C and bhen ext,racted twice &h phenol. 415 DNA showed a sharp boundaq when centrifuged in 1 31 NaCl. The sedimentation coefficient at infinit,e dilution was obtained as s:~,,~ = 23.3 f 0.4 S by extrapola.tion of plot by the reciprocal of sedimentat,ion coefficient to concentration of DNA. When this value of sZo.,~~ is substit,ut,ed in t,he equation given by Freifelder (1970), s~~.,~,= 2.8 + 0.00834 Al 0.478, the RIW of 415 DNB is 11.9 f 0.24 X lo6 daltons. In neutral sucrose gradients 415 DNA sediment,s at, about, 23 S (Fig. 8) relative to 4X17-1 RF1 DNA which sediment,s at, 21 S
Fro. 6. Bacteriophage $1.5 negatively stained with potassium phosphotungstate. (a) Empty virions j wit.h tail and tail appendages. (bj Empty virions wit,h head fibers. (c) Intact phage. Cd) Tail wit,h appendages. (e) Empty virions, empty head, tail, and tail appendages.
BACTERIOPHAGE
415
117
daltons/pm). These results are in good agreement with the molecular weight determined by velocity sedimentation and together they indicate that the molecular weight of $115 DNA is about 12 X lo6 daltons. Nucleotide composition of 415 DNA. 415 DNA bands as a single peak in CsCl density gradients and has a buoyant density of 1.6917 g cmd3. Using the equation = 1.66 + 0.098 (G + C) (Schildkraut et al., 1962), the buoyant density corresponds to a G + C content of 35 %#.This value closely resembles those values reported by Reilly (1965) and Szybalski (1968). 1000+m The absorbancy at 260 nm of 415 DNA in 1 X SSC was determined as a function of temperature. The midpoint of the thermal transition (Twx-) was 85.78 C. This corresponds to a G + C content of 40.2 %#(Marmur and Doty, 1962). The difference between the values obtained from the Tm and t,he buoyant density cannot be explained at present, but may be due to the presence of methylated or other unique basis. Reilly (1965) also observed these differences in @29 and 415 DNA. Similar situations have been reported for Nf phage DNA by Shimizu et al. (1970). Structural proteins of $15. Structural proteins of 415 were dissociated and analyzed Fraction No. bv SDS-polyacrylamide gel electrophoresis. FIG. 7. Equilibrium centrifugation of 615 and Figure 10 shows the acrylamide gel patterns 629 DNA in CsCl. Phages [‘4C]@15 and [3H]&9 were mixed and CsCl was added to the density of of proteins of 415. Six proteins were clearly 1.42981. The solutions were centrifuged at 70,OOOg visible and when a large amount of protein an additional band for 48 hr at 20 C in a SWSOL rotor. [‘%]@15: was electrophoresed, “E” was observed between bands E and F. (--O-o-), [3H]@29: (-.-a--).
(Roth and Hayashi, 1966). In alkaline sucrose gradients, single-stranded 415 DNA sediments at 26 S, indicating that 415 DNA does not contain single strand break. Electron microscopy of 415 DNA. The molecular weight of $15 DNA was also determined by electron microscopy. The lengths of the molecules range from 5.6 pm to 6.3 pm (Fig. 9). The mean length is 6.1 pm, which corresponds to an average molecular weight of 12.2 X 10” dahons (using 2 X lo6
r -r--7--
Fraction
No.
Fro. 8. Sedimentation profiles of 415 DNA in sucrose gradient centrifugat,ion. DNA and [“C]C X174, RF1 DN.4 was centrifuged at 73,ooOgfor 2 hr at 20 C.
A mixture of [SH]+15
118
The molecular weights of each polypeptidcs were estimat,ed by comparing their electrophoretic mobilities with t,hose of marker proteins of known molecular weight (Table 1). DNA-DNA hybridization. In addition to the differences in growth characteristics of 41.5 and $29, it was recently shown that 41.5 tolerant mutant,s (toZA) were st,ill sensitive t,o @9 and that $29 tolerant mutants (tolB) were tolerant to bot,h phages (Ito, 1973). Furthermore, it was observed that t,hcse two phages do not easily hybridize irl, Go (Reilly and Ito, unpublished observations). Therefore, the relationship of 915 and 629 n-as examined by DNA-DNA hybridization. The data in Fig. 11 show that, there is some hy-
bridization between $13 and &9 DNA and only a small amount,, if any, hybridization with host, DKA. No atkmpts were made to determine the precise degree of homolog? between 415 and $29 DNA. Such studies can be performed by using reannealing techniques developed by Britten and Kohn (1968) and by the heteroduplex met,hod described by Davis and Hyman (1971). L)ISCU8SION
Reilly (1965) has shown that ~$15does not produce plaques on B. subtilis 168, but can in some asporogenous mutant,s of B. subtilis 168. We have extended his studies and have shown that 415 actually replicates at. certain
BACTERIOPHAGE
stages of B. subtilis 168 growth, particular13 in germinated and outgrowing spores. B. Rutberg has made a similar observation (personal communication). The highest burst size for 41.5 was obtained during the midlogarithmic &age of cell growth. In subsequent stages of growth, the replication of $11.5 is markedly decreased. Similarly, the burst size of 429, which is closely related to 415, is also decreased at later stages of growth. After 8 hr of germination, no intact 415 are produced, but. 429 is produced at a reduced rate. We feel this difference is sufficient to account for the fact that $15 cannot make plaques on the wild type of B. subtilis, while qU9 is able to form plaques under conventional plating conditions (Reilly, 1965). It was demonstrated in our studies that both 41.5 and 929 genomes are incorporated into spores when cells are infected at a ccrtain critical time during host, cell development . Trapping of phage genomes by mature spores has been shown previously for larger virulent Bacillus phages, such as f13 and $e (Yehel and Doi, 1967; Sonrnshein and
119
ml5 TABLE
1
MOLECULAR WEIGHTS OF THE STRUCTURAL POLYPEPTIDES OF 615 Polypeptide A B c D E E’ F
Molecular weight 80,000 71,000 54,ow 46,000 39,ooo 34,ooo 29,ooo
F 32P-DNA added (CPM x 163)
E D C
6 A
FIG. 10. SDS-polyacrylamide of whole 615.
gel electrophoresis
FIG. 11. Hybridization of 3zP-labeled 415 and $29 DNA with unlabeled @15DNA and B. sublilis 16% DNA. A ronstant amount of denatured unlabeled 4115DNA (20 pg) and B. subtilis DN.4 (100 pp) were immobilized on the membrane filters (Sartorius Membrane filter) and hybridized with various amounts of the 32P-labeled +15 and @29 DNA, respectively. [32P]@15DNA (7500 rpm.‘pg IINA) and [3*P]&9 DNA (10,000 cprnpbg DNA) were sheared by sonic treatment and then denat,ured by boiling for 10 min. 415 DNA-[3*P]415 DNA, (-0-o-j; $15 DNA-[32P]429 DNA, DNA-[=P]415 DNA, C-@-O-) ; B. mblilis (-A-A-) ; B. subtilir DNA-[“*PI+29 I )N -4, (-A-A--).
Roscoe, 1969). Incorporation of phagc DNA into spores is a well-known phenomena as a carrier stage in temperate phages in Bacilli systems (Thorne, 1969; Takahashi, 1964; Bott and St,rauss. 1965; Thornr, 196s:
120
ITO ET AL.
Kawakami and Landman, 196s; Romig, 196s). Our findings indicate that the incorporation of virulent phage genomes into spores may be a rather common phenomenon in Bacilli systems. The differences of growth properties between $15 and 429 are not understood. If the reduction of burst size and the incorporation of phage genomes into spores are due to host RNA polymerase changes as demonslrated for 4e (Losick and Sonenshein, 1969), then we must assume that host RNA polymerase has a different template specificity- for 915 and $29. We are currently examlmng this possibility. Preliminary results indicate that host RNA polymerase, isolated at different &ages of cell growth, does not distinguish 415 DNA from @29DNA in ,nitro (unpublished data). It appears that some other factors in addition to RNA polymerase alterat#ions might be involved in determining the burst size and incorporation of phage DNA into spores. Sonenshein and Roscoe (1969) have shown that the optimum time for incorporation of $e DNA into spores is 5-6 hr after the end of logarithmic growth (7.5-5.5 hr after inoculation). Our data (Fig. 4) indicates that, t.he optimum time for trapping 915 DNA is at the end of the logarithmic growth stage (3 hr after inoculation). These differences may be partly due to different experimental conditions, but most likely are caused by the differences of the bacteriophages. Since 415 contains only about one-tenth of the genetic informat’ion of 4e, for example, it is reasonable t,o assume t,hat replication of this phage is highly dependent on host-cell functions. Yehel and Doi (1967) studied the differential expression of two virulent Bacillus phages, P3 and PZZ, in sporulating cells of B. subtilis W23. They showed that Pa-infected cells are able to sporulate, resulting in the incorporation of & DNA. On the other hand, &z-infected sporulating cells are lysed. Therefore, it was suggested that multiplication of phage-DNA or incorporation of phage genomes into spores is due to the ability of the phages t,o repress the expression of host genomes. The morphology of $15 appears to be identical to that of 429 (Anderson et al., 1966; Wndez et al., 1971) and Nf (Shimizu
et al., 19iO). Since no evidence of tail contraction was obtained from elect,ron micrographs, 415 can be considered to belong to group C of Bradley’s classificat,ion of bacteriophages (Bradley, 1967). 415 DNA is a linear molecule having a mean lengt,h of 6.1 pm. This length corresponds to an average molecular weight of 12.2 X lo6 daltons which is in agreement with the molecular weight obtained by sedimentation experiments. Although the DNAs of 929 and Nf have been reported to be nonpermuted, linear duplex molecules, the formation of circular molecules have also been described, suggesting the existence of cohesive ends (Anderson and Xlosharrafa, 1968; Hagen et al., 1971; Shimizu et al., 1970). Attempts were made t’o show the formatsion of circular molecules of 415 DNA in annealing experiments in which DNA was heated and cooled slowly. Full length, linear duplex molecules but no circular molecules were obtained (Rleinke, unpublished data). Recently, Ortin et al. (1971) have demonstrated that a protein is involved in t’he formation of circular 429 DNA. These results would conform to the concept t’hat $15 and 429 DNA are linear, nonpermuted duplex, similar to T3 and T7 DNA (Anderdon and Mosharrafa, 1968; Thomas and MacHabtie, 1967). The disagreement between the G + C content of $15 DNA as determined by buoyant density analysis and from the Tm are not understood at present. Reilly (1965) also observed this anomalous nature of 915 and 429 DNA. These results may be due to the presence of a methylated base or other minor odd bases. Recently, unique physical properties of DNA isolated from SP-15, which is a large, generalized transducing Bacillus subtilis phage, have been reported (Marmur, Brandon, Neufort, Erlish, Mandel, and Konmicka, 1972). This phage DNA has a low melting temperature and a high buoyant density in neutral CsCl. It was demonstrated that the DNA contained a modified pyrimidine, 5-(4’) 5dihydroxypentyl)-uracil which partially replaces t,hgmine. The composition of 415 DNA is under invest,igation. There are at least, seven prot,eins in 915. The sum of the molecular weights of 415 structural proteins is approximately 354,000
BACTERIOPHAGE
daltons. This requires a coding capacity in double-stranded DNA of about 7.1 X lo6 daltons. Since the molecular weight of 915 DNA is 12 X lo6 daltons, this value corresponds to about 59 % of the entire chromosome. Difference in growth characteristics of 415 and @9 indicate t#hat these small phage systems offer a useful experimental system to study evolution on the basis of nucleotide sequence of DNA. Our preliminary DNADNA hybridization experiments clearly indicate a partial homology between t#hesetwo phage DNA. The precise degree of homology between 415 and $29 DNA can be determined by bhe method similar to those described for t,he T3-T7 system (Davis and Hyman, 1971) and for the SPOZ-4105 system (Chow et CL, 1972). ACKNOWLEDGMENTS We are grat.eful to Dr. M. Hayashi for providing us with [1dC]+S174 RF1 DNA. We thank Dr. B. Reilly for his useful discussion. We also t,hank G. Mildner, C. Svensson, and M. Morgenstern for their excellent assistance. This work was partially supported by American Cancer Society Grant (NP-39). REFERENCES ALONI, I’., WINOCOUR, E., S.ZCHS, L., and TORTEN, J. (1969). Hybridization between SV-40 DNA and cellular DNA’s. J. 11fol. Biol. 44,333-345. ANDERSON, D L., HICKM.~N, D. D., and REILLY, B. E. (1966j. Structure of Ba.cillus subtilis bacteriophage &9 and the lengt,h of 629 deoxyribonucleic acid. J. Bacterial. 91,2081-2089. ANDERSON, D. L., and MOSH.4RR.4Fh4, E. T. (1968). Physiology and biological propert.ies of phage 429 deoxyribonucleic acid. J. r’irol. 2,11X5-1190. BRADLEY, D. (1967). Ultrastructure of bact.eriophages and bact,eriocins. Bacterial. Rev. 31, 230314. BOTT, K., and STR.IUSS, B. (1965). The carrier state of Bacillus subtilis infected with the transducing bacteriophage SPlO. ?,Ti;ro20gy 25, 212-225. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of virus. Biochipn,. Biophys. Acfa 34, 103-110. BRITTEN, R, J., and KOHNE, D. E. (1968). Repeated sequences in DN.4. Science 161, 529-540. CHOXV, L. T., B~ICE, L., and D.IVIDSON, N. (1972). Map of the partial sequence homology between DNA molecules of Bacillus sublilis bacteriophages SPO2 and 6105. J. .Ilol. Bid. 68.391400.
415
121
DAVIS, R. W., and HYMBN, R. W. (1971). A study in evolution, the DNA base sequencehomology between coliphage T7 and T3. J. Mol. Biol. 62, 287-301. DENH.~RDT, D. T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. DUBIN, S. B., BENEDSK, G. B., B.INCROFT, F. C., and FREIFELDER, D. (1970). Molecular weights of coliphages and coliphage DNA. II. Measurement of diffusion coefficients using optical mixing spectroscopy and measurement of sedimentation coefficients. J. Mol. Biol. 54,547-556. FREIFELDER, D. (1970). Molecular weight.s of coliphages and coliphage DNA. IV. Molecular weight,s of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J. Mol. Biol. 54,567-577. HAGEN, E. W., ZEECE, V. RI., and ANDERSON, D. L. (1971). A genetic study of temperaturesensitive mutants of the Bacillus subtilis bacteriophage 429. Virology 43, 561-568. HOCH, J. A., and SPIZIZEN, J. (1969). Genetic cont.rol of some early events in sporulation of Bacillus subtilis 168. In “Spores IV” (L. L. Campbell, ed.), pp. 112-120. American Society for Microbiology, Bethesda, Md. ITO, J., and SPIZIZEN, J. (1971). Abortive infection of sporulating Bacillus subfilis 168 by @2 bacteriophage. J. Ti’rol. 7, 515-523. ITO, J. (1973). Pleiotropic nature of bacteriophage tolerant mutant,s obtained in early blocked asporogenous mutant.s of Bacillus subtilis 16X. Mol. Gen. Genet. (in press). ITO, J., and SPIZIZEN, J. (1971). Increased rate of asporogenous mutations following treatment. of Bacillus subtilis spores with ethyl methane sulfonate. Mufat. Res. 13, 93-96. ITO, J., MEINKE, W., H.~TH.I~.~Y, G. M., and SPIZIZEN, J. (1972). Cont,rol of bacteriophage +15 in sporulating Bacillus sublilis 168 during development. Ba.cferiol. Proc., 1972, 318. K~~.~KAMI, M., and LANDMAN, 0. E. (1968). Nature of the carrier state of bacteriophage SPlO in Bacillus subtilis. J. Bacterial. 95, 1804-1812. LAEMMLI, U. K. (1970). Cleavage of structural prot,eins during the assembly of t,he head bacteriophage T4. Nature (Londo,~) 227, 680-685. LAMERS, K., PUTNET, F., STEINBERG, I. Z., and SCH.~CHM.~N, H. K. (1963). Ultracentrifuge studies with absorption opt,ics. 3. A split.-beam photoelectric, scanning absorption system. Arch. Biochem. Biophys. 103, 379400. LEIGHTON, T. J., and DOI, R. H. (1971j. The stability of messenger ribonucleic acid during sporulat,ion in Bacillus mtbtilis. J. Biol. Chem. 246, 3189-3195. LOSICB, R.,and SONENSHEIN, A. L. (1969j. Changes
122
ITO
in the template specificity of RNA polymerase during sporulation of Bacillus subtilis. :\*aturu (Lodotij 224, 35-37. MURMUR, J.? BR.XNDC)N, C., NEUFORT, S., ERLIBH, M., and KONVICK.~, J. (1972). M., MANDEL, Unique propert.ies of nucleic acid from Bacillus subtilis phage SP-15. Suture (London) New Biol. 239, 68-70. MARMUR, J., and DOTT, P. (1962). Determination of the base composition of deoxyribonucleic acid from it.s thermal denaturation t,emperature. J. Jfol. Biol. 5, 109-11X. MANDEL, J. D., and HERSHEY, A. D. (196Oj. 4 fractionating column for analysis of nucleic acids. Anal. Biochem. 1.66-77. MEINKE, W.? a.nd GOLDSTEIN, D. A. (1971). Studies on the structure and format.ion of polyoma DNA replirative intermediates. J. Mol. Biol. 61, 543563. M~NDEZ, E., RAM~REZ, G., &L.XS, M., and ~I~QJELI, E. (1971j. Structural proteins of bacteriophage $29. Virology 45, 567-576. OaTfN, J., VI~UI:LI, E., Sa~.is, M., andVasqu~:z, C. (1971j. DN.4 protein complex in cirrular DNA from phage $29. Na.ture (London) NM Bid. %34, X5-27i. RFILLY, B. E. (1965). Astudyof the bacteriophages of Bacillus subtilis and their infect.ious nucleic acids. Ph.D. Dinsertdiotl, Department of hZicrobiology, Western Reserve Universit.y, Cleveland, Ohio. ROMIG, W. R. (1968j. Infectivity of Bacillus nubtilis bact,eriophage deoxyribonucleic acid extracted from mature particles and fromlysogenic host.s. Bacterial. Ror. 32, 349-357. ROTH, T., and H;\T.~sHI, M. (1966). Allomorphic forms of bacteriophage +X174 replicative DN.4. Scirnrc 154, 658-660. SCHILDKR.~UT, C. L., MAR~~UR, J., and DOTT, P. (1962). Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J. Uol. Biol. 4,430343. SH.~PIRO, A. L., T’IRUELA, E., andMAIzEL, J. V. JR. (196ij. Molecular weight estimation of poll= pept,ide chains by elect.rophoresis in SDSpolyacrylamide gels. Biochem. Biophys. Res. Conlir/.un. 28, 815-820. SHI~IIZU, N., MIURI, K., and AOKI, H. (1970). Ch aracterlzation ’ of Bacillus subtilis barteriophage. II. Isolation and morphology of phage Nf and properties of its DNA. J. Biochem. 68, 277-286.
ET rl I, X. L.,and Roscor,:, I). H. (1969). The course of phage $e infection in sporulating pells of Bacilllcs wbtilia strain 3610. TYiro20gy 39, 26b276. QPIZIZEN, J. (1958). Transformatiorl of bicwhemitally deficient strains of Bacillus ntrbtilis by deoxyrihonucleatc. Prw. .\‘at. Acad. Sci. I’S 41, 1072-10X STUDIER. F. W. (19653. Sedimentation studies of the size and shape of DNA. J. iVo/. Biol. 11, 373-390. SZYB.1LsK1, W. (196Sj. Use of cesium sulfate for equilibrium density gradient, cent.rifugabion. I,a “hlet.hods in Enzymology” (L. (Grossman and K. Moldave, eds.), Vol. 12, Part B, pp. 330-360. Academic Press, Inc., New York. T~K.\H.~~HI, I. (1963 j. Transducing phage for Bacillus subtilis. J. Get?. Microbial. 31, 211-217. TIH.~HAJHI, I. (1964). Incorporation of bacteriophage gennme by spores of Bacillmn wblilis. J. Barferiol. 87, 1499-1502. T.\MIR, H., and GILV.IRCI, G. (1966). 1)ensit.y gradient centrifugation for the separation of sporw lating forms of bacteria. J. Biol. Chenr. 241, 1085-1090. THOMAS, C. A. JK., and hI.wH.%TTIE, L. A. (1967). The anat.omy of viral DNA molecules. d )o(. Ret,. Biorhena. 36, 485-518. THORNE, C. B. (1962). TransducCion in Bacillrcs subtilis. J. Baderiol. 83,106-111. THORNE, C. B. (1968). Transduction in Bacillus cereus and Bacillus owthraris. Barterid. Reu. 32, 358-361. VAN ALSTYNE, L)., (:R.~NT, G. F., and SIMON, M. (1969). Synthesis of bacterial flagella: Chromosomal synchrony and flagella synthesis. J. Bacterial. 100, 2R3-28i. VINOGR.~D, J., BRUNER, K., KENT, R., and WEIGLE, J. (1963). Band centrifugation of macromolecules and viruses in self-generating density gradients. Proc. -Vat. Aca.d. Sci. US 49,902-910. WEBER, K., PRINGLF,, J.R.,~~~OSBORN, hi. (1972). hleasurement, of molecular weights by elect.rophoresis on SDS-acrylamide gel. IIL “Methods in Enzymology” (C. H. W. Hira and Serge N. Timasheff, eds.), Vol. 26, Part C, pp. 3-27. Academic Press, Inc., New York. YEHEL, C. O., and DOI, R. H. (1967). Differential expression of bacteriophage genomes in vegetative and sporulating cells of Bacillus subfilis. J. T’irol. 1, 935-947. YONF:N~HC:IN,
VIROLOGY
(1973)
Studies JUNETSU Department
on Bacillus
subtilis
MEINKE,
GARY
ITO, WILLIAM of Microbiology,
Scripps
Clinic
Bacteriophage HATHAWilY,’
a.nd Research
Foundation,
415 AND
La Jolla,
JOHN
SPIZIZEN
Calijornia
9%OSY
Accepted August 8, 19YS The growth characteristics and physicochemical properties of Bacillus sztbtilis bacteriophage 415 have been investigated and compared with +29. Differences in growth characteristics of t.hese phages were found in various stages of spore germination and outgrowth which may account for the absence of plaque formation by 615 on wild-type cells. Both 415 and 429 genomes were incorporat.ed into spores when cells were infected at a certain critical time during host cell development.. 415 is morphologically indistinguishable from 429. The DN.4 of $115 is a linear duplex molecule having a mean length of 6.1 pm. The S value of this DNA is 23 & 0.4 S. These values correspond to a molecular weight of approximately 12 X lo6 daltons. At least seven structural proteins of 615 were recognized in SDS-acrylamide gel electrophoresis. The sum of the molecular weights of 615 proteins is approximat,ely 354,000 daltons. This corresponds to about 595 of the coding capacity of doublestranded DNA of 415. 415 DNA was found to hybridize with $29 DNA.
genous mutants (Reilly, 1965; Hoch and Spizizen, 1969; It,o and Spizizen, 1971). In an effort to determine the basis of this selectivit,g, we initiated a study of 415. In this report,, some of the growth characteristics as well as physicochemical properties of 915 and its DNA will be described. A preliminaq report of some of these studies was presented and Spizizen, uto, Bleinke, Hathaway, 1972).
INTRODUCTION
Certain phage-host interactions in Baci1.h have been found to provide useful systems for investigations of cell development. Thus, infection of sporulating cells by some virulent phages results in the incorporation of phage genomes into spores, whereas phagc multiplication takes place in exponentially growing cells. Bacillus subtilis phages /33and 4e (Yehel and Doi, 1967; Sonenshein and Roscoe, 1969) are examples of this relationship. These studies led to the finding that alt,erations in the template specificity of bacterial RNA polymerase occur during sporulation (Losick and Sonenshein, 1969). Similar effects have been observed for the related phages $15 and $29, two of t,he smallest known phages containing doublestranded DNA (Reilly, 1965 ; Anderson, Hickman, and Reilly, 1966; Szybalski, 1968). Phage 915, unlike +Y9, does not produce plaques on wild-type Bacilbus subt~ilis and late-blocked asporogenous mutants, but does plaque on certain early blocked asporo’ Department of Biochemistry, California, Riverside, California.
University
of
MATERIALS
@l 193 by Academic Press. of reproduction in any form
Inc. reserved.
METHODS
Bacteria and pkge. Bacdlus subtilis 168 (trpCd), SR-32 (spoAl2 IrpCd), and Bacillus amyloliquefaciens H were used in this study and described previously (Ito and Spizizen, 1971). Strain SCRlOO (spodlb Zeu- and SCR114 (spoAl2 thyX1 t,hTl2) were con&ructed by PBS-1-transduction (Takahashi, 1963). Recipient cells were BR&% (trpC2 Zeu-) and B. subtil~is 168 th;y-(trpC2 thyX1 thyYl)? which were provided by Drs. Reilly and Anagnostopoulos, respectively. 31edia. Minimal medium was described previously (Spizizen, 1958). Penassay broth [antibiotic medium no. 3 (Difco)] and tryp-
110 Copyright All rights
BND
BACTER.IOPHAGE
tose blood agar base (Difco) were used for t#he propagation of bacterial strains. Plating conditions. Bacteria were plated on TBAB plates and phages were plated on minimal medium agar supplemented with casein acid hydrolysate (0.05 %) and 5 X lo-” di MnC12 with 3 ml of soft agar composed of t,ryptone (Difco), 10 g; NaCl, 8 g; agar (Difco) 0.6 ?J; glucose, 6 g; per liter of dist#illed water. Sporulation medium. Sporulation medium was essentially t,he same as that of Schaeff er’s medium modified by Leighton and Doi (1971). The medium consists of Difco INtrient brot#h (16 mg/ml), 2.5 X lo-* llrl KCl, 2 X lo+’ M CaC12, 1 X lo+’ II1 FeC12, 1 X lo+ Jf RInS04, 1 X 1w4 M lJIgSO4, and 5 X 1O-3M glucose. Preparation. of spores. Bacillus subtilis 168 was grown in sporulation medium at 37 C for 70 hr. Spores were collected by centrifugation, washed once with water, and resuspended in 0.01 M Tris-HCl (pH 7.5) buffer. The spore suspension was then heated for 10 min at 80 C. After cooling to room temperature, spores were treat,ed with lysozyme (20 pg/ml) and deoxyribonuclease I (DNase I) (5 pg/ml) for 20 min at 37 C. Spores were washed again with water and further purified by sedimentation kough Renografin gradients, according to the modified method of Tamir and Gilbarg (1966), (Van Alstyne, Grant, and Simon, 1969). Purified spores were stored in water at 4 C. Preparation and pw$catim of r#d5 aid 429. Unless otherwise st,ated, Bacillus amyloliquefaciens H was used as a host in preparation of lysates of both 415 and #29. Cells were grown in 2.8 1 flasks containing 500 ml Penassay broth supplemented with 0.5 % glucose to a final concentration of 2 X 10’ cells/ml. The cells were pelleted by centrifugat,ion and then resuspended in adsorption medium (0.1 111NaCl, 10-*ill MgS04 in 0.1 ,I;C pot#assium phosphate buffer at pH 7.0). The bacteria were mixed wit,h eit,her 415 or $29 to a multiplicit,y of 5. After 10 min at room temperature the phage-bacteria complex was resuspended in fresh Penassay broth containing 0.5 % glucose to the original volume and shaken at 37 C. Usually after 2 hr the cells were completely lysed. Lysat’es were centrifuged at 80009 for 30 min to remove
415
111
cell debris. The supernatant fluid was treated with DNase (5 pg/ml) and pancreatic RNase (5 pg/ml) at 37 C for 20 min. Phage particles were sediiented by centrifugation at 25,000g for 2 hr at 4 C in a 19 rotor of the Spinco Model L2 preparative ultracentrifuge. Phage pellets were resuspended in phage adsorption medium and centrifuged for 30 min at SOOOgat 4 C. Solid CsCl (the Harshaw Chemical Co., Soton, Ohio) was added to the concent’rated phage suspension until the refractive index reached 1.375. The mixture was then centrifuged for 48 hr at 70,OOOg in the SW5OL rot’or in a Spinco L-2 preparative ultracentrifuge. The peak fractions, with a density of 1.45 g cm+, were pooled and dialyzed against adsorpt.ion medium overnight at 4 C. A rapid purification of phage particles could also be achieved by centrifugation in a three-layered gradient of CsCl as follows. The bottom layer was 1.5 ml of a CsCl solution (1.5 g cme3) ; the middle layer, 1.0 ml (1.3 g cme3) and bhe top layer, 0.5 ml (1.1 g cmd3). Phage suspensions (1 .Oor 1.5 ml) were layered on top and tubes centrifuged in the SW-50L rot,or for 3 hr at 70,OOOg.The phage band was collected t’hrough a hole in the bottom of the tube. Phage particles were diluted with adsorption medium and centrifuged in a 50 Ti rotor at. 80,OOOgfor 30 min at 4 C. The phage pellet was resuspended in phage adsorption medium. Preparation 0j ~raclioadive 415 and +29. Strain SCR 100 (spodld leu-) was grown in minimal casein acid hydrolysate medium consisting of Spizizen’s minimal salt, 0.1 J1 NaCl, lo-* M RInC12 , 10e3 M RIgCl2 ,0.5 ‘% glucose, 20 pg/ml n-leucine, and 0.05 % casein acid hydrolysate (Difco). Cells were grown to about 1 X 10” cells/ml, centrifuged, and resuspended in phage adsorption medium (10 X cone). Phage (either 915 or @29) was added to cell suspensions at a multiplicity of 10 and incubated for 10 min at room t,emperat,ure. The bacterium-phage complex was inoculated into medium consisting of minimal casein acid hydrolysate (above) except that the amount of L-leucine was reduced to 3 pg/ml and [3H]leucine (5 &%/ml, 5 Ci/nid1) or [14C]leucine (1 #Zi/ml, 311 Ci,/mili) added. Infected cells were shaken at 37 C for 2 hr. Radioactive phage
112
ITO ET AL.
particles were isolated and purified as described above. Preparation of 3H-labelecl 415 DNA. Strain SCR114 (spoAl.2 thyX1 thyY1) was grown to 1 X IO8 cells/ml in mmlmal casein acid hydrolysate medium (above) supplemented with 5 pg/ml of [3H]thymidine (15 pCi/mg thymidine), infected with 415 at a multiplicity of 5, and shaken for 3 hr. 3H-415 was purified as described above. DNA was extracted from phage by a phenol method (Mandel and Hershey, 1960). Preparation of 32P-labebed 415 and $829. Bacillus amyloliquefaciens H was grown in low phosphate broth containing 20 PCi of 32Pper ml. Dephosphorylated brot.h was prepared as described previously (Ito and Spizizen, 1971). When the cell density reached 1 X 10” cells/ml cultures were infected with 415 or 429 at a multiplicity of 10, and shaken for 3 hr at 37 C. “P-labeled phage particles were collected and purified as described. [32P]DNA was extracted twice with phenol. The aqueous layer was dialyzed extensively against standard saline citrate (SSC) in 0.01 111Tris-HCl buffer at pH 7.0. Electron microscopy-~15 DNA. Samples of 415 DNA were prepared for electron microscopy as described previously (Rleinke and Goldstein, 1971). Electron micrographs were taken with a Hitachi HU-11 electron microscope. Contour length of 915 DNA molecules were measured w&h a Keuffel and Essen map measurer from tracing of projected negatives. Magnifications were determined by photographing a standard grating replica (Ladd). 415 P&ides. $15 was negatively stained with 1% potassiumphosphotungstate (W,/Vj (Brenner and Horne, 1959). Electron micrographs and magnifications are as described above. Determination of molecular weight by seclimentation velocity. Band sedimentation was performed in 1 M NaC1, pH 7 solvent essentially as described by Studier (1965). Samples of 415 DNA (10 pliters) in 1 X SSC were pipetted into the filling holes of double sector centerpieces (Vinograd et al., 1963) and layered at low speed on the 1 M NaCl solvent. Centrifugation at 88,000 rpm and 20 C was commenced after the disappearance of the initially sharp refractile band.
R.ecordings were made at 4-min int,ervals using the split-beam mode of the phot80elect,ric detector and scanner (Lamers et al., 1963) lvith t,he monochromator set, at 265 nm. Molecular weight was estimated using t#he equations of Freifelder (1970) and t,he sedimentation coefficients calculated from peak positions of the absorption tracings. Sucrose gradient centrifugalion. Linear (520%) sucrose gradients were made by dissolving sucrose in 0.01211 Tris-HCI buffer at pH 7.5 containing 1 M NaCl and 10d3 M EDT-4 (neutral gradients) or by dissolving sucrose in 0.9 II1 NaCl, 0.1 M NaOH, and 10e3 111 EDTA (alkaline gradients). Samples (0.1 ml) were centrifuged in a SW5OL rotor in a Spinco Model L2 ultracentrifuge at 73,000g for 2 hr. To measure the radioactivity in a gradient fraction, seven drops per fraction were collected on 2.0 cm Whatman glass filter paper. Aft,er drying, the filters were treated with 10% cold trichloroacetic acid (TCA) and then with ethanol (95 %). Air dried filters were placed in vials containing 3 ml of toluene-Omnifluor (New England Nuclear) fluid and counted. Equilibrium
Ceretrifugation in CsCl
(a) Phage particles. Solid CsCl was added to phage suspensions and refractive indexes were adjusted to 1.3745. (b) 415 DNA. The refractive index was adjusted to 1.3944 by addition of solid CsCl to DNA solutions containing 5 pg [3”P]915 DNA and 5 pg [3H]BacilZus subtilis DNA (p = 1.7034) as a density reference. Solutions were cent#rifuged for 48 hr at 70,OOOgat 20 C in a SW5OL rotor. Ten drops per fraction were collected into tubes and portions (50 Jiters) assayed for radioactivity and for measurement of densit,ies. Optical melting of 915 DNA. DNA melting profiles were done in a Gilford 2000 continuous recording spectrophotometer equipped with automatic temperature and blank compensation. DNA solutions were pretreated to 50 C to remove dissolved air, then capped with Teflon stoppers before being placed in the sample holder. The melting profile of 25 E.rg/ml of 41.5 DNA was performed in SSC buffer, pH 7.0. The temperature was increased 0.5 C every 7 min.
BACTERIOPHAGE
DNA-DNA hybridization. A modification of the method of Denhardt (1966) was employed as described by Aloni, Winocour, Sachs, and Torten (1969). Polyacrylamide gel electrophoresis of stmctural polypeptides of $15 a&, 429. Purified 415 were suspended in 0.02 M Tris-HCI buffer, pH 7.5 and made 1% (v/v) with sodium dodecyl. sulfate (SDS) and 2-mercaptoethanol. Phage suspensions were heated for 5 min in boiling water and cooled to room t,emperature. SDS-polyacrylamide gel electrophoresis was carried out in 10 % gels according to the methods of Laemmli (1970). Electrophoresis was at room temperature (2 mA per tube) using a buffer containing 0.025 A1 Tris, 0.192 Jr glycine, and 0.1% SDS, pH 8.3. Prior t.o staining, SDS was removed from gels by dialysis overnight against 7 % acet,ic acid. Gels were then immersed for 4 hr in a solution of 0..25 % Coomassie brilliant blue, freshly made in 50 % methanol and 7 ‘% acet#ic acid. Gels were destained overnight against several changes of 30 % met#hanol in 7 % acetic .acid an.d stored in 5 % methanol in 7 4”o acetic acid. Estimation of molecular weigh.ts of polypeptide chaim. The molecular weights of the polypeptide chains were determined by SDS-polyacrylamide gel electrophoresis according to the method of Shapiro, Venuela, and Maize1 (1967). Standard proteins used were muscle phosphorylase a (MW, lOO,OOO), bovine serum albumin (MW, 68,000), ovalbumin (RlW, 43,000), t’rypsin (MW, 23,800), lysozyme (MW, 14,300), cytochrome c (RIW, 11, 700) (Weber, Pringle, and Osborn, 1972). Standard proteins were heated at 100 C for 5 min in 1% SDS and 2-mercaptoethanol and subjected to electrophoresis as above. Chemicals. [h,Iethyl-3H]thymidine (51 Ci/ mM>, and H33”P0, , carrier-free, were obtained from New England Nuclear Corp. RESULTS
Growth characteristics of 415. One-step growth curves of 915 on Bacill,us subtilis 168 and an early blocked asporogenous mutant (spoAl2) are shown in Fig. 1. The latent period of 415 in spoAld strain at 37 C was 45 min and the burst size was about 300. Similar results with a slightly higher burst
113
$15
Time
(mid
FIG. 1. One-step growthof phage+l5 on Bacillus subtilis 168 and spoA12. The bacteria were grown in Penassay broth (Difco) t.o 2 X lo8 cells/ml, centrifuged, and resuspended in an adsorption medium. The bacteria were mixed with 415 at a multiplicity of 0.1 as determined by previous titration of the phage on Bacillus amyloliquefacierls H. After a lo-min incubation period to allow for adsorption, t.he cult,ure was centrifuged and diluted to 10mi into prewarmed brot.h containing 0.5’$Zoglucose and incubated at 37 C wit.h aeration. At indicated times, samples were withdrawn and assayed for the number of infective centers on B. amyloliquejaciens H.
size were obtained when Bacillus amyloliquefaciens H strain was used as host. During this study, it was often observed that some active 415 particles were produced in B. subtilis 168, although plaques were not seen. The burst size of 415 was about 4 (Fig. 1). This value, however, varies from experiment to experiment. In order to study $15 infection of B. subtilis 168 in detail, it was necessary t,o establish a relatively simple but highly reproducible svstem. We have found that infection follo&g spore germination and outgrowth can be pyrformed under reproducible conditions. Spores purified by Renografin gradient centrifugation (Tamir and Gilvarg, 1966) are germinated in sporulation medium and endospores were subsequently formed 8-9 hr after
IT0
114
the inoculation. However, the t.iming of sport formation depends on the size of the inoculum. Figure 2 shows the time course of 41s infection following germination and outgrowth of spores of B. subtilis 168. At. selected times. indicat,ed by the arrows, 415 was added and the optical absorbancy followed. Cells were lysed to a limited extent when infection occurred during the first 120 min of germination, but after 120 min cells were no longer susceptible to lysis. However, 415 adsorbs to these cells and t,hey are capable of forming spores which contain the phage genomc. Similarly, we have found that, qG9 DNA4 is also incorporated into spores when infection oscurs at a lat,er stage of cell development. The phage genome can bc recovered upon germination of such spores. Figure 3 shows grow’th patterns of $15 and +29 whose genomes were incorporated into spores during germination. The latent periods of both phages were 55 min. Sonenshein and Roscor (1969) reported the existence of a crit,ic.al period in host de-
240
300
360
Time (mid
FIG. 2. Infection of 415 on germinat.ed spores of Bacillus subtilis 168. Spores (1 X l@!ml) were inoculated into 20 ml of sporulation medium in 250 ml naphalo culture flasks and shaken in water bath at 3i C. At the times indicated by the arrows, +15 was added (2 X 10g;‘ml) and the optical density was read with a Klett Summerson calorimeter equipped with a 66 filter
h’7’ Al.
Time
(mid
FIG. 3. Recovery of active phages incorpornt,ed into spores upon germination. Spores with trapped +15 or $29 genomes were inoculated into Penassay broth containing 0.5(“. glucose and shaken at 37 C. At indicated times, samples were wit.hdraxn and assayed for t,he number of infective centers on B. ar,~yloliq~cqfaciarrs H.
velopment,al processes during lvhich phage genomts are trapped by spores. We have also determined the optimum t,ime of infect,ion for incorporation of intact 415 DNA into spores by infecting cells at different stages of growt,h. Inf&ed cultures were incubated for 20 hr to allow for completion of spore formaGon. Spores wrs harvested, xvashed, and neutralized with phage antiserum. The percentage of spores n-hich incorporated phage DN.\ was then determined (Fig. 4). The opt,imum time of infect,ion for incorporat8ion of +l5 DNA int,o spores was 3 hr aft.er germination. This time is significantly earlier as compared to t,he +e system which \vas 5-6 hr aft.er t,he end of logarithmic growth (‘7% S.5 hr aft,er inoculation) (Sonenshein and Roscoe, 1969). Compcwison of bwst size of415 and 429. To understand the differences in growth of 015 and 429 and to examine t,he relationship between phage multiplication and incorporation of phage genomes into spores, the avcrage phagc burst size \vas determined in
BACTEKIOPHAGE
0 0
2
4
6
8
10
Time(hours) FIG. 4. Optimum t.ime of infection for incorporation of the +15 genome into spores. Spores were inoculated (1 X 108spores ‘ml) into200 ml of sporulat,ion medium and shaken in 2 lit.er side-armed flasks at 37 C. At the indicated times, 10 ml samples were removed and mixed with $15 (1 X 109 phages’ml). The mixture was shaken at 37 C for 24 hr in 125 ml flask. The spores were harvest,ed, treated with Iysozyme, washed with water, and determined for tot,al spores and infective centers of @15.
single-step growth experiments for cells infected at different stages of growth. In this experiment, fewer spores (5 X lo6 spores/ml) were inoculated into sporulation medium. ,Uhough the appearance of endospores was delayed about 80 min, better synchronization n-as achieved. A high burst size of both phages was obkned in midlogarit~hmic phase cells but t#heyield of 415 then decreased more ra,pidlg when cells at, later stages were infected than that of @29 (Fig. 5). An accurate estimate of burst, size cannot be made because midlogarithmic st#age B. subtilis cells tend to form long chains. Thus, the dat,a indicat,e onlv relative burst size values. The diffrrences’in average burst size of 415 and $29 ma\- account for the fact, t,hat 415 cannot,
115
415
2
4
Time
6
8
10
(hours)
FIG. 5. Average burst size of phage ~15 and $29 for infection at various t.imes during growth of Bacillus subtilis 168. Purified spores were inoculat,ed into 150 ml of sporulat,ion medium containing O.lO;Oglucose (5 X 106,/ml spores). At the indicated times 5 ml samples were removed. -4fter the optical density was read with a Klett Summerson calorimeter equipped wit,h a 66 filter, the culture was used for single-step growth of 615 and $29. Single-step growth experiments were carried out as described in Fig. 1.
form plaques on \vild type or late-blocked asporogenous mutan@ while @29 forms plaques under convrrkional plating conditions. If the decrease in phage burst, size is due to changes in t,he template specificity of bacterial RNA polymerase as demonstrat,ed by Losick and Sonenshein (1969) for de, t#hen we must consider the possibility that host RNA polymerase has different t,emplate specificities for 415 and 629 DNA. Preliminary studies indicate that, host RNA polymerase, isolated at different stages of cell growth, does not distinguish 41.5 DNA from 629 DN-k in llitro. These studies will be presented in a separate publication. These observations
on the
difference
behxveen
415
116
ITO ET -AL.
and @29led us to characterize 415 in greater detail. Morphologica. appea:ralece of 415. Typical electron micrographs of 415 are shon-11in Fig. 6. The phage is morphologically veq similar to $29 (Anderson et ab., 1966) and t#o Nf (Shin~zu et al., 1970). 415 has a hexagonal head with a flattened base and is characterized by head projections. The head is approximately 45 nm long and 33 11111 wide. The tail is 30 nm long and complex. When the tail is dissociated from t,he head it appears to contain two collars at the upper end and a dist’al knob at the lower end. Several tail appendages are seen at#tached near the tail collars. Twelve t,ail appendages have been shown for phages 629 and Nf (Anderson et al., 1966; RKndez et al., 19Tl; Shimizu et al., 1970). The exact number of appendages of 415 are not, known at, present, but there appears to be at least ten. Sedimentat.io?l am1 buoyant density of $115. Purified 415 particles sediment as a single component in sucrose gradients. 415 has a sedimentation coefficient of 260 S, relat,ive
t,o T7 phage which sediments at 453 d (Dubin ct a.l., 1970). A value of 257 S 1la.s been reported for $29 by Undez et al. (1971). The buoyant density of 415 is est,imated as 1.155 g crnm3by CsCl equilibrium centrifugatAion (Fig. 7). Properties of 415 DNA. Purified 415 was made with 1% SDS and the DNA released by heating at 50 C for 10 min, followed by rapid cooling. DNA was treated with DNase free pronase (Ca.lbiochem, 30 pg/ml) for 20 min at 30 C and bhen ext,racted twice &h phenol. 415 DNA showed a sharp boundaq when centrifuged in 1 31 NaCl. The sedimentation coefficient at infinit,e dilution was obtained as s:~,,~ = 23.3 f 0.4 S by extrapola.tion of plot by the reciprocal of sedimentat,ion coefficient to concentration of DNA. When this value of sZo.,~~ is substit,ut,ed in t,he equation given by Freifelder (1970), s~~.,~,= 2.8 + 0.00834 Al 0.478, the RIW of 415 DNB is 11.9 f 0.24 X lo6 daltons. In neutral sucrose gradients 415 DNA sediment,s at, about, 23 S (Fig. 8) relative to 4X17-1 RF1 DNA which sediment,s at, 21 S
Fro. 6. Bacteriophage $1.5 negatively stained with potassium phosphotungstate. (a) Empty virions j wit.h tail and tail appendages. (bj Empty virions wit,h head fibers. (c) Intact phage. Cd) Tail wit,h appendages. (e) Empty virions, empty head, tail, and tail appendages.
BACTERIOPHAGE
415
117
daltons/pm). These results are in good agreement with the molecular weight determined by velocity sedimentation and together they indicate that the molecular weight of $115 DNA is about 12 X lo6 daltons. Nucleotide composition of 415 DNA. 415 DNA bands as a single peak in CsCl density gradients and has a buoyant density of 1.6917 g cmd3. Using the equation = 1.66 + 0.098 (G + C) (Schildkraut et al., 1962), the buoyant density corresponds to a G + C content of 35 %#.This value closely resembles those values reported by Reilly (1965) and Szybalski (1968). 1000+m The absorbancy at 260 nm of 415 DNA in 1 X SSC was determined as a function of temperature. The midpoint of the thermal transition (Twx-) was 85.78 C. This corresponds to a G + C content of 40.2 %#(Marmur and Doty, 1962). The difference between the values obtained from the Tm and t,he buoyant density cannot be explained at present, but may be due to the presence of methylated or other unique basis. Reilly (1965) also observed these differences in @29 and 415 DNA. Similar situations have been reported for Nf phage DNA by Shimizu et al. (1970). Structural proteins of $15. Structural proteins of 415 were dissociated and analyzed Fraction No. bv SDS-polyacrylamide gel electrophoresis. FIG. 7. Equilibrium centrifugation of 615 and Figure 10 shows the acrylamide gel patterns 629 DNA in CsCl. Phages [‘4C]@15 and [3H]&9 were mixed and CsCl was added to the density of of proteins of 415. Six proteins were clearly 1.42981. The solutions were centrifuged at 70,OOOg visible and when a large amount of protein an additional band for 48 hr at 20 C in a SWSOL rotor. [‘%]@15: was electrophoresed, “E” was observed between bands E and F. (--O-o-), [3H]@29: (-.-a--).
(Roth and Hayashi, 1966). In alkaline sucrose gradients, single-stranded 415 DNA sediments at 26 S, indicating that 415 DNA does not contain single strand break. Electron microscopy of 415 DNA. The molecular weight of $15 DNA was also determined by electron microscopy. The lengths of the molecules range from 5.6 pm to 6.3 pm (Fig. 9). The mean length is 6.1 pm, which corresponds to an average molecular weight of 12.2 X 10” dahons (using 2 X lo6
r -r--7--
Fraction
No.
Fro. 8. Sedimentation profiles of 415 DNA in sucrose gradient centrifugat,ion. DNA and [“C]C X174, RF1 DN.4 was centrifuged at 73,ooOgfor 2 hr at 20 C.
A mixture of [SH]+15
118
The molecular weights of each polypeptidcs were estimat,ed by comparing their electrophoretic mobilities with t,hose of marker proteins of known molecular weight (Table 1). DNA-DNA hybridization. In addition to the differences in growth characteristics of 41.5 and $29, it was recently shown that 41.5 tolerant mutant,s (toZA) were st,ill sensitive t,o @9 and that $29 tolerant mutants (tolB) were tolerant to bot,h phages (Ito, 1973). Furthermore, it was observed that t,hcse two phages do not easily hybridize irl, Go (Reilly and Ito, unpublished observations). Therefore, the relationship of 915 and 629 n-as examined by DNA-DNA hybridization. The data in Fig. 11 show that, there is some hy-
bridization between $13 and &9 DNA and only a small amount,, if any, hybridization with host, DKA. No atkmpts were made to determine the precise degree of homolog? between 415 and $29 DNA. Such studies can be performed by using reannealing techniques developed by Britten and Kohn (1968) and by the heteroduplex met,hod described by Davis and Hyman (1971). L)ISCU8SION
Reilly (1965) has shown that ~$15does not produce plaques on B. subtilis 168, but can in some asporogenous mutant,s of B. subtilis 168. We have extended his studies and have shown that 415 actually replicates at. certain
BACTERIOPHAGE
stages of B. subtilis 168 growth, particular13 in germinated and outgrowing spores. B. Rutberg has made a similar observation (personal communication). The highest burst size for 41.5 was obtained during the midlogarithmic &age of cell growth. In subsequent stages of growth, the replication of $11.5 is markedly decreased. Similarly, the burst size of 429, which is closely related to 415, is also decreased at later stages of growth. After 8 hr of germination, no intact 415 are produced, but. 429 is produced at a reduced rate. We feel this difference is sufficient to account for the fact that $15 cannot make plaques on the wild type of B. subtilis, while qU9 is able to form plaques under conventional plating conditions (Reilly, 1965). It was demonstrated in our studies that both 41.5 and 929 genomes are incorporated into spores when cells are infected at a ccrtain critical time during host, cell development . Trapping of phage genomes by mature spores has been shown previously for larger virulent Bacillus phages, such as f13 and $e (Yehel and Doi, 1967; Sonrnshein and
119
ml5 TABLE
1
MOLECULAR WEIGHTS OF THE STRUCTURAL POLYPEPTIDES OF 615 Polypeptide A B c D E E’ F
Molecular weight 80,000 71,000 54,ow 46,000 39,ooo 34,ooo 29,ooo
F 32P-DNA added (CPM x 163)
E D C
6 A
FIG. 10. SDS-polyacrylamide of whole 615.
gel electrophoresis
FIG. 11. Hybridization of 3zP-labeled 415 and $29 DNA with unlabeled @15DNA and B. sublilis 16% DNA. A ronstant amount of denatured unlabeled 4115DNA (20 pg) and B. subtilis DN.4 (100 pp) were immobilized on the membrane filters (Sartorius Membrane filter) and hybridized with various amounts of the 32P-labeled +15 and @29 DNA, respectively. [32P]@15DNA (7500 rpm.‘pg IINA) and [3*P]&9 DNA (10,000 cprnpbg DNA) were sheared by sonic treatment and then denat,ured by boiling for 10 min. 415 DNA-[3*P]415 DNA, (-0-o-j; $15 DNA-[32P]429 DNA, DNA-[=P]415 DNA, C-@-O-) ; B. mblilis (-A-A-) ; B. subtilir DNA-[“*PI+29 I )N -4, (-A-A--).
Roscoe, 1969). Incorporation of phagc DNA into spores is a well-known phenomena as a carrier stage in temperate phages in Bacilli systems (Thorne, 1969; Takahashi, 1964; Bott and St,rauss. 1965; Thornr, 196s:
120
ITO ET AL.
Kawakami and Landman, 196s; Romig, 196s). Our findings indicate that the incorporation of virulent phage genomes into spores may be a rather common phenomenon in Bacilli systems. The differences of growth properties between $15 and 429 are not understood. If the reduction of burst size and the incorporation of phage genomes into spores are due to host RNA polymerase changes as demonslrated for 4e (Losick and Sonenshein, 1969), then we must assume that host RNA polymerase has a different template specificity- for 915 and $29. We are currently examlmng this possibility. Preliminary results indicate that host RNA polymerase, isolated at different &ages of cell growth, does not distinguish 415 DNA from @29DNA in ,nitro (unpublished data). It appears that some other factors in addition to RNA polymerase alterat#ions might be involved in determining the burst size and incorporation of phage DNA into spores. Sonenshein and Roscoe (1969) have shown that the optimum time for incorporation of $e DNA into spores is 5-6 hr after the end of logarithmic growth (7.5-5.5 hr after inoculation). Our data (Fig. 4) indicates that, t.he optimum time for trapping 915 DNA is at the end of the logarithmic growth stage (3 hr after inoculation). These differences may be partly due to different experimental conditions, but most likely are caused by the differences of the bacteriophages. Since 415 contains only about one-tenth of the genetic informat’ion of 4e, for example, it is reasonable t,o assume t,hat replication of this phage is highly dependent on host-cell functions. Yehel and Doi (1967) studied the differential expression of two virulent Bacillus phages, P3 and PZZ, in sporulating cells of B. subtilis W23. They showed that Pa-infected cells are able to sporulate, resulting in the incorporation of & DNA. On the other hand, &z-infected sporulating cells are lysed. Therefore, it was suggested that multiplication of phage-DNA or incorporation of phage genomes into spores is due to the ability of the phages t,o repress the expression of host genomes. The morphology of $15 appears to be identical to that of 429 (Anderson et al., 1966; Wndez et al., 1971) and Nf (Shimizu
et al., 19iO). Since no evidence of tail contraction was obtained from elect,ron micrographs, 415 can be considered to belong to group C of Bradley’s classificat,ion of bacteriophages (Bradley, 1967). 415 DNA is a linear molecule having a mean lengt,h of 6.1 pm. This length corresponds to an average molecular weight of 12.2 X lo6 daltons which is in agreement with the molecular weight obtained by sedimentation experiments. Although the DNAs of 929 and Nf have been reported to be nonpermuted, linear duplex molecules, the formation of circular molecules have also been described, suggesting the existence of cohesive ends (Anderson and Xlosharrafa, 1968; Hagen et al., 1971; Shimizu et al., 1970). Attempts were made t’o show the formatsion of circular molecules of 415 DNA in annealing experiments in which DNA was heated and cooled slowly. Full length, linear duplex molecules but no circular molecules were obtained (Rleinke, unpublished data). Recently, Ortin et al. (1971) have demonstrated that a protein is involved in t’he formation of circular 429 DNA. These results would conform to the concept t’hat $15 and 429 DNA are linear, nonpermuted duplex, similar to T3 and T7 DNA (Anderdon and Mosharrafa, 1968; Thomas and MacHabtie, 1967). The disagreement between the G + C content of $15 DNA as determined by buoyant density analysis and from the Tm are not understood at present. Reilly (1965) also observed this anomalous nature of 915 and 429 DNA. These results may be due to the presence of a methylated base or other minor odd bases. Recently, unique physical properties of DNA isolated from SP-15, which is a large, generalized transducing Bacillus subtilis phage, have been reported (Marmur, Brandon, Neufort, Erlish, Mandel, and Konmicka, 1972). This phage DNA has a low melting temperature and a high buoyant density in neutral CsCl. It was demonstrated that the DNA contained a modified pyrimidine, 5-(4’) 5dihydroxypentyl)-uracil which partially replaces t,hgmine. The composition of 415 DNA is under invest,igation. There are at least, seven prot,eins in 915. The sum of the molecular weights of 415 structural proteins is approximately 354,000
BACTERIOPHAGE
daltons. This requires a coding capacity in double-stranded DNA of about 7.1 X lo6 daltons. Since the molecular weight of 915 DNA is 12 X lo6 daltons, this value corresponds to about 59 % of the entire chromosome. Difference in growth characteristics of 415 and @9 indicate t#hat these small phage systems offer a useful experimental system to study evolution on the basis of nucleotide sequence of DNA. Our preliminary DNADNA hybridization experiments clearly indicate a partial homology between t#hesetwo phage DNA. The precise degree of homology between 415 and $29 DNA can be determined by bhe method similar to those described for t,he T3-T7 system (Davis and Hyman, 1971) and for the SPOZ-4105 system (Chow et CL, 1972). ACKNOWLEDGMENTS We are grat.eful to Dr. M. Hayashi for providing us with [1dC]+S174 RF1 DNA. We thank Dr. B. Reilly for his useful discussion. We also t,hank G. Mildner, C. Svensson, and M. Morgenstern for their excellent assistance. This work was partially supported by American Cancer Society Grant (NP-39). REFERENCES ALONI, I’., WINOCOUR, E., S.ZCHS, L., and TORTEN, J. (1969). Hybridization between SV-40 DNA and cellular DNA’s. J. 11fol. Biol. 44,333-345. ANDERSON, D L., HICKM.~N, D. D., and REILLY, B. E. (1966j. Structure of Ba.cillus subtilis bacteriophage &9 and the lengt,h of 629 deoxyribonucleic acid. J. Bacterial. 91,2081-2089. ANDERSON, D. L., and MOSH.4RR.4Fh4, E. T. (1968). Physiology and biological propert.ies of phage 429 deoxyribonucleic acid. J. r’irol. 2,11X5-1190. BRADLEY, D. (1967). Ultrastructure of bact.eriophages and bact,eriocins. Bacterial. Rev. 31, 230314. BOTT, K., and STR.IUSS, B. (1965). The carrier state of Bacillus subtilis infected with the transducing bacteriophage SPlO. ?,Ti;ro20gy 25, 212-225. BRENNER, S., and HORNE, R. W. (1959). A negative staining method for high resolution electron microscopy of virus. Biochipn,. Biophys. Acfa 34, 103-110. BRITTEN, R, J., and KOHNE, D. E. (1968). Repeated sequences in DN.4. Science 161, 529-540. CHOXV, L. T., B~ICE, L., and D.IVIDSON, N. (1972). Map of the partial sequence homology between DNA molecules of Bacillus sublilis bacteriophages SPO2 and 6105. J. .Ilol. Bid. 68.391400.
415
121
DAVIS, R. W., and HYMBN, R. W. (1971). A study in evolution, the DNA base sequencehomology between coliphage T7 and T3. J. Mol. Biol. 62, 287-301. DENH.~RDT, D. T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. DUBIN, S. B., BENEDSK, G. B., B.INCROFT, F. C., and FREIFELDER, D. (1970). Molecular weights of coliphages and coliphage DNA. II. Measurement of diffusion coefficients using optical mixing spectroscopy and measurement of sedimentation coefficients. J. Mol. Biol. 54,547-556. FREIFELDER, D. (1970). Molecular weight.s of coliphages and coliphage DNA. IV. Molecular weight,s of DNA from bacteriophages T4, T5 and T7 and the general problem of determination of M. J. Mol. Biol. 54,567-577. HAGEN, E. W., ZEECE, V. RI., and ANDERSON, D. L. (1971). A genetic study of temperaturesensitive mutants of the Bacillus subtilis bacteriophage 429. Virology 43, 561-568. HOCH, J. A., and SPIZIZEN, J. (1969). Genetic cont.rol of some early events in sporulation of Bacillus subtilis 168. In “Spores IV” (L. L. Campbell, ed.), pp. 112-120. American Society for Microbiology, Bethesda, Md. ITO, J., and SPIZIZEN, J. (1971). Abortive infection of sporulating Bacillus subfilis 168 by @2 bacteriophage. J. Ti’rol. 7, 515-523. ITO, J. (1973). Pleiotropic nature of bacteriophage tolerant mutant,s obtained in early blocked asporogenous mutant.s of Bacillus subtilis 16X. Mol. Gen. Genet. (in press). ITO, J., and SPIZIZEN, J. (1971). Increased rate of asporogenous mutations following treatment. of Bacillus subtilis spores with ethyl methane sulfonate. Mufat. Res. 13, 93-96. ITO, J., MEINKE, W., H.~TH.I~.~Y, G. M., and SPIZIZEN, J. (1972). Cont,rol of bacteriophage +15 in sporulating Bacillus sublilis 168 during development. Ba.cferiol. Proc., 1972, 318. K~~.~KAMI, M., and LANDMAN, 0. E. (1968). Nature of the carrier state of bacteriophage SPlO in Bacillus subtilis. J. Bacterial. 95, 1804-1812. LAEMMLI, U. K. (1970). Cleavage of structural prot,eins during the assembly of t,he head bacteriophage T4. Nature (Londo,~) 227, 680-685. LAMERS, K., PUTNET, F., STEINBERG, I. Z., and SCH.~CHM.~N, H. K. (1963). Ultracentrifuge studies with absorption opt,ics. 3. A split.-beam photoelectric, scanning absorption system. Arch. Biochem. Biophys. 103, 379400. LEIGHTON, T. J., and DOI, R. H. (1971j. The stability of messenger ribonucleic acid during sporulat,ion in Bacillus mtbtilis. J. Biol. Chem. 246, 3189-3195. LOSICB, R.,and SONENSHEIN, A. L. (1969j. Changes
122
ITO
in the template specificity of RNA polymerase during sporulation of Bacillus subtilis. :\*aturu (Lodotij 224, 35-37. MURMUR, J.? BR.XNDC)N, C., NEUFORT, S., ERLIBH, M., and KONVICK.~, J. (1972). M., MANDEL, Unique propert.ies of nucleic acid from Bacillus subtilis phage SP-15. Suture (London) New Biol. 239, 68-70. MARMUR, J., and DOTT, P. (1962). Determination of the base composition of deoxyribonucleic acid from it.s thermal denaturation t,emperature. J. Jfol. Biol. 5, 109-11X. MANDEL, J. D., and HERSHEY, A. D. (196Oj. 4 fractionating column for analysis of nucleic acids. Anal. Biochem. 1.66-77. MEINKE, W.? a.nd GOLDSTEIN, D. A. (1971). Studies on the structure and format.ion of polyoma DNA replirative intermediates. J. Mol. Biol. 61, 543563. M~NDEZ, E., RAM~REZ, G., &L.XS, M., and ~I~QJELI, E. (1971j. Structural proteins of bacteriophage $29. Virology 45, 567-576. OaTfN, J., VI~UI:LI, E., Sa~.is, M., andVasqu~:z, C. (1971j. DN.4 protein complex in cirrular DNA from phage $29. Na.ture (London) NM Bid. %34, X5-27i. RFILLY, B. E. (1965). Astudyof the bacteriophages of Bacillus subtilis and their infect.ious nucleic acids. Ph.D. Dinsertdiotl, Department of hZicrobiology, Western Reserve Universit.y, Cleveland, Ohio. ROMIG, W. R. (1968j. Infectivity of Bacillus nubtilis bact,eriophage deoxyribonucleic acid extracted from mature particles and fromlysogenic host.s. Bacterial. Ror. 32, 349-357. ROTH, T., and H;\T.~sHI, M. (1966). Allomorphic forms of bacteriophage +X174 replicative DN.4. Scirnrc 154, 658-660. SCHILDKR.~UT, C. L., MAR~~UR, J., and DOTT, P. (1962). Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J. Uol. Biol. 4,430343. SH.~PIRO, A. L., T’IRUELA, E., andMAIzEL, J. V. JR. (196ij. Molecular weight estimation of poll= pept,ide chains by elect.rophoresis in SDSpolyacrylamide gels. Biochem. Biophys. Res. Conlir/.un. 28, 815-820. SHI~IIZU, N., MIURI, K., and AOKI, H. (1970). Ch aracterlzation ’ of Bacillus subtilis barteriophage. II. Isolation and morphology of phage Nf and properties of its DNA. J. Biochem. 68, 277-286.
ET rl I, X. L.,and Roscor,:, I). H. (1969). The course of phage $e infection in sporulating pells of Bacilllcs wbtilia strain 3610. TYiro20gy 39, 26b276. QPIZIZEN, J. (1958). Transformatiorl of bicwhemitally deficient strains of Bacillus ntrbtilis by deoxyrihonucleatc. Prw. .\‘at. Acad. Sci. I’S 41, 1072-10X STUDIER. F. W. (19653. Sedimentation studies of the size and shape of DNA. J. iVo/. Biol. 11, 373-390. SZYB.1LsK1, W. (196Sj. Use of cesium sulfate for equilibrium density gradient, cent.rifugabion. I,a “hlet.hods in Enzymology” (L. (Grossman and K. Moldave, eds.), Vol. 12, Part B, pp. 330-360. Academic Press, Inc., New York. T~K.\H.~~HI, I. (1963 j. Transducing phage for Bacillus subtilis. J. Get?. Microbial. 31, 211-217. TIH.~HAJHI, I. (1964). Incorporation of bacteriophage gennme by spores of Bacillmn wblilis. J. Barferiol. 87, 1499-1502. T.\MIR, H., and GILV.IRCI, G. (1966). 1)ensit.y gradient centrifugation for the separation of sporw lating forms of bacteria. J. Biol. Chenr. 241, 1085-1090. THOMAS, C. A. JK., and hI.wH.%TTIE, L. A. (1967). The anat.omy of viral DNA molecules. d )o(. Ret,. Biorhena. 36, 485-518. THORNE, C. B. (1962). TransducCion in Bacillrcs subtilis. J. Baderiol. 83,106-111. THORNE, C. B. (1968). Transduction in Bacillus cereus and Bacillus owthraris. Barterid. Reu. 32, 358-361. VAN ALSTYNE, L)., (:R.~NT, G. F., and SIMON, M. (1969). Synthesis of bacterial flagella: Chromosomal synchrony and flagella synthesis. J. Bacterial. 100, 2R3-28i. VINOGR.~D, J., BRUNER, K., KENT, R., and WEIGLE, J. (1963). Band centrifugation of macromolecules and viruses in self-generating density gradients. Proc. -Vat. Aca.d. Sci. US 49,902-910. WEBER, K., PRINGLF,, J.R.,~~~OSBORN, hi. (1972). hleasurement, of molecular weights by elect.rophoresis on SDS-acrylamide gel. IIL “Methods in Enzymology” (C. H. W. Hira and Serge N. Timasheff, eds.), Vol. 26, Part C, pp. 3-27. Academic Press, Inc., New York. YEHEL, C. O., and DOI, R. H. (1967). Differential expression of bacteriophage genomes in vegetative and sporulating cells of Bacillus subfilis. J. T’irol. 1, 935-947. YONF:N~HC:IN,