BACTERIOLOGICAL REVIEWS, Dec. 1968, p. 349-357Copyright © 1968 American Society for Microbiology

Vol. 32, No. 4, Pt. 1Printed in U.S.A.

Infectivity of Bacillus subtilis BacteriophageDeoxyribonucleic Acids Extracted fromMature Particles and from Lysogenic

HostsW. R. ROMIG

Department ofBacteriology and Molecular Biology Institute, University of California,Los Angeles, California 90024

INTOODUUCNO................................................................MATERIALS AND METHODS .....................................................

Bacterial and Phage Strains..................................................Media........Preparation of Phage DNA...................................................Transfection and Transformation ...........................................Shearing ofDNA...........................................................Sucrose Gradient Centrifugation ............................................

RESULTS AND DISCUSsioN...................................................Stability ofB. subtilis Lysogenic for SP02.....................................Immunity of 168B Lysogenic for SP02........................................Transfection with Prophage DNA..............................................Comparison of Properties of Prophage DNA and SP02 Phage DNA...............

LITERATru"E CrUED ..........................................................

349349349349350350350350350350354354355357

INTRODUCnIONThe isolation of more than 75 phages active

on transformable strains of Bacillus subtilis hasrecently been reported, but many of these re-ports have proved to represent independent iso-lations of the same phage or closely related phages(17; J. Marmur, unpublished data). On the basisof their relationship with the host cell, B. subtilisphages can be grouped into the following fourclasses. (i) Virulent phages are those which areincapable of establishing a lysogenic associationwith their host. Most of the isolated B. subtilisphages fall into this category (Marmur, un-published data). (ii) Pseudolysogenic phages (2,3, 8, 12, 13, 19, 20) are those which are capableof establishing a carrier state in host culturesand, in several instances, able to act as generalizedtransducing agents. (iii) Defective temperatephages are those which form particles with all themorphological attributes of normal phage butare incapable of replication. All authentic B.subtilis strains thus far examined harbor at leastone kind of inducible defective prophage (7, 18;F. A. Eiserling, Ph.D. Thesis, Univ. of Cali-fornia, Los Angeles, 1964; Eiserling and Romig,Bacteriol. Proc., p. 118, 1964). (iv) True tem-perate phages are those which are capable ofreplication and can lysogenize their host cells.Published reports on phages belonging to this lastclass include only the phage SPO2 isolated byOkubo (11).

Okubo reported some of the properties ofSPO2, including those which led him to classifyit as temperate. We have studied SPO2 in greaterdetail and report here on its biological properties,the nature of its deoxyribonucleic acid (DNA),and its association with the bacterial genomewhich it lysogenizes. Our data are consistent withOkubo's conclusion that SPO2 is temperate, andthat it forms lysogenic associations with B.subtilis similar to those formed by X withEscherichia coli.

MATERIALS AND METHODSBacterial and Phage Strains

B. subtilis 168 was the parent for all strainsused in these experiments (16). Strain 168Bwas derived from 168 by irradiating spores withultraviolet (UV) light and is a double auxotrophrequiring indole and leucine (3). Lysogenic de-rivatives of these strains were prepared bymethods described in a later section.

Bacteriophage SPO2 was isolated from soil byShunzo Okubo and was briefly described in aprevious publication (11). The clear plaquemutant SPO2cl was isolated by UV irradiation ofSPO2. Temperature-sensitive mutants were ob-tained from SPO2cl by the methods describedby Nishihara for SP3 (10).

MediaBacteria were grown and phage were propa-

gated and assayed in the nutrient broth described349

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by Okubo and Romig (11). Media for transfor-mation and transfection were those described byAnagnostopoulos and Spizizen (1).

Preparation of Phage DNAPhage DNA was prepared from concentrated,

purified phage suspensions by phenol extractionaccording to previously published methods (11,12). Bacterial DNA was extracted by the meth-ods described by Saito and Miura (14).

Transfection and TransformationCompetent cells of B. subtilis were prepared

by the procedures of Anagnostopoulos andSpizizen (1), and transformation and transfec-tion experiments were performed by methodspreviously used (11).

Shearing of DNA

Solutions of DNA used in shearing experi-ments were adjusted to a DNA concentrationof 10 ,ug/ml in 0.15 M NaCl-0.015 M sodiumcitrate (SSC). Amounts of 5 ml of these prepara-tions were placed in plastic microcups of a Sor-vail Omninixer, the cups were immersed in anice bath, and the solutions were stirred for 1 hrat speeds indicated in the text. At the higheststirring rate, very little frothing of the solutionswas observed.

Sucrose Gradient CentrifugationAmounts of 0.5 ml of the DNA solutions to be

analyzed were layered onto 28 ml of 5 to 20%linear sucrose gradients made up in 0.01 M tris-(hydroxymethyl)aminomethane buffer and 0.1 MNaCl (pH 7.1). They were centrifuged for 5 to 6hr at 24,000 rev/min at 8 C in the SW25 rotorof a Spinco L2 centrifuge. Fractions from 0.25to 0.75 ml were collected through the bottomof the tube.

Additional experimental procedures are de-scribed in later sections.

RESULTS AND DIscUSSIONCharacteristics of the intact SPO2 particles and

their DNA are presented in Table 1. As can beseen, SPO2 DNA is indistinguishable, by themethods used here, from DNA isolated fromB. subtilis. It was also determined that the buoyantdensity of denatured SPO2 DNA in CsCl isthe same as that of B. subtilis DNA, and likewisegives a unimodal band.

Molecular weight determinations are based onmeasurements of SPO2 DNA isolated frommature particles and prepared for electronmicroscopy by the method of Kleinschmidt,

TABLE 1. Properties of SP02 phage

DNA properties Biological properties

520,w , 29 to 31 Stably lysogenizes B.subtilis 168B

Molecular wt, Lysogenic bacteria are-26 X 106 inducible

Tm, 87 C No detectable trans-duction by SPO2

Density (CsCl), Does not replicate on1.703 g/cc B. subtilis W23

Guanine + cyto-sine, 43%

No "unusual"bases

Lang, and Zahn (9). These measurements andall other electron microscopic observations wereperformed in F. A. Eiserling's laboratory in theDepartment of Bacteriology, University of Cali-fornia, Los Angeles, and permission to presentthese unpublished results is greatly appreciated.

Additionally, we have found that DNA mole-cules extracted from mature SPO2 can be cir-cularized by heating them to 75 C in 0.6 M NaCland slowly cooling them to room temperatureby the methods of Hershey, Burgi, and Ingra-ham (6). Molecular weight determinations madefrom these circularized molecules agree withthose made on linear molecules directly isolatedfrom intact particles. One such circular moleculeis shown in Fig. 1.

Electron microscopy of mature particles re-vealed that SPO2 is morphologically quite simi-lar to phage X. In Fig. 2, an electron micrographshowing both SPO2 and X stained with phos-photungstic acid, it is apparent that the head ofSPO2 is smaller than that of X, a fact consistentwith its smaller DNA complement. More de-tailed studies on the morphology of SPO2 andits DNA will be published elsewhere.

Stability of B. subtilis Lysogenic for SP02Previous studies with the transducing phages

PBS1 (19) and SPIO (2, 8) and with phage SP13(3) have shown that all three can set up a carrierstate with their host cultures in which they aretransmitted in a heat-insensitive form throughthe spores of B. subtilis. It has also been foundthat such carrier cultures are rapidly cured whenvegetative cells are cultured for short periods oftime in antiserum-containing medium. We there-fore studied in some detail the biological aspectsof the phage-bacterium association betweenSPO2 and its lysogenized host. Most of thesestudies were done on B. subtilis 168B.To study the nature of the SPO2-bacterium

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FIG. 1. "Circular" molecule ofSP02 DNA. Phages were subjected to osmotic shock according to the generalprocedures first described by Kleinsch,nidt et al. (9). Determinations nmade from measurements of these and linearmolecules gave a molecular weight for SP02 DNA of approximately 26 X 106 daltons. The marker represents1.0/Am.

association, bacteria from centers of turbidplaques formed by SPO2 on indicator B. subtilis168B were purified by repeated single-colony iso-lation. Cultures from these purified bacteriawere incubated on nutrient agar at 37 C untilsporulation was completed. Bacterial growth wasthen suspended in distilled water and incubatedwith 200 jug of lysozyme per ml to destroy re-maining vegetative cells; the spores were washedsix to eight times with distilled water. Thesepreparations were heated to 80 C for 10 min, atreatment which completely inactivates free SPO2

particles, but which has little effect on B. subtilis168B spores. The pasteurized spores were countedin a bacterial counting chamber, and appropriatedilutions of the suspension were added to twoseries of tubes containing molten soft agar.One series of tubes was poured directly ontohard-agar plates for determination of the num-bers of colony-forming units, and indicator168B cells were added to the other series for de-termination of numbers of plaques formed by thelysogenic bacteria. Both series of plates wereincubated at 37 C, and after 1.5 hr the plates with

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FIG. 2. Virions ofSP02 and X. The head ofSP02,which is on the left, is slightly smaller than that of X.

The two phage preparations were mixed and stainedwith phosphotungstate, and the two phages shown herewere photographed in the same field. The print was

afterwards cut to facilitate comparison. The markerrepresents 0.1 pnm.

added indicator were removed, exposed to 150ergs/mm2 of light (from a 15-w GeneralElectric germicidal lamp) to induce SPO2prophage, and returned to the incubator for12 to 14 hr. The preincubation at 37 C allowed a

lower induction dose, since germinated sporesrequire much less irradiation for inducing phagedevelopment than do the corresponding dormantspores. From 90 to 96% of spores prepared in thismanner were capable of producing phage afterUV induction. This fraction was not changedby exposure of the pasteurized spores to anti-serum against SPO2 (K = 150) for 1 hr beforethey were tested for phage production by theabove procedures. These results showed clearlythat SPO2 genomes can be incorporated withinthe spore and that in this state they have thestability associated with other spore constituents.To determine stability of bacteria lysogenic for

SPO2, nutrient broth containingantiserum againstSPO2 (K = 142) was inoculated with 2 X 107pasteurized spores per ml and incubated at 37 C.After 8 hr of growth, samples of the bacteriawere removed, suitably diluted, and plated todetermine numbers of colony formers and UV-inducible lysogenic bacteria by methods describedabove. Also at this time, a fresh culture was

started by inoculating about 3,000 of these di-luted bacteria per ml into fresh antiserum-

containing broth; incubation was continued for24 hr. This procedure was repeated at 24-hrintervals for a total of 80 hr, allowing over 70bacterial divisions to be tested. The data ob-tained (Table 2) show that, after 8 hr of con-tinuous incubation in antiserum, the fraction oflysogenic, inducible bacteria was approximatelythe same as that originally present in the inocu-lum; that is, negligible prophage loss occurred.However, prolonged continuous growth of thesebacteria in the presence of antiserum resulted ina progressive loss of lysogens compared to totalcell numbers. It is not clear why this progressiveloss of prophage occurred, but, none the less,these data indicate that the lysogens formed afterinfection with SPO2 are strikingly more stablethan carrier cultures formed by other B. subtilisphages. Bott and Strauss (2) reported that culti-vating spores carrying SP10 in the presence ofantiserum resulted in a complete loss of phage-carrying bacteria after about 8 hr. Essentiallyidentical results were reported by Romig andBrodetsky for bacteria carrying SP13.We also determined numbers of free phage

produced by cultures started from pasteurizedlysogenic spores. In studies with SP10 (2, 8)and PBS1 (19), it was shown that culturesstarted with carrier spores soon contained from10- to 100-fold more free phage than bacteria,and further it was found that UV irradiation ofbacteria carrying these phages did not increasethe numbers of free phage particles. In our ex-periments, spores lysogenic for SPO2 were in-troduced into nutrient broth, and at hourly in-tervals during incubation at 37 C samples werediluted in nutrient broth with and without addedchloroform. The chloroform-treated sampleswere plated by the soft-agar method with addedindicator for determination of numbers of free

TABLE 2. Retention ofprophage by sporesa of 168B(SP02) continuously cultivated in antiserum

Results of assays after incubation inaZ.za ~~~~~~antiserum

No. of bacteria § .

8 2 X107 3 X109 2.6 X 109 86(spores)

24 3 X103 4 X109 8 X 108 2024 3 X10' 6 X108 1.25 X108 2524 3 X10' 4 X109 6.4 X 108 16

_~~~~~~~_q

v Of the 168B (SPO2) spores used for inocula-tion, 92% produced SPO2 after UV induction.

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phage; the other samples were spread on nu-trient agar plates for determination of numbersof colony formers. The results presented inTable 3 show that the frequency of free phage

TABLE 3. Production offree phage by 168B (SP02)spores" during growth in nutrient broth

Timeof No. of freeincuba- No. of cells/ml phage/ml Cells/free phage

hr0 5.1 X 107 <101 4.8 X 107 <102 5.6 X 107 1.1 X 104 4.5 X 103 7.0 X 101 1.2X 104 5.8 X 1024 3.3 X 107 5 X 1O 6.6 X 1035 9.1 X 107 3 X104 3 X 1036 1.4 X 108 1.9 X 105 7.2 X 102

a Of the spores used as inoculum, 96% producedSP02 after UV irradiation.

10 -

10

10 -

increases during bacterial growth, probably as aresult of spontaneous phage induction, but thatthe ratio of bacteria to free phage is alwaysgreater than 100:1.

Studies on inducibility of bacteria lysogenicfor SP02 were performed with UV light andmitomycin C used as inducing agents. Results ofone such experiment performed with mitomycinC are presented in Fig. 3. Bacteria lysogenic forSP02 were grown in nutrient broth at 37 C to aconcentration of about 108/ml. They were washedthree times with nutrient broth to remove freephage and were resuspended in nutrient brothwarmed to 37 C. After samples of the bacteriawere removed for determination of cell con-centration, total plaque-forming units (PFU),and chloroform-resistant PFU (free phage),0.4 ,g/ml of mitomycin C was added and in-cubation was continued at 37 C. Samples wereremoved at 10-min intervals, and total PFU andfree phage were determined as above. A burst

I

C HC13 treated 'I

II

III

0 4

> Normal lysis

to = 0.3 % of total

10 20 30 40 50Min. after adding M.C.

60 70 80

FIG. 3. One-step growth curve of mitomycin C-induced B. subtilis 168B lysogenic for SP02. Before adding0.4 ,ug of mitomycin C per ml to the washed culture of 168B (SP02), approximately three free phage per 1,000bacterial colony formers could be detected in the chloroform-treated samples.

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size of about 40 was found in this experiment,with a latent period of about 55 to 60 min pre-ceding normal lysis. The first intracellular phagebegan to appear about 10 min before normalburst. Similar experiments with UV and otherdoses of mitomycin C gave essentially identicalresults except that in several experiments higherburst sizes were found. We also found that, be-fore addition of mitomycin C, fewer than threephages per 1,000 bacteria could be released fromwashed bacteria by treating them with CHCI3.Mass lysates of SPO2 prepared by mitomycin

C induction of lysogenic 168B also contain ap-preciable numbers of defective SP a particles(Eiserling, Ph.D. Thesis, Univ. of California,Los Angeles, 1964; Eiserling and Romig, Bac-teriol. Proc., p. 118, 1964). It is not knownwhether a given induced bacterium lysogenic forboth SPO2 and SP a produced both kinds ofphage particles. Our inability to assay smallnumbers of SP a particles has precluded attemptsto perform single-burst experiments, whichwould be required to elucidate this point. Wehave also found significant numbers of SP aparticles in lysates prepared by infecting B.subtilis 168B with nonlysogenizing, clear plaquemutants of SP02. We have thus found it neces-sary to purify by CsCl density gradient centrifu-gation all SPO2 preparations for purposes forwhich contamination must be avoided.

Immunity of 168B Lysogenic for SP02Indicator bacteria prepared from cultures of

168B (SPO2) do not form plaques when used forassaying SP02 phage. Such lysogenic culturesare also immune to clear plaque mutants (e.g.,SPO2cl) at moderate multiplicities of infection.At multiplicities of infection higher than about20, immunity is overcome, and superinfectingphage, which adsorb normally to lysogenicbacteria, replicate and release infective particles.We did not, however, observe any differencesbetween efficiencies of plating for other B.subtilis phages when 168B (SP02) was used ashost and their efficiencies of plating when 168Bwas used. Likewise, we have found no differencesin the abilities of cultures of competent 168B(SP02) and 168B to undergo transformationfor a number of bacterial markers or to be in-fected with other B. subtilis phage DNA prepa-rations.The results of these biological experiments,

taken together, support the designation of SP02as a temperate phage. They do not, however,give any information concerning the nature ofthe association formed between the bacterialgenome and prophage in lysogenic bacteria.

First attempts in this direction were made byOkubo, who showed that lysates of SPO2 pre-pared from prototrophic bacteria, either byinduction of lysogenized cultures or by infectingsensitive bacteria, were unable to transduce anyof the bacterial mutants he tested (11). This sug-gested either that SP02 is unable to form trans-ducing particles or that it might be a specializedtransducing phage similar to X and thus able totransduce only those genes directly adjacent toits attachment site.

Attempts to map the prophage attachmentsite directly have been made by using bothtransformation and transduction techniques.For this purpose, either DNA was extracted from,or the generalized transducing phage PBSI wasgrown on, nonlysogenic prototrophic B. subtilis.These preparations were used to transform ortransduce a variety of bacterial mutants pre-viously rendered lysogenic for SP02. The trans-formed or transduced colonies were then testedfor retention of SP02 prophage. These experi-ments are still in progress, but results thus farobtained have not revealed linkage of prophageto any of the bacterial genes tested.

Transfection with Prophage DNAOther attempts to elucidate the nature of the

phage-bacterium association were made bytaking advantage of the fact that DNA extractedfrom 168B (SP02) is infective when added tocompetent 168B cultures. This property isanalogous to the results reported by Harm andRupert (5), who showed that DNA extractedfrom the Rd strain of Haemophilus influenzaelysogenic for phage HPI was infectious for trans-formable Rd cells. Like Harm and Rupert, wehave found that most of the infections obtainedwhen competent bacteria are exposed to pro-phage-containing DNA culminate in lysis ofthe infected cell, and we also assume that thisresults from the "zygotic induction" of prophageDNA entering bacteria lacking repressor.We attempted to isolate bacteria rendered

lysogenic by exposure to prophage DNA byfirst allowing all "zygotically induced" bacteriato lyse in the presence of antiserum to inactivatefree phage. After removing antiserum, SPO2cl at amultiplicity of infection of 8 was added to lysenonimmune bacteria, and surviving bacterialcolonies were tested for the presence of SP02prophage. In several such experiments, only onelysogenic colony was detected. Since this couldhave resulted from SP02 contamination, weconcluded that establishment of lysogeny byprophage DNA under standard transfectionconditions is a very rare event, if it occurs at all.

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In our preceding experiments, we assumed, asthe simplest hypothesis, that the prophage DNAin bacteria lysogenic for SP02 was integratedinto the bacterial genome in a manner similar tothat occurring in X lysogens (4). However, ourfailure to detect linkage of SP02 prophage tobacterial genes forced us to consider alternativehypotheses, including the possibility that in B.subtilis the SP02 prophage might replicate in acoordinated manner within the bacterial cell,but unattached to the bacterial chromosome. Totest these possibilities, several experiments wereperformed in which the properties of prophageDNA were compared to those ofDNA extractedfrom mature SP02 particles.

Comparison of Properties of Prophage DNA andSP02 phage DNA

Infectious DNA extracted from lysogenicbacteria and DNA from mature phage particleswere tested for their relative resistance to shear-ing. In these experiments, the prophage-contain-ing DNA extracted from 168B (SP02) had anaverage molecular weight, estimated by sedi-mentation through sucrose, of about 108, whichis approximately four times that of the DNA frommature particles.We assumed that if SP02 prophage was not

inserted into the bacterial chromosome it mightreplicate within the lysogenic bacterium as aconcatenated structure (15), or as a moleculesimilar to those extracted from mature phage,although in the latter case it might assume aclosed configuration (21). The shear sensitivitiesof these different possible forms of prophageDNA should be quite different from the sensitiv-ity of the linear DNA extracted from maturephage.

It has been shown that circular DNA is moreresistant to controlled shear than the correspond-ing linear form (6, 21). We assumed that a con-catenated molecule made up of multiple phagegenomes connected end-to-end would retainbiological activity as long as the structure was notbroken into fragments of less than one genome.Thus, this form might be more resistant thanmature phage DNA to hydrodynamic shear.We further assumed that if the prophage DNA

is inserted into the bacterial chromosome theextracted infectious DNA, with an average mo-lecular weight four times that of mature phageDNA, should consist of "molecules" one-fourthof which is SP02 prophage and three-fourthsof which is bacterial DNA. If such fragments arerandomly formed, i.e., there are no preferentialbreak points, the prophage DNA should occurnear the center of such "molecules" more often

TABLE 4. Eflects ofshearing on1 biological activitiesof 168 (SP02) and SP02cl DNA

Remaining activity (1 hr)

Rate ofi8(P2stirring 168 (SP02)

SPO2CI

PFU Transformants (PFU)

rev/min % % %0 100 100 100

1,000 70 891,500 20 30 802,000 3.3 14 252,500 1.2 10 113,000 0.4 3.3 1.3

that at either end. Thus, a break near the centerof such "molecules," which should occur atstirring rates lower than those affecting thesmaller phage DNA molecules, would oftenoccur within the prophage region. Therefore,transfecting activity of such "molecules" shouldbe more sensitive to shear inactivation thanthat ofDNA from mature phage particles.

Accordingly, we sheared these two idnds ofpreparations with the microattachment of aSorvall Omnimixer for 1 hr over a range ofspeeds. These preparations were then tested forremaining biological activity under standardconditions. Mature phage DNA was tested onlyfor transfecting activity, but prophage DNAwas tested both for transfecting activity and(since the major part of such preparations isDNA from the bacterial chromosome) for re-sidual transforming activity. The results pre-sented in Table 4 show that prophage transfect-ing activity is more sensitive to shear than DNAfrom mature particles, whereas transformingactivity is more resistant. These results are con-sistent with, but do not prove, the hypothesisthat SP02 prophage is inserted into the B.subtilis chromosome.

Finally, we compared the relative sedimenta-tion rates of prophage transfecting activity withthat of DNA extracted from mature phage. Theprophage DNA used in these experiments wasthe same as that used in tests on shear resistancediscussed above. Mature phage DNA was ex-tracted from a clear, plaque-forming, tempera-ture-sensitive mutant, SP02cl-ts23. This madepossible the independent assay of the biologicalactivities of these two preparations from mix-tures of both. The mature phageDNA was heatedto 75 C for 10 min and quickly cooled immedi-ately before use. Mixtures of the two DNApreparations were sedimented through linear5 to 20% sucrose gradients, and fractions were

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TU+

Leu' transformants

10 15Fraction

20 25

FiG. 4. Sucrose sedimentation of a mixture of "prophage DNA" extracted from 168B (SP02) and maturephage DNA extracted from the temperature-sensitive, clearplaque mutant, SP02cl-ts23. The DNA mixture wassedimented through 5 to 20% sucrose gradients andfractionated as described in the text. Fractions were testedfortransfecting and transforming activity by mixing them with competent, nonlysogenic bacteria under standard con-ditions. The solid line marked TU+ refers to activity from transfecting DNA ofprophage origin; the dashed linemarked leu+ transformants refers to transforming activity of bacterial DNA; the dotted line labeled Cl refersto clear, temperature-sensitive plaques attributable to transfection with DNA from mature phages.

assayed for biological activity by transfection andtransformation. Transfection assays were per-formed at 30 C, which allowed both the turbidplaques formed by prophage DNA and the clearplaques produced by DNA from the tempera-ture-sensitive mature phage to be assayed on thesame plates. In doubtful cases, their origin wasconfirmed by picking plaques to seeded indicatorplates which were incubated at 46 C, a tempera-ture that inhibits plaque production by tempera-ture-sensitive mutants. Since prophage DNApreparations consist primarily of bacterial DNA,transforming activity of the fractions could besimultaneously assayed by spreading the exposedcompetent recipients on suitable selective agarplates. Results of one such experiment arepresented in Fig. 4. They show that transfectingactivity characteristic of prophage sediments atabout the same rate as transforming activity,but both of these activities sediment faster than

transfecting activity ofDNA from mature phages.Because of lack of sensitivity, more DNA thanoptimal was used in these sedimentation experi-ments, which probably accounts for the spread-ing observed. These data indicate that prophagetransfecting activity is associated with bacterialDNA in a manner that resists extraction pro-cedures, as well as forces operating duringsedimentation through sucrose solutions.We conclude that SP02 is an inducible tem-

perate phage in the generally accepted use of theterm. B. subtilis lysogenized with SP02 is im-mune to superinfection, it retains phage-pro-ducing capacity after growth in antiserum, andcultures are inducible with either UV light ormitomycin C. Despite our inability to demon-strate linkage of the SP02 prophage to bacterialgenes, the data on shear resistance of prophageDNA and persistent association of bacterialDNA with prophage transfecting activity indi-

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cate that SP02 prophage is probably insertedinto the B. subtilis chromosome in lysogenicbacteria.

ACKNOWLEDGMENTS

It is a pleasure to acknowledge the contributionsof my collaborators, for whom I am spokesman. Asnoted in the text, Shunzo Okubo made many of theinitial observations on the biology of SP02. LuBelleBoice contributed to isolation of phage mutants andpreparation of several bacterial strains. Technicalassistance was ably given by Diane Garrard andNita Kom.

Parts of the work reported here were supported bya contract from the Office of Naval Research andby grants from the National Science Foundation andthe National Institutes of Health.

LITERATUE CiTE1. Anagnostopoulos, C., and J. Spizizen. 1961.

Requirements for transformation in Bacillussubtilis. J. Bacteriol. 81:741-746.

2. Bott, K., and B. Strauss. 1965. The carrier stateof Bacillus subtilis infected with the transducingbacteriophage SP10. Virology 25:212-225.

3. Brodetsky, A. M., and W. R. Romig. 1965.Characterization of Bacillus subtilis bacteri-ophages. J. Bacteriol. 90:1655-1663.

4. CAMPBELL, A. M. 1962. Episomes. Advan. Genet.11:101-145.

5. Harm, W., and C. S. Rupert. 1963. Infection oftransformable cells of Haemophilus influenzaeby bacteriophage and bacteriophage DNA.Z. Vererbungslehre 94:336-348.

6. Hershey, A. D., E. Burgi, and L. Ingraham. 1963.Cohesion of DNA molecules isolated fromphage lambda. Proc. Natl. Acad. Sci. U.S.49:748-755.

7. lonesco, H., A. Ryter, and P. Schaeffer. 1964.Sur un bacteriophage herbege par la soucheMarburg de Bacillus subtilis. Ann. Inst. Pas-teur 107:764-776.

8. Kawakami, M., and 0. E. Landman. 1968.Nature of the carrier state of bacteriophageSP-10 in Bacillus subtilis. J. Bacteriol. 95:1804-1812.

9. Kleinschmidt, A., D. Lang, and R. K. Zahn.1961. Ober die intrazellulire Formation vonBakterien-DNS. Z. Naturforsch. 16:730-739.

10. Nishihara, M., and W. R. Romig. 1964. Tem-perature-sensitive mutants of Bacillus subtilisbacteriophage SP3. I. Isolation and char-acterization. J. Bacteriol. 88:1220-1229.

11. Okubo, S., and W. R. Romig. 1965. Comparisonof ultraviolet sensitivity of B. subtilis bac-teriophage SP02 and its infectious DNA. J.Mol. Biol. 14:130-142.

12. Okubo, S., M. Stodolsky, K. Bott, and B. Strauss.1963. Separation of the transforming and viraldeoxyribonucleic acids of a transducing bac-teriophage of Bacillus subtilis. Proc. Natl.Acad. Sci. U.S. 50:679-686.

13. Romig, W. R., and A. M. Brodetsky. 1961.Isolation and preliminary characterization ofbacteriophages for Bacillus subtilis. J. Bac-teriol. 82:135-141.

14. Saito, H., and K. Miura. 1963. Preparation oftransforming DNA by phenol treatment.Biochim. Biophys. Acta 72:619-629.

15. Smith, M. G., and A. Skalka. 1966. Some prop-erties of DNA from phage-infected bacteria,p. 103-125. In Proceedings of a Symposium onMacromolecular Metabolism, New YorkHeart Association. Little, Brown and Co.,Boston.

16. Spizizen, J. 1958. Transformation of biochemi-cally deficient strains of Bacillus subtilis bydeoxyribonucleate. Proc. Natl. Acad. Sci.U.S. 44:1072-1078.

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