JOURNAL OF VIROLOGY, Jan. 1977, p. 147-152Copyright © 1977 American Society for Microbiology

Vol. 21, No. 1Printed in U.S.A.

Initiation and Termination Mutants ofBacillus subtilisBacteriophage SPO1

JEFFREY GLASSBERG,* MICHELLE FRANCK, AND CHARLES R. STEWARTDepartment ofBiology, Rice University, Houston, Texas 77001

Received for publication 2 August 1976

Mutants affected in cistrons 21 and 32 of bacteriophage SPOl are defectivespecifically in the initiation of DNA replication. Mutations in cistron 32 alsospecifically affect the termination of replication.

In several bacterial and viral systems, cer-tain gene products are known to be necessaryspecifically for the initiation of DNA replica-tion (see, for example, references 12, 13, 21, and23). In this paper, we use density transfer anal-ysis to demonstrate such a role for two geneproducts of Bacillus subtilis phage SPOl. Inaddition, we show that one of these gene prod-ucts plays a specific role in the termination ofDNA replication.SPOl has nine cistrons whose products are

known to be necessary for DNA synthesis (16).We have previously analyzed temperature-sen-sitive (ts) mutations in eight of these nine cis-trons by temperature shift experiments. Weconcluded that none of the mutations affected agene product that was necessary only for initia-tion of the first round of replication. However,the products of five of the cistrons are candi-dates for a specific role in the initiation of eachround of replication (8). To determine which, ifany, of these five gene products are actuallyinitiation proteins, we have combined the tem-perature shift with a density transfer, as fol-lows.B. subtilis, growing in heavy medium, is

infected at permissive temperature with a tsmutant carrying light-density DNA. Midwaythrough the first round of replication, the cul-ture is shifted to the restrictive temperature.With a mutant that affects propagation of thegrowing point, replication will arrest as soon asthe ts protein has been inactivated, and thepositions of the growing points will be frozen.Since synchrony is imperfect, growing pointswill be distributed throughout the genome. Thecloser a genetic marker is to the origin of repli-cation, the higher will be the proportion of thatmarker to be found on replicated, and thushybrid density, portions of the genome. On theother hand, if the mutant affects only initia-tion, all molecules on which replication hasbeen initiated before the temperature shift willreplicate to completion. All molecules will be

either completely replicated or completely un-replicated, and so each genetic marker will bereplicated to the same extent. The latter resultis obtained with mutations in cistrons 21 and32.

MATERIALS AND METHODSBacterial strains. B. subtilis CB-10 has been de-

scribed as SB-1 by Nester and Lederberg (15). CB-312, -313, and -314 were provided by M. Mandel andwere described (22) as strains 129, 151, and 135,respectively. CB-10 is suppressor-, his-, trp-; CB-312 is suppressor-, his-, met-; CB-313 is sup-3+;and CB-314 is sup-1 +, his-, met-. CB-312, -313, and-314 are otherwise isogenic. CB-312 was our stand-ard host strain, and CB-313 was used to grow mostof the suppressor-sensitive (sus) phage mutants.Some of the sus mutants grew only on CB-314. CB-327 was created by transforming CB-312 to proto-trophy. It grows at 20°C, whereas CB-10 does not.Phage strains. All sus mutants were generously

provided by E. P. Geiduschek and have been previ-ously described by Okubo et al. (16). For clarity ofpresentation, each mutant is given a standardizeddesignation, the first term of which indicates thephenotype, the second term the cistron number, andthe third term the specific mutation within thatcistron. Thus, hs30-2 refers to a heat-sensitive (hs)mutant, affected in cistron 30 and designated no. 2.Table 1 shows the correspondence between the no-menclature used in this paper and the original des-ignations of the sus mutants used. All hs and cold-sensitive (cs) strains used have been described (8).Although these ts strains were originally isolatedusing permissive temperatures of 20 and 37°C, re-spectively, 30°C was also permissive for all strains.Media used. Bacterial and phage growth was gen-

erally in VY medium. Platings used TBAB bottomand top agar. These media, the deuterated media,and Spizizen minimal medium have been described(17, 19). Deuterated algal extract and deuteratedsugars were gifts of H. Crespi.

Preparation of 3H-labeled phage lysates. CB-312,growing at either 30 (for hs mutants) or 37°C (for csmutants), was infected with the appropriate mutantphage at a multiplicity of infection of about 10:1.Shortly thereafter, [6-H3]uridine was added to about10 uCi/ml. After lysis, the culture was centrifuged

147

148 GLASSBERG, FRANCK, AND STEWART

TABLE 1. sus mutants used

Designation in this Original designa- Referencework tion

sus1-1 F12 16sus2-1 F47 16sus3-1 F37 16susll-l F6 16sus14-1 Fll 16sus23-1 N5 16sus27-1 HA20 16sus30-2 052 16sus32-1 F38 16sus33-1 F14 16sus34-1 F4 16sus35-1 F24 16sus36-1 N34 16

at 3,000 x g for 10 min. The supernatant fractionwas then recentrifuged at 13,300 x g for 90 min. Thesupernatant fraction was discarded, and the pelletwas resuspended overnight in one-fifth volume ofdeuterated medium.

Preparation of deuterated B. subtilis. B. subtilisCB-327 was grown for about 16 h at 37°C in deuter-ated medium. Samples of this culture were frozen inliquid nitrogen and were used to inoculate freshdeuterated medium.

Density transfer, temperature shift experiments.For hs mutants, deuterated CB-327 was grown at37°C to a cell density of about 1.2 x 10" cells/ml indeuterated medium. The culture was shifted to 43°Cand then infected at a multiplicity of infection ofabout 3:1 with the appropriate hs mutant (lightdensity, 3H-labeled, and resuspended in deuteratedmedium). At 25 min postinfection, 5-ml samples ofthe infected culture were transferred to 250-mlflasks, precooled at 20°C. The above protocol servedto partially synchronize the initiation of DNA repli-cation. At various times after the shift to 20°C, DNAreplication was stopped either by pouring the cul-ture onto 10 ml of frozen Spizizen minimal mediumor by pouring the culture into flasks already at 43°C(restrictive temperature) and incubating for 15 minbefore pouring onto the Spizizen minimal mediumice.

For cs mutants, the procedure was basically thesame, except that 20°C was the restrictive tempera-ture and 37°C was the permissive temperature. At150 min after infection at 20°C, 5-ml samples wereshifted to 37°C, and the infections were then stoppedat various times as before.The DNA extraction procedure was essentially

that of Okun (Ph.D. thesis, Stanford University,Stanford, Calif., 1969) as modified by Stewart et al.(20). Cells were washed three times, frozen andthawed, and then lysed with egg white lysozyme.After successive treatment with RNase and Pro-nase, samples were made 1% with respect to Sarko-syl and incubated at 37°C for 30 min. After overnightincubation with sodium perchlorate, they were di-alyzed against two changes of SSC (0.15 M NaCl,0.015 M sodium citrate).

Shearing of DNA. DNA was sheared by passing

three times through a 30-gauge hypodermic needle(10).

Equilibrium CsCl centrifugation. The procedurefor equilibrium CsCl centrifugation was as de-scribed (18), with the following modifications. Cen-trifugation was in a Beckman L2-65B, using a type65 rotor at 32,000 rpm for about 66 h. Each polyal-lomer tube contained 4.1 ml of sample plus water,plus 5.55 g of CsCl. This mixture produced a densityof about 1.731 g/cm3. Ten-drop fractions (about 0.11ml) were collected from the bottom. Fractions wereassayed for radioactivity by placing 10-,ul samples ofeach on Whatman glass-fiber filters. The filterswere dried, toluene-based scintillation fluid wasadded, and the samples were counted in a PackardTri-Carb scintillation counter.

Preparation of competent cells. Competent cellswere prepared freshly each day as described (18).Marker rescue. The concentration, in each gra-

dient fraction, of DNA carrying specific geneticmarkers was determined by a marker rescue assay.This was performed in a manner similar to thatdescribed by Green (9). Each pair of adjacent frac-tions from the CsCl gradient was pooled. A 0.45-mlamount of competent cells was added to a bovineserum albumin-coated (to prevent the DNA fromsticking to the glass) culture tube already contain-ing 10 Al of one of the pooled fractions. The culturetube was incubated at 37°C with shaking for 4.5 minand then superinfected with 0.1 ml of an SPOl mu-tant carrying a sus mutation in the cistron of inter-est (giving a multiplicity of infection of roughly 5:1).Incubation was continued for another 10 min, andthe culture was then plated on CB-10 (suppressor-)(three plates per culture tube using 0.2, 0.2, and 0.1ml of the sample, respectively). Plates were in-cubated at 30°C for about 20 h, and PFUs werecounted. CB-10 was used as the bacterial indicatorbecause, unlike CB-312, it was insensitive to theconcentrations of CsCl used. With the DNA con-centrations used, there was no measurable trans-fecting activity, so plaque formation depended uponrecombination between the sus- genome and DNAcarrying its sus+ allele.

Calculation of percentage of replication. Foreach CsCl gradient, it was determined, by inspec-tion of the 3H counts per minute profile, which frac-tions constituted the light, hybrid, and heavy re-gions of the gradient. For any given marker, theactivity in each region was determined by markerrescue assay. The percentage of replication was thencalculated as described by Copeland (2), using theformula [(HL/2)/(LL + HL/2)] x 100%, where HLrepresents the total activity for a given marker inthe hybrid region and LL represents the total activ-ity in the light region.

Chemicals. [6-3H]uridine was purchased fromNew England Nuclear Corp. (Boston). RNase, Pro-nase, and lysozyme were purchased from Calbi-ochem (San Diego).

RESULTSInitiation. Deuterated B. subtilis, growing

in heavy medium, was infected at restrictive

J. VIROL.

SPOl INITIATION AND TERMINATION MUTANTS 149

temperature with temperature-sensitive, repli-cation-deficient SPOl whose DNA was of lightdensity. After the time at which replicationwould normally have begun (but had not, dueto the ts block), the culture was shifted to per-missive temperature. (This preliminary incu-bation at restrictive temperature served to par-tially synchronize the initiation of DNA repli-cation.) While the first round ofreplication wasstill in progress, the culture either was rapidlychilled or was shifted to restrictive temperaturefor 15 or 60 min. (Stopping replication by chill-ing served as a control, since this would main-tain the existing distribution of growing pointsand thus should result in a higher proportion ofreplication for markers closer to an origin, nomatter which type ofmutant is used.) The DNAwas then extracted, sheared, and centrifuged toequilibrium in CsCl. Fractions from the CsClgradient were assayed for marker rescue activ-ity with a series of SPOl suppressor-sensitive(sus) markers. For each marker, the proportionreplicated was determined from the proportionof that marker's activity found in the hybridregion.

Figure 1 gives an example of the differentdensity shifts oftwo marker rescue activities inthe same CsCl gradient, showing that cistron30 has replicated before cistron 3. By makingsimilar comparisons for all markers, we haveestablished the sequence of replication of the

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various markers on the SPOl genonfe. An ex-ample of this is shown in Fig. 2, in which thepercentage of replication for each marker isplotted against its position on the genetic map.The cistrons on the SPOl map are arranged innumerical order, from 1 to 34 (16). Replicationof the major portion of the genome begins nearcistron 32 and proceeds leftward to a terminusin the vicinity of cistron 3 (Fig. 2).At least one, and possibly two, additional

origin is used for the replication of the leftmostand rightmost portions of the genome (cistrons1, 2, 33, 34) (data to be published elsewhere).For convenience ofpresentation, this paper con-cerns itself only with the effect of mutations onreplication of the major portion of the genomeand two unlinked markers. The effect of themutations on the replication of the ends of thegenome was no different from their effect on themajor portion.Using the experiment described above, we

tested ts mutants affected in each of the fivepossible "initiator" cistrons, looking for the abo-lition of the cistron 32 to cistron 3 replicationgradient. Figures 3 and 4 show that, for mu-tants affected in three of these cistrons, 22, 30,and 31, a gradient of replication is obtainedeven after a shift to restrictive temperature.(The variations in the details of these curvesare not significant, since there is frequentlyconsiderable variation from experiment to ex-

Cistron

FIG. 1. Differential density shifts of SPOl FIG. 2. SPOl replication gradient. Deuterated B.markers in the same CsCl gradient. 3H-labeled hs3O- subtilis CB-327, growing in heavy medium, was in-1 was allowed to replicate in heavy medium for 10 fected with hs31-1 at 43°C. The culture was shifted tomin at 20°C and then was shifted to 43°C for 15 min. 20°C 25 min later (the preliminary incubation atThe DNA was then extracted, sheared, and centri- restrictive temperature imposes some synchrony onfuged to equilibrium in CsCl. Pairs ofadjacent frac- the initiation of replication). After incubation for 20tions of the gradient were pooled, and samples ofthe min at 20°C, the culture was poured onto Spizizenpooled fractions were used in marker rescue assays minimal medium ice. The DNA was extracted,with either sus3-1 or sus30-2. The number ofplaques sheared, and centrifuged to equilibrium in CsCl.due to reversion ofthe sus marker (19 for sus3-1 and After assaying fractions for radioactivity, pairs of14 for sus30-2) has been subtracted from all values, adjacent fractions were pooled and samples were as-Note that the hs3O-1 mutation is irrelevant in the sayed for marker rescue activity against sus markersmarker rescue assays, since these are done at permis- in the indicated cistrons. The calculation of the per-sive temperature. See Materials and Methods for de- centage of replication for each marker was as de-tails. Symbols: (0) sus30-2; (-) sus3-1. scribed in Materials and Methods.

VOL. 21, 1977

150 GLASSBERG, FRANCK, AND STEWART

co 40 -

,2

r-

O; 20 e

3 1 4Cistron

FIG. 3. Replication of cs22-1. Dtilis CB-327, growing in heavy medwith cs22-1 at 20°C. The culture w150 min later (the preliminary inctive temperature imposes some syntiation of replication). The cultureto 20°C 6 min later. After incubad200C, the culture was poured ontomedium ice. The DNA was extraccentrifuged to equilibrium in CsCfractions for radioactivity, pairs ofwere pooled and samples were asrescue activity against sus markercistrons. The calculation of the pertion for each marker was as descrand Methods. The slope of the reiwhen cs mutants were used as thwas generally less steep than wherused (compare relevant data in T(example). This is probably due to tsynchrony achieved with the cs mz

70 L

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periment.) We conclude that proteins coded bythese cistrons probably are directly involved inDNA chain elongation (see Discussion). In con-trast, mutants in cistrons 21 and 32 yielded nogradients of replication when replication wasstopped by shifting to restrictive temperature,but gave the expected replication gradientswhen stopped by chilling (Tables 2 and 3).The lack of replication gradients in these

mutant-infected cultures, after a shift to re-27 30 32 strictive temperature, is not due to some arti-

23 27 30 32 factual inability of the phage DNA to replicatefurther. In parallel infected cultures in which

euterated B. sub- replication was allowed to proceed for a greaterlum, was infected length of time before the shift to restrictiveas shifted to 37"C temperature, the DNA was replicated to anubation at restric-chrony on the i_ appropriately greater extent. We conclude thatwas shifted back cistrons 21 and 32 code for proteins whose activ-

tion for 60 min at ities are required for the initiation of eachSpizizen minimal round of replication, but not for polymerization.'ted, sheared, and Termination. The sus35-1 and sus36-11l. After assaying markers show a high frequency of recombina-adjacent fractions tion with each other and with all other knownssayed for marker SPO1 mutants (16; our unpublished data), and-sin the indicate their map location is, therefore, not known.centage of replica- They are located very near a terminus of rep-.,ioed in Ma4terialsplication gradientwe infecting phagez hs mutants wereables 2 and 3, forhe lesser degree ofutants.

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11 14Cistron

FIG. 4. Replication of hs3O-1 and hs31-2. Deuter-ated B. subtilis CB-327, growing in heavy medium,was infected with either hs3O-1 or hs31-2 at 43°C.The culture was shifted to 20°C 25 min later (thepreliminary incubation at restrictive temperature im-poses some synchrony on the initiation of replica-tion). Ten (hs3O-1) or twenty (hs31-2) minutes later,the culture was shifted back to 43°C. After incubationfor 15 min at 43°C, the culture was poured ontoSpizizen minimal medium ice. The DNA was ex-tracted, sheared, and centrifuged to equilibrium inCsCl. After assaying fractions for radioactivity, pairs

TABLE 2. Replication of a cistron 21 mutanta% Replication of various markers when

replication was stopped by:

A B

Shift to Shift toChill- restric- Chill- restric-ing tive ing tive

Marker temp temp

sus3-1 15 38 32 53susll-l NDb NDb 31 44sus 14-1 14 38 29 49sus23-1 18 40 40 56sus27-1 15 ND 47 51sus30-2 25 32 59 54sus32-1 30 34 56 45

sus35-1 11 33 28 46

a The procedure was the same as that described inthe legend to Fig. 3, except that infection was withcs21-1 and, after 6 min (A) or 8 min (B) at 37°C,replication was stopped by either rapid chilling orshifting the culture to 20°C (restrictive tempera-ture) and keeping it there for 60 min before chillingand extracting DNA.

b ND, Not determined.

of adjacent fractions were pooled and samples wereassayed for marker rescue activity against susmarkers in the indicated cistrons. The calculation ofthe percentage of replication for each marker was asdescribed in Materials and Methods. Symbols: (0)hs3O-1; (-) hs31-2.

J. VIROL.

SPOl INITIATION AND TERMINATION MUTANTS 151

TABLE 3. Replication of cistron 32 mutantsa% Replication of various markers when

replication was stopped by:A B

Shift to Shift toChill- restric- Chill- restric-ing tive ing tive

Marker temp temp

sus3-1 3 46 39 55susll-1 18 48 39 50susl4-1 20 49 46 58sus23-1 37 48 68 56sus27-1 31 41 62 54sus30-2 38 49 89 56sus32-1 66 42 NDb 50

sus35-1 13 24 29 27sus36-1 11 29 ND 18

a The procedure was the same as that described inthe legend to Fig. 4, except that infection was withhs32-1 (A) or hs32-2 (B) and, after 20 min at 200C,replication was stopped by rapid chilling or, after 15min at 2000, replication was stopped by shifting theculture to 43°C (restrictive temperature) and keep-ing it there for 15 min. In similar experiments, thesusl-l, sus2-1, sus33-1, and sus34-1 markers be-haved like the bulk of the markers above.

b ND, Not determined.

lication, since in density transfer, tempera-ture shift experiments like those above they arealways the last, or nearly the last, markers tobe replicated (see, for instance, Tables 2 and 3).Tentative results obtained by James Cregg inthis laboratory suggest that they are near theterminus in the rightmost segment of the ge-nome.When initiation of a new round of replication

is blocked by shifting cs21-1 to restrictive tem-perature, the sus35-1 marker is replicated tothe same extent as all other markers (Table 2).In a similar experiment (data not shown),sus36-1 behaved in the same way. Thus, cs21-1,although prohibiting initiation, has no effect onthe replication of these terminal markers. Incontrast, when reinitiation is blocked by shift-ing either hs32-1 or hs32-2 to restrictive temper-ature, both the sus35-1 and the sus36-1 markersremain apparently less replicated than allother markers (Table 3). Thus, the cistron 32product seems to play a specific role in thetermination of replication.

DISCUSSIONInitiation. We have shown that mutants in

cistrons 21 and 32 of SPOl are affected specifi-cally in the initiation ofDNA replication. Thatthese mutants are affected in the initiation ofeach round of replication can be deduced from

the following. (i) When infection is allowed toproceed at restrictive tem:perature, there is nofirst round of replication (our unpublisheddata). Therefore, the proteins coded by cistrons21 and 32 are necessary for the initiation of thefirst round of replication. (ii) If cells infectedwith these mutants are shifted to restrictivetemperature while still in the first round ofreplication, the second round of replication isnot initiated. Therefore, these proteins are nec-essary for the initiation of the second round ofreplication. (iii) If cells infected with these mu-tants are allowed to replicate through manyrounds of replication and then shifted to restric-tive temperature, DNA synthesis is rapidlyshut off (8). Thus, the affected proteins arenecessary for later rounds ofreplication as well.

Elongation. Mutants in cistrons 22, 30, and31 exhibit a rapid shut off of DNA synthesiswhen shifted to restrictive temperature (8) butfail to abolish the replication gradient. Takenin conjunction, these data indicate that theproducts of these cistrons are probably directlyinvolved in the elongation of nascent DNAchains.

Termination. Most interestingly, mutants incistron 32 are affected in the termination of around of replication. By "termination," wemean the replication of the terminal region aswell as any specific events occurring at theterminus itself. One might be tempted to turnthis around and propose that reinitiation de-pends upon termination. However, this is prob-ably not so, because under restrictive condi-tions cistron 32 mutants fail to initiate the firstround of replication (for which, presumably, notermination event is necessary). It can also beseen that termination of one round of replica-tion is not dependent per se upon initiation ofthe next, because under conditions in which amutant in cistron 21 prohibits reinitiation itallows replication of all tested markers, includ-ing those nearest to a terminus. We thereforeconclude that the activity of the cistron 32 pro-tein is necessary both for initiation and forsome process associated with termination. Asfar as we are aware, this is the first mutant inany system in which the termination of a roundofDNA replication is specifically affected. Ma-runouchi and Messer (14) did report, however,that replication of the Escherichia coli termi-nus required continued protein synthesis.The fact that markers located near a termi-

nus of replication show high frequencies of re-combination with each other, and with all othermarkers, suggests that there is a hot spot for aspecific sort of recombination in the terminusregion.Although the cistron 32 mutants prevent

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152 GLASSBERG, FRANCK, AND STEWART

much of the density shift of cistrons 35 and 36,some replication is still permitted (see Table 3).All, or part, of this may be replication thattakes place during the time at permissive tem-perature. However, it is possible that the situa-tion is more complicated and that a portion ofthe normal replication of the terminus regioncan take place even without the participation ofthe gene 32 protein.

It is of interest that an origin of replicationlies either within, or very close to, cistron 32and that cistron 32 codes for a protein necessaryfor initiation and termination. This is reminis-cent of the situation with kX174, whose replica-tion origin lies within cistron A, which specifiesa protein that introduces a nick necessary forinitiation (5, 6, 11). Although this protein dif-fers from the SPOl gene 32 protein in that it iscis active, it is possible that the gene 32 proteinmight also function in initiation by introducinga nick in the DNA molecule.

ACKNOWLEDGMENTSThis work was supported by Public Health Service re-

search grant no. GM02221 from the NIH and Career Devel-opment Award no. GM70044 to C.R.S., both from the Na-tional Institute of General Medical Sciences.

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