JOURNAL OF VIROLOGY, Dec. 1974, p. 1463-1469Copyright O 1974 American Society for Microbiology

Vol. 14, No. 6Printed in U.S.A.

Mixed Infections of Bacillus subtilis Involving BacteriophagesSP82 and #22

H. R. WHITELEY, P. E. KOLENBRANDER,' AND H. E. HEMPHILL2Department of Microbiology, University of Washington, Seattle, Washington 98195

Received for publication 19 July 1974

Progeny yields and the synthesis of nucleic acids have been investigated in twostrains of Bacillus subtilis mixedly infected with two unrelated phages, SP82 andp22. When B. subtilis strain 168 was the host, the first phage added dominatedthe infection; when B. subtilis strain SB11 was the host, ,B22 produced progenyeven when added to cells 5 min after infection with SP82. Dominance in thesemixed infections could be correlated with qualitative and quantitative differ-ences in the synthesis of phage-specific RNAs.

It has generally been observed that when twounrelated bacteriophages simultaneously orconsecutively infect the same host cell only oneis able to produce significant numbers of prog-eny. This phenomenon has been called mutualexclusion (3, 4). In previous communications (9,11) we reported that two virulent Bacillussubtilis phages, SP82 and 022, suppressed thedevelopment of SP02c,, a clear-plaque mutantof the temperate phage SP02, when either ofthe former phages was added to cells before,simultaneously, or shortly after infection withSP02c,. In both SP82-SP02c, and l22-SP02c,mixed infections, failure of SP02c, to produceprogeny was associated with a greatly reducedcapacity of this subordinate phage to transcribeand replicate its DNA.

In the present study, we have examined thesynthesis of nucleic acids in B. subtilis cellsmixedly infected with ,B22 and SP82, the twodominant phages in mixed infections withSP02c,. These infections have been of specialinterest for two reasons: first, although these areboth large, virulent phages, ,22 and SP82 arecompletely unrelated. They also differ withrespect to DNA base substitution: SP82 DNAcontains 5-hydroxymethyluracil in place of thy-mine, whereas the DNA of ,22 is unsubstituted.We were interested to know if the presence ofthe unusual base might favor survival andfunctioning of the SP82 genome in mixed infec-tions. Secondly, previous observations (11) indi-cated that dominance, measured as the abilityto produce progeny, was dependent on thebacterial host. When B. subtilis SB11, a deriva-tive of the nontransformable strain W23, wasmixedly infected with SP82 and p22, the latter

1Present address: Department of Microbiology, Pennsyl-vania State University, University Park, Pa. 16802.

'Present address: Department of Biology, Syracuse Uni-versity, Syracuse, N.Y. 13210.

suppressed development of the 5-hydroxymeth-yluracil-containing partner even when addedto cells 5 min after infection with SP82. How-ever, when the transformable strain of B.subtilis 168 was the host, dominance was de-pendent on the order of addition. When cultureswere preinfected with SP82, j322 developmentwas blocked; when the order of infection wasreversed, ,22 progeny were released and fewSP82 were produced.The results of the present investigation indi-

cate that dominance in SP82-f322 mixed infec-tions is correlated with qualitative and quanti-tative differences in the expression of the viralgenomes.

MATERIALS AND METHODSBacterial strains, phages, and growth condi-

tions. B. subtilis 168 (try-) and B. subtilis SB11(try-) were obtained from E. W. Nester. Phages ,22and SP82 were supplied by R. H. Doi and C. H.Stewart, respectively. A modified M medium (16) wasused for cell growth, isotopic labeling of infected cells,and preparation of phage lysates as described previ-ously (9). Titering of phage, determination of prog-eny, and multiplicity of infection (MOI) were outlinedearlier (11).

Nucleic acid hybridization. The method of Gilles-pie and Spiegelman (7) was used for RNA-DNAhybridization and that of Denhardt (5) for DNA-DNAhybridization. The procedure described earlier (9) forperforming competition hybridization was modifiedas follows: all reaction mixtures contained 10 lAg of[H3JRNA, competitor RNAs as indicated and 40 jig ofdenatured DNA adsorbed to filters in a final volumeof 0.3 ml of 0.3 M NaCl plus 0.03 M sodium citrate (2x SSC). After incubation at 65 C for 15 h, the filterswere removed, treated for 30 min with 10 gg ofRNase A (Sigma Chemical Co., St. Louis, Mo.) per ml,washed thoroughly in 2 x SSC, and dried, and theradioactivity was determined using a toluene-basedscintillant. Chloramphenicol-RNA (CM-RNA) wasprepared by adding chloramphenicol (20 Ag/ml final

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concentration) 5 min before adding phage; cells wereharvested 15 min after phage addition. The hybridiza-tion reactions are highly specific (9). For example,addition of 1 mg of 322 RNA (RNA extracted from B.subtilis 5, 15, or 30 min after single infection with ,B22)produced a decrease of 3 to 5% in the binding ofSP82-specific [H3]RNA to SP82 DNA. The samepercentage of decrease was obtained on addition of 1mg of completely heterologous RNA (sea urchinRNA).

RESULTS

Progeny release and time of lysis in SP82-f22 mixed infections. Table 1 summarizes theresults of studies of progeny released in themixed infections discussed in the present paper.These data show that dominance was straindependent, with fl22 always blocking develop-ment of SP82 in mixed infections in B. subtilisSB11. In contrast, considerable numbers ofSP82 progeny were released when SP82 wasadded to B. subtilis 168 cultures before or

simultaneously with p22. It should be noted,however, that the dominant phage in eitherinfection never produced the same yield ofprogeny in mixed infections as in single infec-tions. Our previous studies (11) have shown thatthe progeny yields in these mixed infections arelargely independent of MOI and that bothphages adsorb with high efficiency to cellswhich have been infected simultaneously or

consecutively with both phages. The time oflysis after infection with SP82 was 35 to 40 minfor strain 168 and 40 to 50 min for strain SB11;there were no significant differences in the time

TABLE 1. Relative number of progeny released inSP82-g22 mixed infections in Bacillus subtilis strains

168 and SBlla

Host Time of Time of

strain addition of addition of SP82 #22of B. SP82 (min) ,22 (min) progeny progeny

subtilis

0 1000 100

168 0 0 30 40 5 59 15 0 8 20

0 1000 100

SB11 0 0 <1 780 5 <1 505 0 <1 21

a A value of 100 = 125 x 108 PFU per ml for #22 inB. subtilis 168 and 46 x 108 PFU per ml in strainSB11. The corresponding values for SP82 were 93 x

108 and 214 x 108 PFU per ml. The MOI was 6 for eachphage.

of lysis (50 to 55 min) of the two host strains byf22. The time of lysis of mixed infections ineither strain was 40 to 50 min; hence, it was notapparent whether lysis was controlled by one ofthe phages. However, the plaque-forming effi-ciency of SP82 was significantly lower on strainSB11 than on strain 168, and the progeny yieldat low MOI (0.2) was markedly less in infectionsof SB11 than 168 although equally high progenyyields were obtained from infections at MOI of 5to 10 (11). Comparisons of the two strainsinfected with f22 showed no significant differ-ences either in the plaque-forming efficiency orthe progeny yield at low MOI.DNA synthesis in SP82-f22 mixed

infections. Replication of DNA in bacteriasingly or mixedly infected with f322 and/or SP82was studied by pulse-labeling cells with[H3 ]adenosine, isolating the DNA and hybridiz-ing it to phage or host DNA immobilized onnitrocellulose membrane filters. The results(Table 2) show that very little host DNA wasmade in either single or mixed infections 20 to30 min into the latent period whereas largeamounts of f22 and SP82 DNA were synthe-sized in single infections of both host strains.

Control of DNA synthesis in mixedly infectedcells corresponded closely to observations madein progeny studies. In B. subtilis 168, the firstphage entering the cell was able to replicate itsDNA whereas replication of the second phage

TABLE 2. Host and phage DNA synthesis in SP82-(322mixed infectionsa

adTime of Radioactivity (%) hy-

Strain addition of Time of bridized to DNA from:"Strainphage(min)pulse"SP82 B22 SP82 322 Host

168 0 20-30 58.7 0.40 20-30 38.0 0.1

0 5 25-30 35.8 5.4 0.145 0 25-30 2.3 26.0 0

SB11 0 20-25 53.0 0.230 20-25 44.5 0.11

0 5 25-30 9.3 25.6 0.225 0 25-30 0.36 27.0 0

a The experiments were performed at an MOI of 10for each phage.

'Minutes after addition of the first phage.c One-hundred percent radioactivity in these exper-

iments ranged from 1,200 to 5,000 counts/min. Thevalues given in the table were determined by plottingthe counts per minute hybridized under conditionsdescribed in Materials and Methods when threeconcentrations of each labeled nucleic acid wereincubated with 40 gg of each filter-bound DNA.

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genome was greatly reduced. Quantitatively,the proportions of the two phage DNAs corre-

sponded to the yields of progeny (Table 1). Viralgenome replication in mixed infections con-

ducted in B. subtilis SB11, on the other hand,was dominated by f322 regardless of the order ofaddition of the phages, although both SP82 and,22 grew in this host in single infections. How-ever, the amounts of SP82 DNA synthesized instrain SB11 when this phage preceded,22 (ca.9% of the input radioactivity hybridized) waslarge in relation to the negligible ( < 1%)progeny yield. Thus, it seems unlikely thatreplication of SP82 DNA can be attributed tosingle infection of a small proportion of the cellsand suggests that some replication of SP82DNA occurred in cells infected with f322.RNA synthesis in SP82-,B22 mixed

infections. Transcription in mixed infectionswas investigated by pulse-labeling cultures with[H3]uridine at an early and a late time duringthe latent period, extracting the RNA andhybridizing it to filter-bound denatured SP82,,22, or host DNA. The data in Table 3 showthat in mixed infections in B. subtilis 168 thefirst phage added to the culture largely con-

trolled transcription with relatively little syn-thesis of RNA specific to the subordinate phage.Conversely, when B. subtilis SB11 served ashost (Table 4), the order of addition had no

effect: RNA specific for ,22 was producedpreferentially whether #22 was added before orafter SP82. However, as in the case of DNAsynthesis, a greater proportion of SP82-specificRNA was detected relative to the progeny yield( < 1%) when SP82 was added before ,B22.Again, these results probably cannot be attrib-uted to synthesis of SP82 RNA by a smallpopulation of cells singly infected with thisphage.Analysis of SP82-specific RNA synthesized

in mixed infections. The results of the preced-ing experiments indicate that dominance inSP82-,B22 mixed infections was correlated withthe ability of the successful phage, and an

inability of the subordinate partner, to directthe synthesis of near normal amounts of nucleicacids. These results do not, however, provideany information concerning the nature of thephage-specific RNAs synthesized in the mixedinfections. In several such infections, for exam-ple, the subordinate phage was able to producesmall but significant amounts of RNA and itwould be of interest to know whether thetemporal sequence of transcription was thesame as in single infections. Moreover, in allmixed infections, the yield of the dominantphage was less than that observed in single

TABLE 3. Host and phage RNA synthesized inSP82-fl22 mixed infections in B. subtilis 168&

Time of addition Radioactivity (%) hy-of phage (min) Time of bridized to DNA from:'

pulsebSP82 ,22 SP82 #22 Host

0 5-10 9.1 5.50 20-25 26 4.2

0 5-10 15 2.00 20-25 22 2.8

0 5 10-15 22 2.4 5.00 5 25-30 38 3.6 1.95 0 10-15 8 31 4.25 0 25-30 10 35 2.8

a The experiments were performed at an MOI of 10for each phage.

b Minutes after addition of the first phage.c One-hundred percent radioactivity in these exper-

iments ranged from 2,000 to 40,000 counts/min;percent hybridized to each DNA was calculated as de-scribed for Table 2.

TABLE 4. Host and phage RNA synthesized inSP82-fl22 mixed infections in B. subtilis SBl la

Time of addition Radioactivity (%) hy-of phage (min) Time of bridized to DNA from:'

pulse'SP82 ,22 SP82 622 Host

0 5-10 14.2 4.10 20-25 31.0 0.01

0 5-10 25.4 3.00 20-25 30.0 4.5

0 5 10-15 4.3 34.0 6.80 5 25-30 12.5 26.0 3.25 0 10-15 2.9 27.0 4.95 0 25-30 2.0 37.0 5.7

a The experiments were performed at an MOI of10 for each phage.

b Minutes after addition of the first phage.c One-hundred percent radioactivity and calcula-

tion of percent hybridized to each DNA was as de-scribed for Table 3.

infections of the same phage. It is possible thatthis "depressor effect" reflected qualitativeand/or quantitative changes in the transcrip-tion of the dominant phage resulting from thepresence of the subordinate phage.Studies of the populations of phage-specific

RNAs synthesized in mixed infections centeredon SP82 because the transcriptional program ofthis phage has been studied more thoroughlythan that of I22. Moreover, it seemed reasona-ble to assume that transcription of 022 in mixedinfections in strain SB11 would parallel that insingle infections since ,22 dominated the infec-tion with regard to synthesis of nucleic acids

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and progeny yield. Previous investigationsshowed that six temporal classes ofSP82-specific RNA can be distinguished duringinfection of B. subtilis 168 (13) and SB11(Whiteley, unpublished data); these classes aredefined on the basis of the onset and cessationof synthesis as measured by competition hy-bridization. A similar program of transcriptionhas also been established for the related phageSPOl (6). It should be noted, however, thatcompetition hybridization using total phageDNA is a relatively insensitive method espe-cially when applied to transcription of largephages such as SP82 and ,B22, which undoubt-edly have highly complex transcriptional pro-grams. However, direct comparisons of RNApopulations produced in single and mixed infec-tions should permit detection of large changesin the transcriptional program or marked quan-titative differences in the RNA classes.The analysis of SP82 transcription in mixed

infections utilized three competitor unlabeledRNA preparations obtained from two strains ofsingly infected B. subtilis: CM-RNA, 15-minRNA, and 30-min RNA. CM-RNA was isolatedfrom either strain 168 or strain SB11 15 minafter infection with SP82 in the presence ofchloramphenicol; such RNA is known to con-tain only early transcripts synthesized by un-modified host RNA polymerase (13, 14). Theother two competitor RNAs were extracted fromSP82-infected cells harvested at the indicatedtimes during the latent period; these are ex-pected to contain middle and late RNAs, re-spectively.The data in Fig. 1 come from an analysis of

radioactive RNAs isolated at two intervals fromB. subtilis 168 cells infected first with SP82 andthen with (322. The competition curves for thismixed infection show that CM-RNA was amoderately good competitor for the 10- to 15-min pulse-labeled RNA but only slightly re-duced the binding of the 27- to 30-min pulse-labeled preparation. The 30-min unlabeledRNA, on the other hand, was an excellentcompetitor for the latter transcripts, but was apoor competitor for the 10- to 15-min radioactiveRNA. As expected, 15-min RNA was a bettercompetitor when tested with the 10- to 15-minlabeled RNA than were either CM-RNA or 30-min RNA, indicating that a distinctive popula-tion of transcripts was present in the 10- to15-min interval of the latent period. These dataare consistent with the view that, as in singleinfections, early, middle, and late classes ofSP82 RNA were made in this mixed infection inwhich SP82 was dominant and successful inproducing progeny.

-J8060WM CM-RNA

15' RNA20-= 30' RNA

0 0.2 Q4 0.6 0.8 1.0mg COMPETlITOR RNA

FIG. 1. Competition between unlabeled RNA iso-lated from B. subtilis 168 singly infected with SP82and radioactive RNA from B. subtilis 168 infectedwith SP82 5 min before addition of (22. Hybridizationreactions were performed as described in Materialsand Methods. The specific activities and percenthybridization in the absence of competitor RNA were400 counts/min per ug and 18% for the 10- to 15-min[H3]RNA ( ) and 1,418 counts/min per ,ug and 34%for the 27- to 30-min [H3]RNA (-----).

Even though only a small amount ofSP82-specific RNA was synthesized in the re-ciprocal experiment in which ,B22 was added 5min before SP82, the classes of transcriptsproduced by the latter were qualitatively simi-lar to those obtained in the preceding experi-ment (Fig. 2). Once again, a population ofRNAmolecules containing less late RNA and moreearly classes of RNA was found in the 10- to15-min pulse-labeled RNA and the reverse wasobserved in the 25- to 30-min labeled prepara-tion. It should be noted that the times oflabeling do not correspond precisely to those inthe previous experiment because SP82 wasadded 5 min after ,B22, but the data show thatthe most easily distinguished possible changesin transcription (i.e., continuation of early RNAsynthesis into late times or production of lateRNA at an earlier time in the latent period) didnot occur. The above observations imply thatany modification of the RNA polymerase ortranscription factors by the dominant ,B22 phage(i.e., the first added phage) did not interfere, atleast qualitatively, with the transcription of thesecond phage genome.The analyses of SP82-specific RNA synthe-

sized in mixed infections in B. subtilis SB11were limited to the infection in which SP82 wasadded 5 min before (322 since only negligibleamounts of SP82 RNA were found in thereciprocal experiment. The results presented in

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80 -C

C) -A\ CM-RNAo . -~~~CM-RNA

O 40 _ N

Ne -A~~~~N20 - ---3&30 RNA

. , , , . . . I I

0 0.2 0.4 0.6 0.8 1.0mg COMPETITOR RNA

FIG. 2. Competition between unlabeled RNA iso-lated from B. subtilis 168 singly infected with SP82and radioactive RNA from B. subtilis 168 infectedwith jf22 5 min before addition of SP82. Hybridizationreactions were performed as described in Materialsand Methods. The specific activities and percenthybridization in the absence of competitor RNA were300 counts/min per lAg and 6% for the 10- to 15-min[H3 ]RNA ( ) and 550 counts/min per ug and 10%for the 25- to 30-min [H3]RNA (-----).

Fig. 3 show some qualitative and quantitativedifferences from the competition curves ob-tained in the analogous experiment in B.subtilis 168 (Fig. 1). First, it may be noted thatthe 10- to 15-min pulse-labeled RNA from theinfection in strain SB11 was almost completelycompeted by CM-RNA whereas its binding toDNA was only slightly affected by 30-min RNA.Although similar to the results with the corre-sponding radioactive RNA from mixedly in-fected B. subtilis strain 168, the enhanced effec-tiveness of CM-RNA in the present experimentsuggests that RNA synthesized 10 to 15 minafter infection in SB11 contained mostly earlytranscripts rather than middle RNAs. Thisconclusion was also supported by the competi-tion with 15-min RNA. In the SP82-022 mixedinfection in strain 168 (Fig. 1), the latter RNAwas the most efficient competitor of 10- to15-min pulse-labeled RNA, whereas in the cor-responding experiment in strain SB11 15-minRNA was not as effective as CM-RNA. To-gether, these experiments suggest a delay in thesynthesis of middle classes ofRNA in the mixedinfections in SB11.

Competition experiments with 25- to 30-minpulse-labeled RNA obtained from mixedly in-fected SB11 cells demonstrate, however, that atleast some classes of late SP82 RNAs weresynthesized by this subordinate phage. How-ever, CM-RNA remained a more effective com-

petitor of 25- to 30-min pulse-labeled RNA thanof the corresponding RNA from the identicalinfection in B. subtilis 168 in which SP82 wasdominant.Time of synthesis of SP82 RNA in the two

host strains. The above finding that CM-RNAwas a significantly more effective competitor inthe binding of early [H13]RNA synthesized instrain SB11 (Fig. 3) than in the same experi-ments performed with [H3 ]RNA produced afterinfection of strain 168 (Fig. 1) suggests that thetemporal program of SP82 RNA synthesis in theformer strain must be altered in some way. Todetermine whether this was a feature of mixedinfections or reflected some inherent aspect ofthe infection of each host by SP82, a compari-son was made of the extent of synthesis ofSP82-specific RNA in single infections of thetwo host strains. There was markedly less SP82RNA detected in [H3]RNA extracted early inthe infection when strain SBll was the host,suggesting that an early step in the phageinfection was affected (Table 5). In contrast,synthesis of SP82 RNA began promptly afterinfection of strain 168. However, approximatelyequal levels of SP82 RNA were found in RNAsextracted from both strains later in the infec-tion. No significant differences could be foundin the over-all incorporation of [H3]uridine intoRNA or in the specific activities of host RNApolymerases either in crude extracts or in puri-fied fractions obtained from uninfected cells ofthe two B. subtilis strains (Spiegelman and

100

O 80

0

0 20

0 0.2 0.4 0.6 0.8 1.0mg COMPETITOR RNA

FIG. 3. Competition between unlabeled RNA iso-lated from B. subtilis SBll singly infected with SP82and radioactive RNA from B. subtilis SBII infectedwith SP82 5 min before addition of ,822. Hybridizationreactions were performed as described in Materialsand Methods. The specific activities and percenthybridization in the absence of competitor RNA were1,040 counts/min per ug and 4% for the 10- to 15-min[H3 ]RNA ( ) and 810 counts/min per Ag and 12.5%for the 25- to 30-min [H3]RNA (-----).

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TABLE 5. Synthesis of SP82 RNA in two strains ofB. subtilis

Radioactivity (%) hybridizedto SP82 DNA when pulse-

Time of pulseb labeled in:"

B. subtilis 168 B. subtilis SB11

-2-1.5 1.37 0.25-2-3 2.4 0.250-5 8.2 1.55-10 9.1 14.210-15 12.4 17.820-25 26.0 31.0

a The experiments were performed at an MOI of 10for each phage.

Minutes, relative to addition of phage.One-hundred percent radioactivity in these exper-

iments ranged from 17,300 to 80,000 counts/min;percent hybridized to SP82 DNA was calculated as de-scribed for Table 2.

Whiteley, unpublished data). Thus, it does notseem likely that the decreased level of SP82transcription in strain SB11 can be attributedto the presence of a less active host RNApolymerase. It will be recalled that the synthe-sis of early RNA classes in this infection, as inother phage infections (1, 2), is catalyzed by theunmodified host polymerase (13).

DISCUSSIONFrom the results of the present study of

SP82-f22 mixed infections and previous investi-gations of f22-SP02c, and SP82-SP02c, infec-tions (9), it can be concluded that the subordi-nate phage is unsuccessful because it cannotefficiently replicate and transcribe its DNA. Itis also possible, of course, that other steps indevelopment of this phage are inhibited, but thereduced capacity to synthesize nucleic acidswould, in itself, be sufficient to explain subordi-nation. The dominant phage, on the other hand,carries out a program of DNA replication andtranscription which appears to be qualitativelysimilar over-all to that occurring in single infec-tions but which may be quantitatively less andthus correlated with the depressor effect.

In 322-SP82 mixed infections in which B.subtilis 168 was the host bacterium, dominancewas dependent on the order of addition and it istempting to speculate that the first phage,perhaps reflecting a temporal advantage insynthesizing the first proteins, interfered withexpression of the second phage. Such interfer-ence could occur at several points. We havepreviously considered the possibility that thefirst phage saturates all receptor sites, thus

preventing attachment of the second phage, buthave found that the super-infecting phage bindswith high efficiency (11).A more likely locus of competition may be the

membrane replication positions or transcriptionsites. Snustad (15) has shown that in T4 infec-tions of E. coli only a limited number of viralgenomes can be replicated or phenotypicallyexpressed in multiply infected cells. One inter-pretation of this finding is that each cell con-tains a finite number of compartments, perhapsmembrane sites, one of which must be occupiedby a phage genome before viral-directed macro-molecular synthesis can occur. Evidence for anexclusion from genetic activity, suggesting simi-lar compartmentalization, has been obtained instudies of sequential infections of B. subtilis bymutants of phage q1 (C. W. Rettenmier and H.E. Hemphill, unpublished data). It is notknown, however, whether unrelated phagessuch as SP82 and 122 compete for the samesites.Another early step in phage development

which might affect the yield of the second phageare viral-directed changes in the host DNA-dependent RNA polymerase. Infection of B.subtilis 168 with SP82 results in the appearanceof a modified RNA polymerase as early as 5 to10 min after infection (14; unpublished data).This altered enzyme has a higher specific activ-ity than the host enzyme and has a differentsubunit composition: it contains the f, ,B', and asubunits found in the host enzyme but has agreatly diminished quantity of a subunit andcontains small polypeptides not found in thehost enzyme. The modified polymerase synthe-sizes early and middle classes of SP82 RNA invitro, whereas the host polymerase producesonly early RNA in vitro (13). Although thepolymerase isolated from ,322-infected cells hasnot been examined in similar detail, it is knownthat the subunit composition is also differentfrom that of the host enzyme: it has a lowercontent of the a subunit and has small polypep-tides whose molecular weights are differentfrom the small polypeptides found in SP82-modified polymerase (Whiteley et al., unpub-lished data). Preliminary investigations of en-zyme specificity in vitro indicate that purifiedSP82-modified RNA polymerase transcribesonly early RNAs from 122 DNA. If the modified,B22 polymerase has the same type of specificity,dominance in B. subtilis 168 could result fromthe inability of the second phage to produce asufficient quantity of modified polymerase. Asmall amount of the latter enzyme could besynthesized, however, since host protein synthe-sis, including the production of core polymerase

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subunits, continues until at least the mid-pointof the latent period (Spiegelman and Whiteley,unpublished data). Such synthesis could ac-count for the observed production of smallamounts of SP82-specific middle and late RNApopulations in infections where this phage wasthe subordinate partner.Perhaps the most puzzling aspect of the

,22-SP82 mixed infections was the ability offy22 to block development of SP82 when B.subtilis SB11 was the host. In single infectionsof SB11, at the high MOI used in the presentstudy, SP82 yielded progeny, modified the po-lymerase (Spiegelman and Whiteley, unpub-lished data), and carried out a program oftranscription which was similar to that found instrain 168. One might, therefore, expect thatSP82 would produce progeny under the sameconditions in which it expressed dominance inmixed infections of strain 168 (i.e., when SP82was the first phage infecting the cell). Thiswould be especially true if dominance dependson a competition for sites of genetic expression.Interference in the production of SP82 progenycould also occur at the translational level or inthe final stages of phage assembly. These possi-bilities receive some support from the findingthat an appreciable amount of SP82-specificRNA (ca. one-third the amount synthesized insingle infections) is produced at a late time inthe mixed infection even though very few intactphages are released.

It is also known that B. subtilis SB11 andother derivatives of W23 possess an interferencemechanism which causes abortive infection ofseveral 5-hydroxymethyluracil-containingphages at low MOIs (12). For example, theplating efficiency of SP82 is 10,000-fold lower inSB11 than in 168. This interference phenome-non is not seen at high MOI in liquid media andthe development of the phage appears normalby the criteria studied in the present investiga-tion. However, the requirements for high MOIfor productive infection in SB11 and the presentfinding that the transcription of the SP82 ge-nome is reduced early in infection suggests thatsome cooperative effect between several SP82genomes may be needed for the infection tosucceed. A similar type of phenomenon hasbeen noted previously for transfection of B.subtilis by SP82 DNA (8). Several molecules ofDNA are required for transfection apparentlybecause the entering DNA is inactivated byhost nuclease(s). McAllister and Green (10)reported that this inactivation process is inhib-ited in phage infection of a transformable strain

and that maximal inhibition occurs at approxi-mately 6 min after addition of the phage. Thus,in,22-SP82 mixed infections in strain SB11,,22 could either interfere with some cooperativeprocess required for successful infection of SB11by SP82 or the temporal delay required toorganize the cooperative process could provide asufficient advantage so that ,22 can dominatethe infection.

ACKNOWLEDGMENTSThis investigation was supported in part by American

Cancer Society grant VC-46B. H. R. Whiteley is a recipient ofResearch Career Award K6-GM-442 from the National Insti-tute of General Medical Sciences.

LITERATURE CITED1. Bautz, E. K. F. 1972. Regulation ofRNA synthesis. Prog.

Nucl. Acid Res. Mol. Biol. 12:129-160.2. Calendar, R. 1970. Regulation of phage development.

Annu. Rev. Microbiol. 24:241-296.3. Delbruck, M. 1945. Interference between bacterial vi-

ruses. III. The mutual exclusion effect and the depres-sor effect. J. Bacteriol. 50:151-170.

4. Delbruck, M., and S. E. Luria. 1942. Interference be-tween bacterial viruses. I. Interference between twobacterial viruses acting upon the same host and themechanism of virus growth. Arch. Biochem. 1:111-117.

5. Denhardt, D. T. 1966. A membrane-filter technique forthe detection of complementary DNA. Biochem. Bio-phys. Res. Commun. 23:641-46.

6. Gage, L. P., and E. P. Geiduschek. 1971. RNA synthesisduring bacteriophage SPOl development: six classes ofSPOl RNA. J. Mol. Biol. 57:279-300.

7. Gillespie, D., and S. Spiegelman. 1965. A quantitativeassay for DNA/RNA hybrids with DNA immobilizedon a membrane. J. Mol. Biol. 12:829-842.

8. Green, D. M. 1966. Intracellular inactivation of infectiveSP82 bacteriophage DNA. J. Mol. Biol. 22:1-13.

9. Kolenbrander, P. E., H. E. Hemphill, and H. R. White-ley. 1973. Mixed infections of Bacillus subtilis involv-ing bacteriophage SP02c,. J. Virol. 11:25-34.

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