JOURNAL OF BACTERIOLOGY, Apr. 1990, p. 1948-1953 Vol. 172, No. 40021-9193/90/041948-06$02.00/0Copyright C) 1990, American Society for Microbiology

Bacteriophage-Enhanced Sporulation: Comparison of Spore-Converting Bacteriophages PMB12 and SPlO

TAMI H. SILVER-MYSLIWIEC AND MICHAEL G. BRAMUCCI*

Department of Microbiology and Immunology, Hahnemann University, Philadelphia, Pennsylvania 19102

Received 16 August 1989/Accepted 29 December 1989

The previously characterized bacteriophage SP10 enhanced the frequency of wild-type sporulation byBacillus subtilis W23 and 3-13. Comparison of SP10 with the spore-converting bacteriophage PMB12 indicatedthat both bacteriophages significantly increased the sporulation frequency of an oligosporogenic mutant thatcontained spoOJ::Tn917 flHU261. SP10 and PMB12 caused wild-type bacteria to sporulate in a liquid mediumthat initially contained enough glucose to inhibit the sporulation and expression of cL-amylase by uninfectedbacteria. SP10 also induced the expression of a-amylase in the presence of glucose, whereas PMB12 had nodetectable effect. These observations were consistent with the conclusion that SP10 is a spore-convertingbacteriophage and that SP10 and PMB12 relieve glucose-mediated catabolite repression of sporulation bydifferent mechanisms.

Bacteriophage genomes are frequently "trapped" inspores; i.e., lytic bacteriophage development is repressed ina sporulating cell and the bacteriophage DNA segregatesinto the developing spore (8). After germination of the spore,the bacteriophage genome may direct lytic development insome progeny of the newly emerged cell. Bacteria that areinfected with a spore-converting bacteriophage sporulate athigher frequencies than uninfected cells, resulting in morefrequent entrapment of the spore-converting bacteriophageDNA. Spore-converting bacteriophages have been isolatedfor Bacillus pumilus, B. subtilis, B. thuringiensis, and Clos-tridium perfringens (4, 5, 10, 19, 21). All of the knownspore-converting bacteriophages are pseudotemperate; i.e.,they form turbid plaques and enter an unstable carrier statein the host cell.

In addition to enhancing wild-type sporulation, spore-converting bacteriophages significantly increase the sporu-lation frequencies of Spoc mutants (4, 5, 10, 11, 19). Spocmutants are oligosporogenic or sporulation negative (Spo-).The B. subtilis bacteriophage PMB12 suppresses the stage 0sporulation mutations spoCM-J, spoOJ93, and spoOK141 (5,11). This observation suggests that PMB12 must influencehost cell development before or during the earliest stage ofsporulation. Isolation of PMB12 mutants that do not sup-press Spoc mutations demonstrated that PMB12-enhancedsporulation is not an incidental consequence of bacterio-phage infection, such as preferential killing of vegetativecells. Complementation analysis of the PMB12 mutantsindicated that PMB12 has at least three genes (scn genes)that are required for it to enhance host cell sporulation (11).Since none of the scn mutants enhance the sporulation ofany of the tested Spoc mutants (11), it is unlikely that PMB12suppresses Spoc mutations by complementation.The ability of PMB12 to enhance sporulation has not

previously been compared with that of any other spore-converting bacteriophage, because no suitable host strainwas available. In addition, none of the other previouslydescribed B. subtilis bacteriophages were characterized asspore-converting bacteriophages. Keggins et al. (10) re-ported that bacteriophage SP10 (2, 24) is immunologically

* Corresponding author.

cross-reactive with the B. pumilus spore-converting bacte-riophage PMB1, but could not demonstrate SP10-enhancedsporulation for any of more than 50 independently isolatedB. subtilis W23 Spo- mutants. The data in the present reportestablish SP10 as a spore-converting bacteriophage. Com-parison of PMB12 and SP10 in a common host strainprovided evidence for the conclusion that these bacterio-phages relieved glucose-mediated repression of sporulationby different mechanisms.

MATERIALS AND METHODS

Bacterial strains, bacteriophages, and culture conditions.The bacterial strains used in this study are described in Table1. Penassay broth (Difco Laboratories) was used for routineliquid cultures. Nutrient broth-salts medium (NBS) was usedto propagate SP10 and was prepared as previously described(2). Sporulation experiments were performed in phosphate-buffered 2XSG, which was the same as 2XSG (13), exceptthat FeCl2 was omitted and the medium contained 14 g ofK2HPO4 and 6 g of KH2PO4 per liter (pH 7.0). All liquidcultures were incubated at 37°C with gyratory shaking atapproximately 100 rpm.SP10 plaque assays were performed by standard tech-

niques (1), with tryptone semisoft agar (4) overlaid ontotryptose blood agar base (TBAB; Difco). SP10 lysates wereprepared by inoculating a single SP10 plaque into 10 ml ofNBS and incubating the cultures overnight. The cultureswere clarified by centrifugation, and the culture supernatantswere filter sterilized. SP10 productively infects strains W23and ATCC 7003, but not strain 168 (10, 24, 25). SP10 lysatesprepared on W23TL formed plaques with equal efficiency onstrains W23TL and 3-13. PMB12 lysates were prepared aspreviously described (5). PMB12 productively infects strains168, W23, and ATCC 7003 (5). PMB12 lysates prepared onstrain 1S28 (11) formed plaques with equal efficiency onstrains 1S28, W23TL, and 3-13.

Bacteriophage-infected carrier colonies were isolated andmaintained by streaking infected bacteria onto TBAB thatwas no more than 24 h old.

Cross-streak assay for bacteriophage-enhanced sporulation.The cross-streak assay for bacteriophage-enhanced sporula-tion has been described previously (11, 16) and was per-

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TABLE 1. B. subtilis strains

Strain Descriptiona Source orreference

W23TL Thi- Lys- derivative of W23 Kenneth Bott3-13 His- derivative of ATCC 7003 15KS261 spoOJ::Tn9J7 fQHU261 20MGB3004 Ermr Spoc recombinant obtained This study

by transforming 3-13 withKS261 DNA

BR151 Iys-3 metB1O trpC2 2693.2(4i105J113) spoOJ93 trpC2, lysogenic for 6

il05J113

aThe following phenotypic designations are used: auxotrophic require-ments for histidine, lysine, and thiamine are abbreviated His-, Lys-, andThi-, respectively; Ermr designates the erythromycin-lincomycin resistanceconferred by Tn9O7; and Spol indicates an oligosporogenic or Spo- pheno-type that was suppressed during infection by a spore-converting bacterio-phage.

formed as follows. Bacteriophage lysate was applied as anarrow band across the center of a petri plate containingTBAB that was no more than 24 h old, and bacteria werestreaked across the dried lysate. After incubation at 37°C forthe specified time, bacteria from the uninfected and bacte-riophage-infected regions of the cross-streaks were viewedin wet mounts under a phase-contrast microscope. Thefrequency of sporulation was determined by calculating themean percentage of phase-bright bodies in samples, contain-ing at least 100 cells, that were counted in five different fieldsof the same wet mount.

Bacteriophage-mediated sporulation in media supplementedwith glucose. An SP10-infected or PMB12-infected carriercolony of strain 3-13 was inoculated into phosphate-buffered2XSG or phosphate-buffered 2XSG that contained 2% glu-cose. Sporulation was obtained by allowing the bacteria toenter the stationary phase of growth and exhaust the me-dium. For experiments in which sporulation and a-amylaseactivity were evaluated after overnight incubation, 10-mlcultures were incubated at 37°C with gyratory shaking (100rpm) in 125-ml Erlenmeyer flasks. Cultures for time courseexperiments were incubated under similar conditions, ex-cept that 30-ml cultures were incubated in 300-ml side-armErlenmeyer flasks and 1-ml samples were periodically re-moved for a-amylase and glucose assays. The turbidity ofthe cultures was measured with a Klett-Summerson color-imeter (green filter). The frequency of sporulation wasdetermined by phase-contrast microscopy, as describedabove, or by heat shocking (at 70°C for 20 min) cells that hadbeen removed from the culture by centrifugation and sus-pended in a volume of sterile water equal to the originalsample volume. The levels of a-amylase activity were mea-sured in samples of the cultures that were clarified bycentrifugation. The ox-amylase assay was performed as de-scribed by Nicholson and Chambliss (17), except that en-zyme activity was expressed as units of ox-amylase permilligram (wet weight) of cells. A commercial kit (no. 510;Sigma Chemical Co.) was used as specified by the manufac-turer to determine the concentration of glucose in thesamples of culture supernatants.

Genetic techniques. Strains 3-13 and BR151 were madecompetent for transformation as previously described (3,15). Ermr transformants were selected on TBAB containingerythromycin (1 ,ug/ml) and lincomycin (25 ,ug/ml). Ermrtransformants of BR151 were tested for complementation by4l05J113 as described by Errington and Jones (7). 4l05J113

TABLE 2. Cross-streak assay for bacteriophage-enhancedsporulation by B. subtilis W23TL and 3-13

Strain Phage % Sporulation'

W23TL None <0.2SP1o 5.1PMB12 <0.2

3-13 None <0.2SP1O 3.7PMB12 14.2

aDetermined as the percentage of phase-bright bodies.

lysates for the complementation assays were prepared bymitomycin C induction (9).

RESULTS

Establishment of SP10 as a spore-converting bacteriophage.The turbid plaque morphology of SP10 on strain W23TLinitially suggested that SP10 is a spore-converting bacterio-phage. Microscopic examination of the bacteria from theplaque assay plates after overnight incubation revealed thatthe plaque centers contained considerably more spores thanthe uninfected lawn surrounding the plaques. This observa-tion was similar to previous reports concerning plaqueformation by PMB1 on the wild-type B. pumilus NRS576 andplaque formation by PMB12 on the B. subtilis 168 auxo-trophic mutant BR151 (4, 5, 16).

Since SP10 and PMB12 have overlapping host ranges (5,10, 24), several wild-type B. subtilis strains that are sensitiveto infection by both bacteriophages were tested for bacte-riophage-enhanced sporulation in a cross-streak assay. Onlysporulation by strain 3-13 was enhanced by both bacterio-phages. The data in Table 2 were obtained from a cross-streak assay and indicated that after 18 h neither uninfectedstrain W23TL nor uninfected strain 3-13 had sporulated at adetectable level on TBAB. Although PMB12 infection didnot significantly affect W23TL sporulation, it increased 3-13sporulation at least 70-fold. Nearly 4% of the SP10-infected3-13 cells sporulated. However, in contrast to the PMB12-infected W23TL cells, 5% of the SP10-infected W23TL cellssporulated.

It was previously reported that SP10-infected carrier col-onies could be maintained for strain W23 by repeatedsingle-colony isolation and that each restreaking of an SP10-infected carrier colony resulted in segregation of severalcured colonies (2, 24). SP10-infected carrier colonies couldbe similarly maintained for strain 3-13. Uninfected 3-13colonies had a wrinkled appearance on TBAB, whereas,SP10-infected carrier colonies were smooth. Microscopicexamination revealed a high level of sporulation in SP10-infected carrier colonies and few, if any, spores in uninfectedcured colonies after overnight incubation on TBAB. A 3-13SP10-infected carrier colony and a cured derivative wereinoculated into separate Penassay broth cultures. Penassaybroth was used for this experiment because strain 3-13 doesnot sporulate well in this medium. If a sporulation mediumsuch as 2XSG were used for this experiment, uninfected 3-13cells would sporulate at a frequency of 80 to 100% in 24 h,and little or no difference in sporulation frequencies wouldbe observed between the infected and uninfected bacteria.After 72 h, only a small percentage of the cells derived froma cured colony had sporulated, whereas the level of sporu-lation was more than 100-fold higher for the SP10-infected

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TABLE 3. Sporulation in cultures inoculated with a 3-13SP10-infected carrier colony or a cured derivative

Viable count % CFU infectedTime (x 108 CFU/ml) % Sporu- with SplObCulture (h) Het lation'"Ha

Total resistantc Total resistant

3-13 cured 24 1.7 0.00074 0.0433-13 carrier 24 1.4 0.0012 0.086 97.0 100.0

3-13 cured 48 1.3 0.0013 0.103-13 carrier 48 0.72 0.0015 0.21 93.2 92.8

3-13 cured 72 0.97 0.0019 0.203-13 carrier 72 0.48 0.13 27.1 95.8 97.7

a Determined as the percentage of the total viable count that was heatresistant.

b Determined by counting SP10-infected carrier colonies.' 70°C for 20 min.

cells (Table 3). The higher frequency of sporulation forSP10-infected cells was not merely due to selective killing ofvegetative cells, because the total viable counts and heat-resistant viable counts for both cultures were similar after 24and 48 h. The SP10-induced spores could not be detected asPFU in an assay with 3-13 as the indicator strain. However,it was evident that more than 90% of the CFU in the 3-13SP10-infected carrier culture were infected with SP10throughout the experiment, because of the percentage ofCFU in the SP10-infected carrier culture that gave rise todistinctively smooth SP10-infected carrier colonies on theviable-count assay plates for each time point. The presenceof SP10 in the smooth colonies was verified by detectingSP10 plaque-forming activity in the supernatants of severalcultures that were inoculated with individual smooth colo-nies from the assay plates.

Since all of the known Bacillus spore-converting bacte-riophages improve the sporulation frequency of at least oneSpo' mutant, the effect of SP10 on Spoc mutants wasexamined. Since SP10 transduces genetic markers fromstrain W23 to strain 168, it is evident that it can introduceDNA into strain 168, even though it cannot be propagated onstrain 168 (24). However, SP10 had no detectable effect onthe numbers of phase-bright bodies or the level of heat-resistant CFU produced by the strain 168 Spoc mutantsCM-1 (5, 11) and BGSC 1S28 (11) in cross-streak assays(data not shown). Therefore, a strain 3-13 Spoc mutant wasconstructed by transferring a strain 168 sporulation mutationto strain 3-13. KS261 has transposon Tn9J7 inserted in ornear the spoOJ locus (20). The sporulation mutant MGB3004was isolated by transferring spoOJ::Tn9J7 QlHU261 fromKS261 to 3-13 by transformation with selection for Ermr.SP10-infected carrier colonies could be maintained forMGB3004. Microscopic examination revealed a high level ofsporulation in SP10-infected carrier colonies but no spores inuninfected cured colonies after overnight incubation onTBAB.MGB3004 and MGB3004 bacteriophage-infected carrier

colonies were inoculated into phosphate-buffered 2XSG andincubated for 16 h. The data in Table 4 indicated thatMGB3004 was oligosporogenic. Although the bacteriophage-infected MGB3004 cells did not sporulate as well as unin-fected wild-type cells, SP10 and PMB12 increased the fre-quency of sporulation by MGB3004. Since KS261 sporulatesat a frequency of less than 0.01% (20), the level of sporula-tion by MGB3004 during 16 h of incubation in phosphate-

TABLE 4. Bacteriophage-enhanced sporulation of the Spocmutant MGB3004

Strain Phage % Sporulation'

3-13 None 96.2MGB3004 None 20.6MGB3004 SP1O 69.0MGB3004 PMB12 40.0

' Determined as the percentage of phase-bright bodies.

buffered 2XSG was considerably higher than expected.Therefore, the presence of spoOJ::Tn917fQHU261 inMGB3004 was verified as follows. East and Errington haveisolated a nondefective derivative of 4105, designated4105J113, that contains cloned DNA from strain 168 thatcomplements spoOJ93 (6). We found that in addition tocomplementing spoOJ93, 4i105J113 complemented spoOJ::Tn9J7 fQHU261 in KS261. Since 3-13 and MGB3004 wereresistant to infection by 4105J113, Ermr was transferred tothe strain 168 auxotrophic mutant BR151 by transformationwith DNA from MGB3004. Twenty Ermr transformants thathad the auxotrophic requirements of BR151 were examinedand found to be Spo-. The mutation causing the Spo-phenotype of all 20 transformants was complemented by4105J113. Hence, it was most likely that MGB3004 wasoligosporogenic because of the spoOJ: :Tn917fQHU261 inser-tion.

Relief of catabolite repression by SP10 and PMB12. Theenhancing effect of infection with SP10 or PMB12 on wild-type sporulation suggested that these bacteriophages mightcounteract catabolite repression of sporulation. Although allof the CFU in a culture of uninfected 3-13 cells weredetected as heat-resistant spores after 16 h in phosphate-buffered 2XSG, less than 0.4% of the cells in phosphate-buffered 2XSG that was initially supplemented with 2%glucose sporulated during the same time (Table 5). Hence,strain 3-13 sporulation was repressed by glucose. In contrastto uninfected cells cultured in medium containing glucose,all of the SP10-infected cells were detected as heat-resistantspores in medium containing glucose. All of the CFU in theSP10-infected culture were carriers of SP10, as determinedby their smooth colony morphology. Only about 1% of theCFU in the PMB12-infected culture appeared to haveformed heat-resistant spores in medium with glucose. How-ever, two observations indicated that the level of sporulationfor the PMB12-infected cells was actually about 10-foldhigher than the percentage of heat-resistant CFU suggested.First, the frequency of phase-bright bodies in the PBM12-infected culture was 10.3%. Second, a plaque assay indi-

TABLE 5. Bacteriophage-enhanced sporulation by strain 3-13 inphosphate-buffered 2XSG containing 2% glucose

Viable count2% Glucose (x108 CFU/ml)

present Phage Tt % Sporulation'resistant

- None 7.4 8.8 100.0+ None 0.43 0.0016 0.37+ SP1O 8.4 8.9 100.0+ PMB12 2.3 0.031 15.8

aThe percent sporulation for the uninfected and SP10-infected cells wasdetermined as the percentage of the total viable count that was heat resistant.The percent sporulation for the PMB12-infected cells was determined as thepercentage of PMB12 PFU that were heat resistant, as described in the text.

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HOURSFIG. 1. Growth curve and a-amylase production of uninfected and SP10-infected strain 3-13 cells. (A) Growth (K) and ca-amylase activity

(*) of uninfected 3-13 in 2XSG; (B) growth (O) and a-amylase activity (U) of uninfected 3-13 in 2XSG that initially contained 2% glucose;(C) growth (A) and oa-amylase activity (A) of SP10-infected 3-13 in 2XSG that initially contained 2% glucose.

cated that a sample of cells taken from the PMB12-infectedculture and suspended in the original sample volume ofsterile water had 3.6 x 108 PMB12 PFU/ml. Since theconcentration of PFU in the sample was approximately thesame as the concentration of CFU in the PMB12-infectedculture, it was most likely that all of the cells in the culturewere infected with PMB12. Furthermore, since 15.8% of thePFU in the resuspended sample were heat resistant, approx-imately 15% of the PMB12-infected cells had sporulated andall of the spores were infected with PMB12. Taken together,these observations and the data in Table 5 supported theconclusion that SP10 and PMB12 significantly relieved glu-cose-mediated catabolite repression of sporulation.SP10 and PMB12 might counteract catabolite repression

of sporulation by specifically inducing sporulation in thepresence of glucose or, alternatively, by causing a generalderepression of functions that are normally repressed byglucose. Therefore, the effect of these bacteriophages onsynthesis of a.-amylase was examined. Synthesis of a-amy-lase and sporulation are similar in strain 168, because bothactivities are repressed by glucose and occur after vegetativegrowth (17). Glucose repressed the expression of a-amylaseby uninfected strain 3-13 cells cultured in phosphate-buffered 2XSG (Fig. 1; Table 6). The level of a-amylaseactivity of PMB12-infected cells cultured in medium contain-ing glucose was comparable to the low level of a-amylaseactivity of uninfected cells in medium containing glucose,even though the frequency of sporulation was significantlyhigher for the PMB12-infected cells than for the uninfectedcells (Table 6). This observation suggested that PMB12 didnot cause a general derepression of catabolite-repressedfunctions in host cells. In contrast, the cx-amylase activity ofSP10-infected cells was approximately fourfold higher than

TABLE 6. Bacteriophage-induced a-amylase activity for strain3-13 in phosphate-buffered 2XSG containing 2% glucose

2% Glucose Phgc Souato' s-Amylase activitypresent Phage % Sporulation (U/mg of cells)

- None 95.7 11.3+ None <0.2 1.8+ PMB12 38.3 1.9+ SP1O 61.3 7.4

aDetermined as the percentage of phase-bright bodies in each culture.

the ca-amylase activity of uninfected cells cultured in phos-phate-buffered 2XSG containing glucose. The time courseexperiment illustrated in Fig. 1 indicated that expression ofao-amylase by SP10-infected cells in medium containingglucose (Fig. 1C) increased after vegetative growth andremained markedly higher than that in uninfected cells inmedium containing glucose (Fig. 1B).

Sporulation and production of a-amylase by SP10-infectedcells were not attributable to the depletion of glucose. Thedata in Fig. 2 were obtained by measuring the frequencies ofsporulation and the concentrations of glucose in the samplescollected for the time course experiment presented in Fig. 1.These data indicated that the amounts of glucose remainingin the culture supernatants of uninfected and SP10-infectedcells were approximately the same. Therefore, it was evidentfrom the data in Fig. 1 and 2 that the SP10-infected cells weresporulating and expressing et-amylase in the presence of aconcentration of glucose that repressed both activities inuninfected cells.

DISCUSSIONSP10 enhanced the wild-type sporulation of strains

W23TL and 3-13. In addition, infection with SP10 substan-tially enhanced sporulation by the oligosporogenic mutantMGB3004. Hence, the data in this report supported theconclusion that SP10 is a spore-converting bacteriophage.Demonstrating that SP10 enhanced sporulation by strain3-13 provided the first opportunity to compare PMB12 withanother spore-converting bacteriophage in the same hoststrain. Two observations were consistent with the conclu-sion that SP10 and PMB12 enhanced sporulation by differentmechanisms.One difference between the two bacteriophages was that

SP10 enhanced W23TL sporulation, but PMB12 did notsignificantly affect sporulation by this strain. We have ob-served that in addition to strain W23TL, there are severalwild-type strains of B. subtilis and B. pumilus that are withinthe PMB12 host range but do not experience enhancedsporulation as a result of PMB12 infection (Bramucci, un-published data). The inability of PMB12 to enhance W23TLsporulation provided additional support for a previous con-clusion based upon isolation of PMB12 mutants that do notenhance sporulation (11), i.e., that PMB12-enhanced sporu-lation is not an artifact caused by preferential killing ofnonsporulating bacteria.

I

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60 1 155c

v1 40 io~

P4 20

5 10 5 105 10

HOURSFIG. 2. Sporulation and utilization of glucose by uninfected and SP10-infected strain 3-13 cells. (A) Sporulation (*) by uninfected 3-13 in

2XSG; (B) sporulation (E) and glucose concentration (O) in a culture of uninfected 3-13 cells in 2XSG that initially contained 2% glucose; (C)sporulation (A) and glucose concentration (A) in a culture of SP10-infected 3-13 in 2XSG that initially contained 2% glucose.

The second difference between SP10 and PMB12 con-cerned the abilities of these bacteriophages to relieve catab-olite repression of sporulation and a-amylase in strain 3-13.Sporulation and a-amylase are similar in that both functionsare subject to catabolite repression and temporal regulation(14, 17, 18, 22, 23). The gra-10 mutation, which allowsa-amylase to be expressed in the presence of glucose,demonstrates that catabolite repression and temporal regu-lation of ot-amylase are genetically distinct (17). Similarly,the crs mutations, which make sporulation resistant tocatabolite repression, demonstrate that catabolite repressionand temporal regulation of sporulation are also geneticallydistinct (22, 23). Since Laoide and McConnell have demon-strated that the crsA47 mutation relieves catabolite repres-sion of sporulation and of a P-galactosidase gene that is fusedto B. licheniformis a-amylase regulatory sequences, it isevident that some components of the catabolite repressionsystem affect both activities (12). Our observations concern-ing the effects of SP10 and PMB12 on strain 3-13 areconsistent with a model in which some regulatory compo-nents are involved in catabolite repression of ox-amylase andsporulation whereas other regulatory components are spe-cific for catabolite repression of ot-amylase or sporulation.SP10 and PMB12 caused infected wild-type cells to sporulatein medium that was initially supplemented with enoughglucose to repress sporulation by uninfected bacteria. How-ever, SP10 also derepressed expression of ao-amylase under

CATABOLITEREPRESSION

COMMON ELEMENTS - SPlO

SPECIFIC ELEMENTSL/ 'tg PMB12

oa-Amylese Sporulation

FIG. 3. Possible interactions of SP10 and PMB12 with catabo-lite-repressed bacteria.

these conditions, whereas PMB12 did not affect the expres-sion of this enzyme. Although SP10 caused ot-amylase to beexpressed in the presence of glucose, the enzyme apparentlycontinued to be under temporal regulation. Therefore, itseems that SP10 interfered with catabolite repression, result-ing in expression of at-amylase and induction of sporulationafter vegetative growth as if no glucose were present. SP10may have induced sporulation in medium with glucose byinteracting with components that regulate catabolite repres-sion of sporulation and ot-amylase, and PMB12 may haveinduced sporulation by interacting with the sporulation-specific components of catabolite repression (Fig. 3).

ACKNOWLEDGMENTS

We thank K. Bott, C. Thorne, and P. Youngman for generouslyproviding various bacterial and bacteriophage strains. We are par-ticularly grateful to J. Errington for providing il05J113 prior topublication. We also thank K. Bott for helpful suggestions.

This work was supported by Biomedical Research Support grantno. 1S07RR07241-01.

LITERATURE CITED1. Adams, M. H. 1959. Bacteriophages, p. 450-451. Interscience

Publishers, Inc., New York.2. Bott, K., and B. Strauss. 1965. The carrier state of Bacillus

subtilis infected with the transducing bacteriophage SP10. Vi-rology 25:212-225.

3. Bott, K., and G. A. Wilson. 1967. Development of competencein the Bacillus subtilis transformation system. J. Bacteriol.94:562-570.

4. Bramucci, M. G., K. M. Keggins, and P. S. Lovett. 1977.Bacteriophage conversion of spore-negative mutants to spore-positive in Bacillus pumilus. J. Virol. 22:194-202.

5. Bramucci, M. G., K. M. Keggins, and P. S. Lovett. 1977.Bacteriophage PMB12 conversion of the sporulation defect inRNA polymerase mutants of Bacillus subtilis. J. Virol. 24:194-200.

6. East, A. K., and J. Errington. 1989. A new bacteriophage vectorfor cloning in Bacillus subtilis and the use of 4i105 for proteinsynthesis in maxicells. Gene 81:35-43.

7. Errington, J., and D. Jones. 1987. Cloning in Bacillus subtilis bytransfection with bacteriophage vector b105J27: isolation andpreliminary characterization of transducing phages for 23 sporu-lation loci. J. Gen. Microbiol. 133:493-502.

8. Hemphill, H. E., and H. R. Whiteley. 1975. Bacteriophages ofBacillus subtilis. Bacteriol. Rev. 39:257-315.

9. Jenkinson, H. F., and J. Mandelstam. 1983. Cloning of the

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Bacillus subtilis lys and spoIIIB genes in phage 4105. J. Gen.Microbiol. 129:2229-2240.

10. Keggins, K. M., R. K. Nauman, and P. S. Lovett. 1978. Sporu-lation-converting bacteriophages for Bacillus pumilus. J. Virol.27:819-822.

11. Kinney, D. M., and M. G. Bramucci. 1981. Analysis of Bacillussubtilis sporulation with spore-converting bacteriophage PMB12.J. Bacteriol. 145:1281-1285.

12. Laoide, B. M., and D. J. McConnell. 1989. cis sequencesinvolved in modulating expression of Bacillus licheniformisamyL in Bacillus subtilis: effect of sporulation mutations andcatabolite resistance mutations on expression. J. Bacteriol.171:2443-2450.

13. Leighton, T. J., and R. H. Doi. 1971. The stability of messengerribonucleic acid during sporulation in Bacillus subtilis. J. Biol.Chem. 246:3189-3195.

14. Losick, R., P. Youngman, and P. J. Piggot. 1986. Genetics ofendospore formation in Bacillus subtilis. Annu. Rev. Genet.20:625-669.

15. Lovett, P. S., and M. G. Bramucci. 1975. Plasmid deoxyribonu-cleic acid in Bacillus subtilis and Bacillus pumilus. J. Bacteriol.124:484-490.

16. Lovett, P. S., M. G. Bramucci, and K. M. Keggins. 1978. Controlof bacterial sporulation by nonchromosomal replicons: a novelexample of bacteriophage conversion, p. 227-230. In D. Schles-singer (ed.), Microbiology-1978. American Society for Micro-biology, Washington, D.C.

17. Nicholson, W. L., and G. H. Chambliss. 1985. Isolation andcharacterization of a cis-acting mutation conferring catabolite

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