of (CAMPBELL kauA - Genetics of lysis is due to a bacteriocin ("betacin") produced by SPp lysogens that is active on non- lysogens and on SPpdcitK, lysogens. Mutagenesis of strain

THE PROPHAGE OF SPpc2dcitKl, A DEFECTIVE SPECIALIZED TRANSDUCING PHAGE OF BACILLUS SUBTZLZS

RICHARD ROSENTHAL, PATRYCE A. TOYE, RUTH Z. KORMAN AND STANLEY A. ZAHLER

Division of Biological Sciences, Cornell University, Ithaca, New York 14853

ManuscriFt received September 7, 1978 Revised copy received January 16, 1979

ABSTRACT

The defective specialized transducing phage SPpc2dcitK1 carries two known bacterial genes, kauA and citK, as well as SPP phage markers including the heat-sensitive repressor allele, c2. Some phage genes (including essential ones) are missing. When SPpcZdcitK, transduces SPp-sensitive cells of Bacillus subtilis, the defective prophage is inserted into sites in the homologous bacterial DNA of the attSPp-kauA-citK region of the recipient chromosome. During the growth of these transductants, occasional excisions occur that result in the loss of the phage genes and OI the heterogenotic state. These excisions increase greatly in frequency during growth at repressor-inactivating temperatures. The kinds of insertions and excisions seen suggest that a Campbell-type (CAMPBELL 1962) circular phage genome may occur transiently. If the transductants are superinfected by SPpcZ or by the clear-plaque mutant SPPc1, the resulting double lysogen can be heat induced to release high-frequency- of-transduction (HFT) lysates for kauA and citK.

M O S T strains of Bacillus subtilis derived from Spizizen's transformable strain 168 (SPIZIZEN 1958) are lysogenic for phage SPp (WARNER et al. 1977).

We have show that the prophage lies between the iZuA and kauA genes on the bacterial chromosome. Lysates iiiduced from SPp lysogens by Mitomycin C can mediate specialized transduction of the bacterial genes flanking the prophage: kauA, citK and gZtA to the right of the prophage (ZAHLER et al. 1977; this paper), and ilvA, thyB, iluD and metB to its left (P. FINK and S ZAHLER, unpublished results). Some double lysogens, lysogenic for a defective transducing phage and for a whole phage, release large numbers of transducing phage particles when induced to lyse. These high-frequency-of-transduction (HFI") lysates usually contain about one transducing particle per 700 plaque-forming particles (ZAHLER et al. 1977), although we have seen ratios as high as 1 : l O O and as low as 1:10,000.

In this paper we describe the genomes of some cells that have been transduced for the kauA and citK genes. To help in these studies, we have isolated a mutant of SPp that can be induced from the prophage state by a brief heat shock. The mutation responsible for this phenotype, c2, was incorporated into a defective phage genome that carries the citK+ and kauA+ bacterial genes. This defective Genetics 92: 721-739 July, 1979

722 R. ROSENTHAL et al.

phage, SP@2dcitK1, is the subject of most of this report (the subscript designates the particular SPpc2dcitK phage under study).

We will show that the DNA of defective transducing particles of SPP often integrates into the bacterial chromosome in the region homologous to the bacterial genes carried by the phage particle, rather than into the SPP attachment site. Characteristics of such defective lysogens, and the results of superinfecting them with various SPP mutants, will be discussed.

MATERIALS A N D METHODS

Bacterial strains used: The bacteria used were derived from B. subtilis strain 168. They are listed with a description of their genetic markers in Table 1.

Media: The liquid media used were TB (1% Bacto-tryptone with 0.5% NaC1) and Anti- biotic Medium #3 (Difco) . Tryptose blood agar base (TBAB; Difco) was used as plating medium for phage and bacteria. When CitK- strains were cultured, TBAB and TB were supplemented wiih 0.2% glucose. The medium used for phage overlays was TBA (TE containing 0.7% agar). The synthetic medium was that of ANAGNOSTOPOULOS and SPIZIZEN (1961), supplemented as necessary; unless otherwise specified, synthetic media contained 10 pg of tryptophan and 1 fig of biotin per ml.

Culture conditions: AI1 incubations were at 37" unless otherwise noted. Liquid cultures were aerated in test tubes on a rocking platform shaker fitted with a constant temperature block.

Assays of SPp lysates: Plaque-forming units were assayed by placing 0.1 ml samples G f

appropriate dilutions (in TB) into 3 ml of molten TBA at 45" with about IO7 growing cells of strain CU1050, and plating the overlay mixture on TBAB plates.

SPp transducing particles were assayed by transduction of strain CUI325 with selection for MetB+, KauA+, CitK+ or GltA+. This strain is lysogenic for SPpc2. (See method in ZAHLER et al. 1977.)

PBSl transductions: Phage PBSI lysates were produced, and transductions carried out, as previously described (WARD and ZAHLER 1973).

Phage induction: Mitomycin C inductions were performed by the method reported earlier (ZAHLER et al. 1977) except that the concentration of Mitomycin C was increased to 1 pg per ml.

Heat inductions were performed by growing app-opriate lysogens in TB at 37" to a concentration of IO' to 108 cells per ml. The temperature of the culture was raised by the rapid addition of 0.33 vol of boiling TB. The final volume was usually 5 ml. After aerating for 5.5 min at 50", the tubes were shifted to 37" and aerated until lysis occurred. This took 45 min for light cultures, 60 min for more turbid ones. All lysates were sterilized by filtration through 0.4.5 pm Millipore filters.

Tests for SPp lysogeny: Three different tests have been used. (1) Immunity to SPpcl, a clear-plaque mutant of SPp, was tested as described previously (ZAHLER et al. 1977). ( 2 ) Re- lease of SPp was tested by inducing lysis with Mitomycin C or, if appropriate, with heat shock, and assaying for plaque-forming units. ( 3 ) The third method was carried out as follows. Strain CU1050, our usual indicator strain, was spread in an overlay on a TBAB plate. Individual colonies of putatite lysogens were then touched with a sterile toothpick and transferred to the overlay, as many as 52 per plate. The plate was incubate2 at 37" overnight. All lysogens described in this paper that can release infective phage particles produce a clear zone of killing; nonlysogens do not, nor do bacteria carrying the defective prophage SPpdcitK,. Unpublished work done in this laboratory and by H. E. HEMPHILL at Syracuse University indicates that the zone of lysis is due to a bacteriocin ("betacin") produced by SPp lysogens that is active on nonlysogens and on SPpdcitK, lysogens.

Mutagenesis of strain C U l l l l : T o isolate SPpc2 and c3, we treatec! strain C U l l l l with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) by the method described earlier (WARD and

DEFECTIVE TRANSDUCING PHAGE 723

ZAHLER 1973), except that the mutagenized cells were plated on TBAB and allowed to grow overnight before use so that induced prophage mutations had the opportunity to segregate.

Isolation of SPpc2: The heat-inducible mutants of SPP were isolated from the lysogen CU1111, which carried the gtaB76 allele. This marker makes C U l l l l unable to adsorb SPP because of a defect in its teichoic acid (YOUNG, SMITH and REILLY 1969). This serves to reduce the loss of phage particles due to adsorption of SPP to cells or cell debris. We reasoned that cells carrying heat-inducible prophage mutants would lyse following heat indwtion, and that b o - gens of heat-inducible phage mutants would either be unable to grow at inducing temperatures or would be cured of prophage at inducing temperatures. NTG-h-eated C U l l l l cells (see above) were inoculated into TB at a final cell concentration of about 108 per ml, and aerated at 37" for 60 min. The culture was then placed in a 50" incubator for 12 min t o cause induction of any heat-inducible mutants that had arisen. Since SPp is not produced at 50" (unpublished observations), the culture was next aerated at 37" for 180 min to permit the release of phage from induced cells. The culture was centrifuged, and the supernatant sterilized by filtration.

The filtered supernatant mas plated on the indicator strain CUI050 and incubated at 45", a temperature at which only about 10% of the phage particles produce plaques. Seventy-thousand plaques were examined after incubation, and phage particles were tested from 350 plaques that looked partly clear at 45". They were plated on strain CU1050 at 33". Lysogens of 200 phage particles that formed turbid plaques at 33" were isolated and grown on TBAB plates at 50". Of the lysogens tested, two gave rise to SPp-sensitive clones when grown at 50". Cultures of these two lysogens were then grown in TB at 37" and heat induced at 50" for five min. Upon further aeration at 37", both cultures lysed, yielding phage titers of about IO9 plaque-forming units per ml. The mutations present in the two heat-inducible phages were labelled c2 and c3; our studies have concentrated on SPpc2. We have evidence (see below) that the c2 mutation lies in the same cistron as the clear-plaque mutation c1. We believe it causes production of a temperature- sensitive phage repressor.

Differentiating plaques formed by SPpcl and SPpc2: When we wanted to enumerate the SPPCZ and SPpc2 phage released by double lysogens that carried both alleles, we heat induced the strain (CUI227 or CU1376) and plated the diluted lysate on CU1050. Roughly equal numbers of clear and turbid plaques were seen, but some small plaques could not be identified un- equivocally. We therefore picked cells directly from plaque centers with sterile toothpicks and cross-tested them for sensitivity to SPpc1 (ZAHLER et al. 1977). SPpc2 plaques gave solid growth of lysogens; SPpc1 plaques contained only sensitive cells.

Differentiating plaques formed by SPpc2 and SPpc+: Phage released spontaneously or after induction were assayed on indicator strain CU1050. Lysogens were isolated from plaque centers. Purified lysogens were streaked on TBAB and incubated at 50". Colonies were tested after growth at 50" for sensitivity to SPpcl at 37" as described earlier. Lysogens carrying SPpc2 lost their prophages and became sensitive after growth at 50"; lysogens carrying SPpc+ did not.

Isolation of strain CU1225: We have previously reported (ZAHLER et al. 1977) that strain CU1087 is doubly lysogenic for SPP and for the defective prophage SPpdcitK,, which carries the citK+ and kauA+ genes. When induced by Mitomycin C, CU1087 produces high-frequency- of-transduction (HFT) lysates far the citK+ and kauA+ genes. We used such a lysate to transduce CUI170 to CitK+ and KauA+ simultaneously. CUI170 is a citK1 kauAl strain lysogenic for Sppc2. An isolated transductant was purified and labelled CU1191. This strain segregates CitK- colonies on TBAB, and has the genetic composition iZuBA1 citK1 kauA1 (SPpc2) (SPpdcitK,)-that is, it is doubly lysogenic for SPPc.2 and for the defective prophage SPpdcitK,. It cannot be heat induced. When it is induced by Mitomycin C, it produces HFT lysates.

We wished to produce a recombinant transducing phage carrying the c2 allele. Therefore, we grew strain CUI191 in TB to a cell concentration of about 108 cells per ml. The culture was centrifuged, and the supernatant sterlized by filtration. Spontaneously-released phage found in the supernatant of strain Cul l91 were used to transduce CU1086, a phage-sensitive citK1 kauA1 strain, to CitK+ KauA+. A transductant that was CitK+ and KauA+, was immune to



+

h z 8

2 8 % 2 2 2 5 5 5

B .3 .I-

m 3

m ... W W 2 su

31 QT

C L

w m o h h m 2 2 2 5 5 5


SPPcl, did not release plaque-forming particles and segregated CitK- clones at high frequency when grown on TBAB at 50" was purified and labelled strain CU1225. As we shall show, this strain is heterogenotic for citK and for kauA, and is lysogenic for the recombinant defective prophage SPpc2dcitKI.

Production of strain CU1226: We wished to superinfect strain CUI225 with SPpc2 to learn if the double lysogen could produce heat-inducible HFT lysates. A culture of CU1225 in TB containing about 2 x IO7 cells per ml was heated at 50" for five min to inactive phage repressor and then diluted 1:5 into a suspension containing about 108 SPPc2 phage per ml. The culture was incubated at 37" for 16 hr. It was then streaked on TBAB and incubated at 37" overnight. CitK+ colonies were purified and tested for the ability to release SPP phage. About 95% of the colonies tested after this regimen were able to release SPPc2 (unpublished data). One of these was purified and labelled CU1226. It is doubly lysogenic for SPPc2 and for SPpc2dcitK, and heterogenotic for citK and kauA.

Production of strain CU1227: The same regimen was used to make CU1227 as was used for CU1226, except that the phage used to superinfect CUI225 was the clear-plaque mutant, SPPcI.

RESULTS

Characterization of SPpc2: A heat-inducible mutant of phage SPP was isolated (see MATERIALS AND METHODS) and named SPPc2. Bacteria lysogenic for SPPc2 can be heat induced by raising the temperature of a culture to 50" for a few minutes. Since SPP is unable to multiply above about 45", the temperature of the culture must then be lowered. After 45 to 60 minutes at 37", the cells in the culture lyse, releasing SPpc2 phage. If the culture is kept well aerated at 50", no lysis is observed.

Since the prophage is induced at 50", we examined the survival of SP&2 lysogens plated at 50". As shown in Table 2, when a growing 37" culture of an SP&2 lysogen was assayed for colony formers at both 50" and 37", the fraction of cells able to form colonies at 50" depended to some extent on the age of the culture. Forty percent or more of the cells could form colonies at 50" until well into the stationary phase of growth, when only 5 to 15% of the cells could dos so. Of the colonies that grew at 50", more than 95% had lost their immunity to SPpcl and were cured of the prophage. Neither the inability of stationary-phase cells to form colonies at 50" nor the curing was found with cells lysogenic for wild-type SPP (unpublished observations).

TABLE 2

Ability of lysogens of SPpc2 and of SPP to form colonies a t 50"

Hourj of growth in TB broth at 37"

Colony formers (millions per ml) * Strain Cull47 Strain CU1379 trpC2 (SP/3c2) trpC2 (SPP)

at 37' at 50" at 37" at 50"

4 5 6

225 100 169 234 268 190 361 374 230 27 392 328

* The cells grew as long filaments until the cell concentration reached about 108 ml-1. Then the filaments broke up into short motile cells. Filaments had about the same plating efficiency at 37" and at 50". Dilutions were spread on TBAB plates pre-heated to 37" or 50" and incubated immediately.


The relationship between the c2 mutation and the wild-type c+ allele: Strain C u l l 9 1 carries two prophages, one with c+ and one with c2, and we can determine some properties of the c2 allele by examining this strain. C u l l 9 1 was con- structed by transducing a citKl kauAl strain lysogenic for SPPc2 with an HFT lysate containing many SPpdcitK, particles (see MATERIALS AND METHODS). C u l l 9 1 is heterogenotic for the citK and kauA genes. This can be demonstrated by streaking C u l l 9 1 on TBAB; occasional colonies show asporogenous C i a - sectors, which are morphologically detectable on this medium. Upon testing, most CitK- sectors are also KauA-. When C u l l 9 1 was induced with Mitomycin C, about equal numbers of SPpc2 and recombinant SPPc+ particles were released. Of eight plaques tested, four were SPpc2 and four were SPpc+. However, strain C u l l 9 1 could not be heat induced. We conclude that the c+ wild-type allele is dominant to c2, as would be expected if c2 encoded a temperature-sensitive repressor and C+ encoded a temperature-stable one.

Characterization of the SPpc2.dcitK1 lysogen, CU1225: As we have seen, strain C u l l 9 1 is lysogenic for SPpc2 and for the defective prophage SPpdcitK,, and it releases both SPPc2 and the recombinant SPpc+. We expected that its HFT lysates would contain two kinds of specialized transducing phage as well: SPpdcitK, and the recombinant SPpc2dcitK,. We transduced strain CU1086, a kauAl citKl phage-sensitive strain, to KauA+ and CitK+ with phage released spontaneously by CU1191. We examined those transductants that segregated CitK- sectors, were immune to SPpcl and did not release plaque-forming particles. Two classes containing about equal numbers of transductants were found. One class (five of eight transductants tested) gave occasional CitK- sectors when grown on TBAB at 37" or at 50". These are apparently lysogenic for SPpc+dcitK1. The other class (three of eight transductants tested), of which strain CU1225 is an example, gave occasional CitK- sectors at 37"; but at 50" every colony either contained CitK- sectors or was completely CitK-. Whether they arose at 37" or at 50", the CitK- clones were sensitive to SPpcl. We interpret the instability of the CitK+ character and of the prophage at 50" to mean that CUI225 and other transductants with the same characteristics are lysogenic for a defective prophage that carries the c2 allele. that is, €or SPpcPdcitK,. Lysis does not occur when CU1225 is subjected to the heat-induction protocol.

We isolated and purified clones derived from strain CU1225 after growth on TBAB at 50", and tested them for their CitK, KauA, and SPP-immunity phenotypes. Table 3 shows that among these survivors were several classes that corre- spond to clones that have lost part or all of the defective prophage. The classes include cells that have lost immunity to SPPcl (i.e., they have lost the c2 gene), immunity and kauA+, immunity and citK+, and immunity and both citK+ and kauA+. We assume that these classes have arisen via deletions, since each of the last three classes has lost at least two genes. If these gene losses are accepted as deletions, the order of the three defective prophage genes we can detect must be citK+-c2-kauA +.

Although strain CU1225 is phenotypically KauA+ and CitK+, it still contains the kauAl and citKl markers. This can be shown by their linkage to other bac-


TABLE 3

Phenotypes of colonies derived from strain CUI225 [ilvBAI kauAl citKl (SPpc2dcitK1)] after growth at 50°*

Phenotype ~~

Number found Deletion class:

Clones purified KauAf CitK- SPPS from CitK- sectors KauA- CitK- SPPS

Total

Clones purified KauAf CitK+ SPPI from CitK+ portions KauA- CitK+ SPPS of colonies KauA- CitK+ SPPI

KauAf CitK+ SPPS

Total

36 a 13 b

40

23 parental 71 C

1 d 4 e

-

99

* All CUI225 colonies were C i a - or contained C i a - sectors after overnight growth on TBAB at 50". Forty-nine CitK- sectors were purified on TBAB at 37" and tested. Ninety-nine isolations were made from the periphery of the CitK+ portions of colonies and purified at 37". SPPS means sensitive to SPPcl. SPPI means immune to SPPcI.

-f See Figure 3.

terial markers by means of phage PBSl transduction. PBSl is a virulent phage capable of mediating generalized transduction in B. subtilis (TAKAHASHI 1961). The map distames (in terms of PBSl transduction) for the markers surrounding the normal SPP attachment site (attSPP) are shown in Figures 1A and 1B. The metB5 and gltA2 markers surround attSPP, and each is linked by PBSl transduction to both citK2 and kauA2 in SPP-sensitive strains. The SPP prophage in- creases map distances across the attachment site by 0.55 (ZAHLER et al. 1977). The most likely structure for the genome of strain CU1225, as deduced from the observations that follow, is shown in Figure 1C.

PBSl phage produced in strain CU1225 could be used to make a number of observations. (1) The kauA2 allele in strain CU1225 was linked to its metB+ gene (Table 4, cross I ) , but we did not see co-transduction of kauA2 with gltA+ (cross 11). A likely explanation is that kauA+ lies between gZtA+ and kauA2; whenever gltA+ and k m A 2 are co-transduced, the k a u A f gene is also co-transduced, and the transductant is phenotypically KauAf. (2) The citKl allele of CU1225 was linked to its gltA+ gene (cross 11) , but we did not see co-transduction of citK2 with metB+ (cross I). This suggests that the citK+ gene lies between metB+ and citK2. (3) The kauA+ allele of CU1225 was frequently transferred with &A+ (cross IV), although the chK+ allele was not. (4) The citK+ allele of CU1225 was frequently transferred with metB+ without concommitant trans- fer of k a u A f (cross 111). Taken together, these observations support the gene order metB + -kauAl -citK'-kauA +-citK2 -,@A+ in CU1225.

In crosses with CU1225 as PBSl phage donor and a lysogen of SP@2 as recipient (crosses I, I1 and I11 of Table 4), transductants fell into three classes with respect to their SPP contents: some were sensitive to SPpcl; some were immune and released plaque-forming particles like the recipient; and some were immune but did not releasn plaque-forming particles, like the donor. The pheno-


(*I 15 .IO .I2 .66 I

m - m gl;Af - aJSP@

; : I I

g l tA+ - kouAl c i tK l c2 - - - S P B g d c x ,

I I 1 I (C) ......... ..... ........... I I

-- koiA+ c'i + K I - gl;A+

FIGURE 1.-Genetic map of the Bacillus subtilis chromosome near attSPp. All strains also carry the unlinked iluBA1 mutation.

(A) Map of the region in a typical SPP-sensitive strain, CUI086 (kauAl citKl SPpS). Distances are in phage PBSI transduction units (one minus the fraction co-transduced). The iluD, thyB and iluA genes lie between metB and attSPp. For reasons we do not understand, the distance between citK and gltA is less than 0.66 in certain crosses.

(B) Map of the region in a typical SPpcZ lysogen, strain CUI170 [kauAl citKl (SPpd)] . The prophage lies in the normal SPP attachment site. I t is about 0.55 PBSI transduction units long (ZAHLER et al. 1977).

(C) Map of the region in strain CUI225 [kauAl citKl (SPpcZdcitK,)]. The defective prophage lies between kauA1 and citK1. The stippled region is of SPD origin. Note the unoccupied attSPp.

(D) Map of the region in the double lysogen, CU1226 [kauAl citKl (SPpcZ) (SPpcZdcitK,)].

(E) Map of the region in strain CU134.0 [kauAl citK1 (SPpcZdcitK,)]. The defective prophage Iies between attSPp and kauAl. Note the unoccupied attSPp.

types of the three classes are designated SPps, SPPL, and SPP'. The SPpL class may include double lysogens carrying SP@2 and the defective phage. We assume that any transductant that has lost the ability to release phage in such a cross has received the unoccupied attSPP site from the donor. As can be seen in crosses I and 111 of Table 4, phage-sensitive transductants are frequently found when lysogenic recipients are selected for MetBf , indicating that CUI225 retains the unoccupied phage attachment site linked to metB. The data presented in the table are compatible with the position of the c2 gene of the defective prophage


TABLE 4

Phage PBSI transductions io locate and orient the SPPc2dcitK1 prophage in strain CU1225

Recipient s rain Selection

Phenotypes of transductants.

Number of transductants

CUI316 iluBA1 metB5 ( S P P 4

CUl2.30 iluBA1 metB5 gltA2

(SPPC2)

CUI326 iluBA1 m t B 5 kauA1 citKl (SPPc2)

CUI323 iluBA1 metB5

kauAl citKl gltA2

MetB+

Cross I

GltAf

Cross I1

MetBf

Cross I11

GltA+

Cross IV

Me*+ KauA+ CitK+ SPPS MetB+ KauA- CitKf SPPS M e a + KauA+ CitK+ SPPL MetBf KauAf CitK+ SPPI

MetB- KauAf CitK- GltA+ SPPL MetB- KauA+ CitK+ GltA+ SPPL MetB- KauAf CitK+ GltAf SPPI MetB+ KauA+ CitK- GltA+ SPPI

4 8

12 12

36 tested

12 30

2 1

MetB+ KauA- CitK- SPPL MetBf KauA- CitK- SPBS MetB+ KauA- CitKf SPPI MetB+ KauA+ CitK+ SPPI MetBf KauA+ CitK- SPPS MetBf KauA+ CitK+ SPPS MetBf KauA+ CitK- SPPL

MetB- KauA+ CitI- GltA+ SPPS MetB- KauA- CitK- GltA+ SPPS MetB+ KauA- CitA- GltA+ SPPS

45 test-d

27 55 13 2 4 1 1

103 tested

17 86 1

104 tested

~

* SPPS = sensitive to SPPcl. SPPL = releases phage. SPPI = does not release phage; immune to SPPCI.

lying between its kauA+ and citK+ genes; cross I11 supports this position strongly.

Note that in these PBSI transductions with CUI225 as donor there are large regions of nonhomology between donor and recipient chromosomes. The donor has an unoccupied attSPP and a defective prophage inserted between kauA and citK. The recipient lacks the defective prophage, but carries SPbc2 in its normal attachment site (in crosses 1-111). We cannot rule out the possibility that recombinations may occur during transduction between phage DNA of the defective prophage and the prophage in the recipient. Furthermore, the donor is heterogenotic for the kauA-citK region, which may lead to anomalies as well.

The nature of the deletions found after growth of strain CUI225 at 50" (Table 3) also supports this position of the c2 allele. The CitK+, KauA-, SPPS and CitK+, KauAf, SPPs classes of survivors (classes c and e, Table 3 ) were examined further. When streaked on TBAB at 37" or 50", these clones did not segregate

DEFECTIVE TRANSDUCING PHAGE 73 1

C i t cells. We grew PBSl transducing phage on three class c strains and on one class e strain and used the phage to transduce CU1229 (i2vBAl metB5 gltA2 SPpS) to MetB+ and to GltA+. The citKl allele was not transduced in any case, and kauA2 was not transduced from the class e clone. We conclude that the SPp-sensitive survivors of growth at 50" are no longer heterogenotic.

Double lysogens: Since the defective prophage in strain CU1225 is not at the normal SPj? attachment site, we were able to superinfect the strain with SP@2 or with other SPj? mutants to form double lysogens. For example, CUI226 is a double lysogen carrying SPPc2dcitK and SPj?c2 (see MATERIALS AND METHODS

for details of its construction). By producing PBSl transducing phage in CU1226, we were able to show (Table 5) that its most probable genetic structure is that shown in Figure 1D. The defective prophage still lies between kauAl and citKl. The SPpc2 prophage probably lies in the normal attachment site, since we could not detect co-transduction of phage sensitivity with MetB+ selection.

Unlike strain CU1225, CU1226 lyses after heat induction. The lysates are HFT for the kauA+ and citK+ genes (see Table 6).

SPpcl is a clear-plaque mutant of SPp (WARNER et al. 1977; ZAHLER et al. 1977). Neither H. E. HEMPHILL, who isolated it, nor we have been able to isolate bacteria lysogenic for SPpcl alone. We believe that it is mutant in repressor formation and makes no active phage repressor. Since strain CU1225 does make repressor (it is immune to SPj?cl), we attempted to superinfect CU1225 with SPpcl. We thought that the double lysogen would be stable, since the repressor made by SPpcZdcitK, shculd repress SPj?cl. It proved easy to isolate the double lysogen. One such clone was labeled CU1227.

As can be seen in Table 6, heat-induced lysates of strain CU1227 were HFT for the kauA+ and citK+ genes. These lysates contained about equal numbers of clear-plaque and turbid-plaque phage. Some of the turbid plaques may be cf recombinants of cl and c2; we have not tested for this. Most of them, however, are SPpc2. Of particular interest is the fact that CU1227 is heat inducible. This shows that SPpc2 phage, the clear-plaque mutant, cannot complement the c2 tempera-

TABLE 5

Transduciion of CUI230 [ilvBAl metB5 gltA2 (SPpc2)l by PBSI phage produced in the double lysogen CUI226 [ilvBAl kauAl citKl (SPpc2) (SPpc2dcitK1)]

Selection Phenotype of transductent class. Number in class

MetB f

GltA+

MetB+ KauA+ CitKf GltA- 25 MetBf KauA- CitK+ GltA- 1

Total 26

MetB- KauA+ CitK- GltA+ MetB- KauAf CitK+ GltA+

10 38

Total 48

* All transductants were immune to SPpcI. Phage release was not tested.


TABLE 6

The contents of heat-induced lysates of various strains*

Strain and Plaque-forming particles Transductants (thousands per ml) genotype (millions per ml) MetB+ KauA+ CitK+ GltA+

CUI 147 150 0.20 2.2 0.47 0.20 irpC2 (SPJCW

CU1226 134 0.84 228 1480 0.09 iluBAl kauAl (SPPc2) (SPpc2dcii K,)

iluBAl kauAl citKl (SPPcf) (SPpc2dcitK1)

CU1227 91t

CUI376 56s iluBAl knuAl citKl (SPPC2) (SPpcldcitK,)

iluBAl kauAl citKl

(SPpddciiK,)

CU1390 49

(SPPc2)

0.38 1 4 782 0.03

0.15 510 914 0.02

0.67 314 422 0.04

* All strains except CUI147 are double lysogens and give HFT lysates for kauA+ and citK+. The defective prophage in CU1226 and CUI227 lies between kauAl and citKl. In CU1390, it lies between attSPj3 and kauAl (see text). Transducing particles were assayed on strain CU1325. Plaque-forming particles were assayed on strain CU1050.

+Of 108 plaques tested (see MATERIALS AND METHODS), 56 were SPJCcl and 52 were SPpc2 (or SPpc+).

$ Of 48 plaques tested, 22 were SPpcl and 26 were SPpc2 (or SPpc+).

ture-sensitive repressor; thus we conclude that the c l and c2 mutations lie in the same cistron, the c gene of SPP.

We were also able to show that among the defective transducing particles released by strain CU1227 were recombinants that carried the cl allele. When we transduced a c i tKl kauAl lysogen carrying SFPc2 prophage (strain CU1170) with an HFT lysate from the double lysogen CU1227, some of the Kau+, Cit+ transductants released both clear-plaque (SPPcl ) and turbid-plaque phage particles in about equal numbers in their HFT lysates. One such strain, CU1376 (Table 6), was kept in our stock collection. Its genotype is apparently iluBAl kauAl c i tKl (SPPc2) (SPpcldcitK,) .


Does SP/3c2dcitK1 always integrate between kauA and citK? We wished to learn if the insertion site of the defective prophage could be outside the kauA-citK interval. The defective phage contains the entire kauA and citK genes, some DNA lying to the right of citK, aiid possibly all of the DNA between kauA and attSPP. Is there a favored site for insertion between kauA and citK, or may insertion occur to the right of citK or to the left of kauA?

To answer this question, we transduced a phage-sensitive kauAl citKl strain (CU1086) with spontaneously released phage from the double lysogen CUI 191 and purified five transductants that were immune to SPpcl, but did not release phage. One of thesc no longer segregated CitK- sectors, and we could not find evidence by PBSl transduction that it contained either the citKl or kauAl allele. Two others segregated the citKl allele, but seemed not to have the kauAl allele. We could not find KauA- segregants, nor could we detect kauAl among PBSl transductants (unpublished results). These strains were not analyzed further. The remaining two strains, labelled CU1339 and CUI 340, were heterogenotic for citK and for kauA. CUI339 had the defective prophage inserted between kauA and citK, just as did CUI225 (unpublished data). CU1340, on the other hand, had the defective prophage inserted between kauA and the SPp attachment site.

PBSl transduction data supporting this position of the prophage in CUI340 can be found in Table 7. (1) The kauAl and citKl alleles in CUI340 are both

TABLE 7

PBSl transductions to locate and orient the SPpcWcitK, prophage in strain CUI340

Recipient strain Selection

Phenotypes of transductants

Number of transductants

CUI229 iluBA1 metB5 gltA2

CUI 326 iluBA1 metB5 kauAl citK1 (SPPC4

MetB +

Cross V

MetB+ KauA+ CitK+ SPPs* MetB+ KauA+ CitK+ SPPL MetBf KauA+ CitK+ SPPI MetBf KauA- CitK+ SPPL MetB+ KauA- CitK+ SPPs

GltA+ MetB- KauA+ CitK+ SPPS Met& KauA+ CitK- SPPS

Cross VI MetB- KauA- CitK- SPPs MetB- KauAf CitKf SPpI MetB+ KauA+ CitK- SPpS

MetB+ MetB+ KauA- CitK- SPPL MetBf KauA- CitK- SPPS

Cross VI1 M e a + KauA+ CitK- SPP5 MetBf KauAf C i a + SPPS MetB+ KauA+ CitK+ SPPI

40 21 23 1 1

86 tested

153 26 17 6 1

203 tested

30 30 21

7 17

105 tested

* SPPS = sensitive to SPPcl. SPPL = releases phage. SPPI = immune to S P p d ; does not release phage.

734 R. ROSENTHAL et aZ.

linked to the strain's gltA+ gene (cross VI); they lie in the order kauAl-citKl- &A+. (2) They are essentially unlinked to metB+ (cross V). ( 3 ) Strain CU1340 still contains an unoccupied SPp attachment site, as demonstrated by the ability of PBSl transducing phage grown on CU1340 to cure a lysogen of its prophage (cross V). (4) The order of genes on the defective prophage is kauA+-citK+-c2 (cross VII). From these data we conclude that the SPpc2dcitK, prophage in strain CU1340 lies as shown in Figure 1E.

When cells of CU1340 were plated on TEAB plates and incubated at 50°, all colonies either were CitK-, or segregated detectable CitK- sectors. The phenotypes of a number of purified clones were tested; results are given in Table 8. The classes of survivors that we found support the order of prophage genes kauA+-citK+-c2. Note that no KauA- CitK+ SPpS clones were found; in contrast, this class was frequently found when strain CUI225 was grown at 50" (Table 3).

We tested some of the Kau+ CitK+ SPBS survivors (Table 8, class h) to determine whether they were heterogenotic for kauA or citK. They did not segregate CitK- clones detectably when streaked on TBAB and incubated at either 37" or 50". PBSl phage was produced on four of these clones and used to transduce strain CU1229 (iluBAl metB5 gZtA2 SPps) to both MetB+ and GltA+. We could not detect the kauAl or citKl alleles in any of the transductants. We conclude that the SPj5-sensitive clones from CU1340 after growth at 50" are no longer heterogenotic.

We were able to superiiifect strain CUI340 with SPpc2, using the method described earlier for the construction of CU1226. The resulting double lysogen, strain CU1390. can be heat induced. It produced HFT lysates (Table 6) .

DISCUSSION

Like other temperate phages, SPp has a gene that seems to encode a repressor protein. We call this gene c. Mutations of SPp described in this p'per include the c l mutation (WARNER et al. 1977; ZAHLER et al. 1977) that causes clear-

TABLE 8

Phenotypes of colonies derived from strain CUI340 [ilvAl kauAl citKl (SPpc2dcitK1)] after growth a t 50"*

Phenotype Number found Deletion class+

Clones purified KauA- CitK- SPPS 9 f from CitK- sectors KauA+ CitK- SPPs 12 g

Clones purified KauA+ C i a + SPPI 13 parental from CitK+ portions KauAf CitK+ SPPS 56 h

Total 21

of colonies Total 69 ~~~ ~~

* All CUI340 colonies were CitK- or contained CitK- sectors after overnight growth on TBAB at 50". Twenty-one CitK- sectors were purified on TBAB at 37" and tested. Sixty-" isolations were made from the periphery of CitK+ portions of colonies, and purified at 37".

-f See Figure 3.


plaque morphology and apparently makes lysogenization impossible, and the ~2 allele that permits the heat induction of lysogens. Double lysogens carrying C+

and c2 (strain CU1191) release both types of phage particles; they cannot be heat induced. Double lysogens carrying the c l and c2 alleles (CU1227, CU1376) release both kinds of phage particles and can be heat induced. We interpret these results to mean that c l and c2 are alleles of the same gene, the c gene of SPP, that encodes a repressor protein. The c l allele fails to make active repressor; the c2 allele makes temperature-sensitive repressor.

ROBERT HERENSTEIN in this laboratory has carried out preliminary experiments to determine whether all clear-plaque mutations of SP/3 occur in the c gene or if-as with coliphage lambda (KAISER 1957) , for example-mutations in other phage genes can give a similar phenotype. Of eight independently isolated clear-plaque mutants of SPP, none could complement SPPcl to permit lysogenization. They are probably all mutant in the c gene.

Our ability to produce phage lysates by heat-inducing lysogens of SP@2 simplifies phage production to some extent. It also makes it possible to1 induce lysates by a method that does not simultaneously induce the defective prophage called PBSX (GARRO, LEFFERT and MARMUR 1970) , which is carried by all of our strains.

SPP does not produce plaques at temperatures above about 45". If a strain of B. subtilis lysogenic for SPpc2 is put at 50" to destroy the repressor and then incubated at 37", essentially all of the cells lyse within an hour and release phage. On the other hand, if growing cells of such a lysogen are plated at 50", moist of the cells f m - colonies. Almost all of these colonies consist of phage-sensitive cells that have lost their prophage. Our working hypothesis is that the lysogens are induced at 50" and excision of the prophage takes place. Phage replication (but not cell replication and division) is inhibited at the high temperature, and most of the cells in t he colony are cured of the prophage. The fate of the excised prophage is not known.

It proved easy to produce a bacterial strain doubly lysogenic fo r SP/3c2 and far the defective transducing particle SPPdcitK,, which we described earlier (ZAHLER et al. 1977). The double lysogen, CU1191, could not be heat induced, but phage released from it spontaneously or after Mitomycin C induction included a high proportion of both recombinant plaque-forming particles (SPpc+) and recombinant transducing particles (SP&2dcitK1). These were high-frequency-of-transduction (HFT) lysates.

When the recombinant defective transducing particles were used to transduce sensitive cells for the two bacterial markers known to be carried by the defective particles, kuuA+ and citK+, many of the transductants became hetero- genetic for kauA and citK and immune to SPpcl. If simultaneous infection by SPP particles was avoided by using high dilutions of the transducing phage, the transductants could neither release phage nor produce the phage-associated bacteriocin (see MATERIALS AND METHODS). Of three such transductants examined, two (CU1225 and CU1339) had the defective prophage inserted between the kauA and citK genes. The third transductant, CU1340, had the defective pro-

R. ROSENTHAL et al. 73 6

(A )

I I

c i t K l - &B+ a t t S p B - kauAl

m e t B + a t t s p p - kauAl c i t K l

FIGURE 2.-Possible recombinations between a circular defective transducing form of SP/3c2dcitK1 and the chromosome of strain CUI086 to produce strains CUI225 (above) and CUI 340 (below).

phage inserted between attSPp and kauA. Thus, there is no unique site in the heterogenotic region where the defective prophage always inserts. We suspect that prophage insertion may occur anywhere within the heterogenotic region, probably by a mechanism like that suggested in Figure 2. The orientation of the three defective prophage genes (citK+, kauA+ and c2) in CU1225 and CU1340 strongly supports this model, which is based on that of CAMPBELL (1962). It should be emphasized that there is no physical evidence for circle formation by SPP 01' SPPcBdcitK, DNA.

Some small classes of transductants from the mapping crosses described in Tables 4 and 7 seem to be the result of more than two crossover events. These are class 4 in cross 11; classes 5, 6 and 7 in cross 111; class 3 in cross IV; classes 5 and 6 in cross V; and class 5 in cross VI. Four of these classes (cross 111, class 5 ; cross IV, class 3; cross V, class 5; and cross VI, class 5 ) might have resulted from two crossover events followed by the loss of the defective prophage.

We have been studying a mutant of SPpc2 that is severely deficient in its ability to integrate into and excise from the normal attachment site on the B. subtilis chromosome. This mutant, SPpc2int5 (ROSENTHAL et al. 1978), probably corresponds to the int- mutants (ZISSLER 1967; GINGERY and ECHOLS 1967)

DEFECTIVE TRANSDUCING PHAGE 73 7

of coliphage lambda. We have found (manuscript in preparation) that the defective prophage SP@c2dcitKl cannot complement the int5 mutation. This provides a possible explanation for the fact that we have not found SPpc2dcitKl inserted into the normal SPp attachment site; it lacks the int function needed for recog- nition of and insertion into that site.

Bacteria that are lysogenic only for the defective prophage SPpdcitK, and are heterogenotic for kauA and citK segregate occasional CitK- clones when grown at either 37" or 50". Many of the CitK- clones are also KauA-, and most have become sensitive to SPp.

Heterogenotes that are lysogenic only for SP/3c2dcitK1 segregate occasional CitK- clones when grown at 37", but when they are grown at 50", every colony is either CitK- or contains large CitK- sectors. This is not due to selective death of CitKf cells at 50"; more than 90% of the cells of CUI225 or CUI340 that can farm colonies at 37" can form colonies at 50" (unpublished experiments). We conclude that the increased loss of prophage markers at 50" by lysogens of SPpcBdcitK, is due to the destruction of the temperature-sensitive repressor encoded by the c2 allele. This implies that the deletions are produced with the help of some derepressed phage-coded product.

Two simple models for the loss of genes from the defective prophage during growth at 50" can be considered.

Model I : Some repressor-controlled phage gene product stimulates recombination between homologous sites of the duplicated kauA-citK region. In the upper parts of Figure 3, we present the pairing of the homologs in strains CUI225 and CUI340 and show the losses to be expected. In each case a single act of recombination would result in the loss of a kauA gene, a citK gene and all of the SPP DNA. The surviving cells would be SPp sensitive and no longer heterogenotic.

Model ZZ: A phage-coded gene product stimulates the formation of linear deletions from a site or sites within the SPp DNA of the defective prophage. The deletions extend to the left or right in different cases. The lower portions of Figure 3 show putative classes of linear deletions found in CUI225 and CU1340; quantitative data are presented in Tables 3 and 8.

Model I can explain most of the gene losses described in Tables 3 (CU1225) and 8 (CU1340). Only classes d and e of Table 3 (four of the 148 deletions examined from CU1225) could not have been generated in this way by single recombination events. On the other hand, Model I1 predicts that certain classes of deletions (a, c, d, e, g and h) should still be heterogenotic for kauA or citK, or both. We tested members of classes c, e and h for heterogenosity, but we could not detect the citKl allele in any of them; nor could we detect kauAl in classes e and h. In fact, we have never seen a heterogenote that was sensitive to SPP. Thus, our data strongly support the mechanism of Model I as the origin of a t least most of the prophage deletions seen among the 50" colonies derived from strains CUI225 and CU1340.

Double lysogens that carry a defective prophage with one allele of the c gene and an entire prophage with a different allele of c release roughly equal numbers of plaque-forming particles of the two types. Our data are best for the cl and c2


c u t 2 2 5 cu 1340

+ m e t e + at t kauAt c i t K + - s p a - -

M -

.-.+ gltA+

OD - EL I

-%- m e t E

C L A S S 2: K a u A + C i t K - S P B S - b : K a u A - C i t K - S P P S - c : K a u A - C i t K + S P p S

C L A S S 1 : K a u A - C i t K - S P P S - g : K a u A + C i t K - S P p S - h : K a u A + C i t K + S P P s

M O D E L II t I ......... .......... I I t ......... I I I I

......... , ........ , ........ , aLtspp kauA+ c i t w kauAl c i tKl - aJspp kauAl c i t K ' 9 - k a u A+ c - i t K I

C L A S S Q: K a u A + C i t K - S P P S C L A S S : K a u A - C i t K ' S P B S - g : K a u A + C i t K ' S P B S - h : K a u A + C i t K + S P B S

- b : K a u A - C i t K - S P B S - c : K a u A - C i t K + S P B S - d : K a u A - C i t K ' S P B I - e : K a u A + C i t K + S P P S

FIGURE 3.-Models to explain the loss of SPpcZdcitK, markers from strains CUI225 (left) and C U I 3 4 (right). Model I involves excision by recombination of DNA between homologous sites in the duplicated kauA-citK regions. Model I1 involves linear deletions from the phage DNA t o the left or right. (See Table 3 for the distribution of deletion classes of CU1225, and Table 8 for CU1340.)

alleles of strains CU1227 and CU1376 (Table 6), but similar observations have been made for the C+ and c2 alleles of strain CU1191. This implies that essentially all cells that release phage, release phage with both c alleles. This, in turn, implies that the c allele of the defective prophage is replicated to the same extent as that o€ the whole phage. Reasonable models for this require efficient replication of the entire defective prophage, possibly subsequent to its excision from the bacterial chromosome. Recombination between whole phage genomes and defective phage genomes must then occur frequently to give equal numbers of plaque formers with the different c alleles. The efficient excision otf defective prophage genomes might be mediated by the same phage-encoded function that we have postulated to explain the loss of defective prophage genes in Model I above.

In collaboration with H. E. HEMPHILL and his co-workers, we have isolated a series of temperature-sensitive mutants of SPP unable to fo rm plaques at the


restrictive temperature of 42". We have produced recombinants 04 these ts mutants that carry the c2 allele as well, and lysogenized CU1225 with the recombinant phages by the same methods used for making CU1226 and CU1227. Some of the double lysogens release ts+ phage particles following heat induction, showing that the ts+ allele was present in SPpc2dcitK,. Others release no tsf phage particles, showing that the ts+ allele was missing from the defective prophage. Together with mapping experiments on phage and prophage genomes, these data should enable us to characterize the genetic maps oE the phage, the prophage and the defective prophage in the near future.

This research was supported by National Science Foundation grant 7682221.

LITERATURE CITED

ANAGNOSTOPOULOS, G. and J. SPIZIZEN, 1961

CAMPBELL, A., 1962 Episomes. Adv. Genet. 11: 101-145. GARRO, A. J., H. LEFFERT and J. MARMUR, 1970 Genetic mapping of a defective bacteriophage

GINGERY, R. and H. ECHOLS, 1967 Mutants of bacteriophage A unable to integrate into the host

GOLDSTEIN, B. J. and S. A. ZAHLER, 1976 Uptake of branched-chain or-keto acids in Bacillus subtilis. J. Bacteriol. 127: 667-670.

KAISER, A. D., 1957 Mutations in a temperate bacteriophage affecting its ability to lysogenize Escherichia coli. Virology 3: 42-61.

ROSENTHAL, R., P. KOTARSKI, P. TOYE and S. A. ZAHLER, 1978 Integration-deficient mutants of bacteriophage SPP of Bacillus subtilis. Abstr. Amer. Soc. Microbiol., 1978, p. 259.

SPIZIZEN, J., 1958 Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Natl. Acad. Sci. U.S. 44: 1072-1078.

WARD, J. B., JR. and S. A. ZAHLER, 1973 Geneiic studies of leucine biosynthesis in Bacillus subtilis. J. Bacteriol. 116: 719-726.

WARNER, F. D., G. A. KITOS, M. P. ROMANO and H. E. HEMPHILL, 1977 Characterization of SPP: a temperate bacteriophage from Bacillus subtilis M168. Canad. J. Microbiol. 23: 6-51.

Chromosomal location of genes regulating resistance to bacteriophage in Bacillus subtilis. J . Bacteriol. 98: 1087-1097.

Genetic mapping of alsA, alsR, thyA , kauA and citD markers in Bacillus subtilis. pp. 35-43. In: Microbiology-1976. Edited by D. SCHLESSINGER, Amer. Soc. Microbiol., Washington, D.C.

Bacillus subtilis bacteriophage SPP: localization cf the prophage attachment site, and specialized transduction. J. Bacteriol. 129: 556-558.

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

on the chromosome of Bacillus subtilis 168. J. Virol. 6: 340-343.

chromosome. Proc. Natl. Acad. Sci. U.S. 58: 1507-1514.

YOUNG, F. E., C. SMITH and B. E. REILLY, 1969

ZAHLER, S. A., L. G. BENJAMIN, B. S. GLATZ, P. F. WINTER and B. J. GOLDSTEIN, 1976

ZAHLER, S. A., R. Z. KORMAN, R. ROSENTHAL and H. E. HEMPHILL, 1977

ZISSLER, J., 1967 Integration-negative ( int) mutants of phage A. Virology 31 : 189-189. Corresponding editor: I. P. CRAWWRD

Documents

of (CAMPBELL kauA - Genetics of lysis is due to a bacteriocin ("betacin") produced by SPp lysogens that is active on non- lysogens and on SPpdcitK, lysogens. Mutagenesis of strain