JOURNAL OF VIROLOGY, Oct. 1978, p. 395-402 0022-538X/78/0028-0395$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 28, No. 1 Printed in U.S.A.

Correlated Genetic and EcoRI Cleavage Map of Bacillus subtilis Bacteriophage f105 DNA BARBARA M. SCHER, MING FAN LAW,t AND ANTHONY J. GARRO* Department of Microbiology, Mount Sinai School of Medicine of the City University of New York, New York 10029

York, New

Received for publication 23 February 1978

The seven previously identified EcoRI cleavage fragments of 4105 DNA were ordered with respect to their sites of origin on the phage genome by marker rescue. One fragment, H, did not carry any determinants essential for replication. This fragment was totally missing in a deletion mutant which exhibited a lysogenization-defective phenotype. There is a nonessential region on the 4105 genome which begins in fragment B, spans fragment H, and ends in fragment F. The size of the nonessential region, as estimated by alterations observed in the fragmentation patterns of deletion mutant DNAs, is approximately 2.7 x 106 daltons. Two new EcoRI cleavage fragments with molecular weights of approximately 0.2 x 106 were detected by autoradiography of 32P-labeled DNA. These small fragments were not located on the cleavage map.

4105 is a temperate phage which lysogenizes the transformable strains of Bacillus subtilis 168. Interest in this phage has developed from studies indicating that it differs from the prototype temperate phage A in at least two aspects of its integration-excision cycle. First, while the A att site, through which integration into the host chromosome occurs, is situated in the midregion of a cyclized A genome (for review, see reference 8), the 4105 att site involves the ends of the phage DNA (1, 5). 4105 DNA, like A, can be reversibly cyclized (2, 4), but unlike A, 4105 DNA is circular as extracted from the phage (16). Whether the ends of 4105 DNA act independently during integration or in concert, as would be the case for the circular form, is still an open question. Second, in contrast to A prophage, the derepressed 4105 prophage undergoes extensive replication while still integrated within host DNA (14). With respect to both of these properties, 4105 resembles the mutator coliphage Mu. Mu, however, integrates into a large number of chromosomal sites (for a review of Mu replication, see reference 3), while only a single 4105 integration site has been observed

(13).

Genetic analysis of 4105 is still in its early stages. Eleven essential genes have been identified and mapped in a unique linear sequence (1). Although the number of genes involved in the establishment and maintenance of lysogeny is not known with certainty, a series of lysogenization-defective mutants has been isolated, and several complementation groups have been tent Present address: Viral Oncology and Molecular Pathology Section, National Institutes of Health, Bethesda, MD 20014.

tatively identified (M. F. Law and A. J. Garro, Abstr. Annu. Meet. Am. Soc. Microbiol. 1976, S127, p. 225). Nevertheless, considering that the molecular weight of 4105 DNA is in the range of 24 x 106 to 26 x 106 it seems reasonable to assume that the genetic map is far from saturated. As part of the further genetic analysis of 4105, we wished to develop a correlated physical and genetic map of the phage chromosome. In an earlier study (16), eight EcoRI-generated DNA fragments, A through H, were identified. Two of these fragments, C and D, were shown to be derived from the ends of the molecule, D from the left and C from the right end, respectively. These two fragments carry the single-stranded cohesive ends of the DNA and when associated generate the EcoRI cleavage fragment A. In the present study, we have determined the map order of the remaining, previously identified EcoRI cleavage framents. For the most part this was done by marker rescue of genetically mapped conditionally lethal phage mutations. One fragment which carries determinants essential for lysogenization but not replication was mapped by restriction analysis of two recently characterized (6) deletion mutants. The EcoRI cleavage map determined by this methodology agrees with that deduced by Perkins et a4. (11; personal communication) which is based on an analysis of partial digestion products of EcoRIcleaved 4105 DNA.

MATERIALS AND METHODS Phage and bacterial strains. The wild-type and conditionally lethal mutant strains of 0105 were from 395

396

SCHER, LAW, AND GARRO

L. Rutberg. The 4105 deletion mutants DI:1C and DII:6C were generously provided by J. I. Flock. The clear-plaque mutant cng-2 was isolated after Nmethyl-N'-nitro-N-nitrosoguanidine mutagenesis of +105-infected cells (7). The spontaneous clear-plaque mutants csi-1 and csi-6 were isolated from broth cultures of cells lysogenized by 4105 ind-1. Cultures of this ind- lysogen contain several thousand-fold fewer spontaneously released phage than cultures of wildtype lysogens (7). This low frequency of phage induction makes it relatively easy to detect rarely occurring spontaneous mutants which have escaped the effect of the ind-1 mutation as a result of a second mutation interfering with the maintenance of lysogeny. The suppressor-sensitive (sus-) 4105 mutants were assayed on B. subtilis strain MB228, which carries the SU+3 suppressor. This strain also was used as the recipient for all phage crosses. The su- strain, 44A0, from K. Bott was used to assay PFU by sus+ and temperature-sensitive (ts) mutants. The strain used as the competent recipient for most of the marker rescue experiments was GB7044su-, a strain which can be grown to high levels of competence. In the case of 105Ksus7, the competent recipient was MB228 su+. The Ksus7 mutation blocks 4105 DNA replication (1), and this mutant, in contrast to the other 105sus mutants, cannot be rescued efficiently from an suhost. All other strains used for the induction and propagation of phage have been previously described (16). Chemicals and enzymes. Agarose (electrophoretic grade) and mitomycin C were purchased from Sigma Chemical Co., and the restriction endonucleases EcoRI and HindlIl were purchased from Miles Laboratories Inc. Preparation of phage DNA. All the phage DNAs used were phenol extracted from CsCl-purified phage by using previously described methods and strains (16). The specific activity of the 32P-labeled 4105 DNA was 30,000 cpm/jig. Restriction endonuclease digestions. For analytical experiments, EcoRI reaction mixtures contained, in 0.05 ml: 0.1 M Tris-hydrochloride (pH 7.4); 5 mM MgCl2; 50 mM NaCl; 100 to 140 U of enzyme; and 2 to 10 ,ug of DNA (12). The mixtures were incubated for 18 h at 37°C, and the reactions were terminated by the addition of EDTA to 25 mM followed by heating for 10 min at 65°C. To prepare large quantities of EcoRI-cleaved 4105 DNA for the marker rescue experiments, 0.02-ml reaction mixtures containing 250 U of enzyme and 36 jig of DNA were used. The reactions were terminated as usual and then dialyzed against two changes of 1 liter each of 10 mM Trishydrochloride (pH 7.4) and 25 mM EDTA. After dialysis, the DNA solutions were heated for 10 min at 65°C to dissociate as much of fragment A as possible into its component fragments, C and D. The 0.05-ml HindIII reaction mixtures contained 6 mM Tris-hydrochloride (pH 7.9), 6 mM MgCl2, 6 mM 2-mercaptoethanol, 50 mM NaCl, 30 U of enzyme, and 2 to 3 jig of DNA. The reaction mixtures were incubated and the reactions were terminated as described above. Agarose gel electrophoresis. Analytical agarose

J. VIROL.

gels were run as previously described (16). Preparative agarose gel electrophoresis was performed in a similar manner with the following modifications. The 36-jLg samples of dialyzed and heated EcoRI-cleaved DNA were mixed with an

equal volume of 1.4% agarose in Tris-acetate buffer containing 0.05% bromophenol blue as a dye marker and quickly layered across the top of a vertical slab gel with a 2.4-cm2 cross-sectional area. The sample was moved into the gel at 90 V (9 V/cm) for 5 min and then electrophoresed at 1 V/cm for 24 h at room temperature. The separated fragments were visualized with a long-wave, 366-nm UV light; the gel was sectioned and the DNA was recovered by the method of Thuring et al. (18). The fraction of DNA applied to the gels which was ultimately recovered was estimated, by the use of 3Hlabeled )105 DNA, to be approximately 25%. Marker rescue. The procedure followed was that of Armentrout and Rutberg (1). The EcoRI-generated fragments were added to the competent cells at concentrations equivalent to 6 jig of intact 4105 DNA per ml. Twenty minutes later the cells were superinfected at a multiplicity of 5 with a conditionally lethal phage mutant, and after a further 15-min incubation the resulting phage were titered under restrictive conditions, su- cells or 41°C, and permissive conditions, su+ cells or 30°C. Autoradiography. 32P-labeled, EcoRI-cleaved DNA was electrophoresed on analytical agarose gels. The gel slabs were transferred to a piece of Whatman 3MM filter paper and dried under vacuum at room temperature (17). Kodak no-screen medical X-ray film was used to make the autoradiograms of the dried gels. Complementation analysis. The clear-plaque mutants were tested for their ability to complement each other, with respect to the production of lysogenized cells, by using a procedure similar to that described by Kaiser (9) for clear-plaque A mutants. Portions of exponentially growing cells about 5 x 107 CFU, were first infected at a multiplicity of 5 or more with one mutant, and the infected cells were overlayed on a 4105 assay plate. At this multiplicity the infected cells would form a confluently lysed lawn upon overnight incubation. Before incubation, however, the cells were superinfected by spotting on the overlay, within 10 min of its hardening, 10-Al samples containing 5 x 106 PFU of the other test phages. If complementation occurred, the lysogens formed grew and produced colonies in the area of the spotted lysate. Phage crosses. Exponentially growing MB228su+ cells from a VY broth (2.5% veal infusion [Difco]-0.5% yeast extract-0.01 M NaCl) culture were infected with a mixture of two mutants at a multiplicity of 5 for each parental type. After a 15-min incubation at 37'C to allow for adsorption, the unadsorbed phage were removed by pelleting the cells and resuspending them in VY broth, prewarmed to 37°C, which also contained anti-¢105 antisera at K = 2.5. After 10 min of further incubation, the culture was diluted I0O- with VY broth and incubated for 20 min. A drop of CHCl3 was then added to complete lysis, and the lysates were analyzed for PFU under permissive and restrictive plating conditions. For crosses involving ts mutants, all broth incubations were at 31°C.

VOL. 28, 1978

EcoRI CLEAVAGE MAP OF

RESULTS Marker rescue of conditionally lethal mutations. 4)105 DNA was digested with EcoRI and electrophoresed on agarose gels. The seven unique EcoRI cleavage fragments, B through H, were isolated from the gels, and each was tested for its ability to rescue various genetic markers present in 9 of the 11 mapped phage genes. Fragment A, which as noted above consists of the terminal fragments C and D, was not used. The results presented in Table 1 yield a fragment order of D/E/G/B/F/C. It was not possible to position fragment H by these experiments. It will be shown below that fragment H does not carry any of the known essential genes and that it is located on the 0105 chromosome between fragments B and F. With respect to the individual markers tested, each could be unambiguously assigned to a restriction fragment with the exception of Ksus7. This marker appeared to be rescued, albeit at a very low frequency, by fragment C. Fragments B, H, and F, when tested against Ksus7, failed to increase the level of wild-type phage produced above the background levels seen in Ksus7-infected cells which had not been pretreated with phage DNA. The background of spontaneously derived sus+ phage produced during Ksus7 infections was substantially higher than the backgrounds seen with the other mutants (Table 1). It was not possible to reduce this high background by using the su- strain GB7044 as the competent recipient for Ksus7 rescue. Gene K is the only 4105 gene recognized thus far as being essential for phage DNA replication (1). The need to use an su+ host for Ksus7 rescue presumably reflects a need for a pool of replicating DNA for efficient marker rescue, and in the case

0105 DNA

397

of Ksus7 such a pool can be produced only in an su+ cell. Origin of fragment H. A series of 4105 deletion mutants has been isolated and characterized by heteroduplex analysis (6). It was shown in these studies that 4)105 DNA contains a nonessential region which extends over an interval of 55 to 70% from the left end of the genome. We have examined restriction digest patterns of two of these deletion miutants, DII:6C and DI:1C. The deletions carried by these phages taken together essentially span the identified nonessential region. The results obtained, which are presented below, indicate that the nonessential region begins in EcoRI fragment B, spans fragment H, and ends in fragment F. The restriction fragments produced by EcoRI cleavage of DII:6C and DI:1C, which are deleted for the intervals between 54.7 to 64.5% and 65.0 to 70.3%, respectively (6), are shown in Fig. 1. In the case of DII:6C, only the mobility of fragment B is altered, the deletion reducing its size to that of fragment D. This corresponds to a loss of about 1.9 x 106 daltons of DNA from fragment B. In the case of DI:1C, fragments B, H, and F are missing and a new fragment larger than B is seen. From the known sizes of fragments A and B (16), the size of the new fragment was estimated to be approximately 9 x 106 daltons. The effect of the DI:lC deletion was further analyzed by digestion with HindIII. Cleavage of wild-type DNA with this enzyme generated 12 fragments which were resolved by agarose gel electrophoresis (Fig. 2). HindIII fragment F, which has a molecular weight of 1.5 x 106, is missing in the DI:1C digest, and a new fragment with a molecular weight of 0.8 x 106 is seen migrating between fragments H and I. The segment of DNA deleted in the DI:1C mutant amounts therefore

TABLE 1. Marker rescue of conditionally lethal mutations from isolated EcoRI-generated fragments of 4105 DNA" No. of infectious centers

DNA

fragment B sus14 D E G B H F C

3,500

C susl9

E tsN9

11,000

0

90 50 50

5,100

100

0 0 0

36,000

35,000

100

0 0 200

H tsN34

Jsusll

K sus7

400 500

L su.ss 200

500

25,000

400 700

F susl2

D sus13

30

20 30

5,300 40

0 0 0 500

200

8,600 "Competent Su- bacteria were incubated at 37'C with purified EcoRI fragments equivalent to 6,ug of intact 4105 DNA per ml, and 20 min later the cells were superinfected, at a multiplicity of 5, with phage carrying the sus and ts markers indicated above. The infected cells were assayed for PFU under restrictive conditions as described in Materials and Methods. The background levels of infectious centers produced by the sus and ts phages in the absence of added DNA were subtracted from the above values. Depending on the marker used, these background values ranged from 0 to 200 PFU/ml except in the case of Ksus7, where the backgrounds were about 2,500 PFU/ml. The markers, Bsusl4-Lsus9, are listed according to their map order (1).

398

SCHER, LAW, AND GARRO

J. VIROL.

Lysogenization/defective mutants. Most of the aforementioned 4105 deletion mutants, including DII:6C and DI:1C, have clear-plaque phenotypes (6), suggesting that the deletions have removed a region of the chromosome involved in the regulation of lysogeny. To further explore this question, the map loci of three independently derived mutations, cng-2, csi-1, and csi-6, all of which produce clear-plaque phenotypes, were examined. The genetic map order of

FIG. 1. Electrophoresis of EcoRI cleavage fragments of wild-type and deletion mutant p105 DNAs in 0. 7%o agarose gels. (Slot 1) Wild-type DNA; (slot 2) DI:1C DNA; (slot 3) DII:6C DNA. The samples were electrophoresed at 90 V (9 V/cm) for 2.5 h at room temperature. The figure is a composite of two electrophoreses. The samples electrophoresed to display fragments A through F contained 1.5, 1.4, and 1.2 ,ug of DNA in slots 1 through 3, respectively. The samples electrophoresed to display the smaller, and hence more difficult to visualize, fragments G and H contained 3.6, 4.0, and 2.5 pg of DNA, respectively. To further aid in visualizing G and H, the lower portion of the figure was photographically overexposed to enhance the contrast. The previously determined (16) sizes of the fragments in millions of daltons are: (A) 11.0; (B) 7.1; (C) 5.8; (D) 5.2; (E) 3.5; (F) 2.1; (G) 0.6; and (H) 0.4.

to approximately 0.7 x 106 daltons. The size of the DI:1C deletion, as determined by the HindIII result, is clearly large enough to span the entire 0.4 x 106-dalton EcoRI fragment H. The effect of the DI:1C deletion on EcoRI fragments B, H, and F can be explained therefore by postulating that fragment H is situated on the phage genome between fragments B and F. In the DI:1C mutant, the segment of the genome containing fragment H is deleted, as are the EcoRI cleavage sites between B and H and H and F. The remaining portions of fragments B and F are thus fused, generating the 9 x 106dalton fragment seen in the EcoRI digests.

FIG. 2. Electrophoresis of HindIII cleavage fragments of (p105 wild-type and DI:1C DNAs. (Slot 1) 1.3 Mg of wild-type DNA; (slot 2) 1.8 Mg of DI:1C DNA; (slot 3) EcoRI-cleaved A DNA included as size marker. The samples were electrophoresed as described in Fig. 1.

EcoRI CLEAVAGE MAP OF

VOL. 28, 1978

these mutations with respect to two of the endproximal markers was determined by a series of four-factor crosses (Table 2) to be Bsusl4, cng2, csi-1, csi-6 Lsus9. A series of three-factor crosses involving cng-2, csi-6, and conditionally lethal mutations in the vicinity of the nonessential region was then conducted to determine the relative positions of these markers. The results of these crosses (Table 3) suggest that both cng2 and csi-6 are located between Jsusll and HtsN34. This suggestion was supported by experiments which measured the frequency of cng2 and csi-6 co-rescue, with conditionally lethal mutations, from EcoRI-cleaved DNA. In these experiments it was assumed that linkage between markers located on separate restriction fragments would be disrupted by the EcoRI cleavage. The results (Table 4) show that cng-2 co-rescues with HtsN34 and Fsusl2, both of which are located on EcoRI fragment B, and that csi-6 co-rescues with Jsusll, which is located on EcoRI fragment F. In these experiments, the frequency of phage producing turbid plaques under the nonrestrictive conditions was

0105

DNA

399

used to determine the frequency of spontaneous reversion of the clear-plaque mutation. It can be seen that cng-2, when linked to the Jsusll mutation, reverted at a substantially higher frequency than when it was linked to either HtsN34 or Fsusl2. This was observed for several independently derived Jsusll cng-2 isolates, but the reasons for it are not known. Unmapped fragments. In their analysis of the partial digestion products of EcoRI-cleaved 4105 DNA, Perkins et al. (11; personal communication) observed two EcoRI partial cleavage fragments which differed in size from the corresponding limit digest fragments by approximately 0.2 x 106 daltons. No fragments this small were observed in our initial study of the EcoRI fragmentation pattern; however, two factors may have led to these fragments being missed. In the initial study, the separated EcoRI-generated fragments were visualized by ethidium bromide staining. Since the intensity with which ethidium bromide-stained fragments fluoresce is proportional to their size, very small fragments would fluoresce very weakly. In ad-

TABLE 2. Percentage of wild-type phage produced in four-factor crosses of sus clear double mutants % Wild-type Parental genotypes phagea + + Bsusl4 cng-2 0.27 + + csi-1 Lsus9

Bsus14

+

csi-1

+

cng-2

+

Bsusl4

csi-1

+

+

+

+

csi-6

Lsus9

Bsusl4

+

+

csi-1

csi-6 +

Lsus9

Bsus14

cng-2

+

+

+

+

csi-6

Lsus9

+ Lsus9

+

017

0.03 0.27

+ + csi-6 0.08 + Lsus9 cng-2 a Determined as the ratio of turbid PFU on su- cells to the total PFU on su+ cells. The map order of the clear-plaque mutations was determined from the relative frequency of wild-type phage produced in each set of crosses according to the following rationale:

Bsus14 +

Cross Bsusl4

cx

+

+

+

+

cy

Lsusl4

Bsusl4

+

cy

+

+

cx

+

Lsusl4

Frequency of wild type

Implied order

Cross 1 > cross 2

Bsusl4 cx cy Lsus14

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SCHER, LAW, AND GARRO

400

TABLE 3. Results of three-factor crosses involving cng-2 and csi-6 % Clear plaques on sucells at 40°C

Parental genotypesa

cng-2 +

+ Jsusll

Ksus7

+ cng-2

+

Ksus7

Jsusll

+

csi-6

+

Ksus7

+

Jsusll

+

+

+

+

Ksus7

csi-6

Jsusll

+

cng-2 +

+

Lsus9

Jsusll

+

+

+

Lsus9

cng-2

Jsusll

+

+ csi-6

+ Jsusl 1

Lsus9

HtsN34 +

+

+

cng-2

Lsus9

+

HtsN34

+

+

+

csi-6

Lsus9

Fsusl2

+

+

+

cng-2

Jsusll

Fsusl2

cng-2

+

+

+

Jsus1l

Fsusl2

+

+

+

csi-6

Jsusll

Fsusl2

csi-6

+

+

+

88.5

EcoRI-generated )105 DNA fragments with respect to the phage genetic map. The composite map shown in Fig. 4 has been depicted as a circle, since this is the preferred configuration of the phage DNA (16). The relative distances between the conditionally lethal mutations shown approximate the recombinational map units separating them (1). There are at least two

12.3 96.8 7.6 90.1

12.6 6.4

51.6

57.3 14.7

89.9 6.7

92.4 Jsusll a For the relative positions of the conditionally lethal mutations on the 4105 map, see Fig. 4.

dition, fragments this small would essentially have co-migrated with the bromophenol blue dye maker used in these experiments to follow the progress of the electrophoresis. To circumvent these problems, the fragmentation pattern of 32P-labeled DNA was examined by using autoradiography to visualize the separated fragments. The results of this experiment (Fig. 3) show the presence of what appear to be two small fragments, labeled I and J, which have molecular weights of approximately 0.25 x 106 and 0.20 x 106, respectively. DISCUSSION As a result of the experiments reported here, it has been possible to order the seven major

G.

H

J FIG. 3. Autoradiograph of electrophoresed EcoRIcleaved 32P-labeled c105 DNA.

EcoRI CLEAVAGE MAP OF 0105 DNA

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401

TABLE 4. Co-rescue of conditionally lethal and clear-plaque mutations from EcoRI-cleaved 4i105 DNA' % Turbid plaques PFU/ml

Superinfecting phage

Restrictive conditions 8.2 x 104 2.9 x 105 6.1 x 103 3.9 x 103 1.2 x 103

Nonrestrictive conditions 2.1 x 10"

Restrictive conditions

Nonrestrictive conditions

% Co-rescue

4.2 0.1 4.3 Fsusl2 cng-2 5.1 0.1 1.4 x 108 5.2 HtsN34 cng-2 0.5 1.0 1.5 2.5 x i08 Jsusll cng-2 0 0 2.8 x 108 0 Bsusl4 csi-6 0 0 0 3.1 x 108 EtsN9 csi-6 0 0 0 1.9 x 105 2.3 x 10" Fsusl2 csi-6 21 21 0 1.1 x 10 1.1 x 105 Jsusll csi-6 0.2 0 1.2 x 10" 0.2 5.0 x 104 Ksus7 csi-6 a Competent Su- bacteria were incubated at 37°C with 6 ug of 4105 DNA per ml previously digested with EcoRI, and 20 min later the cells were superinfected, at a multiplicity of 5, with phage carrying either sus and clear markers or ts and clear markers as indicated above. The infected cells were assayed for PFU under both restrictive and nonrestrictive conditions as described in Materials and Methods, and the percentage of turbid plaques in each population was determined. Background levels of PFU produced by the sus and ts phages under restrictive conditions in the absence of DNA were subtracted from the above values. Depending on the markers used, these background values ranged from 0 to 200 PFU/ml except in the case of phage Ksus7 csi-6, where the background value was about 3,500 PFU/ml.

Jsus II.

Hts N34

GtsN1O FIG. 4. Correlated genetic and EcoRI cleavage map ofb105 DNA. The sizes of the EcoRI fragments are proportional to their molecular weights. The localization of the genetic markers to specific EcoRI fragments is based on the data presented in Tables 1 and 4. The distances between markers are based on the recombination frequencies obtained by Armentrout and Rutberg (1). The positions of the deletions DI:JC and DII:6C are based on the data of Flock (6).

additional EcoRI cleavage fragments, about onehalf the size of fragment H, which have not been located on the map. Because of their small size, which is in a range where transformation efficiencies are very low (10), and the paucity of additional genetic markers, methods other than marker rescue, such as an analysis of restriction endonuclease-generated partial digestion products, will have to be used to map these fragments.

Two genetic markers, AtsN15 and GtsNlO,

which were not included in the marker rescue studies are included on the map. AtsN15 is shown as being located on fragment D, since this mutation is the left-most marker on the linear genetic map (1) and fragment D contains the intact left end of the molecule (16). GtsNlO was placed on fragment B on the basis of the genetic studies which mapped this mutation between Fsusl2 and HtsN34 (1). It should be noted that it is not known how close the markers AtsN15 and Lsus9, which terminate the genetic map, are to the cohesive ends of the DNA. Ksus7 has been tentatively positioned on fragment C, but the possibility that it may be located on one of the small unmapped fragments, I or J, has not been eliminated. A location on fragment C is consistent with genetic data (1) which positioned Ksus7 between the fragment C cistron L and the fragment F mutation Jsusll. Fragment F, however, was totally inactive for Ksus7 rescue, whereas fragment C did show activity. Assuming that Ksus7 is on fragment C, the reason for the relatively low level of Ksus7 rescue by fragment C is not known. Although it has been reported that markers situated near the ends of 4105 DNA, such as gene L mutations, are rescued at much lower efficiencies than internal markers (15), there is no indication that Ksus7 is near the end of a fragment. It has been noted previously, however, that the Ksus7 mutation also rescues very poorly even from undigested 4105 DNA (15). Further evidence against the possibility that Ksus7 might be located on fragment F is derived from the observation that the csi-6 mutation, which co-rescued with the fragment F marker, Jsusll, failed to show any significant rescue with Ksus7 (Table 4).

402

SCHER, LAW, AND GARRO

The location on the fragmentation map of the DII:6C and DI:1C deletions is in good agreement with the heteroduplex analysis which positioned these deletions between 54.7 to 64.5% and 65.0 to 70.3% from the left end of the genome (6). Assuming that the DII:6C deletion is situated in the fragment H-proximal portion of fragment B, the sum of the D, E, G, and nondeleted fragment B molecular weights would amount to 14.5 x 106 or 58% of the total molecular weight. Similarly, assuming that the DI:1C deletion begins at the H end of fragment B, the sum of the D, E, G, and B fragment molecular weights, 16.4 x 106, would place the start of the DI:1C deletion at a site 65.6% from the left end of the DNA. The cng-2 and csi-6 mutations were localized to fragments B and F, respectively, on the basis of their co-rescue with markers located in these fragments. The 0.5% co-rescue of cng-2 with Jsusll and the 0.2% co-rescue of csi-6 with Ksus7 were not considered significant with respect to linkage. The EcoRI-cleaved DNA used in these experiments was not fractionated, and the low-level rescues referred to above probably derived from independent events in which the competent cells took up multiple DNA fragments. Similar effects have been seen with sheared DNA preparations where markers on different halves of the cleaved molecules co-rescued at a level approximately 5% that of markers on the same half (1). The location of the cng-2 and csi-6 mutations within their respective fragments is not known with certainty. The csi-6 mutation was placed outside of the DI:1C deletion since these two mutations complement each other in the spot test described in Materials and Methods. Both csi-6 and DI:1C failed to complement either cng2 or DII:6C, but the csi-1 mutation, which mapped between cng-2 and csi-6 by four-factor crosses (Table 2), did complement both cng-2 and csi-6. This complex complementation pattern suggests that there are several genes, located in this region of the chromosome, which are involved in the regulation of lysogeny. The mutations carried by a number of clear-plaque mutants have been linked to Jsusll (1), and the size of the nonessential region, which approaches 4 x 106 daltons (6), is large enough to encompass several genes. The identification of the number and function of genes involved in 4i105 lysogeny, however, will require further investigation. ACKNOWLEDGMENTS We thank D. H. Dean, J. B. Perkins, and C. D. Zarley for providing the results of their analysis of the partial digestion products of EcoRI-cleaved 4105 DNA before publication and J. I. Flock and L. Rutberg for sharing the results of their

J. VIROL. heteroduplex analysis of the 0105 deletion mutants and for making these mutants available to us. We gratefully acknowledge the expert technical assistance of V. Lima. This investigation was supported by Public Health Service

research grant GM21541 from the National Institute of General Medical Sciences.

LITERATURE CITED 1. Armentrout, R. W., and L. Rutberg. 1970. Mapping of prophage and mature deoxyribonucleic acid from temperate Bacillus bacteriophage 4105 by marker rescue.

J. Virol. 6:760-767. 2. Birdsell, D. C., G. M. Hathaway, and L. Rutberg. 1969. Characterization of temperate Bacillus bacteriophage p105. J. Virol. 4:264-270. 3. Bukhari, A. I. 1976. Bacteriophage Mu as a transposition element. Annu. Rev. Genet. 10:389-412. 4. Chow, L. T., L. B. Boice, and N. Davidson. 1972. Map of the partial sequence homology between DNA molecules of Bacillus subtilis bacteriophages SP02 and 4105. J. Mol. Biol. 68:391-400. 5. Chow, L. T., and N. Davidson. 1973. Electron microscope study of the structures of the Bacillus subtilis prophages, SP02 and 4105. J. Mol. Biol. 75:257-264. 6. Flock, J. I. 1977. Deletion mutants of temperate Bacillus 7.

8.

9. 10. 11.

12.

13. 14.

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