Vol. 28, No. 1

JOURNAL OF VIROLOGY, Oct. 1978, p. 403407 0022-538X/78/0028-0403$02.00/0 Copyright © 1978 American Society for Microbiology

Printed in U.S.A.

NOTES Restriction Endonuclease Mapping of Bacteriophage j105 and Closely Related Temperate Bacillus subtilis Bacteriophages plO and p14 JOHN B. PERKINS, C. DAVID ZARLEY, AND DONALD H. DEAN* Department of Microbiology, The Ohio State University, Columbus, Ohio 43210

Received for publication 23 February 1978

Cleavage maps of the three similar Bacillus subtilis temperate bacteriophages, 4105, plO, and p14, were constructed by partial digestion analysis utilizing the restriction endonuclease EcoRI. Comparison of the topography of these maps indicates that all phage DNAs possess cohesive ends and a number of EcoRI restriction sites; the fragments are conserved, and the estimated base substitution/nucleotide divergence between these phages is 0.03 to 0.07 based on conserved fragments or between 0.03 and 0.11 based on conserved cleavage sites. These lines of evidence indicate that 4105, plO, and p14 are closely related. Double-enzyme digestion analysis reveals that p14 DNA has unique SalGI and BglJI restriction sites and 4105 DNA has a unique SalGI restriction site, making these phages possible cloning vectors for B. subtilis.

With the advent of restriction endonucleases, physical maps of various bacteriophages have been constructed: OXX174 (9, 13), G4 (5), S13 (6), fl (11, 24), fd (18, 24), A (8, 22), T5 (14, 25), Z J/2 (24), P4 (7), 415 (12), and 429 (12). One use of restriction maps is in the analysis of related phage DNAs (15). Van den Hondel and Schoenmaker (24) used extensive mapping in quantitating the similarities and differences of the closely related filamentous bacteriophages M13, fd, fl, and Z J/2. More recently, Godson (5, 6) used the same technique to determine the base sequence divergence between the phages OX174, S13, and G4 and suggested that S13 was more related to 4X174 than to G4. We have used this approach to analyze a number of new temperate phages of Bacillus subtilis and have found that two new phages, plO and p14, are closely related to the well-known phage 4105. Phages and phage DNAs were isolated and purified as previously described (1, 3), as was restriction enzyme isolation (20; R. J. Roberts, personal communication) and digestion of phage DNA (3, 10). DNA digestion with SalGI and BglII (New England Biolabs) was identical to EcoRI digestion except for the buffers: SalGI, 0.006 M Tris-hydrochloride-0.006 M MgCl2-0.1 M NaCl-0.006 M 2-mercaptoethanol, pH 7.9; BgllI, 0.02 M Tris-hydrochloride-0.007 M MgCl2-0.007 M 2-mercaptoethanol, pH 7.4. Re-

actions were terminated by addition of B.J. solution (20) amounting to 20% of the total reaction mixture. All mixtures, unless specified, were heated at 65°C for 20 min before electrophoresis. The molecular weights of EcoRI-generated fragments of 4)105, plO, and p14 are given in Table 1. In comparing EcoRI fragments of one phage with those of another, we observed that some fragments of 4105 had molecular weights identical to those of some fragments of plO and p14. This has been substantiated by mixing the fragments from two phages at a time and demonstrating that these fragments co-migrate during gel electrophoresis (data not shown). Scher et al. (18) have recently shown by enzymatic methods that 4105 DNA possesses cohesive ends located on the EcoRI fragments C and D. These two fragments can anneal to form another band, EcoRI-A. Cohesive ends may also be demonstrated by heating (65°C, 20 min) the EcoRI fragments of 4105 DNA before electrophoresis (Fig. 1, slot 1). When compared with the unheated sample of EcoRI-fragmented 4105 DNA (Fig. 1, slot 2), the intensity of fluorescence of the end join decreases as it dissociates into the two end fragments, resulting in a greater fluorescence of these two fragments than of the unheated control. Cohesive ends can also be seen for plO (Fig. 1, slot 3, heated; and 4, unheated) and p14 DNA by heating EcoRI-gener403

404

NOTES

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J. VIROL.

TABLE 1. Endonuclease R EcoRI fragments of group Iphage DNAs 4105 Mol Wtb ± 0.51 ± 0.32 ± 0.16 ± 0.13 ± 0.08 ± 0.08 ± 0.05 ± 0.05

Fragmenta

Ae B

Fragment

Ae B C1i L DR

10.98 7.17 5.73 4.91 3.48 1.95 0.71 0.60 0.25 0.20

CR DL E2 F3 G H If

if

E2 F3

G4 H

plO

p14

Mol wtf 9.40 ± 0.85 7.78 ± 0.39

4.85 ± ± ± ± ± ±

4.25 3.53 1.99 1.89 0.97

0.07

0.07 0.04 0.06 0.06 0.04

Fragment A Be Ci' L D ER F3

G4 H

Mol wtd

7.90 ± 7.36 ± 4.72 ± 4.40 ± 2.48 ± 1.93 ± 1.84 ± 1.56 ±

0.18 0.40 0.173 0.08 0.06 0.07 0.07 0.05

Total 24.93 ± 0.51 25.30 ± 0.73 24.70 ± 0.49 a R, end fragment, right; L, end fragment, left. Fragments that co-migrate have the same superscript number. 'Average of eight determinations x 10-6 Mdal. 'Average of two determinations x 10-' Mdal. d Average of four determinations x 10-6 Mdal. 'End join fragment, not included in totals. f Data of Scher et al. (19).

ated fragments (Fig. 1, slot 5, heated; and 6, unheated). End join and end fragments for all three phages are identified in Table 1. Partial digestion mapping was undertaken to determine the EcoRI fragment order of 4i105 and p14 DNAs. These DNAs were digested by EcoRI enzyme under limiting conditions for 1, _ _ 3, 7, and 13 min. Tables 2 and 3 (p14 and 4)105, respectively) list the molecular weights and fragment orders for each partial digestion product. These data allow the construction of the physite cal maps of p14 and 4)105 (Fig. 2). The fragment order of EcoRI fragments A through G of (105 deduced by partial digestion analysis is the same as that derived from marker rescue analysis (19; B. Scher, M. F. Law, and A. J. Garro, personal communication), again substantiating this map order. Comparison of the physical maps of 4105 and p14 reveals that co-migrating fragments of these phages (Table 1) map relative to each other (Fig. 2). Therefore, a tentative physical map of plO DNA can be constructed by comparing the position of plO fragments with that of co-migrating fragments in the 4105 and p14 maps (Fig. 2 and Table 1). End fragment data (Table 1) are also used to construct this map by orienting the ends of the plO genome. By these methods, EcoRI fragments D, E, F, G, and C can be mapped, leaving EcoRI-H and -B to be positioned. The orientation of fragments H and B can be either HB or BH. The former is chosen to conserve the Effect of heating on EcoRI-generatedfrag- EcoRI site, at map position 14.1 kilobases, be-

te

FIG. 1.

3

4

5

6

~

of 4105, plO, and p14 DNAs. (Slot 1) 105 DNA, heiated; (slot 2) 4105 DNA, unheated; (slot 3) plO DlVA, heated; (slot 4) plO DNA, unheated; (slot 5) pl'4 DNA, heated; (slot 6) p14 DNA, unheated; (slot meents

7) 3T DNA as standard (3). Heating was at 65°C for 20 min. Equal amounts of DNA were put in each slot. (0) End fragments.

VOL. 28, 1978

NOTES

TABLE 2. Partial digestion fragments of p14 Proposed bands in Mol (10-6) Fragment partial fragment 1 24.4 CDAFGHE 23.0 2 CDAFGH 21.5 3 CDAFG 19.0 4 DAFGHE 18.5 CDAF 5 15.5 6 CDA 7 14.5 AFGHE 13.0 DA 8 12.0 9 AFG 11.0 10 GHEC 11 9.50 CD 8.80 HEC 12 A 8.10 7.60 CE B 13 6.25 GHE 14 5.65 FGH 4.90 C D 4.50 EH 15 4.15 16 4.03 FG 17 3.65 GH E 2.55 1.92 F 1.85 G 1.55 H Wt

tween the three phages.

Digestion of 4105 and p14 DNAs with various enzymes has revealed that

4)105 DNA is cut once by SalGI, producing a 23.5-megadalton (Mdal) fragment and a 0.9-Mdal fragment. The sum of these two is the unit-length 4105 genome. Double digestion of 4105 with EcoRI and SalGI produces the same pattern listed in Table 1 with the exception that the 5.73-Mdal C band is cleaved into a 0.9-Mdal band and a 4.83-Mdal band. This clearly places the single SalGI site at 0.9 Mdal from the right (C fragment) end of the phage (Fig. 2). Similar double-digestion analysis (data not shown) has allowed us to locate a single SalGI site within the F fragment and a single BglII site in the H fragment of the p14 genome (Fig. 2). Wilson et al. (26) have previously shown by restriction enzyme analysis that the temperate B. subtilis phages 4105, SP02, and 4)3T are dissimilar. D. H. Dean, C. Fort, and J. Hoch (manuscript in preparation) have found that four new temperate B. subtilis phages (4)105, p6, plO, and p14) are greater than 90% related according to serology, host range, adsorption (receptor) sites, and immunity properties. In this paper we also confirm, by restriction analysis and physical mapping, that the DNAs of three of these phages are similar to one another. The genome size, determined by summation of the EcoRI fragments, is around 24.9 Mdal for all

405

phages (Table 1). This agrees with the sedimentation data of Rutberg and Rutberg (17) for 4105. All three of these phage genomes possess natural cohesive ends (Fig. 1), and we have confirmed these data by electron microscopy (M. Rudinski and D. H. Dean, unpublished observations). It is not known whether the cohesive ends are of the same length and base composition for all three phages, but we have not been able to anneal them to one another (unpublished observation). Phage 0105 possesses att sites at the cohesive ends of the phage genome (2, 16), and it integrates at a unique site (16). Whether or not plO and p14 share these properties is also not known. The co-migrating fragments that result from conserved or similar restriction sites are considered "conserved fragments" (23). We have compared the base sequence divergence/nucleotide according to the methods of Uphold (23) and have found that p14 and plO have a base sequence divergence/nucleotide of only 0.03 when calculated either by conserved restriction enzyme sites or by conserved fragments. The base sequence divergence/nucleotide between 4105 and p14 or between q5105 and plO is 0.07 when calculated by conserved fragments and 0.11 when calculated by conserved site. These values substantiate our classical analysis, which showed a 90% relatedness between these phages (Dean et al., in preparation), and the heteroduplex TABLE 3. Partial digestion fragments of 4105

Fragment

a

Mol wt (10-6)

1 24.9 2 20.0 3 19.0 4 15.2 5 13.5 6 12.3 7 12.0 11.2 A 8.40 8 8.10 9 B 7.40 C 5.80 5.55 10 D 4.95 11 4.50 12 3.75 E 3.50 2.55 13 1.95 F 14 0.99 0.76 G 0.66 H 0.25 Ia ja 0.20 Data from Scher et al. (19).

Proposed band in

partial fragment DIEJGBHFC IEJGBHFC DIEJGBHF BHFC, IEJGBHF EIGBHF FCD, EJGBH IEJGB CD JGB, DE, HFC CF, GB, BH DI

EJG EJ FH GJ

406

J. VIROL.

NOTES DI:1C DX:4c DX: 2c DU:6c :2

DX:29f I

D

E C

a

JG U

6

1 143

13.0

7.477

C

F

H

C

I

0

0

375kb

36.2

A CG

C

6105

2.9

2S.1 26.0

133

G

HH~~~

P14 27.27.0

7.4

28.7

31.5

l33.

0

C

H

E

37 5kb

33.2

0

G

1O 124

0

5

14.1

25.6

10

1S

35.0

20

36.ob

20 a

25 I

10 ' dellems

FIG. 2. Physical maps of group I temperate B. subtilis phages. Large capital letters identify EcoRI fragments for each phage DNA, as shown in Table 3. Small capital letters associated with the q0105 fragments indicate cistrons which occur on those fragments (Scher et al., [19]). The region indicated by lines lettered DI:1c, DI:4c, DI:2c, DI:lt, DI:29t, and DII:6c demonstrate deletion mutants in the 4105 genome (4). EcoRI cleavage sites are indicated as , SalGI as (, and Bgl II as (®). Numbers below the EcoRI cleavage sites indicate the distance in kilobases of that site from the left end, and the scale at the bottom indicates size of the maps in megadaltons.

analysis, which showed 2.8 to 4% heteroduplexing between these phages (Rudinski and Dean, in preparation). Furthermore, preliminary evidence in this lab indicates that these phages recombine in genetic crosses. Restriction endonuclease analysis has also revealed that 4105 and p14 possess single restriction sites, making these phages potential cloning vehicles in B. subtilis. It is desirable for a temperate phage molecular vehicle to have the following properties: a single restriction site in a nonessential region, deletions which allow encapsulation of a large amount of passenger DNA, and a selectable character so that phages carrying a passenger DNA may be readily identified. The phage p14, more so than qb105, may have all these qualities. Inspection of the physical maps reveals that the p14 SalGI site might fall within the nonessential region covered by the 4105 deletions (4). We thank Barbara Scher, Ming Fan Law, and Anthony Garro for sharing unpublished data before publication, Jim Hoch for sending us the Rho phages, Mark Rudinski for

helpful discussions, and Sarah Sieling for typing the manuscript. This work was supported by a grant from The Ohio State University Graduate School and Public Health Service grant 5P30 CA 16048-0351 to The Ohio State University Comprehensive Cancer Center awarded by the National Cancer Institute.

LlTERATURE CITED 1. Adams, M. 1959. Bacteriophages. Interscience Publishers,

New York. 2. Chow, L. T., and N. Davison. 1973. Electron microscope study of the structure of Bacillus subtilis prophage SP02 and 4,105. J. Mol. Biol. 75:257-264. 3. Dean, D. H., J. C. Orrego, K. W. Hutchison, and H. 0. Halvorson. 1976. New temperate bacteriophage for Bacillus subtilis, pll. J. Virol. 20:509-519. 4. Flock, J. I. 1977. Deletion mutants of temperate Bacillus subtilis bacteriophage 0105. Mol. Gen. Genet. 155: 241-247. 5. Godson, G. N. 1975. Evolution of 4X174. II. A cleavage map of the G4 phage genome and comparison with the cleavage map of 4X174 RF DNA. Virology 63:320-335. 6. Godson, G. N. 1976. Evolution of 4X174. III. Restriction map of S13 and its alignment with that of 4X174. Virology 75:263-280. 7. Goldstein, L., M. Thomas, and R. W. Davis. 1975. Eco RI endonuclease cleavage map of bacteriophage

VOL. 28, 1978 P4-DNA. Virology 66:420-427. 8. Haggerty, D. M., and R. F. Schleif. 1976. Location in bacteriophage lambda of cleavage sites of the site-specific endonuclease from Bacillus amyloliquefaciens H. J. Virol. 18:659-663. 9. Hayashi, M. N., and M. Hayashi. 1974. Fragment maps of OX174 replicative DNA produced by restriction enzymes from Haemophilus aphirophilus and Haemophilus influenzae H-1. J. Virol. 14:1142-1151. 10. Helling, R. B., H. M. Goodman, and H. W. Boyer. 1974. Analysis of endonuclease R Eco RI fragments of DNA from lambdoid bacteriophages and other viruses by agarose-gel electrophoresis. J. Virol. 14:1235-1244. 11. Horiuchi, K., G. F. Vovis, V. Enea, and N. D. Zinder. 1975. Cleavage map of bacteriophage fl: location of the Escherichia coli B.-specific modification sites. J. Mol. Biol. 95:147-165. 12. Ito, J., F. Kawamura, and S. Yanofsky. 1976. Analysis of 029 and 415 genomes by bacterial restriction endonucleases Eco RI and Hpa I. Virology 70:37-51. 13. Lee, A. S., and R. L. Sinsheimer. 1974. A cleavage map of bacteriophage 4X174 genome. Proc. Natl. Acad. Sci. U.S.A. 71:2882-2886. 14. Rhoads, M. 1975. Cleavage of T5 DNA by the Escherichia coli RI restriction endonuclease. Virology 64:170-179. 15. Roberts, R. J. 1976. Restriction endonucleases. Crit. Rev. Biochem. 4:123-163. 16. Rutberg, L. 1969. Mapping of a temperate bacteriophage active in Bacillus subtilis. J. Virol. 3:38-44. 17. Rutberg, L., and B. Rutberg. 1970. Characterization of infectious deoxyribonucleic acid from temperate Bacillus subtilis bacteriophage 4,105. J. Virol. 5:604-608.

NOTES

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18. Scher, B. M., D. H. Dean, and A. J. Garro. 1977. Fragmentation of Bacillus bacteriophage 4105 DNA by restriction endonuclease EcoRI: evidence for complementary single-strand DNA in the cohesive ends of the molecule. J. Virol. 23:377-383. 19. Scher, B. M., M. F. Law, and A. J. Garro. 1978. Correlated genetic and EcoRI cleavage map of Bacillus subtilis bacteriophage 4,105 DNA. J. Virol. 28:395-402. 20. Takanami, M., T. Okamoto, K. Suzimoto, and H. Sugisaki. 1975. Studies on bacteriophage fd DNA. I. A cleavage map of the fd genome. J. Mol. Biol. 95:21-31. 21. Tanaka, T., and B. Weisblum. 1975. Construction of a colicin E1-R factor composite plasmid in vitro: means for amplification of deoxyribonucleic acid. J. Bacteriol. 121:354-362. 22. Thomas, M., and R. W. Davis. 1975. Studies on the cleavage of bacteriophage lambda DNA with Eco-RI restriction endonuclease. J. Mol. Biol. 91:315-328. 23. Uphold, W. B. 1977. Estimation of DNA sequence divergence from comparison of restriction endonuclease digests. Nucleic Acid Res. 4:1257-1265. 24. Van den Hondel, C. A., and J. G. G. Schoenmaker. 1976. Cleavage maps of the filamentous bacteriophages M13, fd, fl, and Z J/2. J. Virol. 18:1024-1039. 25. Von Gabain, A., G. S. Hayward, and H. Bujard. 1976. Physical mapping of the Hind III, Eco RI, Sal and Sma restriction endonuclease cleavage fragments from bacteriophage T5 DNA. Mol. Gen. Genet. 143:279-290. 26. Wilson, G. A., M. T. Williams, H. W. Baney, and F. E. Young. 1974. Characterization of bacteriophages of Bacillus subtilis by the restriction endonuclease Eco -RI: evidence for three different temperate phages. J. Virol. 14:1013-1016.