DNA modification induced during infection of Bacillus subtilis by phage φ3T

Gene, 12 (1980) 17-24 © Elsevier/North-HollandBiomedicalPress

17

D N A m o d i f i c a t i o n i n d u c e d during infection of Bacillus subtilis b y phage ¢ 3 T (Recombinant DNA; plasmid-¢3T hybrids; methylation in vitro)

James M. Cregg *, Anh Hoang Nguyen and Junetsu lto ** Department of Cellular Biology, Scripps Clinic and Research Foundation, La Jolla, CA 9203 7 (U.S.A.)

(Received May 8th, 1980) (Accepted July 31st, 1980)

SUMMARY The DNA of the Bacillus subtilis temperate phage ~b3T is not susceptible to cleavage by the restriction endonuclease HaelII, although it is cut by many other restriction enzymes. The host DNA from uninfected cells is cut by HaelII. We show that ¢3T DNA propagated in a restriction-modification-defective Escherichia coli cell can be digested by HaelII. Thus, qb3T DNA does contain the nucleotide recognition sequence of HaelII. We suggest that this phage induces the modification of its own DNA. In support of this mechanism we show that extracts prepared from q~3T-infected ceils contain an activity which in the presence of S-adenosyl-L-methionine (SAM) can modify X DNA against cleavage by HaelII. The same in vitro-modified DNA is still susceptible to cleavage by other restriction endonucleases.

INTRODUCTION Bacteriophage ~3T is a large temperate phage of B. subtilis isolated by Tucker (1969). The phage is

capable of converting thymine auxotrophic hosts to prototrophy upon lysogenization (Tucker, 1969; * Present address: BRL, Inc., 411 North Stonestreet Ave., Rockville, MD 20850 (U.S.A.). ** Please direct correspondence to: Dr. J. Ito, Department of Microbiology,Universityof Arizona, Collegeof Medicine, Tucson, AZ 85724 (U.S.A.). Abbreviations: ApR or ApS, ampicillin-resistantor-sensitive; Md, megadaltons; m.o.i., multiplicity of infection; SAM, S-adenosyl-L-methionine; [3H]SAM, S-adenosyl.L-[Me.3H]methionine; SDS, sodium dodecyl sulfate; TeR or TeS, tetracycline-resistantor -sensitive.

Dean et al., 1976; Williams and Young, 1977), an ability which has been employed in several recent investigations (Ehrlich et al., 1976; Graham et al., 1977; Duncan et al., 1978). ¢3T and its relatives, pl 1 and SP/3, are being developed as cloning vectors for studies in B. subtilis (Kawamura et al., 1979; S. Zahler, personal communication). Previously, we reported on the susceptibility of O3T DNA to cleavage by a large number of restriction endonucleases and found that HaelII could not cut ¢3T DNA (Cregg and Ito, 1979). We estimated from the sequence recognized by HaelII (5'..GGCC..3') (Kelly and Smith, 1970) that the ¢3T genome (mol. wt. 8 X 107, 34% G + C; Ehrlich et al., 1976; Cregg and Ito, 1979; Tucker, 1969; Dean et al., 1976), should contain about lOOHaelII cleavage sites. The molecular mechanism by which 03T DNA is resistant

18 to digestion by HaelII is the subject of this report. Bacteriophages have revealed a number of mechanisms to escape host restriction. Some phages such as B. subtilis phage SPP1, SPO2 and ¢105, depend on the host-modification system to avoid restriction by that host (Bron et al., 1975). However, the B. subtilis 168 host for ¢3T does not normally express a modification system capable of protecting DNA against cleavage by HaelII (Trautner et al., 1974; this publication). A variation of this host modification mechanism is suggested by the fact that B. subtilis 168 when treated with mitomycin C or UV irradiation produces a methylase which modifies DNA within the HaelII recognition sequence (Bron and Murray, 1975; Bron et al., 1975; G(inthert et al., 1976; Mann and Smith, 1977). Thus, 6-(p-hydroxyphenylazo)-uracil used to induce ¢3T prophage, may also induce ~3T DNA modification. However, lytic infection by a ¢3T mutant defective in lysogenization results in progeny, whose DNAs are still resistant to HaelII cleavage (unpublished observation). E. coli phage T7 and T3 (Spoerel et al., 1979) and B. subtilis phage eNR2rH (Makino et al., 1979) induce an "anti-restriction" enzyme which interferes with the host restriction system. This type of mechanism cannot explain the resistance of ¢3T DNA to cleavage by HaelII since restriction enzyme digests are performed in vitro using purified ¢3T DNA. Phages may also avoid restriction by simply not having the nucleotide recognition sequence for that enzyme. For example, bacteriophage ¢29 DNA, which should contain about 20 HaelII sites, does not have the appropriate sequences (Ito and Roberts, 1979). Lastly, many phages induce their own DNA modification system upon infection (Mathews, 1971; Van Ormondt et al., 1975). B. subtilis phage SPO1 produces the modified nucleotide hydroxymethyluracil which it incorporates into its DNA (Okubo et al., 1964). E. coli phage P1 methylates adenines when incorporated into DNA and only within the sequence 5'.. AGmACC.. 3' (B/ichi et al., 1979). In this report we demonstrate that ¢3T DNA does contain the HaelII site by showing that ¢3T DNA can be cut by HaelII when propagated in restrictionmodification-defective (r-m-) E. coli cells. We suggest that ¢3T induces the modification of its own DNA. In support of this suggestion, we show that extracts prepared from ¢3T-infected cells contain a methyltransferase activity capable of protecting any DNA from HaelII cleavage.

MATERIALSAND METHODS

(a) Strains, media and growth conditions Bacterial, bacteriophage and plasmid strains used are listed in Table I. Bacteriophage ¢3T was produced by induction of a ¢3T lysogen using 6-(p-hydroxyphenylazo)-uracil (a gift from Dr. Bernard Langley) as previously described (Cregg and Ito, 1979). ¢3Tca287 was produced by infecting a culture of a SCR910 growing in L-broth (10 g tryptone, 5 g yeast extract and 5 g NaC1 per liter) at 2 × 108 cells/ml and 37°C at an m.o.i, between 5 and 10.

(b) Preparation of DNA ~3T DNA was prepared as described by Cregg and Ito (1979). Plasmid DNA was prepared by a modification of the cleared lysate procedure of Clewell (1972).

(c) Ligation and transformation For DNA ligations, 20/.tl of pBR322 (100/ag/ml) linearized with either PstI or HindlII was added to 20/A of ¢3T DNA (250/ag/ml) also digested with the same restriction endonuclease. The DNA mixture was adjusted to 10 mM Tris • HC1, pH 7.9, 10 mM MgC12, 0.1 mM EDTA, 1 mM dithiothreitol and 2 mM ATE T4-DNA ligase (5/A of 400 units/ml as supplied by Bethesda Research Labs, Inc:) was added and the ligation was allowed to proceed for 18 h at 4°C. Transformations of E. coli strain SCR2199 were performed according to the calcium-shock method of Cohen et al. (1972). When transformants which contained ¢3T DNA recombined into the HindlII sites of pBR322 were to be selected, aliquots (about 100 transformants per 0.01 ml) were spread on L-agar plates containing 2/ag/ml ampicillin. Ap R colonies were picked and streaked into each of two L-agar plates, one with ampicillin and the other with 1/ag/ml tetracycline. For selecting transformants containing ¢3T DNA recombined into the PstI site of pBR322, aliquots (about 100 transformants//A) were spread on tetracycline plates first. Of 200 Tc R colonies selected as possible carriers of hybrid plasmids, 18 were found to be Ap s. Of 200 Ap t~ colonies selected as possible carriers of ¢3T HindIII fragments, about one-third were also Tc s.

19 TABLE I Strains used Strain

Genotype

Source

168 trpC2 (SP# cured)

D. Dean and S. Zahler

W3110 AtrpE5 pBR322 SF8 rk- mk- recBC

D. Helinski R. Davis

Badllussubalis

SCR910 Escherichia coli

SCR 2202 SCR 2199 Bacteriophage O3T O3Tc3267 Plasmids pBR322 pJC1 pJC2 pJC3

Tucker (1969) this laboratory TcRApRderivative of ColE1

Bolivar et al. (1977)

TcRApSpBR322 + ~3T PstI-G or H TcSApRpBR322 + ~3T HindIlI TcSApRpBR322 + ~3T HindlII

this laboratory this laboratory this laboratory

(d) Methyl-transferase assay A culture of SCR910, grown to a cell density of 4 × 10a cells/ml in L-broth at 37 ° was infected with ¢3Tc3267 at a m.o.i, of 5. At the times desired samples were removed, mixed with an equal volume of cold phage adsorption buffer (0.1 M NaCI, 0.05 M TrisHC1, pH 7.4, 0.01 M MgSO4) and washed three times by centrifugation at 4°C. After the first and second centrifugations the cells were resuspended in cold 1 M KC1 and after the third centrifugation the cells were resuspended in one-tenth of the original sample volume of 0.02 M Tris • HC1 (pH 7.4) and i0 mM /3.mercaptoethanol. The cells were disrupted with a sonifier (Branson Instruments, Inc.). Samples were then centrifuged at 0°C for 90 min in a Type 50Ti rotor at 35 000 rev./min. The supernatant solution was stored at -70°C until use. For the methyltransferase assay, 5/al of extract was added to 100 /11 of the following: 100mM Tris-HC1, pH 7.5, 10 mM EDTA, 5mM fl-mercaptoethanol, 10 /aM SAM, 0.1/.tM [°H]SAM at 38 mCi/mg and 60/ag/ml DNA. The mixture was incubated at 37°C for 90 min. The reaction was stopped by adding 0.5 ml of a solution containing 1 N NaOH, 50 mM EDTA and 100/ag/ ml calf thymus DNA. Samples were placed at 100°C for 2 min and then chilled on ice. Next 1 ml of 2 N

HC1 was added and the sample was held on ice for at least 5 rain. Samples were then filtered through GF/A filters (Whatman, Ltd.), washed with 30 ml of cold 0.01 N HC1, followed by 5 ml of cold 95% ethanol, dried and counted.

(e) In vitro modification of DNA To test for the ability of a cell extract to modify DNA, a crude extract (prepared as described above) was added to the methylation reaction mixture described above, containing 6/ag/ml of ~, DNA and no [3H]SAM. After the 90 min incubation SDS was added to 1% and proteinase K to 200/ag/ml and the mixture was incubated at 37°C for 20 min. The DNA was then phenol-extracted, ethanol-precipitated, dialyzed into 0.02 M Tris-HCI (pH 7.4), 1 rnM EDTA. The DNA was digested with restriction endonucleases (Cregg and Ito, 1979).

(f) DNA synthesis assay A culture of SCR910 was grown and infected with ~3Tc3287 as described above for the methyltransferase assay. At specified times 1.0 ml samples of the culture were transferred to tubes containing 0.1 ml of 2.0 #Ci/ml [3H]thymidine at 37°C. Exactly 3 min

20 after transfer, incorporation was stopped by placing the tubes on ice and adding to each, 1 ml of 1 N NaOH. All samples were prepared and counted as described above for the methyl-transferase assay.

(g) Other methods Glycerol gradients were formed by the stepgradient technique previously described for sucrose gradients (Kawamura and Ito, 1977). Centrifugation was for 8 h in an SW56 rotor (Beckman Instruments, Inc.) at 40 000 rev./min and 4°C. Agarose gel electrophoresis was as previously described (Kawamura and Ito, 1977). DNAs were treated with restriction enzymes (Bethesda Research Labs, Inc.) as described in Cregg and Ito (1979). RESULTS

(a) Treatment of O3T DNA with restriction endonuclease Haelll q~3T DNA is not cut by restriction endonuclease HaelII (Fig. 1, lane 2), although the DNA is cleaved by many other restriction enzymes (Fig. 1, lanes 5 and 10, and Cregg and Ito, 1979). DNA extracted from the B. subtilis host (not infected by ¢3T) is digested by HaelII (Fig. 1, lane 4). The most probable explanations for this phenomenon are either that the ¢3T DNA does not contain the HaelII recognition sequence or that ¢3T induces a modification system which protects DNA against HaelII. To distinguish between these two mechanisms, ~3T DNA fragments were inserted into plasmid pBR322 and propagated in SCR2199, a restrictionmodification-defective (r-m-) strain of E. coli. If the HaelII nucleotide recognition sequence exists in ¢3T DNA but is modified, then ~3T DNA maintained in an r-m- host should be sensitive to HaelII. Fig. 1, lane 6 shows the hybrid plasmid, pJC1, containing ¢3T PstI fragment. The plasmid appears to contain either the ¢3T PstI G or H fragments which are 3.4 and 3.3 Md in size, respectively (Cregg and Ito, 1979). Comparison of pBR322 digested with both restriction enzymes PstI and HaelII in lane 8 to the PstI-HaelII digest of pJC1 in lane 7 clearly shows that the ~3T PstI fragment is cut by HaelII into at least four fragments. Fig. I, lanes 11 through 15, show the analogous result with ~3T HindlII fragment

Fig. 1. Agarose gel electrophoresis of DNA. The following DNA samples (0.1-0.5 ~g) treated with the designated restriction endonucleases were electrophoresed through at 1.0% agarose gel: (1) ~3T DNA; (2) ¢3T DNA, HaelII; (3) B. subtiffs DNA; (4) B. subtilis DNA, HaelII; (5) ~3T DNA, PstI; (6) pJCL, PstI; (7) pJCL, Pstl and HaelII; (8) pBR322, PstI and HaelII; (9) pBR322, PstI; (10) ~3T DNA, HindlII; (11) pJC19, HindlIl; (12) pJCL9, HindlII and HaellI; (13) pJC20, HindlII; (14) pJC20, HindlII and HaelII, (15) pBR322, HindllI and HaelII; (16) pBR322, HindlII.

containing plasmids pJC19 and pJC20. The hybrid plasmid pJC20 appears to have two q~3T-HindlII fragments, the larger of which is cut by HaelII (Fig. 1,lanes 13 and 14). T.hus, ¢3T DNA does contain the nucleotide recognition sequence for the restriction enzyme HaelII. Indeed, if the 3.3 Md pJC1 insert, which appears to contain three HaelII cleavage sites, is representative of the ¢3T genome, then that genome should contain about 80HaelII sites. These results suggest that ¢3T induces the modification of its own DNA against cleavage by HaelII. Furthermore, the fact that ¢3T DNA can be cleaved by 22 out of 23 other restriction endonucleases tested (Cregg and Ito, 1979) suggests that a specific nucleotide sequence (or sequences) on the ¢3T genome is modified. (b)Methyl-transferase assay of ~b3T-infected cell extracts Since the B. subtilis 168 host is known to induce under certain conditions an enzyme which methylates

21

within the HaelII recognition sequence (Gtinthert et al., 1976), we considered it likely that the ~3T DNAmodifying enzyme would also be a methyl-transferase (perhaps the same enzyme). To test whether a DNA methyl-transferase is induced during g}3T infections, a culture of SCR910 was infected with ~3Tc3287, a lysogenization-defective deletion mutant of ¢3T. Samples of the infected culture were removed at the time periods shown in Fig. 2, and cell-free extracts were prepared. Each crude extract was tested for its ability to transfer [3H]methyl groups from [sH]SAM to ), phage DNA. Fig. 2 shows that after infection there is an explosive increase in methyl-transferase activity peaking around 25 min past infection. The

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figure also shows that the rise in methylase activity precedes the increase in phage DNA synthesis, sug. gesting the enzyme may be an early gene product. As a control the methylase assay was repeated replacing the ), DNA with an equal amount of ~3T DNA. No radioactivity was incorporated into the ~3T DNA suggesting that an unmodified DNA is required for the reaction and that a methylase which modifies ~3T DNA in vivo is being measured. (c) In vitro modification of ~ DNA We wished to determine whether or not the ¢3Tinfected cell extracts could protect DNA against cleavage by restriction endonuclease HaelII. The crude extract prepared from cells harvested 25 min after infection was added to the same methyl-transferase reaction mixture described above but containing only one-tenth the concentration of )~ DNA. As controls, aliquots of the reaction mixture were also incubated with or without extract from uninfected cells. Fig. 3 shows that, after incubation with the extract from ~3T-infected cells, the )~DNA cannot be cut by HaelII (lane 5), but is sensitive to

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Fig. 2. Transfer of [3H]methyl from [3H]SAM to DNA catalysed by extracts of ~3Tc3267-infected SCR910. The graph presents the number of counts incorporated into alkalistable, acid-precipitable material as a function of time after infection. The abscissa shows the times at which aliquots of culture were placed on ice. The zero time point is activity in extracts of uninfected culture, o o, DNA methyltransfemse activity; • =, [3H]thymidine incorporation into DNA during ~3T infection. In this case the abscissa shows the times at which allquots of culture which had been transferred to a tube 3 rain previously were placed on ice.

Fig. 3. Agarose gel electrophoresis of h DNA. The following ?, DNA samples (0.5 ~g) were electrophomsed into a 0.8% agarose gel. (1) no treatment; (2) treated with HaeIII; (3) treated with EcoRI; (4) DNA incubated with O3Tcs267infected cell extract; (5) incubated with infected cell extract and then with HaelH; (6) incubated with infected cell extract and then with EcoRI.

22 EcoRI (lane 6), whereas unmodified DNA (lane 2)

and DNA treated with the extract from uninfected cells was cut (data not shown). Other restriction endonucleases can also cut the modified DNA (data not shown). Thus, these results with extracts of ¢3T-infected cells simulate the pattern of O3T DNA modified in vivo, i.e., X DNA has been made resistant to digestion by HaelII and yet still susceptible to cleavage by other restriction enzymes. (d) Glycerol gradient fractionation of O3T-infected cell extracts

To determine the mol. wt. of the DNA methylase induced by ~b3T,extracts prepared from cells harvested 25 min after infection were centrifuged through a 5 to 20% linear glycerol gradient. DNA methyl-transferase activity sedimented in between two reference markers, /3-galactosidase and bacterial alkaline phosphatase (see Fig. 4), at 7.8S, i.e. approx. 150000 daltons. The activity protecting X DNA against HaelII cosedimented with the DNA methylase.

DISCUSSION The purpose of this study was to determine the mechanism by which the DNA of B. subtilis temperate phage ¢3T is made resistant to cleavage by the restriction endonuclease HaelII. The results pre-

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40

20

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1 40

Fractions

Fig. 4. Glycerolgradient fractionation of O3Tc3267-infected cell extract. Enzyme activity in aliquots of selected fractions is shown as a percentage of the total activity present in all fractions assayed, o o, methyl-transferase activity; % alkaline phosphatase activity; • •, ~-galaetosidase activity.

sented show that DNA from the noninfected B. subtilis host was susceptible to HaelII digestion. The host, therefore, does not normally express this modification system. It is possible that the antibiotic 6.(p-hydroxyphenyl-azo)-uracil, used to induce ~3 T prophage, may also induce the DNA modification system. However, infection with a lysogenizationdefective mutant of ~3T resulted in phage DNA still resistant to cleavage by HaelII. It is also possible that ~b3T DNA does not contain the HaelII recognition sequence. To eliminate this possibility ¢3T DNA fragments were inserted into E. coli plasmid pBR322 and propagated in an r m E. coli host. HaelII was then able to cut ¢3T DNA. We therefore concluded that ¢3T probably induces the modification of its DNA. In support of this mechanism we presented results which show that a methyltransferase activity is induced after ~3T lyric infections. In addition we showed that an activity exists in crude extracts of ~b3T-infected cells which requires only SAM to modify X DNA in vitro against HaelII cleavage. This modification appeared to be sequence-specific, since the same modified X DNA was susceptible to cleavage by other restriction endonucleases. Finally we demonstrated that the activity which modified X DNA against HaelII attack and the DNA methyl-transferase activity sediment at the same rate during glycerol gradient centrifugation, suggesting the two activities are the result of the same enzyme. The induction of a methyl-transferase during bacteriophage infection is not unusual. The genome of E. coli phage P1 is known to code for its own restriction modification system (Hubacek and Glover, 1970). This system has been studied in considerable detail (Yuan and Reiser, 1978; B~'chi et al., 1979) and found to be different from both Type I and Type II restriction-modification systems. Expression of the E. coli phage Mu-induced methylase requires the interaction of both phage (morn) and host (dam) functions (Toussaint, 1977; Khatoon and Bukhari, 1978). Since -the recognition sequence of the Mu-induced methylase is different from that expressed by the dam function, it was suggested that either the specificity of the dam function is changed by mom, or that the morn function requires dam for activity (Khatoon and Bukhari, 1978). The first phage-induced methylase was described by Gold et al. (1964) with E. coli cells infected with phage T2. An interesting similarity

23 exists between the T2-induced enzyme and that of ~b3T. In both cases the respective hosts appear to produce a methylase with the same sequence specificity as the phage-induced enzyme (van Ormondt et al., 1975; GOnthert et al., 1976; Lacks and Greenberg, 1977). Mutants of T2 have been isolated which induce an altered methylase (Hehlmann and Hattman, 1972) suggesting that the T2-induced methylase is distinct from the E. coli enzyme. It will be interesting to know whether the ~b3T-induced modification system is a host or phage product. It is not known what role or roles the phageinduced methyl-transferase plays in the life cycle of bacteriophage ~3T. At least two possibilities exist. The first is that the methylase may extend the host range of ~3T to strains which constitutively express a restriction-modification system like that of B. subtilis R strains, and the second is that B. subtilis 168 strains express a restriction-modification system with the same sequence specificity as the ¢3T-induced modification system, after exposure to conditions which also induce prophage (Arwert and Rutberg, 1974; Yasbin et al., 1975; Garro et al., 1976; Giinthert et al., 1976; Ganesan, 1979). It may be that after induction, ¢3T DNA is synthesized too rapidly to be completely methylated by the host-induced methylase system. Therefore, the additional methylase (or an increased level of the host-induced methylase) is required to completely methylate the replicating phage DNA. A similar explanation has been suggested for the existence of the modification systems of E. coli phage T2 and plasmid N3 (Lacks and Greenberg, 1977). Both the phage and the plasmid induce their own methylase which recognizes the same nucleotide sequence as that of the host (Van Ormondt et al., 1975; May and Hattman, 1975). Other roles for the ¢3T-induced methylase are also possible, such as the control of transcription through the methylation of sites within a regulatory element or the protection of ¢3T DNA during lytic infections while a ¢.b3T-induced restriction enzyme helps degrade the host DNA. The ¢3T-induced methyl-transferase may be of practical use as well as of academic interest. Ganesan (1979) found that a DNA methylase and a restriction type of activity were induced in B. subtilis 168 by the competency regimen routinely used. Yasbin (1977) found that when B. subtilis phage ~105 DNA transfected competent cells, a proportion of the phage

produced were immune to restriction by a B. subtilis R strain. Progeny phages of infected cells were restricted by the R strain. These results suggest that a restriction-modification system is present in competent cells and that the sequence specificity of this system may be the same as that of B. subtilis R and, therefore, the same as that of the ~3T-induced methylase. Plasmids from Staphylococcus aureus transformed competent B. subtilis 168 at a low frequency (Ehrlich, 1977). The same plasmids had a 50-fold higher transformation frequency after propagation in B. subtilis. It is possible that by methylating unmodified or incorrectly modified DNA, one may increase the transformation efficiency of that DNA. The ability to increase the transforming efficiency of DNA (particularly foreign DNA) would be of value in developing B. subtilis as a cloning system and in other studies as well. ACKNOWLEDGEMENTS This research was supported by NIH Research Grant GM 25081. J.C. was supported by an American Society Postdoctoral Fellowship. J.I. is the recipient of a Faculty Research Award from the American Cancer Society. REFERENCES Arwert, F. and Rutberg, L.: Induction of prophage SPO2 in Bacillus subtilis by 64para)-hydroxyphenylazouracil. J. Virol. 14 (1974) 1470-1475. B~'chi, B., Reiser, J. and Pirrotta, V.: Methylation and cleavage sequences of the EcoPI restriction-modification enzyme. J. Mol. Biol. 128 (1979) 143-163. Bolivar, F., Rodriguez, R.L., Betlach, M.C. and Boyer, H.W.: Construction and characterization of new cloning vehicles, I. Ampiciliin-resistant derivatives of the plasmid pMB9. Gene 2 (1977) 75-93. Bron, S. and Murray, K.: Restriction and modification in B. subtilis. Nucleotide sequence recognized by restriction endonuclease R. BsuR from strain R. Mol. Gem Genet. 143 (1975) 25-33. Bron, S., Murray, K. and Trautner, T.A.: Restriction and modification in B. subtilis. Purification and general properties of a restriction endonuclcase from strain R. Mol. Gen. Genet. 143 0975) 13-23. Clewell, D.B.: Nature of ColE1 plasmid replication in Escherichia coli in the presence of chloramphenicol.J. Bacteriol. 110 (1972) 667-676. Cohen, S.~., Chang, A.C.Y. and Hsu, L.: Nonchromosomal ~ntihintic resistance in bacteria: Genetic transformation

24, of Escherichia coli by R-factor DNA, Proc. Natl. Aead. Sei. USA 69 (1972) 2110-2114. Cregg, J.M. and Ito, J.: A physical map of the genome of temperate phage 43T. Gene 6 (1979) 199-219. Dean, D.H., Orrego, J.C., Hutchison, K.W. and Halvorson, H.O.: New temperate bacteriophage for Bacillus subtills, p l l . J. Virol. 20 (1976) 509-519. Duncan, C.H., Wilson, G.A. and Young, F.E.: Mechanism of integrating foreign DNA during transformation of Bacillus subtilis, Proc. Nat. Acad. Sci. USA 75 (1978) 36643668. Ehrlieh, S.D.: Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc. Natl. Aead. Sci. USA 74 (1977) 1680-1682. Ehrlich, S.D., Bursztyn-Pettegrew, H., Stroynowski, I. and Lederberg, J.: Expression of the thymidylate synthetase gene of the Bacillus subtilis bacteriophage Phi-3-T in Escherichia coll. Proc. Natl. Acad. Sci. USA 73 (1976) 4145--4149. Ganesan, A.T.: Genetic recombination during transformation in Bacillus subtilis: Appearance of a deoxyribonucleic acid methylase. J. Bacteriol. 139 (1979) 270-279. Garro, A.J., Hammer, P. and Recht, B.: Biochemical and genetic analysis of the defective Bacillus subtilis bacteriophage PBSX, in D. Schlessinger (Ed.), Microbiology1976, American Society for Microbiology, Washington, DC, 1976, pp. 340-349. Gold, M., Hansman, R., Maitra, U. and Hurwitz, J., The enzymatic methylation of RNA and DNA, VIII. Effects of bacteriophage infection on the activity of the methylating enzymes. Proc. Natl. Acad. Sci. USA 52 (1964) 292-305. Graham, R.S., Young, F.E. and Wilson, G.A., Effect of sitespecific endonuclease digestion on the thyP3 gene of bacteriophage 43T and the thyP11 gene of bacteriophage p l l . Gene 1 (1977) 169-180. Gtinthert, U., Pawlek, B., Stutz, J. and Trautner, T.A.: Restriction and modification in Bacillus subtilis: Inducibility of a DNA methylating activity in non-modifying cells. J. Virol. 20 (1976) 188-195. Hehlmann, R. and Hattman, S.: Mutants of bacteriaphage T2gt with altered DNA methylase activity. J. Mol. Biol. 67 (1972) 351-360. Hubaeek, J. and Glover, S.W.: Complementation analysis of temperature-sensitive host specificity mutations in Escherichiacoli. J. Mol. Biol. 50 (1970) 111-127. Ito, J. and Roberts, R.J.: Unusual base sequence arrangement in phage 429 DNA. Gene 5 (1979) 1-7. Kawamura, F. and Ito, J.: Studies of Bacillus subtilis phage M2: a physical map of theM2 genome. Virology 83 (1977) 233 -245. Kawamura, F., Saito, H. and Ikeda, Y.: A method for construction of specialized transducing phage p l l of Bacillus subtilis. Gene 5 (1979) 87-91. Kelly, T.J. and Smith, H.O.: A restriction enzyme from Hae. mophilus influenzae, II. Base sequence of the recognition site. J. Mol. Biol. 51 (1970) 393-409.

Khatoon, H. and Bukhari, A.I.: Bacteriophage Mu-induced modification of DNA is dependent upon a host function. J. Bacteriol. 136 (1978) 423-428. Kiss, A., Sain, B., Csordas-Toth, E. and Venetianer, P.: A new sequence-specific endonuclease (Bsp) from Bacillus sphaericus. Gene 1 (1977) 323-329. Lacks, S. and Greenberg, B.: Complementary specificity of restriction endonucleases of Diplococeus pneumoniae with respect to DNA methylation. J. Mol. Biol. 114 (1977) 153-168. Makino, O., Kawamura, F., Saito, H. and Ikeda, Y.: Inactivation of restriction endonuclease BamNx after infection with phage eNR2. Nature 277 (1979) 64-66. Mann, M.B. and Smith, H.O.: Specificity of HpalI and HaelII DNA methylases. Nucl. Acids Res. 4 (1977) 421-422. Mathews, C.K.: Bacteriophage Biochemistry, Van Nostrand Reinhold, New York, 1971. May, M.S. and Hattman, S.: Deoxyribonucleic acid-cytosine methylation by host- and plasmid-controlled enzymes. J. Bacteriol. 122 (1975) 129-138. Okubo, S., Strauss, B. and Stodolsky, M. The possible role of recombination in the infection of competent Bacillus subtilis by bacteriophage deoxyribonucleic acid. Virology 24 (1964) 552-562. Spoerel., N., Herrlich, P. and Bickle, T.A.: A novel bacteriophage defence mechanism: the anti-restriction protein. Nature 278 (1979) 30-34. Toussaint, A.: The DNA modification function of temperate phage Mu-1. Virology 70 (1976) 17-27. Trautner, T.A., Pawlek, B. and Anagnostopoulos, C.: Restriction and modification in B. subtilis. Biological aspects. Mol. Gen. Genet. 131 (1974) 181-191. Tucker, R.G.: Acquisition of thymidylate synthetase activity by a thyrnine-requiring mutant of Bacillus subtilis followhag infection by the temperate phage 43. J. Gen. Virol. 4 (1969) 489-504. Van Ormondt, H., Gorter, J., Havelaar, K.J. and deWaard, A. Specificity of a deoxyribonucleic acid transmethylase .induced by bacteriophage T2, I. Nucleotide sequences isolated from Micrococcus luteus DNA methylated in vitro. Nucl. Acids Res. 2 (1975) 1391-1400. Williams, M.T. and Young, F.E.: Temperate Bacillus subtilis bacteriophage 43T: Chromosomal attachment site and comparison with temperate bacteriophages 4105 and SPO2. J. Virol. 21 (1977) 522-529. Yasbin, R.E.: DNA repair in Bacillus subtilis, II. Activation of the inducible system in competent bacteria Mol. Gen. Genet. 153 (1977) 219-225. Yasbin, R.E., Wilson, G.A. and Young, F.E.: Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent ceils. J. Bacteriol. 121 (1975) 296-304. Yuan, R. and Reiser, J.: Steps in the reaction mechanism of the Escherichia coli plasmid P15-specific restriction endonuciease. J. Mol. Biol. 122 (1978) 433-445. Communicated by W. Szyhalski.