Gene, 7 (1979) 69--77 @l~Juevier/North-Holland Biomedical Press, Amsterdam -- Printed in The Netherlands
69
ISOLATION AND CHARACTERIZATION OF VIABLE DELETION MUTANTS OF Bacfflus subtilis BACTERIOPHAGE SP02 (Heat-EDTA treatment; restriction mapping; nonessential regions of the temperate phage genome; cloning vector)
8. GRAHAM, Y. YONEDA and F.E. YOUNG Del~rtment of Microbiology, University of Rochester Medical School, Rochester, N Y 14642 (~S.A.) (Received March 18th, 1979) ( Accepted June 14th, 1979 )
SUMMARY
Spontaneous deletion mutants of the bacteriophage SP02, which are viable and retain their temperate character, were isolated using a heat-EDTA enrichment step. They were identified by endonuclease digestion and agarosegel electrophoresis of phage DNA. Two of the nine mutants were characterized in d ~ . Both mutants have a 2.3 Md deletion removing the single Bg/II site and two of the XbaI fragments. The deletion extends 1.0 Md to one side of the former BglII site and 1.3 Md on the other side. This region of the $P02 genome is non.essential for either lysogeny or viabie phage production and thus is a suitable region for the insertion of exogenous DNA fragment~. INTRODUCTION
SP02 is a well-characterized temperate bacteriophage of Bacillus subtilis strain 168. This phage has a double-stranded circular DNA genome of approx. 23 Md (Yoneda et al., 1979) or 38.6 kbp (Chow and Davidson, 1973) which is genetically permuted upon integration into the bacterial host chromosome (Chow and Davidson, 1973; Arwe~ et al., 1976). A linear genetic map, consisting of 17 cistrons, was established through complementation of sus mutations (Yasunaka et al., 1970). Yoneda et al. (1979) constructed a circular physical map based on restriction-endonuclease fragment analysis. Several features of $P02 make it attractive as a potential general cloni~lg vehicle for the BaciUi. First, the phage contains single-endonuclease sites for the BglII, 8alI, and Sinai restriction endonucleases. These could serve as potential sites Abbreviations: Md, nu~.adaltons; kbp, kilobase pairs.
70
for the insertion of exogenous DN~L Second, the host range of SP02 has been extended from B. subtilis to other Bacilli (Graham, S., Yoneda, Y., W'dson, G., and Young, F.E., unpublished observation); thereby introducing the possibility for Wtdizeetional inter-specific genetic exchange among Bacilli not known to exchange genetic information either by tzansfonnation or trans~ duetio~ Any phage vehicle is limited in its use by the ~mount of exogenous DNA that may be ~ into the phage genome and still yield viable phage. This ~ n i t is set by the phage bead's DNA storage capacity. For bacteriophage X the phage head capacity is a maximum of 1.09 X genomes (Weisberg and Adhya, 1977); thus, a maximum of 2.8 Md of additional DNA could be •dded to the X genome. Deletion mutants permit more exogenous DNA to be added than in wild-type phage. For example 30% of the X genome can be deleted without substantially affecting lyric phage functions. The use of ~able X deletion strains has greatly i n ~ the usefulness of ~ as a cloning vector. The same should be true for b ~ o p h a g e 81'02. By analogy to X, wild-type SP02 could accept a maximum of 2.4 Md of exogenous DNA without interfering with proper DNA p~kaging by the phage head. To increase the amount of exogenous DNA that might be inserted into the SP02 ~enome, a series o f spontaneous deletion mutants which are viable and
preserve thek temperate ehamet~ were imlated. This communication de~ scribes the enzichment for, the d ~ o n of, and subsequent characterization of such mutants. MATERIALS AND METHOI~
1.8trains and methods of propagation Bacillus subtilis strgm BR151 (lys~, metBlO and trpC2), was grown and maintained on M-broth, M-semisolid agar, or M-agar (Yasbin et aL, 1973). Bacteriophage SP02 lysogens of BR151 were maintained on Tryptose blood agar (TBAB). Bacteriophage were isolated by (1) induction of lysogeus with mitomycin C (Yasbin et al., 1973) or (2) by scraping the lysed bacterial lawn from an agar plate, resuspending the phage, removing the cells and agar by centrifugation, and filtering the phage supematant through a 0.45/~m filter. 2. Enrichment for deletion-mutants by heat-EDTA treatment To a phage lysate containing approx. 1- 109 phage in 1.0 ml M-broth was added Tris, pH 8.0 to 20 mM and EDTA to 20 raM. This solution was then heated for 15 rain at 65°C. Survivors were isolated by plating the treated phage on a bacterial lawn ofB. subtilis BR151. Phage st~ks for subsequent heat-EDTA treatments were o ~ e d by plating survivors at a phage concentration sufficient to almost completely lyse the bacterial lawn. Phage were then ~olated by the scraping method described above. It is important to note two things about this procedure: (1) the pH of the EDTA is not adjusted prior to addition to the phage and (2) the procedure
71
does not involve the isolation and subsequent heat-EDTA treatment of phage from individual plaques until the final isolation. 3. Isolation o f phage DNA Phage DNA was obtained by two methods. (a) Mini-lysate procedure. Sufficient phage DNA (5--10 #g total) for several restriction analyses was obtained from 40 ml of a mitomycin-C-induced lysogen of B. subtilis (SP02). Phage were collected from an induced culture (as described above), concentrated by precipitation with 10% polyethylene glycol 6000, and resuspended in 20 ml TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). The resuspended phage was treated with DNase (Worthington) and RNase A (Sigma), both at 100 ug/ml final concentration, for 1 h at 37°C. The phage suspension was then treated with proteinase K (Boehringer, Mannheim) at 100 ug/ml final concentration for 1 h at 37°C. The DNA was extracted three times with an equal volume of TE-buffered phenol, concentrated by ethanol precipitation, and redissolved in 200--400 #1 TE buffer. Subsequent improvements in this procedure to give a yield of 5 #g phage DNA from 10 rnl (rather than 40 ml) of phage lysate along with increased gel resolution will be described in detail eisew~re (Yoneda, Y., Graham, S., Evans, T., and Young, F.E., in preparation). (b) CsCI purified phage DNA. Phenol~xtracted DNA was obtained from phage concentrated by CsCI equilibrium centrifugation as previous'y described (Graham et al., 1977). This DNA gave better resolution during restriction analysis than the PEG-mini-lysate DNA due to (i) absence of even trace amounts of chromosomal DNA and (ii) absence of PEG fluorescence during photography. 4. Restriction-endonuelease analysis Isolation and purification of the site~specific endonucleases BamHI, BglI, Bg/H,,and E¢oRI was as previously described (Graham et al., 1977). Sinai and XbaI were obtained from New England Biolabs while Bali was obtained from Miles Biochemicals. Conditions for DNA digestion, ethidium bromide gel electrophoresis, DNA fragment visualization and photography were as previously described (Graham et al., 1977). RF.~ULTS AND DISCUSSION
1. Isolation o f 8P02 deletion mutants Spontaneous SP02 deletion mutants were identified by DNA fragment analysis of a lysogenic bacteriophage population which had been enriched for bacteriophages with deletions by heat-EDTA treatment. (For alternative methods of enriching for deletion mutants see Parkinson and Huskey, 1971). Heat-EDTA treatment inactivated 76% of the SP02 phage (Fig. 1). Survivors were still sensitive to inactivation by heat-EDTA. After 5 cycles of treatment the majority of survivors become tolerant to heat-EDTA exposure, although survival never was much greater than 50%. Continued treatment of cycle 5
72
sun~vors for 3 additional cycles did n o t further increase the proportion of survivors (data n o t shown). The relative tolerance of SP02 t o the initial hest-EDTA treatment (as compared t o cycles 2,3,4) was reproducible but n o t readily explainable. Other B. subt///s lysogenic phage as well as several clear-plaque host-range vsriants of SP02 were initially reduced 10000-fold (data n o t shown). Greater than 95% of the c y c l e 5 mtwivors formed clear plaques and were ~ incapable of forming lysogens. Only phage producing cloudy plaques (i.e. premmptive lysogens) were exLmined further. Lysogens were purified by single colony plating and ten lysogens from cycle~4 survivors and thirty
,J
oo~ W
~t O0001,
0,000001,
!
2
3
4
$
IlUldlER OF HE&lr-EOTA CYCLE
Fig.1. Effect of heat-ElYrA treatment on SP02. The heat-ElYrA treatment and subsequent propagation of phage survivors was as described in MATERIAI~ AND METHODS. Phage survival was estimated as follows. After suspend'mg~phage in Tris-EiYrA, the solution was divided into aliquots, one of which was then heated. Survivalwas calculated as the ratio of plaqu~forming units (pfu)after 65°C heating/pfu before heating. At room temperature, Tris-EDTA had no effect on phage viability.
lysogens from cycle-5 survivors were induced with mitomycin C. Phage DNA was isolated and examined by restriction analysis using Bg/I, £¢oR~ and HindH. All ten of the cycle-4 survivors and the majority (21 out of 30) of c y c l e 5 survivors had a ~ e n t pattern identical to SP02. If these phages had a deletion, it was t o o small to detect by alteration of DNA fragment mobility, i.e., less than 0.1 Md. Thus heat-EDTA survivors should not a priori be considered to contain deletions.
73
Of the nine cycle-5 survivors, that did have an altered DNA fragment pattern, only two of them could form stable lysogens upon repeated subcultm~g. These two phage strains, designated SP02A 51 and SP02~ 54, were examined in detail. The seven phage which formed unstable lysogens had similar DNA fragment patterns to each other but different from both SP02A 51 and SP02~ 54 and from SP02. These seven lysogens exhibit high rates of rapid spontaneous induction in both M-broth and on TBAB or M-agar plates. Whether loss of lysogeny represents unstable integration or pseudolysogeny is under investigation.
2. 8ire-specific endonuclease analysis of 8P02 deletion strains DNA from CsCl-banded phages SP02, SP02-41 (a cycle-4 survivor), SP02A 51, and SP02A 54 was examined after digestion with BglI, BglI + BglII, EcoRI and XbaI (Fig. 2). SP02A51 and SP02A54 fragment patterns were identical to each other and different from SP02 and SP02-41 (which are identical). The largest BglI fragment (BglI-A) of SP02A51 and SP02A 54 migrated faster than the comparable SP02 BglI-A fragment; a change in migration equivalent to the loss of 2.3 Md of DNA. No other alteration in the BglI pattern was detectable. The SP02 BglI-A fragment contained the single SP02 BglII site. Digestion of Bgll digested SP02A51 or SP02A 54 DNA with BglII did not alter the BglI fragment pattern. Therefore, SP02A 51 and SP02A 54 lacked the BglII site. The SP02 BglI.A fragment also contained four XbaI sites closely clustered around the Bgill site (Fig.3). Digestion of SP02 DNA with XbaI produced six (A-F) fragments, with fragments C, D, E and F residing within the BglI-A fragment. Digestion of SP02~ 51 and SP02~ 54 DNA with XbaI produced only four fragments. The three largest fragments (A, B, and C) co-migrated with SP02 XbaI-A, -B, and -C. SP02~ 51 and SP02~ 54 were missing XbaI fragments D and E. The SP02~ 51 and SP02~ 54 XbaI-F migrated faster than SP02XbaI-F. Whether this faster migrating F fragment was derived from a truncation of the parental XbaI.E fragment or of the F fragment has not been established. Summation of the SP02~51 and SP02~54 XbaI fragments gave a molecular weight value 2.27 Md smaller than for SP02. Further evidence that SP02~51 and SP02~54 contained similar deletions of approx. 2.3 Md was obtained by the series of combination endonuclease digestions (Fig. 4). The DNA fragment patterns of SP02~51 and SP02A 54 in these complex digestions (e.g., slots 8 and 9, EeoRI plu~ BglI plus SalI plus SmaI) were identical. All the patterns indicating a deletion of 2.3 Md had occurred extending approx. 1.0 Md of DNA to one side of the SP02 BglII site and 1.3 Md to the other. The exact orientation and end points of the deletion still remain to be firmly established. Although the site-specific endonuclease patterns of SP02~ 51 and SP02~ 54 were identical, they produced cloudy plaques distinct from each other. Furthermore, SP02~ 54 spontaneously produced clear-plaque variants at a high rate. Therefore, the possibility exists that the two phages are of independent origin and contain s'nnilar but not identical deletions.
74
Fig.2..Analysis of SP02, SP02-41, SP02A 51, and SP02A54 DNA fragments. SP02 (A), SP02-41 (B), SP02A51 (C), or SP02A54 (D) DNA was digested with the indicated endonuclease and the sample eleetrophoresed as described in MATERIALS AND METHODS. For reference the location and size in Md of the SP02 BglI fragments (left margin) and SP02 EcoRI fragments (right margin) are given. Stool
4.50 Xbol
3.si a.54a i-t5 F O i
r
.
ll.o8 E
a I-sz C
CcoRe a
A B
2-40
Fig. 3. Location of the SP02A 51 and SP02~ 54 deletion on the SP02 restriction-fragment map. Only a portion of the SP02 map around the Bg/II site is represented. The DNA deletion in SP02A 51 and SP02~ 54 is depicted in both possible orientations (A or B).
75
12345
A
B
~
C D £ F
Fig. 4. Comparison of the SP02, SP02A 51, and SP92~ 54 DNA fragment patterns. SP02, SP02A 51, or SP02A 54 DNA was digested with various combinations of endonucleases and electrophoresed as previously described: Slot I (SP02 DNA) with BglI; 2 (SP02) with £ e o R I ; 3 (SP02) and 4 (SP02A54) with E c o R l + BglIl + Bali; 5 (SP02) and 6 (SP02A54 with £ e o R I + SalI + Smal; 7 (SP02), 8 (SP02A 51 ) and 9 (SP02A 51 ) with £eoRI + 8alI + Sinai + BglH; 10 (SP02), 11 (SP02~51) and 12 (SP02~54) with EcoRl + XbaI; 13 (SP02) and 14 (SP02~54) with Xbal; 15 (SP02) with EcoRI. For references, the location of the SP02 Bgll fragments (left margin) and E c o R I fragments (right margin) are given.
Several other potential deletion strains were identified during the initial mini-lysate screening. These strains had an altered EcoRI-B fragment and could not be maintained as stable lysogens. Genetic studies indicate that the 4.74 Md SP02 EcoRI-B fragment (equivalent to 24% of the SP02 chromosome) contains at least two of the 17 SP02 complementation groups (Yasunaka et al., 1970). This more genetically "active" region of the SP02 chromosome is apparently less tolerant of deletion than the BglII area.
3. Relation o f 8PO2A51 and 8PO2A54 to previous 8P02 deletions Chow and Davidson (1973) characterized by heteroduplex analysis two SP02 deletion strains, R90 and $25, originally isolated by R. Skillern. These strains formed ~!ear plaques and did not lysogenize. $25 contained a 3.7% deletion (equivalent to approx. 0.86 Md) and R90 contained a 7.9% deletion (2.2 Md) both located approx. 65% from one end of the SP02 chromosome. Although SP02 exists as a circular molecule within the phage head and during infection (Chow and Davidson, 1973; Arwert et al., 1976), the molecules observed by Chow and Davidson (1973) were linear. During the construction of the SP02 endonuclease fragment map, Yoneda et al. (1979) described an
76
EcoRI fragment which when heated dissociated into two smaller fragments.
They suggested that this heat-labile chromosomal junction might be the point of chromosome circularization. The SP02 BglII site (and hence the SP02~51 and SP02~54 deletion) is located approx. 64% from one end of this heat-labile junction. Further evidence that the SP02~ 51 and SP02A 54 deletions occur in the same chromosomal region as the deletions in 825 and R90 comes from the localization of the phage attachment site at or near both sets of deletions. Chow and Davidson located the phage attachment site 2% and 5.6% of the chromosome away from 825 and R90 respectively. Preliminary genetic studies of SP02 have located the SP02 attachment site in the vicinity of the SP02 BglII site (Graham, S., Yoneda, Y., Wilson, G. and Young, F.E., unpublished). Therefore the SP02 termini observed by Chow and Davidson (1973) and by Yoneda et al. (1979) are probably the same. Furthermore, the deletions in SP02A 51 and 8P02~ 54 and in S24 and R90 most likely occurred in the same region of the SP02 chromosome. Deletion of the 2.3 Md of SP02 DNA around the BglII site in SP02~51 and SP02A 54 caused a change in plaque morphology from wild-type SP02 but did not prohibit lysogeny or phage production. Consistent with the idea that this area of the 8P02 genome is non-essential was the observation that super-infection marker-rescue of SP02 sus mutants with purified wild-type DNA fragments failed to locate any of the 17 known SP02 complementation groups within this area (unpublished observations). Furthermore, a hostrange mutant of SP02 exists that has a DNA rearrangement near the BglII site which is suggestive of a deletion of SP02 DNA with the concomitant introduction of non-SP02 DNA (Graham, S., Yoneda, Y., Wilson, G. and Young, F.E., unpUblished). CONCLUSIONS
Bacillus subtilis bacteriophage SP02 appears to be quite promising as a cloning vehicle for the Bacilli. The accrued genetic and physical information on SP02 shows (1) the phage has several usable (Le., single) endonuclease sites; (2) the BglII site is located in a genetically "silent" region of the chromosome; (3) viable deletion mutants are available thus increasing the potential amount of clonable DNA; and (4) the host-range of SP02 can be extended to Bacilli other than B. subtilis. Further work with this phage should produce a usable cloning vector. ACKNOWLEDGEMENTS
We wish to thank Dr. Junetsu Ito for suggesting the heat-EDTA selection procedure used for isolating the viable deletion mutants. We would also like to thank Henry Baney for his help in preparing phage DNA and endonucleases and also thank Ms. Pat Morrison for her technical assistance. This research was supported by a research grant from Miles Laboratories.
77 REFERENCES
Arwert, F., Bjursell, G. and Rutberg, L., Induction of prophage SP02 in Bacillus subtilis: isolation of exised prophage DNA as a covalently closed circle, J. Virol., 17 (1976), 492--502. Chow, L.T., and Davidson, 1'4., Electron microscope study of the structures of the Bacillus subtilis prophages, SP02 and ~105, J. Mol. Biol., 75 (1973), 257--264. Graham, 8., Young, F.E. and Wilson, G.A., Effect of site-specific endonuclease digestion on the thyP3 gene of bacteriophage ~3T and the t h y P l l gene of bacteriophage p 11, Oene, 1 (1977) 169--180. Parkinson, J.8. and Huskey, R.J., Deletion mutants of bacteriophage lambda, I. Isolation and initial characterization, J. Mol. Biol., 56 (1971) 369--384. Weisberg, R.A. and Adhya, 8 , magitimate recombination in bacteria and bac~riophage, Annu. Rev. Oenet., 11 (1977) 451--478. Yasbin, R.E., Wilson, G.A. and Young, F.E., Transformation and transfection in lysogenie strains of B~illus subtilis 168, J. Bacteriol., 113 (1973) 540--548. Yasunaka, A., Tsukamato, H., Okubo, 8. and Horiuchi, T., Isolation and properties of suppressor-sensitive mutants of Bacillus subtilis bacteriophage SP02, J. Virol., 5 (1970) 819--821. Yoneda, Y., Graham, S. and Young, F.E., Restriction-fragment map of the temperate l ~ i l l u s subtilis bacteriophage gP02, Oene, 7 (1979) 51--68. Communicated by W. Szybalski.
69
ISOLATION AND CHARACTERIZATION OF VIABLE DELETION MUTANTS OF Bacfflus subtilis BACTERIOPHAGE SP02 (Heat-EDTA treatment; restriction mapping; nonessential regions of the temperate phage genome; cloning vector)
8. GRAHAM, Y. YONEDA and F.E. YOUNG Del~rtment of Microbiology, University of Rochester Medical School, Rochester, N Y 14642 (~S.A.) (Received March 18th, 1979) ( Accepted June 14th, 1979 )
SUMMARY
Spontaneous deletion mutants of the bacteriophage SP02, which are viable and retain their temperate character, were isolated using a heat-EDTA enrichment step. They were identified by endonuclease digestion and agarosegel electrophoresis of phage DNA. Two of the nine mutants were characterized in d ~ . Both mutants have a 2.3 Md deletion removing the single Bg/II site and two of the XbaI fragments. The deletion extends 1.0 Md to one side of the former BglII site and 1.3 Md on the other side. This region of the $P02 genome is non.essential for either lysogeny or viabie phage production and thus is a suitable region for the insertion of exogenous DNA fragment~. INTRODUCTION
SP02 is a well-characterized temperate bacteriophage of Bacillus subtilis strain 168. This phage has a double-stranded circular DNA genome of approx. 23 Md (Yoneda et al., 1979) or 38.6 kbp (Chow and Davidson, 1973) which is genetically permuted upon integration into the bacterial host chromosome (Chow and Davidson, 1973; Arwe~ et al., 1976). A linear genetic map, consisting of 17 cistrons, was established through complementation of sus mutations (Yasunaka et al., 1970). Yoneda et al. (1979) constructed a circular physical map based on restriction-endonuclease fragment analysis. Several features of $P02 make it attractive as a potential general cloni~lg vehicle for the BaciUi. First, the phage contains single-endonuclease sites for the BglII, 8alI, and Sinai restriction endonucleases. These could serve as potential sites Abbreviations: Md, nu~.adaltons; kbp, kilobase pairs.
70
for the insertion of exogenous DN~L Second, the host range of SP02 has been extended from B. subtilis to other Bacilli (Graham, S., Yoneda, Y., W'dson, G., and Young, F.E., unpublished observation); thereby introducing the possibility for Wtdizeetional inter-specific genetic exchange among Bacilli not known to exchange genetic information either by tzansfonnation or trans~ duetio~ Any phage vehicle is limited in its use by the ~mount of exogenous DNA that may be ~ into the phage genome and still yield viable phage. This ~ n i t is set by the phage bead's DNA storage capacity. For bacteriophage X the phage head capacity is a maximum of 1.09 X genomes (Weisberg and Adhya, 1977); thus, a maximum of 2.8 Md of additional DNA could be •dded to the X genome. Deletion mutants permit more exogenous DNA to be added than in wild-type phage. For example 30% of the X genome can be deleted without substantially affecting lyric phage functions. The use of ~able X deletion strains has greatly i n ~ the usefulness of ~ as a cloning vector. The same should be true for b ~ o p h a g e 81'02. By analogy to X, wild-type SP02 could accept a maximum of 2.4 Md of exogenous DNA without interfering with proper DNA p~kaging by the phage head. To increase the amount of exogenous DNA that might be inserted into the SP02 ~enome, a series o f spontaneous deletion mutants which are viable and
preserve thek temperate ehamet~ were imlated. This communication de~ scribes the enzichment for, the d ~ o n of, and subsequent characterization of such mutants. MATERIALS AND METHOI~
1.8trains and methods of propagation Bacillus subtilis strgm BR151 (lys~, metBlO and trpC2), was grown and maintained on M-broth, M-semisolid agar, or M-agar (Yasbin et aL, 1973). Bacteriophage SP02 lysogens of BR151 were maintained on Tryptose blood agar (TBAB). Bacteriophage were isolated by (1) induction of lysogeus with mitomycin C (Yasbin et al., 1973) or (2) by scraping the lysed bacterial lawn from an agar plate, resuspending the phage, removing the cells and agar by centrifugation, and filtering the phage supematant through a 0.45/~m filter. 2. Enrichment for deletion-mutants by heat-EDTA treatment To a phage lysate containing approx. 1- 109 phage in 1.0 ml M-broth was added Tris, pH 8.0 to 20 mM and EDTA to 20 raM. This solution was then heated for 15 rain at 65°C. Survivors were isolated by plating the treated phage on a bacterial lawn ofB. subtilis BR151. Phage st~ks for subsequent heat-EDTA treatments were o ~ e d by plating survivors at a phage concentration sufficient to almost completely lyse the bacterial lawn. Phage were then ~olated by the scraping method described above. It is important to note two things about this procedure: (1) the pH of the EDTA is not adjusted prior to addition to the phage and (2) the procedure
71
does not involve the isolation and subsequent heat-EDTA treatment of phage from individual plaques until the final isolation. 3. Isolation o f phage DNA Phage DNA was obtained by two methods. (a) Mini-lysate procedure. Sufficient phage DNA (5--10 #g total) for several restriction analyses was obtained from 40 ml of a mitomycin-C-induced lysogen of B. subtilis (SP02). Phage were collected from an induced culture (as described above), concentrated by precipitation with 10% polyethylene glycol 6000, and resuspended in 20 ml TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA). The resuspended phage was treated with DNase (Worthington) and RNase A (Sigma), both at 100 ug/ml final concentration, for 1 h at 37°C. The phage suspension was then treated with proteinase K (Boehringer, Mannheim) at 100 ug/ml final concentration for 1 h at 37°C. The DNA was extracted three times with an equal volume of TE-buffered phenol, concentrated by ethanol precipitation, and redissolved in 200--400 #1 TE buffer. Subsequent improvements in this procedure to give a yield of 5 #g phage DNA from 10 rnl (rather than 40 ml) of phage lysate along with increased gel resolution will be described in detail eisew~re (Yoneda, Y., Graham, S., Evans, T., and Young, F.E., in preparation). (b) CsCI purified phage DNA. Phenol~xtracted DNA was obtained from phage concentrated by CsCI equilibrium centrifugation as previous'y described (Graham et al., 1977). This DNA gave better resolution during restriction analysis than the PEG-mini-lysate DNA due to (i) absence of even trace amounts of chromosomal DNA and (ii) absence of PEG fluorescence during photography. 4. Restriction-endonuelease analysis Isolation and purification of the site~specific endonucleases BamHI, BglI, Bg/H,,and E¢oRI was as previously described (Graham et al., 1977). Sinai and XbaI were obtained from New England Biolabs while Bali was obtained from Miles Biochemicals. Conditions for DNA digestion, ethidium bromide gel electrophoresis, DNA fragment visualization and photography were as previously described (Graham et al., 1977). RF.~ULTS AND DISCUSSION
1. Isolation o f 8P02 deletion mutants Spontaneous SP02 deletion mutants were identified by DNA fragment analysis of a lysogenic bacteriophage population which had been enriched for bacteriophages with deletions by heat-EDTA treatment. (For alternative methods of enriching for deletion mutants see Parkinson and Huskey, 1971). Heat-EDTA treatment inactivated 76% of the SP02 phage (Fig. 1). Survivors were still sensitive to inactivation by heat-EDTA. After 5 cycles of treatment the majority of survivors become tolerant to heat-EDTA exposure, although survival never was much greater than 50%. Continued treatment of cycle 5
72
sun~vors for 3 additional cycles did n o t further increase the proportion of survivors (data n o t shown). The relative tolerance of SP02 t o the initial hest-EDTA treatment (as compared t o cycles 2,3,4) was reproducible but n o t readily explainable. Other B. subt///s lysogenic phage as well as several clear-plaque host-range vsriants of SP02 were initially reduced 10000-fold (data n o t shown). Greater than 95% of the c y c l e 5 mtwivors formed clear plaques and were ~ incapable of forming lysogens. Only phage producing cloudy plaques (i.e. premmptive lysogens) were exLmined further. Lysogens were purified by single colony plating and ten lysogens from cycle~4 survivors and thirty
,J
oo~ W
~t O0001,
0,000001,
!
2
3
4
$
IlUldlER OF HE&lr-EOTA CYCLE
Fig.1. Effect of heat-ElYrA treatment on SP02. The heat-ElYrA treatment and subsequent propagation of phage survivors was as described in MATERIAI~ AND METHODS. Phage survival was estimated as follows. After suspend'mg~phage in Tris-EiYrA, the solution was divided into aliquots, one of which was then heated. Survivalwas calculated as the ratio of plaqu~forming units (pfu)after 65°C heating/pfu before heating. At room temperature, Tris-EDTA had no effect on phage viability.
lysogens from cycle-5 survivors were induced with mitomycin C. Phage DNA was isolated and examined by restriction analysis using Bg/I, £¢oR~ and HindH. All ten of the cycle-4 survivors and the majority (21 out of 30) of c y c l e 5 survivors had a ~ e n t pattern identical to SP02. If these phages had a deletion, it was t o o small to detect by alteration of DNA fragment mobility, i.e., less than 0.1 Md. Thus heat-EDTA survivors should not a priori be considered to contain deletions.
73
Of the nine cycle-5 survivors, that did have an altered DNA fragment pattern, only two of them could form stable lysogens upon repeated subcultm~g. These two phage strains, designated SP02A 51 and SP02~ 54, were examined in detail. The seven phage which formed unstable lysogens had similar DNA fragment patterns to each other but different from both SP02A 51 and SP02~ 54 and from SP02. These seven lysogens exhibit high rates of rapid spontaneous induction in both M-broth and on TBAB or M-agar plates. Whether loss of lysogeny represents unstable integration or pseudolysogeny is under investigation.
2. 8ire-specific endonuclease analysis of 8P02 deletion strains DNA from CsCl-banded phages SP02, SP02-41 (a cycle-4 survivor), SP02A 51, and SP02A 54 was examined after digestion with BglI, BglI + BglII, EcoRI and XbaI (Fig. 2). SP02A51 and SP02A54 fragment patterns were identical to each other and different from SP02 and SP02-41 (which are identical). The largest BglI fragment (BglI-A) of SP02A51 and SP02A 54 migrated faster than the comparable SP02 BglI-A fragment; a change in migration equivalent to the loss of 2.3 Md of DNA. No other alteration in the BglI pattern was detectable. The SP02 BglI-A fragment contained the single SP02 BglII site. Digestion of Bgll digested SP02A51 or SP02A 54 DNA with BglII did not alter the BglI fragment pattern. Therefore, SP02A 51 and SP02A 54 lacked the BglII site. The SP02 BglI.A fragment also contained four XbaI sites closely clustered around the Bgill site (Fig.3). Digestion of SP02 DNA with XbaI produced six (A-F) fragments, with fragments C, D, E and F residing within the BglI-A fragment. Digestion of SP02~ 51 and SP02~ 54 DNA with XbaI produced only four fragments. The three largest fragments (A, B, and C) co-migrated with SP02 XbaI-A, -B, and -C. SP02~ 51 and SP02~ 54 were missing XbaI fragments D and E. The SP02~ 51 and SP02~ 54 XbaI-F migrated faster than SP02XbaI-F. Whether this faster migrating F fragment was derived from a truncation of the parental XbaI.E fragment or of the F fragment has not been established. Summation of the SP02~51 and SP02~54 XbaI fragments gave a molecular weight value 2.27 Md smaller than for SP02. Further evidence that SP02~51 and SP02~54 contained similar deletions of approx. 2.3 Md was obtained by the series of combination endonuclease digestions (Fig. 4). The DNA fragment patterns of SP02~51 and SP02A 54 in these complex digestions (e.g., slots 8 and 9, EeoRI plu~ BglI plus SalI plus SmaI) were identical. All the patterns indicating a deletion of 2.3 Md had occurred extending approx. 1.0 Md of DNA to one side of the SP02 BglII site and 1.3 Md to the other. The exact orientation and end points of the deletion still remain to be firmly established. Although the site-specific endonuclease patterns of SP02~ 51 and SP02~ 54 were identical, they produced cloudy plaques distinct from each other. Furthermore, SP02~ 54 spontaneously produced clear-plaque variants at a high rate. Therefore, the possibility exists that the two phages are of independent origin and contain s'nnilar but not identical deletions.
74
Fig.2..Analysis of SP02, SP02-41, SP02A 51, and SP02A54 DNA fragments. SP02 (A), SP02-41 (B), SP02A51 (C), or SP02A54 (D) DNA was digested with the indicated endonuclease and the sample eleetrophoresed as described in MATERIALS AND METHODS. For reference the location and size in Md of the SP02 BglI fragments (left margin) and SP02 EcoRI fragments (right margin) are given. Stool
4.50 Xbol
3.si a.54a i-t5 F O i
r
.
ll.o8 E
a I-sz C
CcoRe a
A B
2-40
Fig. 3. Location of the SP02A 51 and SP02~ 54 deletion on the SP02 restriction-fragment map. Only a portion of the SP02 map around the Bg/II site is represented. The DNA deletion in SP02A 51 and SP02~ 54 is depicted in both possible orientations (A or B).
75
12345
A
B
~
C D £ F
Fig. 4. Comparison of the SP02, SP02A 51, and SP92~ 54 DNA fragment patterns. SP02, SP02A 51, or SP02A 54 DNA was digested with various combinations of endonucleases and electrophoresed as previously described: Slot I (SP02 DNA) with BglI; 2 (SP02) with £ e o R I ; 3 (SP02) and 4 (SP02A54) with E c o R l + BglIl + Bali; 5 (SP02) and 6 (SP02A54 with £ e o R I + SalI + Smal; 7 (SP02), 8 (SP02A 51 ) and 9 (SP02A 51 ) with £eoRI + 8alI + Sinai + BglH; 10 (SP02), 11 (SP02~51) and 12 (SP02~54) with EcoRl + XbaI; 13 (SP02) and 14 (SP02~54) with Xbal; 15 (SP02) with EcoRI. For references, the location of the SP02 Bgll fragments (left margin) and E c o R I fragments (right margin) are given.
Several other potential deletion strains were identified during the initial mini-lysate screening. These strains had an altered EcoRI-B fragment and could not be maintained as stable lysogens. Genetic studies indicate that the 4.74 Md SP02 EcoRI-B fragment (equivalent to 24% of the SP02 chromosome) contains at least two of the 17 SP02 complementation groups (Yasunaka et al., 1970). This more genetically "active" region of the SP02 chromosome is apparently less tolerant of deletion than the BglII area.
3. Relation o f 8PO2A51 and 8PO2A54 to previous 8P02 deletions Chow and Davidson (1973) characterized by heteroduplex analysis two SP02 deletion strains, R90 and $25, originally isolated by R. Skillern. These strains formed ~!ear plaques and did not lysogenize. $25 contained a 3.7% deletion (equivalent to approx. 0.86 Md) and R90 contained a 7.9% deletion (2.2 Md) both located approx. 65% from one end of the SP02 chromosome. Although SP02 exists as a circular molecule within the phage head and during infection (Chow and Davidson, 1973; Arwert et al., 1976), the molecules observed by Chow and Davidson (1973) were linear. During the construction of the SP02 endonuclease fragment map, Yoneda et al. (1979) described an
76
EcoRI fragment which when heated dissociated into two smaller fragments.
They suggested that this heat-labile chromosomal junction might be the point of chromosome circularization. The SP02 BglII site (and hence the SP02~51 and SP02~54 deletion) is located approx. 64% from one end of this heat-labile junction. Further evidence that the SP02~ 51 and SP02A 54 deletions occur in the same chromosomal region as the deletions in 825 and R90 comes from the localization of the phage attachment site at or near both sets of deletions. Chow and Davidson located the phage attachment site 2% and 5.6% of the chromosome away from 825 and R90 respectively. Preliminary genetic studies of SP02 have located the SP02 attachment site in the vicinity of the SP02 BglII site (Graham, S., Yoneda, Y., Wilson, G. and Young, F.E., unpublished). Therefore the SP02 termini observed by Chow and Davidson (1973) and by Yoneda et al. (1979) are probably the same. Furthermore, the deletions in SP02A 51 and 8P02~ 54 and in S24 and R90 most likely occurred in the same region of the SP02 chromosome. Deletion of the 2.3 Md of SP02 DNA around the BglII site in SP02~51 and SP02A 54 caused a change in plaque morphology from wild-type SP02 but did not prohibit lysogeny or phage production. Consistent with the idea that this area of the 8P02 genome is non-essential was the observation that super-infection marker-rescue of SP02 sus mutants with purified wild-type DNA fragments failed to locate any of the 17 known SP02 complementation groups within this area (unpublished observations). Furthermore, a hostrange mutant of SP02 exists that has a DNA rearrangement near the BglII site which is suggestive of a deletion of SP02 DNA with the concomitant introduction of non-SP02 DNA (Graham, S., Yoneda, Y., Wilson, G. and Young, F.E., unpUblished). CONCLUSIONS
Bacillus subtilis bacteriophage SP02 appears to be quite promising as a cloning vehicle for the Bacilli. The accrued genetic and physical information on SP02 shows (1) the phage has several usable (Le., single) endonuclease sites; (2) the BglII site is located in a genetically "silent" region of the chromosome; (3) viable deletion mutants are available thus increasing the potential amount of clonable DNA; and (4) the host-range of SP02 can be extended to Bacilli other than B. subtilis. Further work with this phage should produce a usable cloning vector. ACKNOWLEDGEMENTS
We wish to thank Dr. Junetsu Ito for suggesting the heat-EDTA selection procedure used for isolating the viable deletion mutants. We would also like to thank Henry Baney for his help in preparing phage DNA and endonucleases and also thank Ms. Pat Morrison for her technical assistance. This research was supported by a research grant from Miles Laboratories.
77 REFERENCES
Arwert, F., Bjursell, G. and Rutberg, L., Induction of prophage SP02 in Bacillus subtilis: isolation of exised prophage DNA as a covalently closed circle, J. Virol., 17 (1976), 492--502. Chow, L.T., and Davidson, 1'4., Electron microscope study of the structures of the Bacillus subtilis prophages, SP02 and ~105, J. Mol. Biol., 75 (1973), 257--264. Graham, 8., Young, F.E. and Wilson, G.A., Effect of site-specific endonuclease digestion on the thyP3 gene of bacteriophage ~3T and the t h y P l l gene of bacteriophage p 11, Oene, 1 (1977) 169--180. Parkinson, J.8. and Huskey, R.J., Deletion mutants of bacteriophage lambda, I. Isolation and initial characterization, J. Mol. Biol., 56 (1971) 369--384. Weisberg, R.A. and Adhya, 8 , magitimate recombination in bacteria and bac~riophage, Annu. Rev. Oenet., 11 (1977) 451--478. Yasbin, R.E., Wilson, G.A. and Young, F.E., Transformation and transfection in lysogenie strains of B~illus subtilis 168, J. Bacteriol., 113 (1973) 540--548. Yasunaka, A., Tsukamato, H., Okubo, 8. and Horiuchi, T., Isolation and properties of suppressor-sensitive mutants of Bacillus subtilis bacteriophage SP02, J. Virol., 5 (1970) 819--821. Yoneda, Y., Graham, S. and Young, F.E., Restriction-fragment map of the temperate l ~ i l l u s subtilis bacteriophage gP02, Oene, 7 (1979) 51--68. Communicated by W. Szybalski.
