Gene, 7 (1979) 51--68

51

© Elsevier/North-HollandBiomedical Press, Amsterdam -- Printed in The Netherlands

RF~TRICTION-FRAGMENT MAP OF M BACTERIOM(/E

TEMPERATE Bacillus subtilis

SP02

(Endonuclease Bg/I, BglH, EcoRI, SalI, SmaI, XbaI ; chromosome circularization; phage vectors) Y. YONEDA,S. GRAHAMand F.E. YOUNG Del~rtment of Microbiology, University of Rochester Medical Center. Rochester, IVY 14642 (U.S.A.) (Received April 2nd, 1979) (Accepted June 14th, 1979) SUMMARY The endonucleases BglI, BglII, EcORI, SalI, Sinai, and XbaI were used to fragment the phage SP02 DNA. Electrophoretic analysis using ethidiumbromide agarose gels showed the phage to have nine Bgll sites, one BgiII site, four £coRI sites, one SalI site, one Sinai site, and six XbaI sites. Using partial d~'estions, multiple endonuclease digestion, and autoradiography the fragment~ were sized and ordered into a circular map of 23 Md. Such an analysis locates the endonuclease sites, indicates which endonucle.Jses are potentially useful in cloning with SP02, and allows insertions and/or deletions in the SP02 DNA to be characterized.

INTRODUCTION

The temperate bacteriophage SP02, whose normal host is Bacillus subtilis, contains double-stranded DNA of 23 Md size. A partial genetic map is based on the complementation of sus mutants (Y~sunaka et al., 1970). Other SP02 mutants include deletion strains (Graham et al., 1979) and mutants whose host-range has been extmuted to other Bacilli (Graham, S., Yoneda, Y., Wilson, G., and Young, F.E., unpublished observations). As part of a program to evaluate the potential of SP02 as a generalized cloning vector in the Bacilli, a detailed restriction map was constructed. Such an analysis indicates which endonucleases are potentially useful in cloning with bacteriophage SP02, ami p erm~'ts insertions and deletions of SP02 DNA to be characterized. Abbreviation: Md, mepdaltons.

52 MATBRIALSAND METHOI~

1. Bacteriophage 8102 maintenance, induction, and DNA isolation BactefiOldmge SP02 was propagated in B. subtf/is 168 strain BR151 (lys-3 trpC2 metB10, c ~ y i n g the gtaB marker) grown either on M4gar plates or in M-broth (Yasbin et al., 1973). 8P02 was induced by sdding mitomycin C, 5 pglml final concentration, to the lysogen growing in M-broth (Klett~ Summenmn value of 50). After lysis (% 120 min) the remaining cells and cellular debris were removed by centfifugation, at 12 000 × g for 10 rain. Phage were p r e c i p i ~ from the supenmtant by the addition of polyethylene glycol 6000 (Union Carbide) to 10% (w/v) followed by storage at 4 ° C for 12 h and hawested by centfifugation, 12 000 X g for 20 rain at 4* C. The phage pellet was resuspended in phage buffer, treated with DNase and RNase, the DNA phenol extracted, and collected by ,~thanol precipitation as previously described (Yasbin et al., 1973). The DNA was redissolved in TE buffer (10 mM TrJs, pH 8.0 containing I mM EDTA) and stored under chloroform at 4 ° C until further use. 2. Site-specific endonuciease analysis The purification procedures for EcoRl (Wilson et al., 1974), BamHI (WJJson and Young, 1975), and Bill[ and Bgl[l (Duncan et al., 1978a) were previously described. The Sa/Y.,Sinai, and Xbal enzymes were obtained from Miles Biochemicsi. Enzyme reactions were done in TMM buffer (6 mM Tris, pH 7.5 containing 6 mM MgCI2 and 6 mM #-mercaptoethsr.ol). Digestion was terminated either by heat (65 ° C, 10 min) or by adding EDTA to a final con. centratlen of 10 raM. Digestion of the DNA sample with more than one enzyme was performed either by simultaneous digestion or by digestion with the i'n~t enzyme, heat inactivation, and then digestion with the second enzyme. E l ~ p h o r e s i s of DNA was done on a Blaircraft vertical slab gel apparatus using a 20 hole slot former wita gels of 0.8% agarose (Sigma), 1% agarose, or 1.2% agarose in TPE buffer (40 mM Tris, pH 7.9 conb~ting 36 mM K2HPO,, 1 mM EDTA, and 0.5 #g/ml ethidium bromide). Gels were electrophoresed either 12--14 h at 25 NA or for 4 h at 75 mA. The molecular weights of the DNA fragments were estimated by comparison of their mobility in agarose gels with the mobility of internal standazds: (1) an E¢oRI[BamHI dig~t of adenovirus-2 DNA (Duncan et al., 1978b); (2) an EcoRI digest of~ DNA (He]ling et al., 1974); (3) a BamHI digest of DNA (Hsggezty and Schleif, 1977); and an EcoRI/BamHl[Bgl[I digest of pCD1 DNA (Duncan et al., 1977). Adenovirus-2 DNA, Z DNA, and pCD1 DNA were generously prov~.dedby G. Wilson and P. Spear. The gels were photognq)hed under ultra-violet illumination using Polaroid Type PN55 film and a Vivitar red No. 25 A filter.

5~

3. Elution of DNA fragments from agarose gels SP(#2 DNA (100--200/~g) w a s limit digested with the appropriate s'~eSl~eCific endonuclease and the DNA fragments separated on a 1% TPE agarose gel lacking ethidium bromide as previously described. DNA bands were detected by soaking the gel for 10 rain in 100 ml TPE buffer containing 0.5 #g/ml ethidium bromide and viewing under a hand-held long wavelength ultra-violetlamp (Mineralight, Ultra-violetProducts, Inc.). The separv~e bands were excised and the agarose strip squeezed through a 22.5 gauge needle into a conical test tube containing 2.0 ml TE buffer. The agaros.~ slurry was heated 3 to 12 h in a 55 ° C water bath. The agarose was pelleted by centrifugation (8000 X g for 10 min) and the supematant removed and stored at 4 ° C. The agazose pellet was resuspended in 2.0 ml TE buffer and incubated for an additional 3 to 12 h at 55 ° C. The mixture was centrifuged again and the supernatant was removed and pooled with the first supematent. The supematant was adjusted to 0.3 M sodium acetate, mixed with 2 vol. ethanol, and stored for 12 h at - 2 0 ° C. After collecting the DNA precipitate by centrifugation (12 000 X g for 10 rain a t - 2 0 ° C) the DNA was redissolved in 1.0 m! TE buffer. Any remaiving agarose was removed by centrifugation in an Eppendorf centrifuge. The supematant was then extracted 2 to 5 times with an equal volume of TE saturated redtistilled phenol. The water phase was made 0.3 M with sodium acetate and the DNA precipitated by adding 2 vol. ethanol and storage a t - 2 0 ° C for 12 h. The DNA was collected by centrifugation and redissolved in 300--500 #1 of TE buffer. This DNA was susceptible to further site.specific endonuclease cleavage, amenable to ligation with T4 DNA ligase, and active in DNA-mediated tra~mfection.

4. Hybridization of purified DNA fragments to SP02 DNA Purified fragments of SP02 DNA (see above) were labeled with s2p in vitro using the "nicked translation" procedure (Rigby et al., 1977). This DNA was then hybridized to non-radioactive SP02 DNA which had been limit digested, electrophoresed in an agaroseCthidium bromide gel, denatured and eluted onto a cellulose nitrate filter as described by Southern (1975) and modified by Botchan et al. (1976). Hybridization was detected by exposure of the cellulose nitrate filter on X-ray film. RESULTS

1. 8ire-specific endonu¢lease digestion of 8P02 DNA SP02 DNA limit digested singly or in combinations with the site-specific endonucleases BglI, BglII, £co RI, Bali, Sinai, and XbaI was electrcphoresed into an agaroseethidium bromide gel producing the DNA fragmen; patterns shown in Fig. 1. The molecular weights of the various DNA fragments (Table I) were determined by compa~-ison to the electrophoretic mobility of DNA fragments of known size run in the same gel (data not shown). Molecular weight values in parentheses are estimations for fragments whose migra~on is outside the linear migration range of our gels.

9 12 5

6

3 '7 8

BglI EcoRl BglII =

sozP

Xbol BglH + Sail BgIH + Sall a Sm=IC

(18.08) (14.84) (9.85) (13.54)

(o.0.88)

(8.31) (16.04) (16.36)

A

(6.27) (9.85) (8.89) (9.68)

(5.18)

4.11 4.74 (9.4?)

B

5.15

1.30

8.12 1.62 b

C

1.14

2.29 0.66

D

1.07

2.11

E

Frsement size (Md)

0.54

1.62

F

0.58

G

0.46

H

0.45

I

23.40 24.18 23,89 23.17

25.56

23.05 23.06 25.8~

Sum of frqment sizes (Md)

a Sample heated fol° 10 min at 65 ° C prior to electrophoresis. b C~C=. e Derived from SP02 fragment map (Fig. 8) and included here for completeness. Experimentally Smal was used only in combination with other endonucleMes.

.

Number of determinations

Enzyme

SUMMARY OF SITE-SPECIFIC ENDONUCLEASE DIGESTION PROFILES OF SP02

TABLE I

55

Fig. 1. Site-specific endonuclease analysis of SP02 DNA. SP02 DNA was digested with the indicated endonuclease, heated (A) as indicated, and electrophoresed as described in MATERIALS AND METHODS. This particular gel illustrates the various SP02 digestion patterns from which molecular weight determinations were made. The actual determinations were made from gels which included appropriate molecular weight stm~dards.

Digestion with EcoRI yielded four fragments: EcoRI-A, 16.04 Md; Eco RIB, 4.74 Md; EcoRI-CIC2, 1.62 Md; and EcoRI.D, 0.66 Md having an aggregate molecular weight of 23.06. Digestion with BglI yielded nine fragments, BglI-A through BglI.I, having an aggregate molecular weight of 23.05 while digestion with XbaI produced 6 fragments, XbaI-A through XbaI-F having an aggregate molecular weight of ~3.40. These values were slightly smaller than those calculated from contour 1ength, 38.6 kilobases (Chow and Davidson, 1973) or from sedimentation boundary analysis, 24.0 Md (L. Larcom, personal communication). Digestion of SP02 DNA with Bg/II, SalI, or SmaI gave inconsistent results" either a single band with the migration rate of undigested SP02 DNA or two faster migrating bands. SP02 DNA is a circular molecule (Chow and Davidson, 1973; Arwert et al., 1976). Since undigested open circular SP02 DNA co-migrates with linearized SP02 DNA, endonucleases BglII, SmaI, and SalI could have either one site or no sites. To distinguish between these possibilities SP02 DNA was digested with either BglI or EcoRI in combination with either BglII, SalI, or Sinai. Digestion with Bgll/BglII resulted in the disappearance of a single BglI fragment (BglI.A) and the appearance of two additional fragments with faster migration (see Figure 1). BglII also altered

56

a single £coRI fragment (EcoEI-A) with the concomitant appearance of two new fragments (Fig. 1). Therefore SP02 DNA has only a single BgflI recognition site. Likewise, SdI/Bg/I and SmaI/Bgfl digestion caused the disappearance of a single BgH fragment (for Sail, BgH-F disappearS; for Sinai, Bg/I-B disappeared) and both SaH/EcoRI and SmaI/£coRI caused the disappearance of the EcoRI-A f~sgment. In both cases, two new fragments with faster migration appeared. Therefore SP02 has only single recognition sites for SalI and Sinai. 2. 8P02 h~t-labile junction

Digestion of SP02 DNA with £coRI produces four hsgments. Heating E c o R I ~ e s t e d 8P02 DNA at 65 ° C for 15 to 30 rain results m the disappearante of the 1.62 Md EcoRI-CIC~ ~ g m e n t and the appearance of two additional fragments of 0.78 Md (£coRI-CI) and 0.75 bid (£coRI-C~) (Fig. 2).

,f the EcoRI-C, C2 junction. EcoRI digested SP02 DNA or purified E c o R I ~ C ~ fragwent was h~ated (A) as indicated and electrophomsed as described in MATERIALS AND METHODS.

57 Similarly, heating BglII, SalI, or SmaI digested SP02 DNA at 65 ° C for 15 to 30 min changes the DNA fragment pattern from a single band to two faster migrating bands. This heat labile junction probably represents the SP02 termini and therefore the point at which SP02 circularizes. Whether the mcchanism of SP02 circularization involves overlapping cohesive ends, as in ), (Hershey et al., 1963) and B. subtilis bacteriophage ~105 (Scher et al., 1977), or a protein linker, as in B. subtilis bacteriophage ~29 (Ortin et al., 1971), is n o t known. Treatment of the EcoRI-C~C2 fragment with T4 DNA ligase prevented the heat dissociation. Furthermore, the EcoItI-C~C~ frag. m e n t was not dissociated by treatment with phenol, or proteinase K (100 #g/ml final conc., I h 37 ° C), or 10% SDS or 10% sarkosyl (w/v, I h 37 ° C) [Yoneda et al., manuscript in preparation]. Therefore we favor a cohesive-ends model for SP02 circularization.

3. Ordering of EcoRI fragments Partial digestion of SP02 DNA was employed to order the four EcoRI fragments (Table II). From the molecular weight of the partial digestion fragm e n t (pdf), it was often possible t o unambiguously determine the limit digest fragments contained within it. For example EcoRI-pdfl, 7.35 Md, had to consist of the EcoRI.B, EcoRI-CIC2, and EcoRI.D fragments. Similarly EcoRI-pdf4 had to consist of the £coRI.CI, the EcoEI.C2, and the £coRI.D fragments. £coRI-pdf 3 was most likely EcoRI-B and EcoRI-C2 through an EcoRI.B and EcoRI-CI combination could n o t be excluded on the b ~ i s of molecular weight. Therefore the EcoRI fragment order was either A-B.C=C,-D or A-B-CIC~-D. TABLE II $P02 £¢oRI DIGESTION PROFILE

E¢oRI

pdfs

fragment A

Proposed Molecular composition weight (Md) of pdf expected

12

16.04

4

7.35

BC,C2D

7.02

2 3

6 2

6.35 5.48

BC,C, BC2 or BC,

6.36 5.49

14

4.74

6 13

2.35 1.62

C,C2D

2.28

5 3

0.78b 0.75b

7

0.66

4

C2 D

Molecular weight (Md) observed

1 B

C,C2 C,

Number of determinations

Sum:

23.06

a Partial digestion fragment. b Not included in calculations of total molecular weight. The C,C= fragment was counted as a single . f ~ e u t in the summation.

58

4. Location o f the ~ site and the Bglll site Both the ~ Sail and the single Bg/H site were located within the !arge (16.04 Md) EcoRI-A fragment. Treatment of EcoRI ~Ylgested SP02 DNA with Sail resulted in the disappearance of the EcoRI-A fragment and the concomitant appeanmce of two new ~ e n t s of 12.37 Md and 3.67 Md (aggregate = 16.04). Partial digestion of SP02 DNA with EcoRI/SalI (Table m ) indicated t h a t the 3.67 Md fragment was contiguous with the EcoRI-D TABLE HI

8P02 £¢oRllBgllllSall DIGESTIONPROFILE Fragment

Mj 8

M_b

Z

B Ve

xa

C,C=

C! C~ D

pdf

Numberof determln-tions 9 XO 8 1 1 3 1 1 8 2 8

Molecular weight(Md) observed 2.37 a (12.5) 9.55 8.6 8.00 6.95 6.30 5.50 4.82 4.39 3.67

8

3.49

2 7 1 3 2 3

2.28 1.63 1.42 0.78 0.76 0.66 8urn:

Proposed composition of pdf

Molecular weight(Md) expected

XB(C, or C,) XB BCsC~D BCtC~ B(¢~ or C,)

9.07 8.31 7.02 6.36 5.49-'5.51

YD

4.33

C,CsD

2.28

D(C, or C,)

1.42--1.44

23.82

a Seen when 8P02 was digested with only £coRl[$aH. b Seen when 8P02 was digested with only EeoRI/BglII. e Prom EcoEI-A digested with 8a/I; see Table V for details. d From EcoRI-A digested with BgflI;see Table V for details.

fragment. This indicated that the SalI site was approx. 5 Md from the EcoRI-D side of the EcoRI-CzC2 heatAabfle junction (3.67 Md + EcoRI-D (0.66 Md) + EcoRI Cl or C2 (0.77 Md) = 5.10 Md.) HeatingSa/I digested SP02 DNA to split the single Saff fragment at the £CORI-CzC2 junction resuited in two fragments of 20.38 Md and 5.18 Md (Table I), supporting the placement of the Sa/I site approximately 5 Md from the EcoRI-CzC: junction.

59

Treatment of EcoRI digested SP02 DNA with BglII resulted in the disappearance of the EcoRI-A fragment and the concomitant appearance of two new fragments of 12.15 Md and 3.49 Md (aggregate = 15.64). Partial digestion of SP02 DNA with EcoRI/BglII (Table III) indicated that the 3.49 Md fragment was contiguous with the EcoRI-B fragment. This indicated that the BglII site was approx. 9.08 Md to the EcoItI-B side of the EcoRICIC2 ~ c t i o n (3.49 Md + EcoRI-B (4.82) + EcoRI-CI or C2 (0.77) = 9.08). Heaifmg BglII digested SP02 DNA to split the EcoRI-CIC2 junction resulted in two fragments of 16.35 Md and 9.47 Md (Table I), thus supporting placement of the BglII site approx. 9.1 Md from the EcoRI-CIC2 junction. Confirmation of the relationship of EcoRI-B and EcoEI-D to the EcoRICzC2 h s g m e n t and the placement of the BglII and SalI sites was obtained by the partial digestion of SP02 with EcoRI/BglII/SalI (Table III). The further reduction iu the £¢oRI-A fragment size by EcoEI/BglII/SalI (to 9.55 Md) from that seen *~ith EcoRI/BglII (12.15 Md) or EcoRI/SalI (12.37 Md) indicated that: (1) both the BglII and SalI sites were located within the EcoRI-A fragment and (2) the sites were 9.55 Md apart. Our previous placement of the Sa/I site 3.67 Md from the E¢oItI-D fragment and of the BglII site 3.49 Md from the £¢oRI-B fragment indicated that the SalI and BglII site

Xlml

14

Fig. 3 . 8 P 0 2 endonuelease fragment map. The BgII, Bgfll, EeoRI, 8all, Smal, and Xbal sites were ordered as described in the text R g the heat-dissoeiatable junction as an origin. Fragment sizes are in megadaltons.

60

were approx. 8.9 Md apart; a value in reasonable agreement with the £coRU BgHIISaH digest/on data. Utilizing the h ~ e £coR143,C2 junction as an oz~in, the Fz~oRI, ~ ~ f f , and 8maI sites (data presented later) were ordered as shown in the inner two circles of Fig. 3.

5. Order# the nts A limit digest/on of SP02 DNA with BgH produces nine fngments: Bg/I-A to/~fl-l. As with EcoRI utfliz~on of partial digestion concUt/ons revealed some of the BgH fragment interrelationships (Table IV). For example, Bg/Ipdf 2 (5.72 Md) was composed either of fragment BF (5.73 Md) or of fragment ECI (5.68 Md) or ECH (5.69 Md) - - t h e molecular weights of I and H and of BF and EC (H or I) being too ,im,l,r for unambiguous assignment. However, pclf 7 (2.65 Md) can only be composed of fragments FG (H or I). Therefore the relationship of B, F, G, and (H or I) was most likely BFG (H or I). Pdf 4, 6, 8, and 9 were comlmsed of C (H or I), D (H or I), E (H or I), and G (H or I) respectively. Thus fragments C, D, E, and G all flank either H or I. The molecular weJl~t of pdf I was consistent with three possible fragTABIJg IV 8P02/~ff D I G ~ O N PROFILE l~rqpuent

pdf

A

3 B 4 C

§ 6 7 8

D E F 9 G H I

Number of determin~iom

Molecukr Projzzsed weight Odd) eompo~ion obeerved of pdf

9

9.33 s

3 3

7.3 6.72

3 10 3 10 2 4 3 1 10 10 11 3 9 9

4.78 4.11 3.60 3.12 2.96 2.76 2.62 2.44 2.20 2.11 1.62 1.13 0.58 0.46 0.45

5

8urn: a High,due, correct value 8.31.

24.05

Molecular weisht (Md) expected

FGIC~ BF F,CI 1.1]gC D][.IB

7.88 5.78 5.68 5.69 4.86

CI

8.67

? DH lq31 EH

2.75 0..66 2.57

GI

1.03

61

ment combinations: (1) FG (I or H) CF; (2) BC; or (3) BFHI. The only combination consistent with the deduced composition of the other pdfs was that of FG (I or H) CF. With a linkage of CE, then H or I, then FG: the most probable Bg/I fragment order was ABFG (I or H)CF (I or H) DA, an order which was later confirmed.

6. Location of the BgllI site, the Sail site and the Sma/site The single BgllI site was located in the middle of the large (8.31 Md)/BglIA fragment. Treatment of B&II digested SP02 DNA with BgIII resulted in the disappearance of the BglI-A f~agment and the concomitant appearance of 4.15 Md doublet (aggregate = 8.30) (Fig. 1). This was confirmed by treatment of purified individual BgH fragments with BgHI. 0nly the BglI-A fragment was cut and into two fragments of 4.10 Md each (Table V). Placement of the Bg/II site in th e middle of the BgH-A fragment means the BglI-A fragment must overlap to'some degree both the EcoRI-A and EcoRI-B fragments. The single Sa/I site was located within the small (1.62 Md) BglI-F fragment. Treatment of BglI digested SP02 DNA with Sail resulted in the disal~ peamnce of the BglI-F fragment and the concomitant appearance of two smaller fragments of 0.94 and 0.56 Md (Fig. 1). Treatment of purified individual BgH fragments with Sail resulted in only the BglI-F fragment being cut and into two fragments of 0.94 and 0.56 Md. With these data it was not pmsible to precisely locate the position of the Sail site within the BglI-F fragment. The location of the single Sinai site was similarly determined. Treatment of Bgll digested $P02 DNA with Sinai resulted in a slightly faster migrating BglI-B fragment. A change consistent with a reduction in molecular weight from 4.11 Md to 3.89 Md, the small 0.22 Md fragment was not detected (Fig. 1). From a series of digestions of SP02 DNA with either Smal/SalI or SmaI/Bglll it was possible to precisely locate the Sinai site 4.55 Md from the Sail site and 4.53 Md from the BglII site. As will be shown later, this placement positioned the Sinai site very near one terminus of the BglI-B fragment.

7. Location of the EcoRl sites within the Bgll fragments It was possible to locate the four EcoRI sites and the nine BglI sites relative to each other. Treatment of BglI digested SP02 DNA with EcoRI resuited in tlie disappearance of the BglI.A, BglI.C, and BglI.E fragments with the concomitant appearance of new fragments of 7.76, 1.76, 1.27, 0.74, 0.72, 0.68, and 0.61 Md (Fig. 1). Treatment of purified individual BglI fragments with £¢oRI or purified individual £eoRI fragments with BglI allowed the unambiguous determination of the relationship between the EcoRI sites and the BgH sites (Table V). For example, the BglI-A fragment (8.31 Md) was cut once by £eoRI to produce two fragments of 8.00 Md (an abnormally high value from this experiment) and of 0.74 Md. The BglI-C fragment

EcoRI £coRI EcoRl £coRl EcoRI £coRI EooRI EcoRI EooRI BgilI EcoRI/BgilI Sail Sail

Bgn

Bgll BE/I

Xbal

Bgii-A BgH-B Bgil.C Bgil.D Bgil-E BglI-F Bgil-G B&II-H BgiI-I BglI-A ~ii-A BglI-F BglI-E EooRI-A (16.04) EcoRI-B (4.74)

EcoRI~ (1.62) EcoRI-D (0.66)

EcoRI-A

2 8

- -

(a)

0.75 (doublet) No digestion

7.76 2.10 ( ~ i i - D ) 1.80 (Xbal-B)

1.27

4.01 ( ~ n - B )

1.14 (Xbal-C)

1.72 0.75

1.07 (Xbal-D)

1.58 (~n-F) 0.52 (BSII-H)

4

Bend (molQcul~ weight Md)

0.74 8.00 -0.72 ( S c o R l ~ , ) 0.61 (gcoRI-D) 1.76 -0.68 1.27 ----4.80 (doublet) 3.70 0.75 4.80 0.66 0.94

1

a Large fra~nent of undetermined molecular weight.

Bsil

Enzyme

Fragment

RE-DIGESTION OF INDIVIDUAL SP02 DNA FRAGMENTS

TABLE V

0.78

0.58 (~U-O)

5

0.54 (XbaZ-E)

0.46 (BgZI-I)

6

m t~

63

(3.19- Md) was cut twice to produce three fragments of 1.76, 0.72 (ce~migrated with EcoRI-C1), and 0.61 (co-migrated with EcoRI-D) Md. The BSIIE fragment (2.11 Md) was cut once to produce two fragments of 1.27 and 0.68 Md. None of the other BgH fragments contained an EcoRI site. Likewise the BglI sites were located within the purified individual EcoRI fragments. The EcoRI-A fragment was cut by BglI three times to produce four fragments of 7.76, 4.01 (co-migrated with BgII-B), 1.72, and 1.58 (co-migrated with BgH-F) Md. This implied that the B&lI A, B, and F fragments were located within the EcoRI-A fragment. The EcoRI-B fragment was also cut three times by Bgfl to produce four fragments of 2.10 (co-migrated with B&II-D),1.27, 0.75, and 0.52 (co-migrated with BglI-H) implying that the B&H-D and -H fragments were located within the EcoRI-B fragment. The £coRI-C fragment was cut once to produce a 0.75 Md doubJet. The EcoRI-D fragment was not cut by BgH. Unambiguous corzelation of the EcoRI and BglI fragments was obtained using the Southern procedure to hybridize purified individual radioactive fragments (i.e., probes, Bg/I-C, BglI-E, EcoRI-CzC2, ~nd the 9.47 Md BgllI fragment) to SP02 DNA that had been digested (EcoI~I, BglI, XbaI, or E¢oRI/BglII/SalI) and displayed on an agarose gel (Table VI). The EcoRI-A fragment contained portions of the Bgll-C fragment and portions of the 9.47 Md BglII fragment but did not contain any portions of either the BglI.E fragment or the £coRI-C,zC: fragment. The EcoRI-B fragment contained portions of the BgII-E fragment and 9A7 Md Bg/II fragment. And the EcoRI. CzC2 fragment contained portions of both the BglI-C and BglI.E fragments. Hybridization of the probes to an £eoRI digestion revealed the £coRI.C= hagment contained portions of the Bg/I.E and 9.47 Md BglIl fragment while the £eoRI-Ca fragment contained portions only of the B~/II.C fragment. Localization of the £coRI-D fragment within the BglI.C fragment meant the fragment order was £coRI.A-D.Cz.C2-B.A with the BglI.C fragment contiguous with £eoRI-ADCt and the Bg/I-E fragment contiguous with EeoRI-C2B. Thus these lesults enabled us to unequivocally order the Cz and C2 fragments. Hybridization of the probes to a BglI digest of SP02 revealed that both the BglI.C and Bg/I.E fragments contain portions of £coRI-CtC2. The BglI-A, D, E, and H fragments all contained portions of the 9.47 Md BglII fragment. The BglI fragments had been ordered ABFG(I or H)CE(I or H)DA. Since one of the 9.47 Bg/II terminus was located in the middle of the BglI-A fragment, and since the 9.47 Md Bg/II frasment contains portions of Bg/I-E and BglI-H but not BglI.I or B&/I-C;the Bg/I fre~nent order had to be A.B-F-G-I~,-E-HD-A. The £ e o R l and

Bgll sites

were located relative to each other as shown in

Fig. 3. This order was consistent with the internal order of the BglI fragments, the order of the £eoRI fragments, the relationship of BglI.E to the £coRI-C2B fragment, the relationship of BgII.C, to the £¢oRI-ADC~ fragment, and the location of the BgIIl, 8aIH, and Sinai sites within both the £¢0RI fragments and the Bg/I fragments.

64

' r J m z , S , ~w ~alARY

OF 8OUTHERN'8

HYBRIDIZATION _

_~

n

.

"P-~/I-C ,

BooR!

A 4.74 B

16.04

1.62

C,C,

0.78 C,

.

4"

44.

4"

4.

SalI

Xbal

•

4"

4.

4"

4.

4" 4-

4.

4"

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0.78 geoRl29 (Moreno et aI., 1974; Reilly et al., 1975; Ito et aI., 1976) and SPP1 (Spatz and Trautner, 1970; Mcintosh et aI., 1978) and the temperate bacteriophage 1>105 (Scher et aI., 1977; 1978). The physical map reported here extends the list to the temperate B. subtilis bacteriophage SP02. Although SP02 is both similar in size and shape to 1>105 and shares a small degree of DNA homology with ¢105 (Boice et aI., 1969; Chow et al., 1972); it is a distinctly different bacteriophage (Boice, 1969; Rutberg et aI., 1972). Site-specific endonuclease analysis shows SP02 to contain single sites of susceptibility to the BglII, SalI, and SmaI enzymes. The isolation of viable mutants containing a 2.4 Md deletion including the BglII site indicates that this region of the SP02 chromosome is not genetically essential (Graham et

et

67

ai., 1979). The SP02 endonuclease map is circular, reflecting the circular nature of the SP02 chromosome (Arwert et al., 1976; Chow and Daviclson, 1973). Heating SP02 DNA disassociates the circular molecule at the EcoRICIC2 junction to form a linear molecule. Ligation of SP02 DNA with T4DNA ligase prior to site-specific endonuclease digestion preserves the BgII-CE junction, the XbaI.AB junction, and the SacI-AB junction (Yoneda, Y., Graham, S. and Young, F.E., in prep.). Thus, circularization of SP02 is probably due to cohesive overlapping ends as in bacteriophage ~ which have a high probability of dissociating, although the existence of a protein-linker has not been rigorously disproved. The SP02 genetic map is linear (Yasunaka et al., 1970) although it is permuted upon integration into the host chromosome (Chow and Davidson, 1973; Arwert et al., 1976). Recent correlation of the physical and genetic maps of SP02 indicates that one end of the linear genetic map is near the EcoRI-DC~ junction and the other end near the EcoRI-C2B junction. The intervening genetic markers can be ordered counter-clockwise around the SP02 physical map starting at EcoRI-DC~ and ending at EcoRI-C2B (Graham, S., Yoneda, Y. and Young, F.E., in prep.). Given the progress in developing bacteriophage and plasmid cloning vectors, it is probable that B. subtilis will become a widely used organism for recombinant DNA research. Mutant~ of bacteriophage SP02 exist extending its host-range to other Bacilli such as B. amyloliquefaciens and B. natto (Graham, S., Yoneda, Y., Wilson, G. and Young, F.E., unp. obs.). The development of detailed genetic and physical maps of the Bacillus phages will increase the potential usefulness of the B. subtilis molecular cloning system. ACKNOWLEDGEMENTS

We would like to thank Henry Baney for his technical aasistance. The study was enhanced by the thoughtful criticism of Dr. Gary Wilson. This research was s u p p o ~ by a grant from Miles Laboratories. REFERENCES

Arwert, F., Bjumell, G. and Rutberg, L., Induction of prophage $P02 in Bacillus subtilis: isolation of excised prophage DNA as a eovalently closed circle, J. Virol., 17 (1976) 492--502. Boiee, L., Evidenee that Bacillus subtilis bacteriophage 8P02 is temperate and heteroimmune to bacteriophage ~105, J. Virol., 4 (1969) 47--49. Boiee, L., Eiserling, F.A. and Romig, W.R., Structure of Bacillus subtilis phage SP02 and its DNA: similarity of Bat/flus subtilis phage SP02, ~105, and SPPI Biochem. Biophys. Res. Commun., 34 (1969) 398--403. Botehan, M., Topp, W. and Sambrook, J., The arrangement of Simian Virus 40 sequences in the DNA of transformed ceils, Cell, 9 (1976) 269--287. Chow, L.T., Boiee, L. and Davidson, N., Map of the partial sequence homology between DNA molecules of Bacillus subtilis bacteriophage $P02 and ~105, J. Mol. Biol., 68 (1972) 89--400. Chow, L. and Davidson, N., Electron microscope study of the structures of the Bacillus subtilis prophages, SP02 and ~105, J. MoL Biol., 75 (1973) 257--264. I)unean, C.Ho, Wilson, G.A., and Young, F.E., Transformation of Bacillus subtilis and Eseheriehia eoli by a hybrid plasmid pCD1, Gene, 1 (1977) 153--157.

68 Dmmm, C.H., W'dson, G.A., and Young, F.E., Bioehemieai and genetic properties of siteremietlon e n d ~ u d e ~ in ~ # l o ~ , J. ~ o L , 184 (1978) 3~8---844. Dunean, C.IL, W'dson, G.A., and Young, F.E., Meekm~sm of i n ~ foreign DNA durb~ tnmsformnfion of ~ s u b f f H s , Proc. Natl. Acad. 8cL USA, 75 (1978) Graham, S., Yoneda, Y., and Young, F.E., Isolation and chametedzsZ/on of viable dele//on mutants of ~ subtHis baeterioldmge 8P02, Gene, 7 (1979) 69--77. Hsggerty, D.M. and ff~zkif, R~'., Location in ~ p k m g e lmmbda DNA of cleavage sites of the ~ endonueleue from Baeff/us amyloliquefaciens H, J. Virol., 18 (1977) 669-. Helling, R.B., (~oodman, H.M. and Boyer, H.W., Analysis of e n d o n u ~ R~F,coRI fragments of DNA from lambdoid bacteriophages and other viruses by aprose-gel electrophoresb, J. Virol., 14 (1974) 1235--1244. Hemhey, A.D., Burgi, F~ and I n g r ~ L., Cohesion of DN& molecules isolated from Idmge lambda, Proe. Natl. A a d . ~ USA, 49 (1963) 748--q§§. Ito, J., Kawnmura, F. and Yanofsky, S., Analysis of @29 and @16 genomes by bacterial restric4/on endonuclesses, EeoRI and//pal, Virology, 70 (1976) 37---51. Ito, J., Bacteriophage ~29 terminal protein: its ~soeiation with the 5' termini of the d29 genome, J. ViroL, 28 (19"/8) 895--904. Melntosh, P.K., Dunker, R., Mulder, C. and Brown, N., DNA of Baeff/us subtilis bacteriophage m~Pl: physiesl nmpping and localization of the origin of replication, J. ViroL, (1978) 866-676. Moreno, F., Camscho, A., Vinuel8, ~ and 8slss, M~, Suppressor-sensitive mutants and genetic map of Baeff/us subh~ bacteriophage ~29, V'uology. 62 (1974) 1--16. Ortin, J., Vasqnez, O., Vinwla, 9. and ~ M., DNA-protein complex in circular DNA from phage ~29, Nature (London) New Biol., 234 (1971) 275--~77. Reilly, B.~.., Tosi, M.B. and Andemon, D.L., Genetic analysis of baeteriophqe ~29 of subtffb: r r m ~ of the "_ek~_ons~ for struetm~ proteins, J. Viroi., 16 (1975) 1010--1016. Riffoy, P.E., Dieelunann, M., Rhodes, C. and Berg, P., Ldtbeling deoxyribonucleic scld to high spe~fie activity in vitro by nick translation with DNA polymeric I, J. Mol. Biol., 113 (1977) 237---251. Rutberg, L., Armentrout, R.W. and Jonuson, J., Unrelatedness of temperate Bacillus subtilb bacteriophage 8P02 and ~10§, J. Virol., 9 (1972) 732--737. 8chef, B., Dean, D.H. and G i n , A.J., Fragmentation of Bacffhas bacteriophage ¢105 DNA by restrict/on endonucleese geoRl: evidence for complementary single-stnnded DNA in the cohesive ends of the moleeule, J. ViroL, 28 (1977) 377--383. Scher, B., Law, M. and Garro, A., Correlated genetic and £coRI cleavage map of Bacfflus subtilis bacteriophage ~106 DNA, J. Viroi., 28 (1978) 395---402. Southern, E ~ . , Detection of specific sequences among DNA fragnwnts separated by gel electrophoresb, J. Mol. BioL, 98 (1975) 503---517. 8patz, H.C. and Trautner, T.A., One way to do experiments on gene conversion? Tranefection with h ~ u p l e z ~ DNA, Mol. Gen. Genet., 109 (1970) 84--106. W'dson, G.A., W'dlianm,M.T., Baney, H.W. and Youn& F.E., Chnraetedzaflon of temperate bacteriophages of Bacillus subtilb by the restriction endonueleme EcoRI: evidence for three different temperate bacteriophages, J. Virol., 14 (1974) 1013--1016. WRson, G.A. and Younq, F.E., Isolation of a sequenewspeeJfie endonucle~e (Baml) from Bacilbas amy/o//quefae/ens H, J. MoL Biol., 97 (1975) 123--125. Yasbin, R.E., W'dson, G.A. aud Y..oung, F.F~, Transformation and trandeetion in lysogenic strains of Bac///us subtff/s 168. J. Bactmiol,, 113 (1973) 540-548. Yasunaka, A., Tsukamoto, H., Okubo, S. and Horiuehi, T., Isolation and properties of suppressor4ensitive mutants of l~¢fflus subtf~ baetm~ophage 81'02, J. Virol., 5 (1970) 819---821. Communicated by W. Szybalski'

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