J. Mol. Biol. (1977) 110, 219-253

C h r o m o s o m e - M e m b r a n e Association in Bacillus subtilis lll~. Isolation and Characterization of a DNA-Protein Complex Carrying Replication Origin Markers K~ZUO Y~L~--J,GUCHI -~WD ~-~TROS]~IY O ~ H mK A W A

Cancer Research Institute, Kanazawa University, Kanazawa, Japan (Received 13 May 1976, and in revised form 2 November 1976) A chromosomal fragment containing purA, a genetic m a r k e r near the replication origin of the Bacillus subtilis chromosome, was found in two different forms. One was t i g h t l y associated with the m e m b r a n e (M-complex) while the other was a complex (S-complex) containing proteins t h a t was easily solubilized during cell lysis. The S-complex h a d a m a r k e d l y higher sedimentation rate (70 to 120 S) t h a n the b u l k of the s u p e r n a t a n t D N A (40 S). High-salt concentrations, Pronase, and ionic detergents reduced the rate to 40 S, indistinguishable from t h a t of the bulk D N A . I t s characteristic sedimentation rate allowed us to isolate a single D N A fragment carrying genetic markers near the origin (e.g. purA) in a highly purified state. F r o m the ratio of purA to hisA (a middle m a r k e r of t h e chromosome) in the purified complex, the p u r i t y of the p u r A - D N A fragment was e s t i m a t e d to be 70 to 80%. No genetic m a r k e r s other t h a n purA a n d those closely linked to it were found in the S-complex. The origin-labelled D N A was concentrated in it b u t the replicating point was not. Lysates obtained without using detergent a n d mechanical shearing yielded the same a m o u n t of S-complex as these t r e a t m e n t s , suggesting t h a t the S-complex is n o t a p a r t i a l l y degraded p r o d u c t of the m e m b r a n e - b o u n d purA-DNA. Biochemical evidence suggests t h a t the complex is an intermolecular aggregate of purA-DNA-protein complex. This was directly proved b y electron microscopic observation of purified S-complex. Aggregates of several D N A molecules (average 3.4) form a structure containing loops, bushes which are sensitive to RNAase, a n d amorphous materials stained black. These were seen b y electron microscopy when the complex was fixed b y glutaraldehyde. The assumption t h a t t h e D N A in t h e S-complex carries a particular region of t h e chromosome containing the purA m a r k e r was confirmed b y site-specific cleavage of t h e D N A with restriction endonucleases. HindIII and HaeII produced at least four a n d t e n m a j o r fragments, respectively, in a b o u t equal molar ratios. I n b o t h cases the sum of the molecular weights of t h e fragments was approxim a t e l y 2 • 107.

1. Introduction P r e c i s e coupling b e t w e e n cell p r o l i f e r a t i o n a n d c h r o m o s o m e r e p l i c a t i o n in b a c t e r i a is a c h i e v e d t h r o u g h r e g u l a t i o n o f t h e i n i t i a t i o n of D N A r e p l i c a t i o n ( u et al., 1964; M a a l o e & K j e l d g a a d , 1966; Cooper & H e l m s t e t t e r , 1968). M a n y r e p o r t s h a v e s h o w n t h a t one or m o r e specific p r o t e i n s a n d R N A s y n t h e s i s h a v e a d i r e c t role in t h e i n i t i a t i o n o f e a c h r e p l i c a t i o n cycle i n b o t h Bacillus subtilis ( u 1965; L a u r e n t , 1973; M u r a k a m i et al., 1976) a n d Escherichia coli ( L a r k & R e n g e r , 1969; Paper I I in this series is Yamaguchi & Yoshikawa (1973). 219

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Ward & Glaser, 1969; Lark, 1972; Messer, 1972; Hiraga & Snitch, 1974). However, little is known either of the chemical nature of these molecules or of the molecular mechanisms of the initiation event. Major difficulties in analyzing initiation seem to reside in the fact that the chromosome is bound to the cell membrane at the replication origin (Sueoka & Quirm, 1968; Snyder & Young, 1969; Ivarie & Pdne, 1970; Fielding & Fox, 1970; Yamaguchi et al., 1971; Yamaguchi & Yoshikawa, 1973), and that the structural integrity of that binding is required for initiation (Worcel & Burgi, 1974). Furthermore, it is assumed that this binding participates not only in the initiation event but also in the synthesis of the cell envelope and the partition of the replicated chromosome which occur following initiation (Jacob et al., 1963). It is therefore essential to determine the mode of association between the replication origin and the cell membrane, and to characterize cellular components which constitute the origin-membrane complex in order to understand the molecular mechanism of the bacterial cell division cycle. Association of both the origin and the terminus of replication with the cell membrane was demonstrated in B. subtilis first by Sueoka & Quinn (1968) and then by Snyder & Young (1969) by genetic and biochemical methods. These authors supposed that these associations are permanent throughout the cell cycle. Their claim seems reasonable if one assumes that the B. subtilis chromosome is circular and replicates unidirectionaUy as a unit, and also binds to the membrane at the origin-terminus junction. Such a binding would serve not only as a regulatory matrix for initiation and termination but also as a means of segregating replicated chromosomes. Contradictory to these assumptions it was found that the B. subtilis chromosome replicates bidireetionally (Wake, 1972; Hara & Yoshikawa, 1973; Harford, 1975; O'Sullivan et al., 1975), resulting in physical separation of the origin from the terminus by two chromosome segments. In accord with the bidirectional mode of replication, the chromosome was found to be associated with the membrane separately at the origin and at the terminus as reported in a previous paper (Yamaguchi & Yoshikawa, 1973). In the light of this finding, the mode of DNA-membrane association at the origin should be re-examined carefully. Because there are two specific binding sites in each chromosome, in addition to the binding at the replication point, it is reasonable to assume that the binding of the origin plays a role only at the time of initiation and therefore is not required to remain intact throughout the cell cycle. It was recently demonstrated that the E. coli chromosome is dissociated from the membrane after replication has been terminated, and then reassoeiates with the membrane before initiation of the next replication cycle (Worcel & Burgi, 1974). However, contradictory findings were also reported (Dworsky & Schaeehter, 1973; Ryder & Smith, 1974; Korch et al., 1976). In this paper we report that a chromosomal segment carrying several genetic markers near the origin was isolated as two different forms. One was tightly associated with the membrane, and the other, a complex containing proteins, was easily solubilized during cell lysis. Purification of the soluble complex using its characteristic sedimentation rate allowed us to isolate a single DNA fragment, containing several genetic markers near the origin, in a highly purified state. Biochemical properties and electron microscopic observation of the complex show that it is an aggregate of a specific DNA-protein-RNA complex and suggest that the DNA fragment contains the replication origin. A prelimiuary note on the isolation and purification of the complex has been published (Yamaguchi & Yoshikawa, 1976).

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I N B. S U B T I L I S

221

2. Materials and Methods (a) Bacterial strains B. subtilis 168-LTT (leu trpC2 thy) (Yoshikawa, 1967) was used t h r o u g h o u t the study. A temperature-sensitive m u t a n t CRK2005 (leu trpC2 thy dna6087) was isolated from 168-LTT b y N-methyl-N'-nitro-N-nitrosoguanidine t r e a t m e n t (100/~g/ml at 34~ for 15 min) as described previously (Yoshikawa & Haas, 1968). The following B. subtilis strains were used as recipients to assay genetic m a r k e r s b y transformation (Hara & Yoshikawa, 1973; Yamaguchi & Yoshikawa, 1975): CRK3000 (leu8 metB5 purA16 hisA3), CRK3003 (metB5 purA16 h/sA3 sacA321), CRK5001 (metB5 purA16 ts56), CRK5002 (metB5 purA16 ts199), CRK2701 (purA16 leu ts8132). (b) M e d i a Medium C + G is m e d i u m CG (Yoshikawa, 1966) with the addition of 0"05% casein hydrolysate a n d 50/~g L-tryptophan/ml. Medium CG-AA is m e d i u m CG supplemented with a m i x t u r e of 15 L-amino acids (Ala, Arg, Asp, Cys, Gly, His, Ile, Lys, Met, Phe, Pro, Set, Thr, T y r and Val, each at 20/Lg/ml). I n m e d i u m lowS-CG-AA used to label cells with asS, (NI-I4)2SO4, MgSO4 and casein h y d r o l y s a t e in m e d i u m C + G were replaced b y NH4C1 (1.62 g/l), MgC12"2H20 (165 mg/1), Na2SO4 (8.86 mg/1, 2/~g/ml S atom) and a mixture of 13 n-amino acids (Ala, Arg, Asp, Gly, His, Ile, Lys, Phe, Pro, Ser, Thr and Val, each a t 20/zg/ml). Medium L P contained the following ingredients per litre: 1-5 g beef extract, 1-5 g y e a s t extract, 5.0 g Bactopeptone, 12.1 g Tris (pH 7.1), 3-5 g NaC1, 140 m g K2HPO4, 60 m g KI-I2PO4 a n d 5 g glucose. All media were supplemented with 50/~g L-leucine/ml, 50 /~g T,-tryptophan/ml and 5/~g t h y m i d i n e / m l unless otherwise indicated.

(c) Chemicals 18.4 Ci/mmol), [5-3H]uridine (5 Ci/mmol) and [2-all]glycerol (500 mCi/mmol) were purchased from the Radiochemical Centre, Amersham. [2-14C]Thymine (51-0 mCi/mmol) a n d HsasSOa were obtained from Daiichi Pure Chemicals Corp. Egg-white lysozyme was from Seikagaku K o g y o Corp. Pronase (B grade) was from Calbiochem and was autolysed at 37~ for 3 h. Bovine pancreatic R N A a s e A was from Sigma Chemical Corp. a n d was t r e a t e d a t 80~ for 15 rain to inactivate a contaminating DNAase. Triton X100 a n d Brij58 were from N a k a r a i Chemicals a n d Atlas Chemical Industries, respectively. Sarkosyl NL97 was a gift of Geigy I n d u s t r i a l Chemicals. 6-(p-hydroxyphenylazo)-uracil was k i n d i y provided by Dr B. W. Langley of I m p e r i a l Chemical Industries. Nalidixic acid was a gift from D r H. K o g a of Daliehi Seiyaku Corp. N - m e t h y l - N ' - n i t r o - N - n i t r o s o g u a n i d i n e was from Aldrick Chemical Corp. Glutaraldehyde (grade for electron microscopy), formamide a n d eytochrome c (type VI) were from Tokyo Kasei Industries, W a k o Pure Chemicals Industries, and Sigma Chemical Corp., respectively. Agarose and ethidium bromide were from Seakem a n d Boots Pure Drug Corp.

[methyl-aI-I]Thymidine (15.7 to

(d) Radioactive labelling To label DNA, cells were cultured in m e d i u m C + G or L P containing [3H]thymidine (0.5 /~Ci/5 /~g per ml) or [14C]thymine (0.3 /~Ci/2.5 /~g per ml) unless otherwise specified. To estimate lipid, cells were grown in m e d i u m L P with [all]glycerol (3/zCi/100/~g per ml). To label cellular proteins with asS, cells were grown in m e d i u m IowS-CG-AA containing H~asSO4 (40/~Ci/ml).

(e) Preparation of cell lysates (i) Lysis by lysozyme-Brij58 E x p o n e n t i a l l y growing cells (4 • 107 to 5 • 107 colony formers/ml, 40 to 50 K l e t t units) were h a r v e s t e d on a m e m b r a n e filter (Sartorius, pore-size 0.45 ~ n ) , washed with T K E 1 buffer (20 mM-Tris'HC1 (pH 8.1), 0-1 •-KC1 and 1 m ~ - E D T A ) a n d resuspended (2.5 • 109

222

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H. Y O S H I K A W A

to 3"1 • colony formers/ml) in the same buffer containing 2 0 m ~ - N a N s , 10m•-2mercaptoetha~ol and 1 mg egg-white lysozyme/ml. The cell suspension grown in L P medium was incubated at 37~ for 5 to l0 rain, while the cell suspension gro~n in m e d i u m C~-G was incubated for l0 to 15 rain. During the incubation, cells retained a rod shape b u t most of the cytoplasmic material was seen to be released from cells under phase contrast microscopy. The lysate was then mixed with 0.2 vol. 5% Brij58, incubated for an additional 1 to 2 m i n and quickly cooled in ice water. The resultant viscous lysates were sheared b y passing l0 times through a hypodermic needle (1.2 m m in diameter). (ii) Lys/s without the detergent Two different methods were used. I n one method, protoplasts were prepared as described previously (Yamaguchi et al., 1971) and harvested b y centrifugation at 6000g for 10 min at 2~ The protoplasts were lysed b y mixing them ~ i t h TKE1 buffer containing 10 mM-2-mercaptoethanol. Shearing of the lysate was as described above. The other method was the same as the lysis with lysozyme-Brij 58 except for a prolonged incubation (10 to 15 rain for cells grown in medium LP) with lysozyme a n d without the Brij-treatment. (f) Purification of the S-complex The cell lysate (2 to 6 ml) was layered on top of 24 ml of a chilled 5% to 20% linear sucrose gradient in T K E 1 buffer with a shelf of 6 ml of 64~/o sucrose in the same buffer and centrifuged in a Becl~nan SW27 rotor at 20,000 revs/min for 45 m i n at 2~ Two-ml fractions were then collected from the top of the tube b y a ISCO fractionator. Supern a t a n t fractions (4 to 14 ml from the top of the gradient) were pooled, dialysed twice against 5 0 0 m l of PKE1 buffer (20mM-sodium phosphate, 0.1 M-KC1, 1 mM-EDTA, p H 7.0) containing 5 mM-2-mercaptoethanol in an ice bath for about 4 h, and concentrated b y burying the solution, which was inside a dialysis tube, in Sephadex G50 powder. The concentrated s u p e r n a t a n t fraction (2 to 4 ml) was layered on 28 ml of a 10~o to 30% sucrose gradient in P K E 1 buffer with a shelf of 2 ml of 64% sucrose in the same buffer and was centrifuged in the SW27 rotor at 25,000 revs/min for 4 h at 2~ Twenty-five fractions, each of 1.2 ml, were collected from the top of the tube. As described in Results, fractions which show high transforming activities for purA were pooled, dialysed against PKE~ buffer containing 5 mM-2-mercaptoethanol and concentrated as above. The resultant crude S-complex fraction was again sedimented through a sucrose gradient under the same conditions as the second sucrose gradient centrifugation. I f necessary, further purification of the S-complex was achieved b y rebanding it in the fourth gradient under the same conditions as the second gradient. (g) Radioactive labelling of the replication point Cells growing exponentially in 50 ml of medium L P containing [14C]thymine (0.2 ~Ci/2.5 ~g per ml) for several generations were pulse-labelled for 10 s at 34~ with [ZH]thymidine (10 ~Ci/ml). The incorporation was stopped b y pouring the culture into 50 g of frozen TKE1 buffer containing 50 mM-NaN3. (h) Radioactive labelling of replication origin The 168-LTT spores were prepared b y the methods reported previously (Yoshikawa, 1965}. Spores (3.3 • 10~~ were activated b y heating at 70~ for 15 rain and were germinated in 120 ml of medium CG-AA with 7.5 ~g nalidixic acid/ml and without t h y m i n e for 100 m i n at 30~ They were then collected on a m e m b r a n e filter (pore-size 0.45 ~m), washed with 150 ml of the prewarmed medium CG-AA lacking thymine, resuspended in 60 ml of the same medium and then divided into 2 parts. One part (40 ml) was mixed with [3H]thymidine (1.0 mCi) and unlabelled thymidine (20 ~g) to label the replication origin, and further incubated at 30~ After 5 or 10 min, a 20-ml sample of the culture was mixed with a n equal volume of the prewarmed medium containing 2 mg unlabelled thymidine/ml and immediately the cells were washed on a m e m b r a n e filter with 100 ml of the prewarmed culture lacking thymidine. Cells were then resuspended in 60 ml of medium L P containing [14C]thymine (0"1 ~Ci/2.5 ~g per ml) and further incubated for

DNA-PROTEIN

COMPLEX IN B. SUBTILIS

223

3 h at 30~ To pulse-label chromosomal fragnlents other t h a n the one containing the origin, as a control, the other part (20 ml) was incubated with 5 ~g tmlabelled t h y m i d i n e / m l for 30 rain and filtered through a m e m b r a n e filter. The collected cells were labelled first with [3H]thymidine for 5 m i n and then with [14C]thymine for 3 h as described above. The 5-min pulse at the onset of DNA replication during germination labelled less t h a n one-fortieth of the chromosome since completion of the first replication cycle in germinating spores takes more t h a n 3 h under the conditions used (i.e. in medium CG-AA at 30~ (i) Transformation Transforming DNA was extracted from samples obtained from various fractions in sucrose gradients with phenol as reported by Saito & Mittra (1963). Samples were mixed with a n equal volume of 0.2 M-Tris.HC1 (pH 9.0)-2% sodium lauryl sulphate followed b y incubation at 50~ for 5 rain. They were shaken gently with an equal volume of phenol and then the aqueous layer was dialysed twice against 1 1 of 0.15 M-NaC1-0.015 M-sodium citrate. Transformation was carried out b y the method described previously (Haas & Yoshikawa, 1969b) with a minor modification. Recipient cells were grown in medium C-i- G until just before the stationary phase at 37~ Cells were then frozen in liquid nitrogen with 20% glycerol until used. Quicldy thawed cells were diluted 10-fold with medium C-~G', which is medium CG supplemented with 0.01% casein hydrolysate, 5 ~g L-tryptophan/ml, 5 ~g r.-histidine/ml and 100/~g adenine/ml. After cells were grown for about 2 h until over 40 Klett units, 0.05 ml of the DNA solution was added to 0-5 ml of competent cells and incubated for an additional 60 min. Transformed cultures were appropriately diluted in medium CG and plated on selective media. Relative marker frequencies of samples were normalized b y that of spore DNA. (j) Molecular weight of D N A To determine the molecular weight of DNA, 0.5-ml samples from sucrose gradients were incubated with 200/~g Pronase/ml in P K E 1 buffer at 37~ for 5 m i n and then with 1 ~ sodium lattryl sulphate for a n additional 20 min. They were dialysed against 500 ml of 0.15 M-NaC1-0.015 M-sodium citrate-1 mM-EDTA at room temperature and t h e n a 0.2-ml sample was layered on top of 4"8 ml of a 5% to 20% linear sucrose gradient in 0.15 M-NaC1-0.015 M-soditun citrate-1 mM-EDTA. Centrifugation was carried out in a Beckman SW50.1 rotor at 40,000 revs/min for 3 h at 10~ Ten-drop fractions were collected from the bottom of the tube. aH or 14C-labelled DNA from a defective phage P B S H (12 • 106, Mz.) (Haas & Yoshikawa, 1969a) was used as an internal marker. (k) Assay of radioactivity Radioactivity in D N A or protein was assayed b y collecting cold trichloroaeetie acid precipitates after overnight incubation with 1 N-NaOH at 37~ on W h a t m a n GF/C glass fibre filters. The filters were placed in toluene scintillation fluid containing 5 g P P O (2.5-diphenyloxazole)/1 and were counted in a Becl~man LS230 liquid scintillation counter. Bacterial lipids extracted with chloroform/methanol/buffer mixttu'es according to the procedure of Bligh & Dyer (1959) were di'ied in scintillation vials and cotmted as above. (1) Electron microscopy Fractions from sucrose gradients were treated with 0.1% glutaraldehyde for 30 m i n at 30~ to link proteins covalently to D N A and then dialysed twice against 1000 ml of PKE1 buffer to remove sucrose and glutaraldehyde. The samples were spread b y the cytochrome monolayer technique described b y Davis et al. (1971). Ten-/A samples were mixed with 10 ~1 of 1 M-Tris.I-IC1 (pH 8"5), 10/~1 of 0.1 M-EDTA (adjusted with N a O H to p H 8.5), 20 ~l of water, 40 ~1 of formamide and 10 ~l of cytochrome c (1 mg/ml), a n d were spread on a hypophasc containing 20% (v/v) formamide, 0.01 ~-Tris.HC1 (pH 8.5), and 1 mM-EDTA. The cytochrome film was picked up on Parlodion-coated copper grids (150 mesh), stained with 5 • 10 -5 ~ - u r a n y l acetate a n d rotary shadowed with p l a t i n u m palladium. Electron micrographs were taken with an electron microscope model JEM-100B (JEOL Corp.) at a magnification of 8000 • The length of the DNA molecules was detel~nined 15

224

K. Y A M A G U C H I A N D H. Y O S H I K A W A

b y tracing figures magnified on a 1Wikon Profile Projector, model 6C, with a m a p measurer using D N A from a defective phage, P B S H (12 x 106, Mr), as a reference (Haas & Yoshikawa 1969a). (m) Cleavage of DNA by restriction endonueleases The pm'ified S-complex was incubated at 37~ with 10 ~1 pancreatic R N A a s e / m l for 5 min and then with 50 ~g Pronase/ml and 1 ~ sodium lam'yl sulphate for an additional 15 min. I t was mixed with 0"1 vol. 1 M-Tris.HCI-I M-NaC1 (pH 9.0) and shaken gently with an equal volume of phenol s a t u r a t e d with 0.1 M-Tris.HC1-0.1 M-NaCI-1% sodium sulphate ( p i t 9.0). The aqueous layer was adequately dialysed against 50 mM-Tris.HC1-0.1 mM-NaC1 ( p i t 7.4). W h e n necessary, the D N A was concentrated b y precipitation with 2 vol. cold ethanol. Restriction endonucleases, Ecol%I, HindIII and HaeII%, were k i n d l y provided b y Dr M. Takanami. D N A (1 to 3 ~g) was digested with 1 to 3/A of HindIII or HaeII in 10 mM-Tris.HC1 ( p i t 7.5)-7 mM-MgC12-7 mM-2-mercaptoethanol at 37~ for 2 h. One /A of HindIII or HaeII was sufficient to digest 10 ~g of phage l a m b d a D N A completely at 37~ for 1 h. W h e n EcoRI was used, 0" 1 M-NaC1 was a d d e d to the above reaction mixtm'e. Specific D N A fragments generated b y restriction endonucleases will be referred to as EcoRI-fragments, HindIII-fragments and HaeII-fragments. (n) Agarose-gel electrophoresis Agarose-gels (14 cm X 0.6 cm) were prepared in cylindrical glass tubes as described b y Helling et al. (1974). A sample containing 50 ~1 of digested D N A and 5 ~1 of 0.05% bromophenol blue in 80% sucrose was layered on a 0.7~ agarose-gel in T r i s / a c e t a t e / E D T A buffer (40mM-Tris-acetic acid, 20mM-sodium acetate, 2mM-Na2EDTA, pH8-05). Electrophoresis was done first at 100 V for 5 rain and thereafter at 1.5 V/cm of gel (21 V) for 15 h at room t e m p e r a t u r e (approx 24~ Buffer chambers contained T r i s / a c e t a t e / E D T A buffer. After the rtm the gels were soaked in 0.5 ~g ethiditun bromide/ml for 1 h to stain the DNA. Gels were photographed under long wavelength ultraviolet light (UVL56, Ultraviolet Products Inc.) using a yellow filter. Six EcoRI-fragments of l a m b d a D N A (provided b y Dr M. Takanami) were used as standards to estimate the molecular weights of the D N A species. I n order to count r a d i o a c t i v i t y in DNA, gels were cut into slices of 1 to 3 m m width and crushed with 0"5 ml water in vials to which a scintillation fluid (5 g PPO/1000 ml toluene/500 ml Triton X100) was added. The vials were shaken overnight at room t e m p e r a t u r e before counting.

3. Results (a) Sedimentation profile of D N A carrying p u r A marker (an origin marker) W h e n a l y s a t e was p r e p a r e d from e x p o n e n t i a l l y growing cells b y t h e l y s o z y m e Brij58 t r e a t m e n t a t p H 8.1, s h e a r e d b y p a s s i n g t h r o u g h a needle a n d s e d i m e n t e d t h r o u g h a sucrose d e n s i t y g r a d i e n t (for d e t a i l s see M a t e r i a l s a n d Methods), we o b t a i n e d a f a s t - s e d i m e n t i n g m e m b r a n e o u s m a t e r i a l w h i c h c o n t a i n e d 5 to 1 0 % of t h e t o t a l cellular D N A a n d in w h i c h m a r k e r s n e a r t h e r e p l i c a t i o n origin, i.e. purA, o c c u r r e d p r e f e r e n t i a l l y to m i d d l e m a r k e r s such as hisA ( Y a m a g u c h i et al., 1971 ; Y a m a g u c h i & u 1973). This f r a c t i o n was d e s i g n a t e d t h e M - c o m p l e x a n d its p a r t i a l c h a r a c t e r i z a t i o n has been r e p o r t e d p r e v i o u s l y ( Y a m a g u c h i & Y o s h i k a w a , 1973). I n s u m m a r y : (1) t h e i n c r e a s e d e n r i c h m e n t for purA was o b s e r v e d as t h e s h e a r i n g force increased, (2) t h e a s s o c i a t i o n of b o t h origin a n d t e r m i n u s D N A w i t h t h e m e m b r a n e o u s m a t e r i a l was n o t affected b y w a s h i n g t h e c o m p l e x w i t h E D T A or non-ionic d e t e r g e n t s . Thus, m a r k e r s l o c a t e d n e a r t h e origin a n d t e r m i n u s were r e t a i n e d in t h e c o m p l e x while m a r k e r s l o c a t e d on t h e o t h e r p a r t s of t h e b i d i r e c t i o n a l l y r e p l i c a t i n g c h r o m o s o m e were r e a d i l y released from t h e complex. From E. coli, Haemophilus influenzae and H. aegyptlus, respectively.

D I ~ A - P R O T E I N C O M P L E X I N B. S U B T I L I S

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D u r i n g t h e course of these studies we f o u n d t h a t , after cell lysis a n d shearing, a p p r o x i m a t e l y half of t h e t o t a l cellnlar purA m a r k e r was released i n t o a s u p e r n a t a n t f r a c t i o n of t h e sucrose g r a d i e n t as r e a d i l y as m i d d l e m a r k e r s (Fig. 1). Solubilization of m e m b r a n e - a s s o c i a t e d D N A m a y occur if u n a s s o c i a t e d p o r t i o n s of t h e m e m b r a n e b o u n d D N A are f r a g m e n t e d b y shearing. I n fact, middle m a r k e r s a n d some of t h e

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I

Fraction no.

FIG. I. Distribution of purA, an origin marker, and ofhisA, a middle marker, in sucrose gradients. Cells grown in 100 ml of C + G medium were uniformly labelled with [3H]thymidine and lysed by lysozyme-Brij58 as described in Materials and Methods. The cell lysate (2 ml) was centrifuged through a 10% to 30% linear sucrose gradient in TKE1 buffer (pH 8.1) with a shelf of 64% sucrose for 45 min at 20,000 revs/min in an SW27 rotor, and 2-ml fractions were then collected from the top of the tube. Each fraction was assayed for 3H radioactivity in DNA (- - (D - - C) - -) and transforming activity for purA ( - - A - - A - - ) and hisA ( - - A - - A - - ) using CRK3000 as a recipient. DNA in the upper part of the gradient (fractions 1 to 4) was extracted with phenol before the transformation assay because lysozyme present in these fractions inhibits transformation. The relative numbers of transformants, 1 unit for purA and hisA, were 1 • 105 and 5 • l04, respectively. Fractions 1 to 4 were collected (designated as the soluble fraction) and used for further experiments. t e r m i n a l m a r k e r s which s e d i m e n t i n t h e M-complex are released to t h e s u p e r n a t a n t as t h e shearing force is increased. U n l i k e those markers, the a m o u n t of purA i n t h e s u p e r n a t a n t was n o t affected b y increasing t h e shearing force (see Fig. 1 i n Y a m a g u c h i & Yoshikawa, 1973). The u n i q u e n a t u r e of t h e purA m a r k e r in t h e s u p e r n a t a n t f r a c t i o n was clearly shown w h e n it was r e s e d i m e n t e d i n a sucrose g r a d i e n t at a higher centrifugal force. F i g u r e 2(a) shows t h a t D N A c a r r y i n g purA m a r k e r (purA-DNA) sedim e n t s more r a p i d l y t h a n other D N A , for e x a m p l e D N A c a r r y i n g t h e m i d d l e m a r k e r hisA. I n this F i g u r e t r a n s f o r m i n g activities i n fractions 3 to 12 are n o t s h o w n because t h e y are c o m p l e t e l y i n h i b i t e d b y lysozyme a n d Brij58 c o n t a m i n a t i o n i n these fractions. Therefore, t r a n s f o r m i n g activities i n these fractions were assayed after e x t r a c t ing D N A w i t h p h e n o l from each fraction. O n l y 10 to 2 0 % of t h e t o t a l purA a c t i v i t y

SlLIDU.IJOJSUDJJ, ~0 'OU ~A!J,DIS~ OD o

r

T

r

r

I

o

!

0

I

~,7~ ~

~

~.4

41

"o

0

0

,~

0

0

6

6

I

I.

I--'--

~

~

~ ~

~ ~ ~...0 t/0

~=~-i -~ -

2

" ~ ~ ~"'o--- ~

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/_

"6

o .9

or

2

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.'O"Ox

-

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20

15

I0 Fraction

5

I

no.

FIG. 11. Isolation of the S-complex from the cell lysate prepared without Brij58. Exponentially growing cells in L P medium containing [aH]thymidine were lysed (a) by an osmotic shock of protoplasts or (b) b y lysozyme alone as described in detail in Materials and Methods. Crude S-complex fractions were prepared from these lysates by two centrifugations in the sucrose gradient as described in the legend to Fig. 2 and t h e n centTifuged through 10% to 30% linear sucrose gradients at 25,000 revs/min for 4 h. - - O - - O - - , aH radioactivity in DNA; - - A - - A - - , T u f a (l = 1 x 104); - - A - - A - - , h/aA (1 = 1 x 10a).

242

K. YAMAGUCHI

AND H. YOSHIKAWA

FIG. 12. Electron micrograph of the S-complex. The S-complex fraction after the third sucrose gradient centrifugation was prepared from cells grown in L P medium as described in the legend to Fig. 2. After being fixed with 0.1~/o glutaraldehyde for 30 rain at 30~ the sample was spread with cytochrome c by the m e t h o d of Davis e~ aL (1971). A bar in the Figure represents 1 ~m.

D N A - P R O T E I N COMPLEX I N

B. SUBTILIS

243

molecules represented the structure of the S-complex. Figure 12 shows t h a t an aggregate displayed several loops of D N A near the centre, where bushes and often a mass stained black were seen. Some of the D N A strands stretched out from the centre were very short in length, i.e. 78 out of 193 strands measured were less than 2.5 ~m long from the centre area to the ends. This suggests t h a t D N A molecules are not joined end to end because D N A isolated from the purified complex had a molecular weight of 2.9 to 4.0 • 107 and the D N A in the complex was seen to have a 17.3/zm length (a mean value for 20 molecules observed) when they were not fixed with glutaraldehyde. Table 3 summarizes the distribution of DNA molecules according to TABLE 3

Summary of electron microscopic observation of S.comTlex

Preparation~

Free DI~A S-complex S-complex (d- RNAase) S-complex (in PE1 buffer)w

Single molecule Aggregate Stretched Entangled With bush Without bush

80 105

0 9

36

3

30

13

0 33 (2) 2

Total no. No. of of DNA DNA molecular molecules molecule ends forming aggregate aggre- aggregate gates$

0 5

-6.5

-127

-3.3

11

6.8

46

3.5

3

5-0

13

2.6

S-complex and its treatments are described in the legend to Figs 12 to 14. Free DNA is represented by fractions 7 to 9 in Fig. 2(b). :~ Number of DNA molecules forming aggregates was calculated from the number of duplex ends assuming that DNA molecules were joined side by side to each other. wPE1 buffer contains 20 m~-phosphate (pH 7.0) and 1 m~-EDTA. their morphology seen in electron mierographs. The number of molecules in a given aggregate was calculated assuming t h a t D N A molecules were joined side b y side, i.e. if five strand ends were counted in a given aggregate, it was assumed to contain three molecules. On this assumption 53~ (127/127d-105d-9) of the D N A molecules observed in the S-complex fraction formed aggregates. The bushes near the centre of the aggregates were eliminated b y treating them with pancreatic RNAase suggesting t h a t t h e y are mainly R N A (Fig. 13 and Table 3). Some single molecules in the S-complex fraction were also seen to have an unusual entangled structure as shown in Figure 14. Dialysis of the S-complex against the phosphate buffer lacking KC1 increased the number of these entangled single molecules in parallel with reducing the number of aggregates. These results support our assumption that the S-complex is an aggregated form of the lourA-DNA-protein complex and that it is dissociated into a monomeric complex at low-salt concentrations. (vii)

Site-specific cleavage of DNA from the S-complex by restriction endonucleases,

From the evidence described above, we suppose t h a t D N A molecules in the Scomplex (eDNA) contain a common D N A segment on which the purA marker is located. However distal parts of the molecules are not expected to be identical due to

244

K. YAMAGUCHI AND H. YOSHIKAWA

FIG. 13. S-complex treated with ribonuclease. The purified S-complex fraction (the same as in Fig. 12) was incubated with 20 ~g pancreas ribonuelease A]ml for 15 rain at 30~ before fixation with glutaraldehyde. a random shearing during preparation. Such a DNA population is schematically shown in Figure 15. I f the common region is large enough to produce a multiple number of specific fragments generated by a site-specific restriction endonuclease, such fragments would be predominant in quantity and detectable as major fragments with an equal molar ratio. To test this assumption, three [ZH]sDNA preparations with different purity of p u r A - D N A were digested b y HindIII, H a e I I or EcoRI. Figures 16 and 17 show, respectively, HindIII and HaeII fragments separated b y agarose-gel electrophoresis. A sheared whole DNA from the same strain was used as control. In both cases, sDNA produced several fragments of relatively high molecular weight. The higher the purity of the IDurA-DNA, the more clearly the fragments were seen, Tables 4 and 5 present molecular weights and molar ratios of the major HindIII and H a e I I fragments derived from sDNA. In both cases, as the purification o f p u r A DNA proceeded, molar ratios of relatively smaller fragments (e.g. bands 7 and 8 of

DNA-PROTEIN

COMPLEX

I N B. S U B T I L I S

245

r

FIG. 14. S-complex in a low concentration of KC1. The purified S-complex fraction (the same as in Fig. 12) was dialysed overnigh% against 20 m~phosphate-1 m ~ - E D T A (pH 7.0) and then fixed with glutaraldehyde.

HindIII fragments and bands 10 to 12 and probably 13 to 16 of Ha~II fragments) tended to decrease, resulting in an increase in the number of equim01ar fragments. This may be due to the fact that a large number of small fragments derived from contaminating DNA molecules in the complex increased backgroundlevels of the specific fragments of similar molecular sizes. Therefore, it is plausible that radioactivities in small specific fragments derived from the common region are overestimated even for sDNA of the highest purity. Thus, we tentatively assumed that bands 2, 5, 7 and 8 in H~ndIII fragments and bands 6, 7, 9, 10, 11, 12, 13, 14, 15 and 16 in HaeII

I t I I

I I I I i I I I I I

I I I I I

Io

I

I I I I I I I I I I I

t I I I I I I I I I I

I I I t I I I I I I I

I I I i I I I I I I

3~4x10 7

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I t I I I

! ~ 2 x I0

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Fro. 15. Schema of DNA molecules forming S-complex. Solid lines are DNA strands. Broken lines show cleavage-sites by a restriction endonuclease. A

B

C

D

E

F

2

5 -

? 8 --

3

2

:FIG. 16. Eleetrophomsis of HindYlI fragments from sDNA in 0"7 ~o agarose-gels. A, L a m b d a D:NA (0.5/~g); B, sheared whole cellular DNA from 168-LTT (3"2/~g); C, sDNA-I (1.3-/~g); D, sDNA-II (2.3/~g); and E, s D N A - I I I (2.4/~g) were digested b y H/nflTTT a t 37~ for 2 h. F, EcoRI fragments derived from ]ambda DNA.

COMPLEX

DNA-PROTEIN

A

B

C

D

247

I N B. S U B T I L I S

E

F

10

6

q

7

_o

9 - -

x

I0-II 12

- -

13

- -

- -

14 - 1 5 - 16

- -

, 4 84

FIG. 17. Electrophoresis of HaeII fragments from sDNA in 0.7 ~o agarose-gels. A, Lambda DNA (0.5 ~g); B, sheared whole cellular DNA from 168-LTT (3.2 pg); C, sDNA-I (1.3 ~g); D, sDNA-II (2.3 tzg); and E, s D N A - I I I (2.4 pg) were digested by HaeII at 37~ for 2 h. F, EcoRI fragments from ]ambda DNA.

248

K. YAMAGUCHI

AND H. YOSHIKAWA

TABLE

4

HindlII fragments from sDNA Band a 1 2

Mr ( • 106) 8.2 6"45

Radioactivity, cts/min (molar ratio) b sDNA-Ic sDNA-IId sDNA-IIIe --f 161 (1.00)

3

5.75

--

5 7 8

4.85 3.3 2.95

124 (1-02) 244 (2.96) 196 (2.66)

Total

186 606 375 484 735 627

5530

(0.24) (I.00) (0.69) (1.07) (2.37) (2.26)

13,030

180 925 349 547 614 679

(0.15) (1.00) (0.42) (0.79) (1.30) (1.60)

13,650

a Designated in Fig. 16. b Expressed as molar ratio to band-2. Prepared from 168-LTT grown in L P medium. The purA/hisA of this preparation was 47-9 and purity of purA-DNA was estimated as 41% (see Discussion). a Prepared from 168-LTT grown in L P medium. The purA/hisA and the purity of this preparation were 114 and 62~o, respectively. . Prepared from GRK2005 (dna6087) grown in L P medium. The purA/hisA and the purity of this preparation were 200 and 72%, respectively. t Not determined.

TABLE

Hae//fragments Band t 2 3 4 5 6 7 9 10 11 12 13 14 15 16 Total

Mr ( • 106) 5.7 4-9 4-7 4.1 3.95 3.2 2-85 2-35 2-3 2-1 1-6 1-47 1-40 1.24

5

from sDNA

Radioactivity, cte/min (molar ratlo)~ sDNA-I sDNA-II sDNA-III

183 105 214 197 172 155 236 245 192

8290

Designated in Fig. 17. Expressed as molar ratio to band-9. w Not determined,

--w (0.69) (0.41) (0.95) (0.92) (0.98) (1.00) (1.83) (1.95) (1.67) ---

223 416 299 473 585 645 560 600 554 394

(0-21) (0.43) (0.32) (0.59) (0.76) (1.03) (1.00) (1.30) (1.23) (0.96) ---

--

--

--

-17,310

202 248 324 460 764 548 682 604 542 444 552 528 538 776 17,830

(0.15) (0.21) (0.29) (0.47 (0.81 (0.72 (1.00 (1.07 (0.98 (0.88 (1.44 (I.50 (1.61 (2.62)

DNA-PROTEIN COMPLEX IN B. ~ U B T I L I S

249

fragments are derived from the common DNA segment containing purA marker. Total molecular weights of these fragments are 17.6 • 106 and 22.5 • 106, respectively. These indicate that at least half of the DNA molecule is common to all DNA in the S-complex (Fig. 15). When the sDNA was digested by EcoRI, 6 fragments (total 18 • daltons) were observed, but they are not so well separated from minor fragments as to determine their molar ratios. 4. Discussion A genetic marker, purA was estimated to be located on the B. subtilis chromosome within 1 to 2% of the whole chromosome length from the origin (O'Sullivan & Sueoka, 1967; Hara & u 1973). Therefore purA is located within 2 • 107 to 5 • daltons from the origin if the molecular weight of the B. subtilis chromosome is assumed to be 2.0)