The effect of lysozyme on DNA—Membrane association in Escherichia coli

149 Biochimica et Biophysica A cta, 366 (1974) 149--158

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98103 T H E EFFECT OF LYSOZYME ON DNA--MEMBRANE ASSOCIATION IN ESCHERICHIA COLI

SAMUEL SILBERSTEIN and MASAYORI INOUYE Department of Biochemistry, State University of New York at Stony Brook, Stony Brook, N.Y. 11790 (U.S.A.)

(Received January 10th, 1974} (Revised manuscript received May 21st, 1974)

Summary Th e c h r o m o s o m e of Escherichia coli sediments three different ways in sucrose density gradients, depending upon the c o n c e n t r a t i o n of lysozyme used to lyse the cells, as follows: (1) Low-S complex. In the presence of 2--400 pg/ml of egg white lysoz y me with Brij-58-Na d e o x y c h o l a t e or Triton X-100, the c h r o m o s o m e sediments at 5 0 0 - - 1 1 0 0 S (low-S complex). When Brij-58 alone is used, some of the DNA sediments as a low-S com pl e x and some sediments rapidly with the membrane fraction. (2) S eco n d ar y D N A - - m e m b r a n e complex. In the presence of 800 pg/ml of egg white ly s o zy m e, all the DNA sediments with the m em brane fraction at greater than 4000 S regardless of which detergent is used. This DNA--membrane c o m p l e x is due to the high c o n c e n t r a t i o n of lysozyme, for when a pre-formed low-S c om pl ex is exposed to a high c o n c e n t r a t i o n of lysozyme, it sediments with the m e m b r a n e fraction. This association can also be seen in the presence o f poly(L-lysine), ribonuclease, Mg 2÷ or spermidine, indicating that l y s o z y m e mediates the D N A - - m e m b r a n e association non-specifically as a polycation. An artificial association be t w een exogenous coalfish DNA and an E. coli m e m b r a n e fraction is induced by 800 pg/ml of lysozyme. (3) Primary D N A - - m e m b r a n e complex. A c o n c e n t r a t i o n of T4 l ysozym e as low as 0.01 pg/ml, or 20 l y s o z y m e m o l e c u l e s / c h r o m o s o m e , can lyse cells. This is in contrast to 2 • 106 l y s o z y m e m o l e c u l e s / c h r o m o s o m e corresponding to 800 pg/ml of egg white lysozyme. Under these mild lysis conditions, the c h r o m o s o m e sediments with t he m e m b r a n e fraction, suggesting a primary D N A - - m e m b r a n e association.

Introduction It has been r e p o r t e d by m a n y authors [1--3] t hat bacterial c h r o m o s o m e s

150 are associated with membranes. Newly synthesized DNA as well as the origin and terminus of replication [4--7[ have been shown to be preferentially attached to membrane fractions. It has also been reported that the Escherichia coli chromosome is attached to the membrane at multiple attachment sites [8--10]. On the other hand it can also be isolated as a folded complex of 3200 S [11] or 1300--2200 S [12] without any apparent association with the membrane. In all these experiments, egg white lysozyme is used to lyse the cells. However, because it is a basic protein, lysozyme is known to bind to DNA [13] and to mediate a ribosome--membrane complex [14[. Furthermore, Fuchs and Hanawalt found lysozyme in a DNA "growing p o i n t " complex [6]. We have examined the effect of different concentrations of egg white lysozyme, using three different detergents, on the sedimentation of the E. coli chromosome. We found that the chromosome sediments three different ways depending on the lysis conditions. A new method for the isolation of the primary DNA--membrane complex has been developed in order to avoid the secondary effects of a high lysozyme concentration. Materials and Methods

Preparation of a double-labeled culture E. coli strain MX74T2 thy- was grown to stationary phase in M9 minimal salts medium supplemented with 2 pg/ml thymine, 2 pg/ml vitamin B1, 4 mg/ml glucose and 0.8 mM MgSO4. 1 ml of this culture was diluted into 10 ml of M9 medium supplemented with 2 pg/ml thymine, 2 pg/ml vitamin B1, 40 mg/ml glucose and 0.8 mM MgC12 with no SO4~- present. Whe the cells have grown to a concentration of about 4 . 1 0 7 cells/ml, 8 pCi of [ a l l ] t h y m i n e and l pCi of carrier-free H23sSO4 were added. At about 1.5 • l 0 s cells/ml, 0.2 M disodium EDTA, pH adjusted to 7--8, was added to a final concentration of 2 mM and the cells were centrifuged at 5000 × 3" for 5 min at 4 ° C. Lysis procedure The lysis procedure, basically that of Stonington and Pettijohn [11], is as follows: A b o u t 1 . 5 . 1 0 9 cells were resuspended in 0.2 ml of 20% sucrose, 0.1 M NaC1, 0.01 M Tris--HC1, pH 8.2, and 0.01 M NaN3. To the cell suspension, 0.05 ml of egg white lysozyme (Sigma) of varying concentrations or 0.05 /~g/ml T4 lysozyme in 0.05 M disodium EDTA, 0.12 M Tris--HC1, pH 8.2, was added and the mixture incubated for 5 min at room temperature. Then 0.25 ml of either Brij-58 (final concentration, 0.5%), Brij-58 (0.5%) plus sodium deoxycholate (0.2%), or Triton X-100 (0.1%) in 10 mM disodium EDTA, pH adjusted to 8.2, was added. The mixture was further incubated for 20 min at 30°C. Poly(L-lysine) or ribonuclease A were added together with Triton X-100 to a final concentration of 1 mg/ml and 50 #g/ml, respectively. Great care was taken to prevent shearing the DNA. Pulse labeling of the growing point E. coli MX74T2 was grown in 20 ml M9 medium supplemented with 2 pg/ml t h y m i n e , 2 pg/ml vitamin B1, 4 mg/ml glucose and 0.8 mM MgSO4.

151 When the cell c o n c e n t r a t i o n grew to a b o u t 4 • 107 cells/ml, 2 pCi of [ 14C] thymine was added. At a b o u t 1.5 • 108 cells/ml, the cells were collected by centrifugation at 10 000 rev./min for 10 min. T h e y were resuspended in I ml M9 medium prewarmed to 30°C, supplemented as described above except for 0.5 t~g/ml t h y m i n e and 100 pCi [ 3HI t h y m i n e instead of 2 pg/ml t h y m i n e . The cell suspension was incubated for 30 s at 30°C and 0.5 ml was transfered into a test-tube containing 0.025 ml of 0.2 M EDTA--0.2 M NaN3 on ice. T o the remaining 0.5 ml of the cell suspension was added 10 ml M9 containing 20 pg/ml th y mine. The cells were collected by centrifugation at 10 000 rev./ min for 10 min and resuspended in 10 ml M9 medium supplemented with 20 pg/ml t h y m i n e , 2 pg/ml vitamin B1, 4 mg/ml glucose and 0.8 mM MgSO4, and incubated at 37°C for a not he r 30 min to a c o n c e n t r a t i o n of about 2.2 • 108 cells/ml. After the incubation, 0.5 ml of 0.2 M EDTA--0.2 M NaN3 was added. In b o t h pulse and pulse--chase experiments, the cells were centrifuged at 5000 rev./min for 5 min and resuspended in 0.2 ml of 20% sucrose, 0.1 M NaC1, 0.01 M Tris--HC1, pH 8.2, and 0.01 M NAN3. To the-cell suspension, 0.05 ml of 0.05 gg/ml T4 l ys oz ym e in 0.05 M disodium EDTA, 0.01 M NAN3, and 0.12 M Tris--HC1, pH 8.2 was added, and the mixture was incubated for 5 min at 30°C. 0.25 ml of 0.1% T r i t on X-100 in 0.01 M EDTA, and 0.01 M NaN3 pH adjusted to 8.2, was added, and the mixture was incubated for a n o th er 20 min at 30 ° C. T he lysate was syringed t w e n t y times through a 25G needle, and v o r t e x e d at m a x i m u m speed on a Super-Mixer (Lab-Line) for 5 min in order to shear the DNA. T he final lysate was then put ont o a sucrose gradient.

Sucrose density gradient centrifugation The lysates prepared as described above were gently poured ont o 10 ml of a 10--30% sucrose gradient containing 1 mM disodium EDTA, i mM ~-mercapt o e t h a n o l , and 0.1 M Tris--HC1, pH 8.2, layered over a 62% sucrose shelf containing CsCl, p = 1.5. Two gradients were made by layering 9 ml of 12 to 30% sucrose in the same buffer over the shelf; one of these gradients had 1 ml of 1 mg/ml l y s o z y m e in 10% sucrose in the same buffer over the gradient, and the o t h e r had the same layer on t op of the gradient e x c e p t that it did n o t contain lysozyme. 10 pl of T4 phage (2 • 10 s ) were added to the t o p of the lysate as a sedimentation marker. Centrifugation was in a Beckman SW-41 r o t o r for 10 min or 30 min at 23 000 rev./min at 4°C. The gradients were collected from the to p with a Buchler Auto-Densi-Flow F r a c t i o n a t o r into equal fractions containing a b o u t 0.8 ml each. After the fractionation a b o u t 0.8 ml of water was added to the e m p t y centrifuge tube and the tube was v o r t e x e d in order to recover the pellet at the b o t t o m . Each fraction was t h o r o u g h l y mixed and 0.15 ml r emo v ed and placed o n t o filter discs (2.3 cm, Whatman 3MM). These were then soaked in 5% trichloroacetic acid containing 20 pg/ml t h y m i n e and 8 mM MgSO4, three times for 1/2h each. After the third wash the discs were dried with acetone. T h e y were t he n c o u n t e d in t ol uene-om ni fl uor scintillator. The position of the T4 phage used as an internal sedimentation marker was d e t e r min ed by a spot-test on a bacterial lawn. Its sedimentation velocity was taken as 1025 S [15] and the sedimentation velocity of the DNA was estim a t ed by comparison of its distance f r om the t o p of the centrifuge t ube with

152 that of the T4 phage, assuming a linear relationship between distance sedim e n t e d and sedimentation constant.

Formation oT a D N A - - m e m b r a n e complex mediated by lysozyme E. coli MX74T2 thy- was grown in the media described above. To 30 ml of t h e c u l t u r e was added 250 gCi of carrier-free H23 s SO4 for three generations. At a b o u t 109 cells/ml the cells were collected and disrupted by sonication w i t h o u t using lysozyme. T he m e m b r a n e fraction was prepared by differential centrifugation as described previously [ 1 6 ] . T he m e m b r a n e fraction was suspended in 2.5 ml of 10 mM disodium EDTA, 0.01 M Tris--HC1, pH 8.2. To 0.25 ml of the m e m b r a n e suspension was added 0.5 ml of DNA (Sigma t y p e VI from soft roe of pollock or coalfish; dissolved by shaking slowly for several days in 60% sucrose, 10 mM disodium EDTA, Tris--HC1, pH 8.2) to a final c o n c e n t r a t i o n of 10 pg/ml, and 0.25 ml of egg white l ysozym e in 10 mM disodium EDTA, Tris--HC1, pH 8.2 to a final concent rat i on of 800 pg/ml. As controls, th e same Tris--EDTA buffer as above was substituted for either the l y s o z y m e or the m e m b r a n e fraction, or the same Tris--EDTA buffer containing 60% sucrose as above, was substituted for the DNA. The mixtures were incubated for 5 min at r o o m t e m p e r a t u r e and layered o n t o 10 ml of a 40--60% sucrose gradient prepared in the same buffer as the gradients described above, and t he n centrifuged in an SW-41 r o t o r for 10 min at 8000 rev./min at 4°C. T he gradients were collected from the t op into ten equal fractions of a b o u t 1.1 ml each. After the fractionation, the centrifuge tubes were washed with 1.1 ml of water and vort exed to resuspend the pellet. The fractions were t h o r o u g h l y mixed and 0.2 ml was removed from each one for counting 3 sS radioactivity in order to determine the m e m b r a n e distribution. Th e DNA c o n t e n t was det er m i ned by applying the diphenylamine procedure [17] to 0.5 ml f r om each fraction. R eage n ts Poly(L-lysine) and HBr were obtained f r o m Miles Laboratories. Ribonuclease A was obtained f r o m Worthington Biochemicals, and heated in boiling water for 10 min to inactivate deoxyr i bonuc l ease activity before use. Radioisotopes were obtained from Schwarz/Mann. Results Effect of egg white lysozyme concentration The sedimentation of the E. coli c h r o m o s o m e and protein was examined after lysing the cells with varying am ount s of egg white l ysozym e using three d i f f er en t detergent systems. As shown in Fig. 1, regardless of which detergent system is used, the c h r o m o s o m e sediments as a D N A - - m e m b r a n e complex on the shelf when the cells are lysed with 800 pg/ml of lysozyme. The sedimentation velocity of the D N A - - m e m b r a n e com plex was f o u n d by a separate experim e n t with a 10 min centrifugation to be greater than 4000 S (data n o t shown). However, at low concentrations, 2--400 pg/ml of lysozyme, the c h r o m o s o m e sediments at 5 0 0 - - 1 1 0 0 S (low-S complex). T he sedimentation velocity is larger when Brij-58 is used than when Brij-58-deoxycholate or T ri t on X-100 is used.

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FRACTION NUMBER Fig. 1. T h e e f f e c t of l y s o z y m e c o n c e n t r a t i o n o n t h e s e d i m e n t a t i o n b e h a v i o r o f t h e E. coli c h r o m o s o m e . E. coli M X 7 4 T 2 t h y - , d o u b l e - l a b e l e d w i t h H 2 3 5 S O 4 a n d [ 3H] t h y m i n e w a s l y s e d w i t h d i f f e r e n t a m o u n t s o f e g g w h i t e l y s o z y m e in d i f f e r e n t d e t e r g e n t s y s t e m s , as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . L y s o z y m e c o n c e n t r a t i o n s b e f o r e t h e a d d i t i o n o f d e t e r g e n t s are: C o l u m n 1, 2 # g / m h C o l u m n 2, 1 0 0 # g / m l ; Colu m n 3, 4 0 0 ~ g / m l ; C o l u m n 4, 8 0 0 # g / m l . T h e d e t e r g e n t s y s t e m s a r e as f o l l o w s : R o w A, 0 . 5 % Brij-58; R o w B, 0 . 5 % Brij-58 a n d 0 . 2 % s o d i u m d e o x y c h o l a t e ; a n d R o w C, 0 . 1 % T r i t o n X - 1 0 0 . T h e l y s a t e w a s g e n t l y p o u r e d o n t o 1 0 m l o f a 1 0 - - 3 0 % s u c r o s e ~ a d i e n t a n d c e n t r i f u g e d f o r 8 0 r a i n as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . T h e w a s h f r a c t i o n a f t e r f r a e t i o n a t i o n is d e s i g n a t e d b y " W ' . T h e r e s u l t s are e x p r e s s e d as p e r c e n t r e c o v e r y o f t o t a l r a d i o a c t i v i t y : • 0, [ 3 H ] t h y m i n e ; × . . . . . . X, 3 fiS. T h e d o t t e d v e r t i c a l line r e p r e s e n t s t h e p o s i t i o n o f t h e shelf, a n d t h e p o s i t i o n o f t h e T 4 p h a g e m a r k e r is s h o w n as cross-hatched bar.

The transition from the low-S complex to the DNA--membrane complex is sharp for Triton X-100 (between 400 #g/ml and 800 pg/ml of lysozyme) but gradual for Brij~leoxycholate, with both the low-S complex and the DNA-membrane complex coexisting at 400 #g/ml. With Brij-58 both complexes coexist at all lysozyme concentrations used except 800 pg/ml. Lysozyme activity was found in both the low-S complex and the DNA--membrane complex in Figs l(A-2) and l(A-3) (data not shown).

The secondary effect of lysozyme and other polycations In order to examine whether the DNA--membrane complex obtained at 800 pg/ml of lysozyme results from the secondary interaction of the low-S complex with excess lysozyme, the following experiment was done: cells were lysed with 10 pg/ml of lysozyme in 0.1% Triton X-100 (the same as Fig. I(C-1)) and applied to two sucrose gradients, identical except that one of them had a 1 ml layer of 1 mg/ml of lysozyme on top. As shown in Fig. 2A, the low-S complex formed with 10 pg/ml of lysozyme sediments to the shelf after passing through the lysozyme layer. This indicates that a secondary interaction

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F i g . 2. S e c o n d a r y c o m p l e x f o r m a t i o n o f t h e E. coli c h r o m o s o m e b y l y s o z y m e . T h e cells w e r e t r e a t e d a s i n Fig. 1 using 10 #g/ml of lysozyme followed by 0.1% Triton X-100, with these modifications: The lysate was c e n t r i f u g e d f o r 10 rain at 23 0 0 0 r e v . / m i n o n t w o g r a d i e n t s . (A) 1 m l o f 10% sucrose was l a y e r e d on t o p of t h e g~adient, ( B ) T h e same as A e x c e p t t h a t t h e t o p 10% sucrose layer c o n t a i n e d 1 m g / m l lysozyme. • e, [ 3HI t h y m i n e ; X . . . . . . X , 3SS. T h e r e s u l t s a r e e x p r e s s e d a s p e r c e n t r e c o v e r y o f t o t a l radioactivity.

of the low-S complex with lysozyme causes it to sediment as a DNA-membrane complex. Similar effects are observed with poly(L-lysine) (Fig. 3B) and ribonuclease (Fig. 3C). A polyamine such as spermidine converts the low-S complex to a DNA--membrane complex when its concentration in the gradient is 5 mM. When added with the detergent at 1 mM concentration, it condenses the DNA so that it sediments at 2000 S (data not shown). These results indicate that the lysozyme effect is not specific but due to its polycationic character.

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F i g . 3. T h e e f f e c t o f a b a s i c p o l y p e p t i d e a n d p r o t e i n o n t h e s e d i m e n t a t i o n b e h a v i o r o f t h e E. coli c h r o m o s o m e . T h e c e l l s w e r e t r e a t e d as i n F i g . 1 u s i n g 1 0 ~ug]ml o f l y s o z y m e f o l l o w e d b y 0 . 1 % T r i t o n X-IO0, w i t h these m o d i f i c a t i o n s : (A) C o n t r o l . (B) P o l y ( L - l y s i n e ) . H B r was a d d e d w i t h the d e t e r g e n t t o a f i n a l c o n c e n t r a t i o n o f 1 m g / m l . (C) R i h o n u c l e a s e A w a s a d d e d w i t h t h e d e t e r g e n t t o a f i n a l c o n c e n t r a t i o n o f 1 m g / m l . The c e n t r i f u g a t i o n was f o r 10 rain. • e, [ 3H] t h y m i n e ; X . . . . . . X , 3SS. T h e r e s u l t s are e x p r e s s e d as p e r c e n t r e c o v e r y o f t o t a l r a d i o a c t i v i t y .

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Fig. 4. T h e e f f e c t o f M g C l 2 c o n c e n t r a t i o n o n t h e s e d i m e n t a t i o n b e h a v i o r of t h e E, coli c h r o m o s o m e . T h e cells w e r e t r e a t e d as in Fig. 1 u s i n g 10 p g / m l o f l Y s o z y m e f o l l o w e d b y 0 . 1 % T r i t o n X - 1 0 0 c o n t a i n i n g M g C l 2 t o t h e s e final c o n c e n t r a t i o n s : ( A ) 0 m M ; (B) 1 0 m M ; (C) 2 0 m M . • •, [3H]thymine; X ...... × , 3 5 S . T h e c e n t r i f u g a t i o n w a s f o r 3 0 r a i n . T h e r e s u l t s a r e e x p r e s s e d as p e r c e n t r e c o v e r y o f total radioactivity.

Mg 2+ is often used in the lysis of cells [2,18]. To determine whether Mg 2+ has a similar effect to basic polypeptides, we lysed the cells at various MgCl 2 concentrations (at 10 mM EDTA) with 100 pg/ml lysozyme and 0.1% Triton X-100. We obtained the low-S complex in the absence of MgC12, a low-S complex together with a 1500 S c o m p o n e n t at 10 mM MgCl2, and only a DNA--membrane complex at 20 mM MgCl~, as shown in Fig. 4. The 1500 S DNA at 10 mM MgC12 is possibly an intermediate between the low-S and the DNA--membrane complex.

DNA--membrane association mediated by lysozyme In order to examine the direct effect of lysozyme on DNA and membranes, a mixture of purified DNA, E. coli membranes (prepared by sonication and free of DNA and lysozyme) and lysozyme were incubated for 10 min at room temperature and centrifuged on a 40--60% sucrose density gradient. As shown in Fig. 5A, membranes sedimented with the DNA to the b o t t o m of the centrifuge tube. However, when lysozyme was omitted from the mixture, both DNA and the membranes remained at the top of the gradient (Fig. 5B). On the other hand, when DNA was omitted, the membranes sedimented faster than

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Fig. 5. F o r m a t i o n of a D N A - - m e m b r a n e c o m p l e x m e d i a t e d b y l y s o z y m e . A 35S-labeled m e m b r a n e fract i o n w a s p r e p a r e d b y d i f f e r e n t i a l c e n t r i f u g a t i o n f r o m E. coli M X 7 4 T 2 t h y - d e s c r i b e d p r e v i o u s l y [ 1 8 ] a n d in Materials a n d M e t h o d s . T h e m e m b r a n e f r a c t i o n w a s s u s p e n d e d in 2 . 5 m l of 10 m M d i s o d i u m E D T A , 0.1 M T r i s - H C l , p H 8.2. ( A ) T o 0 . 2 5 m l o f t h e m e m b r a n e s u s p e n s i o n w a s a d d e d 0 . 5 m l o f D N A to a final concentration of 1 0 # g / m l and 0.25 ml of egg white lysozyme to a final c o n c e n t r a t i o n of 800/Zg/ml. (B) No l y s o z y m e w a s a d d e d . (C) N o D N A w a s a d d e d . ( D ) No m e m b r a n e s w e r e a d d e d . T h e D N A c o n t e n t w a s d e t e r m i n e d b y a p p l y i n g t h e d i p h e n y l a m i n e p r o c e d u x e [ 1 9 ] to 0 . 5 m l f r o m e a c h f r a c t i o n . • --•, a b s o r b a n c e at 6 0 0 rim. X . . . . . . × , 35S r a d i o a c t i v i t y . T h e r e s u l t s are e x p r e s s e d as p e r c e n t o f t o t a l a b s o r b a n c e at 6 0 0 n m for D N A , a n d p e r c e n t o f t o t a l r e c o v e r y o f r a d i o a c t i v i t y f o r p r o t e i n .

membranes in the absence of lysozyme (presumably because they were aggregated by lysozyme) but much more slowly than the DNA--membrane-lysozyme complex, as shown in Fig. 5C. Without the membrane, D N A itself formed a c o m p l e x with lysozyme which sedimented to the b o t t o m of the gradient (Fig. 5D). Thus, D N A is absolutely required for the membranes to sediment to the b o t t o m of the gradient. When DNA, membranes and lysozyme were mixed together with 1.0 M NaC1, both the D N A and the membranes remained at the top of the gradient, as in Fig. 5B (data not shown). We conclude that lysozyme mediates a DNA--membrane complex at 800 pg/ml lysozyme.

Primary DNA--mem brane complex These results suggest that DNA--membrane c o m p l e x e s obtained with high concentrations of basic polypeptides or Mg 2÷ do not necessarily prove the well-documented DNA--membrane association in cells. In order to avoid the secondary binding of lysozyme to the E. coli chromosome, we used lysozyme in as low a concentration as possible to lyse the cells. Although the cells can be lysed with 2 pg/ml of lysozyme, this is still about 5 • 103 lysozyme molecules/ chromosome. In order to further lower the lysozyme concentration, we used T4 phage lysozyme, since it has a 250-fold higher specific activity towards E. coli than egg white lysozyme [ 1 9 ] . When we lysed the cells with 0.01 pg/ml of T4 phage lysozyme (20 lysozyme molecules/chromosome), followed by 0.1% Triton X-100, the c h r o m o s o m e sedimented to the shelf, as shown in Fig. 6. The

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FRACTION NUMBER Fig. 6. T h e e f f e c t o f a n e x t r e m e l y l o w c o n c e n t r a t i o n o f T 4 p h a g e l y s o z y m e o n t h e s e d i m e n t a t i o n b e h a v i o r o f t h e E. c o l i c h r o m o s o m e . T h e p r o c e d u r e is t h e s a m e as in Fig. 1 e x c e p t t h a t t h e cells w e r e l y s e d with 0.01 pg/ml of T4 phage lysozyme instead of egg white lysozyme, followed by 0.1% Triton X-IO0, and the centrifugation was for 10 rain. • --40 [ 3 H ] t h y m i n e ; X . . . . . . × , 35S. A l l r e s u l t s a r e e x p r e s s e d as percent recovery of total radioactivity.

same result is obtained even when the centrifugation time is reduced to 10 min (data not shown). By pulse labeling newly synthesized DNA, lysing the cells with T4 phage lysozyme, and shearing the DNA before layering the lysate onto a sucrose gradient, we obtained a 7-fold enrichment of the growing point over long-term labeled DNA, at the membrane fraction as shown in Fig. 7A. This enrichment is completely eliminated by chasing the label (Fig. 7B). These results indicate that DNA is associated with the membrane fraction in a specific manner. [

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Fig. 7. E n r i c h m e n t o f t h e g r o w i n g p o i n t o f t h e c h r o m o s o m e a t t h e m e m b r a n e f r a c t i o n . T h e cells w e r e p u l s e - l a b e l e d w i t h [ 3 H ] t h y m i n e as d e s c r i b e d in M a t e r i a l s a n d M e t h o d s . C e n t r i f u g a t i o n w a s c a r r i e d o u t f o r 10 min at 23 000 rev./min (A) Without chase. (B) With chase. • e, [ 14C] thymine (long-term label); × ...... × , [ 3 H I t h y m i n e ( p u l s e l a b e l ) . T h e r e s u l t s a r e e x p r e s s e d as p e r c e n t r e c o v e r y o f t o t a l r a d i o a c t i v i t y .

158

Discussion From these data, we conclude that the E coli chromosome sediments in three different ways, depending on the lysis conditions: (1)Primary DNA-membrane complex. The chromosome sediments with the membrane when the cells are lysed very gently in the presence of extremely low concentrations of lysozyme (Fig. 6). This a t t a c h m e n t is specific since the growing point was enriched at the membrane fraction (Fig. 7). (2) Low-S complex. When the cells are more extensively digested with lysozyme (2--400 ]~g/ml) and lysed with Brij-58-deoxycholate or Triton X-100, the chromosome is released from the bulk of the membranes in a folded form that sediments slower than membranes, as a low-S complex (Figs 1B and 1C). Brij-58 by itself is mild enough so that part of the DNA still sediments with the membrane fraction (Fig. 1A). The low-S complex still has 1--5% of the total cellular protein and analysis of these proteins by sodium dodecylsulfate gel electrophoresis shows that it still contains a substantial a m o u n t of membrane proteins in addition to chromosomespecific proteins (data not shown}. (3) Secondary DNA--membrane complex. When concentrations of lysozyme, other polycations, or Mg 2÷ are increased, the chromosome condenses and membranes reaggregate, and the low-S complex sediments as a secondary D N A - m e m b r a n e complex. We are currently analyzing chromosome-specific proteins using chromosomes isolated by one of the above methods (10 gg/ml egg white lysozyme, 0.1% Triton X-100). Since this m e t h o d avoids the use of 1 M NaC1 and 800 gg/ml lysozyme (= 2 • 106 lysozyme molecules/chromosome) [11,12], it may be more advantageous for the isolation of chromosome-specific proteins. Acknowledgments We thank Dr R. Sternglanz for a critical reading of the manuscript and Mr A. Barton for his skillful technical assistance. This research was supported by grants from the U.S. National Institute of Health (GM19043-02), American Cancer Society (BC-67) and the National Science Foundation (BO42237). References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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