Volume 2 number 9 September 1975
Nucleic Acids Research
Analysis by Isopycnic centrifugatton of isolated nucleolds of Escherichia colt
Ralph Giorno, Ralph M. Hecht and David Pettijohn Department of Biophysics and Genetics, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, CO 80220, USA
Received 8 July 1975 ABSTRACT The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 ± 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 ± 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid. INTRODUCTION The nucleoid of Escherichia coli has been isolated as a structure with similar dimensions and DNA content as the nucleoid observed in vivo8 Methods for preparing the nucleoids with or without attached membranes have been developed and these structures have been characterized1-5 8. The membrane-associated nucleoid sediments as a broad band in sucrose density gradients with an average sedimentation coefficient of about 3200 S and it contains a heterogeneous protein population 3,4 . Protein and DNA have been found to be present in roughly equal amounts in the membrane-associated nucleoid3. The membrane-free nucleoid sediments at about 1600 S and contains only about 10% protein relative to DNA. The major fraction of the protein in membrane-free nucleoids consists of the subunits of core RNA polymerase. The fraction of RNA relative to DNA is about 40% in the mem3 brane-free nucleoid In the present work, we have found that both membrane-free and membraneassociated nucleoids can be banded by isopycnic centrifugation in CsCl density gradients after fixation with formaldehyde. Both nucleoids band at their characteristic densities and remain condensed during isopycnic
centrifugation. MATERIALS AND METHODS 6 E. coli strain D-10 was grown and harvested as previously described
1559
Nucleic Acids Research Protein was labeled with S as described by Worcel & Burgi . DNA was 1 labeled with 3H-methyl -thymidine and C-thymidine as previously described 7 . When protein was labeled with 14 C, cells were grown for one generation in 20 iiCi/ml of 14C-labeled protein hydrolysate (Schwarz-Mann) in medium lacking casamino acids. Cells were lysed by a modification of the procedure of Stonington and Pettijohn 1 . Exponentially growing cells, in 3-5 ml of medium, were centrifuged and resuspended in 0.2 ml of a solution of 1.0 M NaCl and 0.1 M TrisHCl (pH 8.1 at 25 C). The suspension was made 2 mg/ml in lysozyme and 0.01 M in EDTA (ethylene diamine tetraacetic acid, disodium salt) by addition of 0.04 ml of a solution containing lysozyme, 10 mg/ml, 0.05 M EDTA, 0.1 M Tris-HCl, pH 8.1 and 1 M NaCl. The suspension was incubated for 45 sec at 40C; 0.25 ml of a solution containing 1%, Brij 58, 1 M NaCl, 0.01 M EDTA, 1% Sarkosyl and 5 mg/ml sodium deoxycholate was then added to the suspension. The membrane-free nucleoids were prepared by incubating the suspension at 24°C for 6 min and were isolated by sedimentation at 17,000 revs/ min for 40 min at 40C in Spinco SW 50 or SW 27 rotors on 10-30% (w/v) sucrose gradients containing 1 M NaCl, 0.01 M Tris-HCl, pli 8.1, 0.001 M EDTA, 0.002 M B-mercaptoethanol. The membrane-associated nucleoids were prepared by allowing the suspension to stand for 30 min at 40C and were isolated by sedimentation on identical gradients for 5 min. Nucleoids were fixed as outlined by Hecht et al.8, in which fractions (0.3 ml) frorm a preparative sucrose gradient were collected directly into tubes containing 0.03 ml of 3% (v/v) formaldehyde in 0.3 M Na2HP04. Nucleoids were banded by isopycnic centrifugation in CsCl density gradients as follows: A saturated CsCl solution (Pierce, Sequanal grade) was placed in a polyallomer tube which had been soaked overnight in bovine serum albumin (BSA; 10 mg/ml in 1 M NaCl). The CsCl solution was made 0.01 M in Na2HPO4 and 0.004 M in trisodium citrate. To this solution was added 250 ig of BSA and 30-50 pg of heat-denatured salmon sperm DNA to act as carriers and to minimize adsorption of nucleoids to the tube walls. A solution of 0.5-0.9 ml containing purified nucleoids was gently mixed into the CsCl solution. The final density was adjusted to 1.70-1.72 g/cm3 for membrane-free nucleoids and 1.42-1.44 g/cm for membrane-associated nucleoids. The samples were centrifuged at 34,000 revs/min, 20-25 C, 44-48 hr in a Spinco SW 50.1 rotor. Gradients were collected from the bottom and
aliquots of each fraction were taken for liquid scintillation counting and for measurements of refractive index to determine CsCl density.
1560
Nucleic Acids Research RESULTS
Isolated membrane-free nucleoids were fixed with 0.3% formaldehyde and centrifuged to equilibrium in CsCl density gradients. As shown in Figure I~~~~~~~~~~~ la, the membrane-free nucleoid banded at a density of 1.69 ± 0.02 g/cm and was significantly less dense than an internal marker of purified E. coli DNA (p = 1.71 g/cm , ref. 9).
4
0.10
I?
IN
0 0
_
_
x0 < I X~
ON 0.0 4°os
2
0.10 IN4 Cl)
o o x
0
C4
x
00I I
1- "I I
O1 10
20
30
10
F ract ion No.
Figure 1. Isopycnic centrifugation in CsCl of the membrane-free nucleoids. (a) Fixed nucleoids labeled withTH-thymidine, (0 - 0 ); (b) Fixed nucleoids long-term labeled with 14C-thymidine (0 -- 0) and pulse-labeled 0 ); (c) Fixed nucleoids labeled with 3H(30 sec) with 3H-uridine ( 0 thymidine (0 --0) and 14C-amino acids (0 0); (d) Unfixed nucleoids labeled with 3H-thymidine (0 -- 0). X X indicate the density of CsCl through the gradients and A A indicate A260 profile of purified E. coli DNA in all panels.
1561
Nucleic Acids Research In order to analyze the various components of nucleoids during isopycnic centrifugation, double labeling experiments were carried out. The nucleoids were isolated from cells which had been labeled for 20 min with C-thymidine and 30 sec with 3H-uridine, fixed and brought to equilibrium in CsCl gradients. Most of the 3H-uridine labeled material banded with C-labeled DNA in the nucleoids (Fig. lb). As revealed by the 3H:14C the ratio before and after banding, the RNA:DNA ratio in the fixed nucleoids was unchanged (Table 1). Previous studies have demonstrated that most of the 3H-uridine is incorporated into RNA when nucleoids were labeled as in Figure lb 10,11 . Furthermore, about 65% of the 3 H radioactivity could be separated from the 1 C-labeled DNA when unfixed nucleoids were banded in CsCl (Table 1). We conclude that the RNA chains of the fixed nucleoid predominantly remain associated during isopycnic centrifugation. Similar experiments were carried out to analyze the protein components of formaldehyde-fixed nucleoids. Protein was labeled either with 35S (as As shown hydrolysate and DNA with H2 24)4or with of membrane-free the DNA banded with protein C-labeled in Figure ic, nucleoids, although some protein was released and floated to the top of the gradient. An examination of Table 1 indicates that there is a two-fold decrease in the amount of protein banding with the fixed nucleoids. We conclude that some of the protein isolated with the nucleoid bands with the DNA, but there is a dissociation of protein during isopycnic centrifugation.
~~1414C-protein
3H-thymidine.
TABLE 1.
Relative Protein and RNA Contents of Nucleoids Before and After Isopycnic Centrifugation.
C-protein: H-DNA ratio(cpm) H-RNA: C-DNA ratio(cpm) before CsCl after CsCl
gradient gradient Nucleoid 0.07±0.004 fixed 0.14±0.007 embrane-free; ----Membrane-free; unfixed 0.9±0.05 2.0±0.1 embrane-assoc; fixed
before CsCl after CsCl gradient gradient 20.4±1.0 20.0±1.0 21.8±1.0
6.8±0.4
The fixed, membrane-associated nucleoids had a buoyant density in CsCl of 1.46 ± 0.02 g/cm3 (Fig. 2). Although this density was characteristic and reproducible in our hands, some variation can be expected in different preparations, since the amount of membrane which remains attached to the DNA is highly dependent upon the conditions of lysis. In a manner similar to the membrane-free nucleoid, only about half of the protein of the membrane-associated nucleoid remains attached to the structure during the
centrifugation in CsCl (Table 1 and Fig. 2). 1562
Nucleic Acids Research
C~4 e4
0~x
0
cn 0
a. o.
Xxx
I
I I
CL u
If
10
20
30
10
20
30
Fraction No.
Figure 2. Isopycnic centrifugation in CsCl of the membrane-associated *) .and nucleoids. (a) Nucleoids labeled with 3H-thymidine (@ 0 indicate position of 14C-T4 phage marker O0); 0 35S-H2SO4 (O (p = 1.51 g/cm3; see ref. 12); (b) Nucleoids labeled with 3H-thymidine 0). 0) and 14C-amino acids (O (0
Both the membrane-free and the membrane-associated nucleoids exhibited narrow, symmetrical bands in the CsCl gradient characteristic of a monodisperse species. For example, the width of the nucleoid band in Figure 2a is comparable to that of phage T4. This finding demonstrates that the RNA, DNA and protein components are present in nearly constant ratios-in the different nucleoids. Furthermore, the amount of protein dissociated during isopycnic centrifugation must be rather uniform among the different nucleoids. Earlier findings have shown that nucleoids isolated from randomly growing cells are in different stages of DNA replication and that the
breadth of their sedimentation profile is attributable to a heterogeneity in sedimentation rate2 When nucleoids from the peak, leading or trailing edge of the heterogeneous sedimentation profile were banded by isopycnic centrifugation, all fractions had the same buoyant density (Fig. 3). Thus,
1563
Nucleic Acids Research it appears that nucleoids of different sedimentation rate and presumably at different stages of replication also have a constant composition of protein, RNA and DNA. It should be emphasized that the membrane-free nucleoids have protein and RNA contents accounting for approximately 40% of their mass and that minor variations in these contents would be expected to substantially influence the buoyant density of the particles. The formaldehyde-fixed nucleoids remained condensed in particles after centrifugation in CsCl gradients, as indicated from their sedimentation rate and from fluorescence microscopy studies. As shown in Figure 4, the membrane-free nucleoid sedimented at 1300 S after the isopycnic centrifugation, compared to 1500 S before exposure to CsCl (Fig. 4a). The membrane-associated nucleoid sedimented at the same rate (3200 S) before and after the isopycnic centrifugation (Fig. 4b). The nucleoids visualized by fluorescence microscopy had the same size and appearance before and after exposure to CsCl (Plate 1). Fixation of the nucleoids prior to banding in CsCl was required to preserve their structure. The unfixed nucleoids shifted in buoyant density to a value very near that of purified E. coli DNA (Fig. Id). Furthermore, there was a loss of RNA components (Table 1) and the sedimentation rate of the DNA was more characteristic of the unfolded chromosome (Fig. 4a). DISCUSSION The present research demonstrated that isolated bacterial nucleoids, after fixation, can be analyzed by isopycnic centrifugation in CsCl density gradients. While the condensed DNA remained packaged during this procedure and the RIA components of the nucleoid remained attached, there was a significant dissociation of the protein components. Apparently the lost proteins were not essential in the DNA packaging, since the state of condensation of the chromosome did not change significantly. Isopycnic centrifugation may therefore provide a means for purifying the nucleoid while preserving components essential to its structure. For studies requiring preservation of the protein components of the nucleoid, it is possible that a more prolonged treatment with formaldehyde would fix all of the proteins. The membrane-associated nucleoid had a buoyant density substantially less than the membrane-free nucleoid. This difference is consistent with the known protein and lipid compositions of the structures. The membranefree nucleoid has no detectable phospholipids, while the membrane-associated nucleoids have bound phospholipids and about 10 times the amount of protein per particle3.
1564
Nucleic Acids Research
5
10
15
Fraction No.
Figure 3.
of membraneBuoyant density and sedimentation velocit y free nucleoids. Cells were labeled with 3H-methyl -thymidine and nucleoids isolated on a sucrose gradient as described in Materials S Methods. The bottom of the gradient contained a "shelf" of saturated CsCl in 50% sucrose at fraction no. 3 to collect very rapidly sedimenting material. Fractions from leading and trailing edges and peak of the 3H-labeled nucleoid sedimentation profile (0 0) were fixed and brought to equilibrium in CsCl gradients as described in Materials & Methods. (0 0) indicate buoyant densities. 6
a)b) TA(1025S)
TA
10
20
0
a.
E
a. UL
20 10 Fraction No.
Figure 4. Sucrose density gradient sedimentation analysis of nucleoids labeled with bH-thymidine. (a) Membrane-free nucleoids; (b) Membraneassociated nucleoids; (0 0), fixed nucleoids before centrifugation in CsCl (0 - 0), fixed nucleoids after centrifugation in CsCl; (E 0), unfixed nucleoids after centrifugation in CsCl. Arrows indicate position of 14C-phage T4 marker (s = 1025 S).
1565
Nucleic Acids Research The near homogeneity in density of both the membrane-free and membrane-associated nucleoids suggests a rather remarkable constancy in the gross composition of both kinds of particles. This homogeneity was not
evident from sucrose gradient analyses, which exhibit heterogeneous profiles that may indicate different stages of DNA replication in nucleoids as suggested by Worcel S Burgi2 Our data suggest that, regardless of size or sedimentation rate of
El'_
nucleoids, the relative amounts of RNA and protein to DNA are constant.
Plate 1. Fluorescence microscopy of isolated membrane-fiue nucleoids before and after isopycn centrifugation. Nucleoids were visualized as described by Hecht et al.8 (a) Nucleoids from a preparative sucrose gradient; (b) Nucleoids after banding in CsCl as described in Materials and Methods. Scale bar represents 5p.
ACKNOWLEDGEMENTS We thank Ms. Sue Hamilton for preparation of C-amino acid labeled nucleoids. This work was supported by Ui.S. Public Health Service Grant No. GM 18243-04, by U.S. NTational Science Foundation Grant No. GB43358 and by a U.S. Public Health Service Training Grant No. GM 00781-16. This is publication No. 638 of the Department of Biophysics and Genetics, University of Colorado Medical Center.
REFERENCES 1 2 3 4 5
6
Stonington, O.G. and Pettijohn, D.E. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 6-9 Worcel, A. and Burgi, E. (1972) J. Mol. Biol. 71, 127-147 Pettijohn, D.E., Hecht, R.M., Stonington, O.G. and Stamato, T.D. (1973) In DNA Synthesis in Vitro, pp. 145-161, University Park Press, Baltimore Worcel, A. and Burgi, E. (1974) J. Mol. Biol. 82, 91-105 Pettijohn, D.E. and Hecht, R. (1973) Cold Spring Harbor.Symp. Quant. Biol. 38, 31-41 Pettijohn, D.E., Clarkson, K., Kossman, C.R. and Stonington, O.G. (1970) J. Mol. Biol. 52, 281-300
1566
Nucleic Acids Research Giorno, R., Stamato, T., Lydersen, B. and Pettijohn, D. (1975) J. Mol. Biol., in press 8 Hecht, R.M., Taggart, R.T. and Pettijohn, D.E. (1975) Nature 253, 60-62 9 Szybalski, W. and Szybalski, E.H. (1971) In Procedures in Nucleic Acid Research, Vol. II, pp. 311-354, Harper and Row, New York 10 Pettijohn, D.E., Stonington, O.G. and Kossman, C.R. (1970) Nature 228, 235-239 11 Hecht, R.M. and Pettijohn, D.E. (1975) Submitted for publication 12 Mosig, G. (1963) Carnegie Inst. Wash. Yearbook, pp. 586-589.
7
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Nucleic Acids Research
Analysis by Isopycnic centrifugatton of isolated nucleolds of Escherichia colt
Ralph Giorno, Ralph M. Hecht and David Pettijohn Department of Biophysics and Genetics, University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, CO 80220, USA
Received 8 July 1975 ABSTRACT The isolated, formaldehyde-fixed nucleoid of E. coli has been analyzed by isopycnic centrifugation in CsCl density gradients. The membrane-free nucleoid bands at a density of 1.69 ± 0.02 g/cm3. The membrane-associated nucleoid bands at a density of 1.46 ± 0.02 g/cm3. Both species sediment to equilibrium as nearly monodisperse bands in CsCl, suggesting that the nucleoid components of DNA, RNA and protein are present in relatively constant ratios. These ratios are constant regardless of the position of the nucleoids in the heterogeneous sedimentation profile of a preparative sucrose gradient. The fixed nucleoids remain condensed during isopycnic centrifugation and there is no detectable loss of RNA from the nucleoid. INTRODUCTION The nucleoid of Escherichia coli has been isolated as a structure with similar dimensions and DNA content as the nucleoid observed in vivo8 Methods for preparing the nucleoids with or without attached membranes have been developed and these structures have been characterized1-5 8. The membrane-associated nucleoid sediments as a broad band in sucrose density gradients with an average sedimentation coefficient of about 3200 S and it contains a heterogeneous protein population 3,4 . Protein and DNA have been found to be present in roughly equal amounts in the membrane-associated nucleoid3. The membrane-free nucleoid sediments at about 1600 S and contains only about 10% protein relative to DNA. The major fraction of the protein in membrane-free nucleoids consists of the subunits of core RNA polymerase. The fraction of RNA relative to DNA is about 40% in the mem3 brane-free nucleoid In the present work, we have found that both membrane-free and membraneassociated nucleoids can be banded by isopycnic centrifugation in CsCl density gradients after fixation with formaldehyde. Both nucleoids band at their characteristic densities and remain condensed during isopycnic
centrifugation. MATERIALS AND METHODS 6 E. coli strain D-10 was grown and harvested as previously described
1559
Nucleic Acids Research Protein was labeled with S as described by Worcel & Burgi . DNA was 1 labeled with 3H-methyl -thymidine and C-thymidine as previously described 7 . When protein was labeled with 14 C, cells were grown for one generation in 20 iiCi/ml of 14C-labeled protein hydrolysate (Schwarz-Mann) in medium lacking casamino acids. Cells were lysed by a modification of the procedure of Stonington and Pettijohn 1 . Exponentially growing cells, in 3-5 ml of medium, were centrifuged and resuspended in 0.2 ml of a solution of 1.0 M NaCl and 0.1 M TrisHCl (pH 8.1 at 25 C). The suspension was made 2 mg/ml in lysozyme and 0.01 M in EDTA (ethylene diamine tetraacetic acid, disodium salt) by addition of 0.04 ml of a solution containing lysozyme, 10 mg/ml, 0.05 M EDTA, 0.1 M Tris-HCl, pH 8.1 and 1 M NaCl. The suspension was incubated for 45 sec at 40C; 0.25 ml of a solution containing 1%, Brij 58, 1 M NaCl, 0.01 M EDTA, 1% Sarkosyl and 5 mg/ml sodium deoxycholate was then added to the suspension. The membrane-free nucleoids were prepared by incubating the suspension at 24°C for 6 min and were isolated by sedimentation at 17,000 revs/ min for 40 min at 40C in Spinco SW 50 or SW 27 rotors on 10-30% (w/v) sucrose gradients containing 1 M NaCl, 0.01 M Tris-HCl, pli 8.1, 0.001 M EDTA, 0.002 M B-mercaptoethanol. The membrane-associated nucleoids were prepared by allowing the suspension to stand for 30 min at 40C and were isolated by sedimentation on identical gradients for 5 min. Nucleoids were fixed as outlined by Hecht et al.8, in which fractions (0.3 ml) frorm a preparative sucrose gradient were collected directly into tubes containing 0.03 ml of 3% (v/v) formaldehyde in 0.3 M Na2HP04. Nucleoids were banded by isopycnic centrifugation in CsCl density gradients as follows: A saturated CsCl solution (Pierce, Sequanal grade) was placed in a polyallomer tube which had been soaked overnight in bovine serum albumin (BSA; 10 mg/ml in 1 M NaCl). The CsCl solution was made 0.01 M in Na2HPO4 and 0.004 M in trisodium citrate. To this solution was added 250 ig of BSA and 30-50 pg of heat-denatured salmon sperm DNA to act as carriers and to minimize adsorption of nucleoids to the tube walls. A solution of 0.5-0.9 ml containing purified nucleoids was gently mixed into the CsCl solution. The final density was adjusted to 1.70-1.72 g/cm3 for membrane-free nucleoids and 1.42-1.44 g/cm for membrane-associated nucleoids. The samples were centrifuged at 34,000 revs/min, 20-25 C, 44-48 hr in a Spinco SW 50.1 rotor. Gradients were collected from the bottom and
aliquots of each fraction were taken for liquid scintillation counting and for measurements of refractive index to determine CsCl density.
1560
Nucleic Acids Research RESULTS
Isolated membrane-free nucleoids were fixed with 0.3% formaldehyde and centrifuged to equilibrium in CsCl density gradients. As shown in Figure I~~~~~~~~~~~ la, the membrane-free nucleoid banded at a density of 1.69 ± 0.02 g/cm and was significantly less dense than an internal marker of purified E. coli DNA (p = 1.71 g/cm , ref. 9).
4
0.10
I?
IN
0 0
_
_
x0 < I X~
ON 0.0 4°os
2
0.10 IN4 Cl)
o o x
0
C4
x
00I I
1- "I I
O1 10
20
30
10
F ract ion No.
Figure 1. Isopycnic centrifugation in CsCl of the membrane-free nucleoids. (a) Fixed nucleoids labeled withTH-thymidine, (0 - 0 ); (b) Fixed nucleoids long-term labeled with 14C-thymidine (0 -- 0) and pulse-labeled 0 ); (c) Fixed nucleoids labeled with 3H(30 sec) with 3H-uridine ( 0 thymidine (0 --0) and 14C-amino acids (0 0); (d) Unfixed nucleoids labeled with 3H-thymidine (0 -- 0). X X indicate the density of CsCl through the gradients and A A indicate A260 profile of purified E. coli DNA in all panels.
1561
Nucleic Acids Research In order to analyze the various components of nucleoids during isopycnic centrifugation, double labeling experiments were carried out. The nucleoids were isolated from cells which had been labeled for 20 min with C-thymidine and 30 sec with 3H-uridine, fixed and brought to equilibrium in CsCl gradients. Most of the 3H-uridine labeled material banded with C-labeled DNA in the nucleoids (Fig. lb). As revealed by the 3H:14C the ratio before and after banding, the RNA:DNA ratio in the fixed nucleoids was unchanged (Table 1). Previous studies have demonstrated that most of the 3H-uridine is incorporated into RNA when nucleoids were labeled as in Figure lb 10,11 . Furthermore, about 65% of the 3 H radioactivity could be separated from the 1 C-labeled DNA when unfixed nucleoids were banded in CsCl (Table 1). We conclude that the RNA chains of the fixed nucleoid predominantly remain associated during isopycnic centrifugation. Similar experiments were carried out to analyze the protein components of formaldehyde-fixed nucleoids. Protein was labeled either with 35S (as As shown hydrolysate and DNA with H2 24)4or with of membrane-free the DNA banded with protein C-labeled in Figure ic, nucleoids, although some protein was released and floated to the top of the gradient. An examination of Table 1 indicates that there is a two-fold decrease in the amount of protein banding with the fixed nucleoids. We conclude that some of the protein isolated with the nucleoid bands with the DNA, but there is a dissociation of protein during isopycnic centrifugation.
~~1414C-protein
3H-thymidine.
TABLE 1.
Relative Protein and RNA Contents of Nucleoids Before and After Isopycnic Centrifugation.
C-protein: H-DNA ratio(cpm) H-RNA: C-DNA ratio(cpm) before CsCl after CsCl
gradient gradient Nucleoid 0.07±0.004 fixed 0.14±0.007 embrane-free; ----Membrane-free; unfixed 0.9±0.05 2.0±0.1 embrane-assoc; fixed
before CsCl after CsCl gradient gradient 20.4±1.0 20.0±1.0 21.8±1.0
6.8±0.4
The fixed, membrane-associated nucleoids had a buoyant density in CsCl of 1.46 ± 0.02 g/cm3 (Fig. 2). Although this density was characteristic and reproducible in our hands, some variation can be expected in different preparations, since the amount of membrane which remains attached to the DNA is highly dependent upon the conditions of lysis. In a manner similar to the membrane-free nucleoid, only about half of the protein of the membrane-associated nucleoid remains attached to the structure during the
centrifugation in CsCl (Table 1 and Fig. 2). 1562
Nucleic Acids Research
C~4 e4
0~x
0
cn 0
a. o.
Xxx
I
I I
CL u
If
10
20
30
10
20
30
Fraction No.
Figure 2. Isopycnic centrifugation in CsCl of the membrane-associated *) .and nucleoids. (a) Nucleoids labeled with 3H-thymidine (@ 0 indicate position of 14C-T4 phage marker O0); 0 35S-H2SO4 (O (p = 1.51 g/cm3; see ref. 12); (b) Nucleoids labeled with 3H-thymidine 0). 0) and 14C-amino acids (O (0
Both the membrane-free and the membrane-associated nucleoids exhibited narrow, symmetrical bands in the CsCl gradient characteristic of a monodisperse species. For example, the width of the nucleoid band in Figure 2a is comparable to that of phage T4. This finding demonstrates that the RNA, DNA and protein components are present in nearly constant ratios-in the different nucleoids. Furthermore, the amount of protein dissociated during isopycnic centrifugation must be rather uniform among the different nucleoids. Earlier findings have shown that nucleoids isolated from randomly growing cells are in different stages of DNA replication and that the
breadth of their sedimentation profile is attributable to a heterogeneity in sedimentation rate2 When nucleoids from the peak, leading or trailing edge of the heterogeneous sedimentation profile were banded by isopycnic centrifugation, all fractions had the same buoyant density (Fig. 3). Thus,
1563
Nucleic Acids Research it appears that nucleoids of different sedimentation rate and presumably at different stages of replication also have a constant composition of protein, RNA and DNA. It should be emphasized that the membrane-free nucleoids have protein and RNA contents accounting for approximately 40% of their mass and that minor variations in these contents would be expected to substantially influence the buoyant density of the particles. The formaldehyde-fixed nucleoids remained condensed in particles after centrifugation in CsCl gradients, as indicated from their sedimentation rate and from fluorescence microscopy studies. As shown in Figure 4, the membrane-free nucleoid sedimented at 1300 S after the isopycnic centrifugation, compared to 1500 S before exposure to CsCl (Fig. 4a). The membrane-associated nucleoid sedimented at the same rate (3200 S) before and after the isopycnic centrifugation (Fig. 4b). The nucleoids visualized by fluorescence microscopy had the same size and appearance before and after exposure to CsCl (Plate 1). Fixation of the nucleoids prior to banding in CsCl was required to preserve their structure. The unfixed nucleoids shifted in buoyant density to a value very near that of purified E. coli DNA (Fig. Id). Furthermore, there was a loss of RNA components (Table 1) and the sedimentation rate of the DNA was more characteristic of the unfolded chromosome (Fig. 4a). DISCUSSION The present research demonstrated that isolated bacterial nucleoids, after fixation, can be analyzed by isopycnic centrifugation in CsCl density gradients. While the condensed DNA remained packaged during this procedure and the RIA components of the nucleoid remained attached, there was a significant dissociation of the protein components. Apparently the lost proteins were not essential in the DNA packaging, since the state of condensation of the chromosome did not change significantly. Isopycnic centrifugation may therefore provide a means for purifying the nucleoid while preserving components essential to its structure. For studies requiring preservation of the protein components of the nucleoid, it is possible that a more prolonged treatment with formaldehyde would fix all of the proteins. The membrane-associated nucleoid had a buoyant density substantially less than the membrane-free nucleoid. This difference is consistent with the known protein and lipid compositions of the structures. The membranefree nucleoid has no detectable phospholipids, while the membrane-associated nucleoids have bound phospholipids and about 10 times the amount of protein per particle3.
1564
Nucleic Acids Research
5
10
15
Fraction No.
Figure 3.
of membraneBuoyant density and sedimentation velocit y free nucleoids. Cells were labeled with 3H-methyl -thymidine and nucleoids isolated on a sucrose gradient as described in Materials S Methods. The bottom of the gradient contained a "shelf" of saturated CsCl in 50% sucrose at fraction no. 3 to collect very rapidly sedimenting material. Fractions from leading and trailing edges and peak of the 3H-labeled nucleoid sedimentation profile (0 0) were fixed and brought to equilibrium in CsCl gradients as described in Materials & Methods. (0 0) indicate buoyant densities. 6
a)b) TA(1025S)
TA
10
20
0
a.
E
a. UL
20 10 Fraction No.
Figure 4. Sucrose density gradient sedimentation analysis of nucleoids labeled with bH-thymidine. (a) Membrane-free nucleoids; (b) Membraneassociated nucleoids; (0 0), fixed nucleoids before centrifugation in CsCl (0 - 0), fixed nucleoids after centrifugation in CsCl; (E 0), unfixed nucleoids after centrifugation in CsCl. Arrows indicate position of 14C-phage T4 marker (s = 1025 S).
1565
Nucleic Acids Research The near homogeneity in density of both the membrane-free and membrane-associated nucleoids suggests a rather remarkable constancy in the gross composition of both kinds of particles. This homogeneity was not
evident from sucrose gradient analyses, which exhibit heterogeneous profiles that may indicate different stages of DNA replication in nucleoids as suggested by Worcel S Burgi2 Our data suggest that, regardless of size or sedimentation rate of
El'_
nucleoids, the relative amounts of RNA and protein to DNA are constant.
Plate 1. Fluorescence microscopy of isolated membrane-fiue nucleoids before and after isopycn centrifugation. Nucleoids were visualized as described by Hecht et al.8 (a) Nucleoids from a preparative sucrose gradient; (b) Nucleoids after banding in CsCl as described in Materials and Methods. Scale bar represents 5p.
ACKNOWLEDGEMENTS We thank Ms. Sue Hamilton for preparation of C-amino acid labeled nucleoids. This work was supported by Ui.S. Public Health Service Grant No. GM 18243-04, by U.S. NTational Science Foundation Grant No. GB43358 and by a U.S. Public Health Service Training Grant No. GM 00781-16. This is publication No. 638 of the Department of Biophysics and Genetics, University of Colorado Medical Center.
REFERENCES 1 2 3 4 5
6
Stonington, O.G. and Pettijohn, D.E. (1971) Proc. Nat. Acad. Sci. U.S.A. 68, 6-9 Worcel, A. and Burgi, E. (1972) J. Mol. Biol. 71, 127-147 Pettijohn, D.E., Hecht, R.M., Stonington, O.G. and Stamato, T.D. (1973) In DNA Synthesis in Vitro, pp. 145-161, University Park Press, Baltimore Worcel, A. and Burgi, E. (1974) J. Mol. Biol. 82, 91-105 Pettijohn, D.E. and Hecht, R. (1973) Cold Spring Harbor.Symp. Quant. Biol. 38, 31-41 Pettijohn, D.E., Clarkson, K., Kossman, C.R. and Stonington, O.G. (1970) J. Mol. Biol. 52, 281-300
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