JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 878-883 0021-9193/78/0133-0878$02.00/0 Copyright i) 1978 American Society for Microbiology
Vol. 133, No. 2
Printed in U.S.A.
Nucleoid Release from Escherichia coli Cells ERIC C. MATERMAN
AND
AUGUST P. VAN GOOL*
F. A. Janssens Memorial Laboratory of Genetics, University of Leuven, 3030 Heverlee, Belgium Received for publication 6 September 1977
The time course of morphological changes during lysis of Escherichia coli cells was examined with respect to an undisturbed release of nucleoids. The addition of detergents to plasmolyzed, osmotic sensitive cells resulted in the immediate reversal of plasmolysis followed by the appearance of rod-shaped ghost cells without any detectable spheroplast formation. Electron microscopic examination of the rod-shaped ghost cells revealed a zonal gap in the cell envelope, allowing the free release of the nucleoid. Due to the high ionic strength, a suitable cell lysis was shown to require higher incubation temperatures. However, in the absence of an appropriate control this may result in the sphering and vesiculation of ghost cell envelopes and even the unfolding of released nucleoids. To avoid this unfavorable consequence of lysis at high temperatures, a microscopic examination on the course of rod-shaped ghost formation is suggested. In a recent review on the nucleoid of Escherichia coli, Pettijohn emphasized the empirical nature of the methods used in the isolation of bacterial nucleoids. He described their successful isolation as a compromise between two extreme conditions of cell lysis resulting either in unfolded chromosomes or in nucleoids that remain trapped in the partially disrupted cell envelope (8). From the sedimentation analysis of purified nucleoids these extremes were characterized either as membrane-associated or membranefree but more or less unfolded complexes (9, 15). The proportion of both types of nucleoids was shown to depend on the temperature of cell lysis, the former being predominant at 0°C (9, 15). Although initially the association of the nucleoid with the cell membrane was considered to be specific (15), it was subsequently shown to result from a nonspecific binding of the nucleoid to the cell envelope (7). Since these data indicated that a variation in the sedimentation characteristics of isolated nucleoids may be traced back to their release from the cell during lysis, Korch et al. (5) examined the lysis requirements to reach the compromise of successful nucleoid isolation. Using a method for sedimentation analysis of crude lysates, they concluded that an efficient release of nucleoids requires a careful control of cell lysis (5). In this respect we studied the cytological changes occurring in lysing cells, trying to visualize directly the requirements for successful nucleoid release. MATERIALS AND MErHODS Bacterial strain and growth conditions. E. coli
NF 87, D-10 metB argA relA1, a strain obtained from N. Fiil (University of Copenhagen) and closely related to the one used by Stonington and Pettijohn (12), was grown in M9 minimal salts medium (16) supplemented with 0.2% glucose, 50 pug of methionine per ml, and 50 ug of arginine per ml. Lysis procedure. Exponentially growing cells were quickly pelieted at 5,000 x g (00C) and resuspended to a concentration of approximately 109 cells/ml. Cell lysis was carried out by detergent treatment of plasmolyzed, osmotic sensitive cells as described by Stonington and Pettijohn (12). However, to avoid interference with glutaraldehyde fixation, tris(hydroxymethyl)aminomethane buffer was replaced by triethanolamine (TEA) (1), and for similar reasons sodium citrate was used rather than ethylenediaminetetraacetate. To improve the electron microscopic visualization of nucleoid release, attempts were made to trap the nucleoid in the neighborhood of the cell from which it emerged by enmeshment of the cells in 1% agar before lysis. However, under these conditions cell lysis proceeded very slowly and was only successful at incubation temperatures equal to or higher than 200C. Morphokinetic analysis. At 1-min time intervals after the addition of detergents, 0.3-ml samples were quickly removed from the incubation mixture, and the absorbance at 660 nm was measured immediately in a Beckman-25 spectrophotometer. At the same time intervals, samples were also fixed with 2% (vol/vol) formaldehyde in 1 M NaCl and rapidly scanned in a Zeiss GFL phase-contrast microscope to obtain a representative series of micrographs. These micrographs allowed a reproducible quantitative reconstruction of the gross morphological changes observed in the cells during the course of cell lysis. Electron microscopic analysis. Samples were either withdrawn directly from the lysate or derived from the supernatant after a low-speed centrifugation of the lysate (5,000 x g, 5 min, 0°C). They were subsequently prefixed with 1% glutaraldehyde for 10 min
878
VOL. 133, 1978
NUCLEOID RELEASE FROM E. COLI
at the temperature of cell lysis. These conditions of prefixation were selected after examination of the effect of different chemical fixatives on the stability of isolated nucleoids (Table 1). After prefixation the samples were fixed with OS04 at a 0.5% final concentration for 10 min before enmeshment in 1% agar containing 1 M NaCl. Agar blocks were further processed as described by Ryter et al. (11). Sodium chloride at a 1 M final concentration was added to all fixatives. Overnight fixation in the presence of 0.1% tryptone was carried out at 4°C to avoid interference of the visualization of DNA fibers by tryptone precipitation. Samples were sectioned with an LKB III ultramicrotome and examined in a Philips EM 300 electron microscope at 80 kV.
RESULTS Morphokinetics of cell lysis. After a brief exposure to lysozyme and citrate, a twofold dilution of plasmolyzed E. coli cells into a highsalt solution containing deoxycholate and Brij58 led to a gradual clearing of the incubation mixture. At the same time a distinct sequence of morphological changes was observed in the cell population (Fig. 1, transition B to C). The plasmolysis of cells induced in step A, and which remained throughout step B, was rapidly reversed upon addition of the detergent mixture (Fig. 2a and b). This reversal was followed by a
879
TABLE 1. Effect of fixation on nucleoid stability Fixation conditiona Addition after fixation
Glutaraldehyde
None
Glutaraldehyde 0(0°
Control 0.01%
1%
-
-
-
(0.01%)
Glutaraldehyde (1%)
(0.%)4 101)O04(.% +
+ NT5b SDS, 0.5% + + NT + RNase, 0.1% 'The supernatant of a low-speed centrifugation (5,000 x g, 5 min) of a 15°C lysate was fixed for 10 min at 15°C. A qualitative estimate of the increase in viscosity of the supernatant (+), after addition of sodium dodecyl sulfate (SDS) or pancreatic ribonuclease (RNase), was used to test nucleoid fixation. bNT, Not tested. Pr oress of Lysis
Uorphological Effect
Phase Contrest
Micrograph-s
Diagram
A step
5icruse
Flesmclysias
20Q,"
DC; 3 to 4 min
|, step
Flasmolyzed osmotic
L4soZyme 300 ,g/ml
sensitive rods No spheroplasts
5cdium citrate 50 mM
Cu
/
m
I
; 30 sec
C step NaDI; 14W
D Or
1. Reversal of
plasmolysis
2r" C).ZC
# 2.
ertJ-58 0.5s U to
25"C;
10
Rod
shaped ghosts
.
a
min 3. Spherical ghosts 4
.4. Aggregates of
Q
a -
U,
ghosts
FIG. 1. Phase-contrast micrographs and diagrammatic representation of the sequence of gross morphological changes observed throughout the lytic procedure. x2,020.
880
MATERMAN AND VAN GOOL
a
J. BACTERIOL.
b
d
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'; :.I
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e
IL
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4. I
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;
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Ice,:11 410-Al
FIG. 2. Electron micrographs showing in detail the same sequence of morphological changes as in Fig. 1. Bar = 1 ,um. (a) Plasmolyzed cell in which the nucleoplasm is dispersed throughout the cytoplasm. (b) Reversal of plasmolysis after detergent addition. The cell contains a contracted nucleoplasm. (c and d) Rod-shaped ghost cells with characteristic zonal envelope gap either polar (c) or equatorial (d). In (c) the nucleoid has been withheld at the level of the envelope gap, whereas in (d) it has been released. Cell lysis was carried out at 20°C, and in (c) cells were enmeshed in agar before lysis. (e) Sphering ghost cells. (f) Ghost cell after lysis at 0°C. The rod shape is more or less preserved and the DNA is released through the different gaps. (g) Nucleoid trapping vesiculating cell envelope remnants which occur after spherical ghost cell formation. Lysis was carried out at 20°C, and the lysate purified by a low-speed spin.
gradual formation of rod-shaped ghost cells, tent including the sizable nucleoid was released which subsequently became converted to spher- through this envelope gap (Fig. 2c). At these ical ghosts (Fig. 1 and 2). Confirming earlier higher incubation temperatures a vesiculation observations (14), spheroplasts were never ob- of the ghost cell envelopes was observed concurserved either during the lysozyme treatment or rent with the appearance of spherical ghosts after dilution of the osmotic sensitive cells with (Fig. 2g and e). Simultaneously, the ghost cells the detergent mixture. started to aggregate while the lysate became The envelope structure of the rod-shaped highly viscous, indicating nucleoid unfolding. ghost cells was dependent on the lysis temperaAlthough the sequence of cytological changes ture. At 0°C convoluted envelopes were ob- during lysis was invariant, the time ofoccurrence served (Fig. 2f), whereas at higher incubation temperatures (20 to 25°C) the envelopes were smooth and revealed a single gap, frequently located either near the cell equator or at the pole (Fig. 2c and d). Since serial sections did not show additional interruptions in the continuity of the ghost envelope, the entire cell con-
of the respective changes in cell morphology depended on several parameters, including lysis temperature (Table 2, Fig. 3) and salt concentration (Fig. 4). Temperature dependence of cell lysis. Measured on a time course basis, the decrease in absorbance of the lysate after detergent ad-
NUCLEOID RELEASE FROM E. COLI
VOL. 133, 1978
TABLE 2. Time course of gross morphological changes and decrease in absorbance during cell lysis as a function of the incubation temperature Absorb- Reversal Rod-shaped ghost cells Spherical ance eofplasTemp crease molysis (O) (660 rum) (P) (t¶5(tE tSG;j s) _ O_.5 _ ( 5tAI.5 [8]) (tp05 [8]) [SG) 5 a(t [s]) [_ tS(;
881
z
0 J9
(RGhstG)R
0
263 (4)
b
IL
54 _d
138 360 Nonec 318 480 None 180 450 None 25 284 (19) 52 60 180 180 170 90 _ a to.5 values indicate, respectively, the time interval required after detergent addition to obtain a 50% decrease of the original value (tAo.5 and tpo.5) or to reach half the maximum value (tRsGo.5 and tSGo.5)bAverage values with the number of observations indicated in parentheses. c No spherical ghosts were observed at this incubation temperature. d No value available because the morphological change occurred too fast to be measured accurately.
0
2
4
6
8 0
2
4
6
-_
8
TIME (min )
FIG. 4. Effect of high salt concentration on the conversion of plasmolyzed cells to spherical ghosts. Plasmolyzed cells (sucrose, 20%o [wt/vol]) were incubated with lysozyme (800 lg/ml) and sodium citrate (10 mM), respectively, at 0°C (A and C) and 25°C (B and D). To reaction mixtures C and D, NaCl at a I M final concentration was added. At 1-min time intervals, 0.3-ml samples were diluted twofold in distilled water and immediately fixed with 2% formaldehyde before microscopic observation. Symbols: *,
nonplasmolyzed, rod-shaped cells; E, spheroplasts; 0, rod-shaped ghost cells; O, spherical ghost cells.
tion temperature was raised from 0 to 25°C, and a maximum number of rod-shaped ghosts was obtained 7 and 2 min, respectively, after addition of detergents (Table 2, Fig. 3). At the higher incubation temperatures the number of rod0. shaped ghosts decreased beyond its maximum, and a proportional number of spherical ghosts in the lysate (Fig. 3B). This observa. _ A _ _ _~ A A S_appeared _ 0 tion suggests that the spherical ghosts originated )B from rod-shaped ghost cells and did not arise from the lysis of spheroplasts, which were never observed in the lysate. Consequently, incubation z of the lysate beyond the appearance of rodshaped ghost cells is not necessary for nucleoid release and should be avoided because of the _ _-_nonspecific trapping of the DNA in vesiculating envelope fragments (Fig. 2g). 2 0 10 4 6 8 Cell lysis at high ionic strength. The use TIME (min) of a high salt concentration (1 M NaCl) for FIG. 3. Effect of the incubation temperature ofcell stabilization of the released nucleoids was found lysis on ithe decrease in number of rod-shaped nonto have unfavorable consequences with respect plasmoly,zed cells (A) and the appearance of rodto a fast and gentle isolation at low temperature shaped ((O) and spherical (5) ghost cells. (A) Incuof a fragile cel component. bation at 0O'C; (B) incubation at 250(C. A first consequence of this high-ionic-strength dition was observed to be exponential. Therefore environment is illustrated in Fig. 4, which shows the senssitivity of cells to detergent lysis could the effect of 1 M NaCl on the activity of lysobe charsacterized by the time required to reduce zyme measured over the temperature range northe abs(Drbance of the lytic mixture to half its mally used. At high salt concentration the rate of conversion of plasmolyzed cells to spherical original value (tA.5). This parameter and the ghosts was considerably lower, especially at correspcrnding one for reversal of plasmolysis higher incubation temperatures (cf. Fig. 4B and (tPO.5) Wras essentially temperature independent (Table '2). However, despite considerable fluc- D). A further analysis revealed that at high salt tuations3, the rate of formation of rod-shaped concentration the fraction of spheroplasts was ghosts i]ncreased considerably when the incuba- considerably reduced parallel with the appearA
z
0
4
0
0.
>
w
C-)
4
B
0
0
w
U
B
882
MATERMAN AND VAN GOOL
ance of a high percentage of rod-shaped ghost cells (cf. Fig. 4A and B with C and D). To overcome this inhibitory effect on lysozyme ac-
tivity, excessive amounts of this enzyme have been used ranging from final concentrations of 0.8 up to 2.0 mg/ml (3, 12). However, cell lysis became strongly inhibited when cells were incubated with increasing concentrations of lysozyme in the presence of a chelating agent (Fig. 5). Although the extent of cell lysis may not have been appropriately reflected by the decrease in turbidity of the incubation mixture, parallel microscopic observations revealed that rapid and extensive cell aggregation occurred at a lysozyme concentration of 800 ,ug/ml, preventing cell lysis almost completely. When, according to Kornberg et al. (6), 1 M NaCl was replaced by 5 mM spermidine, an immediate and complete cell lysis was observed upon incubation with the same detergent mixture. This suggests that a preincubation of plasmolyzed, exponentially grown cells for 30 s with lysozyme and citrate was sufficient to render all the cells susceptible to detergent lysis. Consequently, a second major inhibitory effect of the high salt concentration on cell lysis was postu-
E 0 0
(0
LU
uJ
5
10
15
J. BACTERIOL.
lated at the level of the detergent action. The lysis of lysozyme-citrate-treated cells by the detergent mixture was considerably slower in the presence of 1 M NaCl over a temperature range extending from 0 to 25°C (Fig. 6). DISCUSSION The examination of morphological changes during cell lysis directly illustrates that the undisturbed release of nucleoids can only result from a compromise between two extremes. On the one hand, genome release may be restrained by a convoluted, unappropriately disrupted cell envelope as it occurs in cell lysis at 00C (Fig. 2f), whereas at the other extreme the nucleoid may unfold and bind nonspecifically to vesiculating envelopes after a prolonged incubation of the lysate at higher temperatures (Fig. 2g). To avoid these extremes, cell lysis should be carried out in correspondence with two essential requirements for an efficient release of folded nucleoids. First, cell lysis should provide the possibility of an undisturbed release of the sizable nucleoid through the cell envelope. Second, the fragile DNA molecule must be protected by a fast and gentle lysis at low temperature in the presence of 1 M NaCl as a counterionic charge (4, 12). These requirements, however, are difficult to reconcile since the high salt concentration was shown to inhibit the lytic effect of both lysozyme (Fig. 4) and the detergent mixture (Fig. 6). Attempts to improve lysis efficiency using higher lysozyme concentrations seemed to be unfavorable (Fig. 5). Cell lysis has to proceed at higher incubation temperatures (20 to 25°C) to obtain a fast and undisturbed release of nu-
20
z co
4 2K
2.8
B 2.4
0 m
(0
0
4
2.0
CD 1,6
1.2
m
0
co
1,2 0.8
0,4
0
10
20
30
40
TIME (min)
FIG. 5. Effect of lysozyme concentration on osmotic sensitivity. Exponential cells were suspended in TEA buffer (10 mM, pH 8.2) at n = 6 x 107/ml. After addition of sodium citrate (10 mM final concentration) and lysozyme the absorbance of the incubation mixture was measured as a function of time. (A) Incubation at 25°C; (B) incubation at 0°C. Symbols: 0, final concentration of lysozyme, 800 ug/ml; E, 400 ,g/ml; A, 200 pg/ml; x, 100ug/ml; 0, 25 pg/ml.
0
L4
12 8 TIME (mn)
16
FIG. 6. Effect of high salt concentration on the detergent lysis of lysozyme-treated, plasmolyzed cells. Plasmolyzed cells were incubated with lysozyme (200 pg/ml) and sodium citrate (10 mM). After 30 s the incubation mixture was diluted twofold with a detergent mixture containing 1% Brij-58 and 0.4% deoxycholate. Symbols: *, 0°C; A, 25°C; 0, 0°C, 1 M NaCl; A, 25°C, 1 M NaCl.
VOL. 133, 1978
NUCLEOID RELEASE FROM E. COLI
cleoids through a single gap in the cell envelope (Fig. 2 and 3). Since unlike the effect of detergents (2), lysozyme activity is temperature dependent, this gap presumably results from a selective interaction of lysozyme with zones of enhanced murein synthesis in the cell wall (13). However, prolonged incubation of the lysate at high temperature results in a partial unfolding of the released nucleoids (9) and the trapping of vesiculating cell envelope fragments within nucleoids (Fig. 2g). In addition, extensive detergent exposure could lead to a denaturation of proteins, contributing to stabilization of the folded nucleoid in situ (10). It is therefore suggested that a microscopic control on the formation of rod-shaped ghosts should occur shortly after addition of the detergents.
bacterial nucleoids. J. Mol. Biol. 96:217-237. 4. Godson, G. N. 1967. A technique of rapid lysis for the preparation of Escherichia coli polyribosomes. Methods Enzymol. 12:503-516. 5. Korch, C., S. Ovreb0, and K. Kleppe. 1976. Envelopeassociated folded chromosomes from Escherichia coli: variations under different physiological conditions. J. Bacteriol. 127:904-916. 6. Kornberg, T., A. Lockwood, and A. Worcel. 1974. Replication of the Escherichia coli chromosome with a soluble enzyme system. Proc. Natl. Acad. Sci. U.S.A. 71:3189-3193. 7. Meyer, M., M. A. De Jong, C. L. Woldringh, and N. Nanninga. 1976. Factors affecting the release of folded chromosomes from Escherichia coli. Eur. J. Biochem. 63:469-475. 8. Pettijohn, D. E. 1976. Prokaryotic DNA in nucleoid structure. CRC Crit. Rev. Biochem. 4:175-202. 9. Pettijohn, D. E., and R. Hecht. 1973. RNA molecules bound to the folded bacterial genome stabilize DNA folds and segregate domains of supercoiling. Cold Spring Harbor Symp. Quant. Biol. 38:31-41. 10. Rouviere-Yaniv, J., and F. Gros. 1975. Characterization of a novel, low-molecular-weight DNA-binding protein from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:3428-3432. 11. Ryter, A., E. Kellenberger, 0. Birch-Andersen, and 0. Maal0e. 1958. Etude au microscope electronique de plasmas contenant de l'acide desoxyribonucleique. Z. Naturforsch. Teil B 13:597-605. 12. Stonington, 0. G., and D. E. Pettijohn. 1971. The folded genome of Escherichia coli isolated in a protein DNA-RNA complex. Proc. Natl. Acad. Sci. U.S.A. 68:6-9. 13. Trentini, W. C., and R. G. E. Murray. 1975. Ultrastructural effect of lysozyme on the cell wall of Caryophanon latum. Can. J. Microbiol. 21:164-172. 14. Voss, J. G. 1964. Lysozyme lysis of gram-negative bacteria without production of spheroplasts. J. Gen. Microbiol. 35:313-317. 15. Worcel, A., and E. Burgi. 1974. Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J. Mol. Biol. 82:91-105. 16. Zussman, D. R., A. Carbonell, and J. Y. Haga. 1973. Nucleoid condensation and cell division in Escherichia coli MX74T2 ts52 after inhibition of protein synthesis. J. Bacteriol. 115:1167-1178.
ACKNOWLEDGMENTS We are grateful to N. Nanninga of the Laboratory for Electron Microscopy, University of Amsterdam, for the use of electron microscope facilities and to M. J. Heuts for critical reading of the manuscript. Special thanks are due to C. L. Woldringh for his profound interest in this study and his direct guidance of E. M. while carrying out the electron microscopic part of this work. This work was supported by the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek (A.V.G.), by the Bilateraal Cultereel Akkoord Belgie-Nederland (E.M.), and partly by a doctorandus-scholarship from the Onderzoeksfonds K.U.Leuven (E.M.).
LITERATURE CITED 1. Delius, H., and A. Worcel. 1974. Electronmicroscopic visualization of the folded chromosome of Escherichia coli. J. Mol. Biol. 82:107-109. 2. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol. 115:717-722. 3. Giorno, R., R. Stamato, B. Lydersen, and D. Pettijohn. 1975. Transcription in vitro of DNA in isolated
883
Vol. 133, No. 2
Printed in U.S.A.
Nucleoid Release from Escherichia coli Cells ERIC C. MATERMAN
AND
AUGUST P. VAN GOOL*
F. A. Janssens Memorial Laboratory of Genetics, University of Leuven, 3030 Heverlee, Belgium Received for publication 6 September 1977
The time course of morphological changes during lysis of Escherichia coli cells was examined with respect to an undisturbed release of nucleoids. The addition of detergents to plasmolyzed, osmotic sensitive cells resulted in the immediate reversal of plasmolysis followed by the appearance of rod-shaped ghost cells without any detectable spheroplast formation. Electron microscopic examination of the rod-shaped ghost cells revealed a zonal gap in the cell envelope, allowing the free release of the nucleoid. Due to the high ionic strength, a suitable cell lysis was shown to require higher incubation temperatures. However, in the absence of an appropriate control this may result in the sphering and vesiculation of ghost cell envelopes and even the unfolding of released nucleoids. To avoid this unfavorable consequence of lysis at high temperatures, a microscopic examination on the course of rod-shaped ghost formation is suggested. In a recent review on the nucleoid of Escherichia coli, Pettijohn emphasized the empirical nature of the methods used in the isolation of bacterial nucleoids. He described their successful isolation as a compromise between two extreme conditions of cell lysis resulting either in unfolded chromosomes or in nucleoids that remain trapped in the partially disrupted cell envelope (8). From the sedimentation analysis of purified nucleoids these extremes were characterized either as membrane-associated or membranefree but more or less unfolded complexes (9, 15). The proportion of both types of nucleoids was shown to depend on the temperature of cell lysis, the former being predominant at 0°C (9, 15). Although initially the association of the nucleoid with the cell membrane was considered to be specific (15), it was subsequently shown to result from a nonspecific binding of the nucleoid to the cell envelope (7). Since these data indicated that a variation in the sedimentation characteristics of isolated nucleoids may be traced back to their release from the cell during lysis, Korch et al. (5) examined the lysis requirements to reach the compromise of successful nucleoid isolation. Using a method for sedimentation analysis of crude lysates, they concluded that an efficient release of nucleoids requires a careful control of cell lysis (5). In this respect we studied the cytological changes occurring in lysing cells, trying to visualize directly the requirements for successful nucleoid release. MATERIALS AND MErHODS Bacterial strain and growth conditions. E. coli
NF 87, D-10 metB argA relA1, a strain obtained from N. Fiil (University of Copenhagen) and closely related to the one used by Stonington and Pettijohn (12), was grown in M9 minimal salts medium (16) supplemented with 0.2% glucose, 50 pug of methionine per ml, and 50 ug of arginine per ml. Lysis procedure. Exponentially growing cells were quickly pelieted at 5,000 x g (00C) and resuspended to a concentration of approximately 109 cells/ml. Cell lysis was carried out by detergent treatment of plasmolyzed, osmotic sensitive cells as described by Stonington and Pettijohn (12). However, to avoid interference with glutaraldehyde fixation, tris(hydroxymethyl)aminomethane buffer was replaced by triethanolamine (TEA) (1), and for similar reasons sodium citrate was used rather than ethylenediaminetetraacetate. To improve the electron microscopic visualization of nucleoid release, attempts were made to trap the nucleoid in the neighborhood of the cell from which it emerged by enmeshment of the cells in 1% agar before lysis. However, under these conditions cell lysis proceeded very slowly and was only successful at incubation temperatures equal to or higher than 200C. Morphokinetic analysis. At 1-min time intervals after the addition of detergents, 0.3-ml samples were quickly removed from the incubation mixture, and the absorbance at 660 nm was measured immediately in a Beckman-25 spectrophotometer. At the same time intervals, samples were also fixed with 2% (vol/vol) formaldehyde in 1 M NaCl and rapidly scanned in a Zeiss GFL phase-contrast microscope to obtain a representative series of micrographs. These micrographs allowed a reproducible quantitative reconstruction of the gross morphological changes observed in the cells during the course of cell lysis. Electron microscopic analysis. Samples were either withdrawn directly from the lysate or derived from the supernatant after a low-speed centrifugation of the lysate (5,000 x g, 5 min, 0°C). They were subsequently prefixed with 1% glutaraldehyde for 10 min
878
VOL. 133, 1978
NUCLEOID RELEASE FROM E. COLI
at the temperature of cell lysis. These conditions of prefixation were selected after examination of the effect of different chemical fixatives on the stability of isolated nucleoids (Table 1). After prefixation the samples were fixed with OS04 at a 0.5% final concentration for 10 min before enmeshment in 1% agar containing 1 M NaCl. Agar blocks were further processed as described by Ryter et al. (11). Sodium chloride at a 1 M final concentration was added to all fixatives. Overnight fixation in the presence of 0.1% tryptone was carried out at 4°C to avoid interference of the visualization of DNA fibers by tryptone precipitation. Samples were sectioned with an LKB III ultramicrotome and examined in a Philips EM 300 electron microscope at 80 kV.
RESULTS Morphokinetics of cell lysis. After a brief exposure to lysozyme and citrate, a twofold dilution of plasmolyzed E. coli cells into a highsalt solution containing deoxycholate and Brij58 led to a gradual clearing of the incubation mixture. At the same time a distinct sequence of morphological changes was observed in the cell population (Fig. 1, transition B to C). The plasmolysis of cells induced in step A, and which remained throughout step B, was rapidly reversed upon addition of the detergent mixture (Fig. 2a and b). This reversal was followed by a
879
TABLE 1. Effect of fixation on nucleoid stability Fixation conditiona Addition after fixation
Glutaraldehyde
None
Glutaraldehyde 0(0°
Control 0.01%
1%
-
-
-
(0.01%)
Glutaraldehyde (1%)
(0.%)4 101)O04(.% +
+ NT5b SDS, 0.5% + + NT + RNase, 0.1% 'The supernatant of a low-speed centrifugation (5,000 x g, 5 min) of a 15°C lysate was fixed for 10 min at 15°C. A qualitative estimate of the increase in viscosity of the supernatant (+), after addition of sodium dodecyl sulfate (SDS) or pancreatic ribonuclease (RNase), was used to test nucleoid fixation. bNT, Not tested. Pr oress of Lysis
Uorphological Effect
Phase Contrest
Micrograph-s
Diagram
A step
5icruse
Flesmclysias
20Q,"
DC; 3 to 4 min
|, step
Flasmolyzed osmotic
L4soZyme 300 ,g/ml
sensitive rods No spheroplasts
5cdium citrate 50 mM
Cu
/
m
I
; 30 sec
C step NaDI; 14W
D Or
1. Reversal of
plasmolysis
2r" C).ZC
# 2.
ertJ-58 0.5s U to
25"C;
10
Rod
shaped ghosts
.
a
min 3. Spherical ghosts 4
.4. Aggregates of
Q
a -
U,
ghosts
FIG. 1. Phase-contrast micrographs and diagrammatic representation of the sequence of gross morphological changes observed throughout the lytic procedure. x2,020.
880
MATERMAN AND VAN GOOL
a
J. BACTERIOL.
b
d
W..
-
#
I"
-4
d
..O
'; :.I
f
e
IL
I
9
A
-) IL
4. I
,.
;
p.-.
Ice,:11 410-Al
FIG. 2. Electron micrographs showing in detail the same sequence of morphological changes as in Fig. 1. Bar = 1 ,um. (a) Plasmolyzed cell in which the nucleoplasm is dispersed throughout the cytoplasm. (b) Reversal of plasmolysis after detergent addition. The cell contains a contracted nucleoplasm. (c and d) Rod-shaped ghost cells with characteristic zonal envelope gap either polar (c) or equatorial (d). In (c) the nucleoid has been withheld at the level of the envelope gap, whereas in (d) it has been released. Cell lysis was carried out at 20°C, and in (c) cells were enmeshed in agar before lysis. (e) Sphering ghost cells. (f) Ghost cell after lysis at 0°C. The rod shape is more or less preserved and the DNA is released through the different gaps. (g) Nucleoid trapping vesiculating cell envelope remnants which occur after spherical ghost cell formation. Lysis was carried out at 20°C, and the lysate purified by a low-speed spin.
gradual formation of rod-shaped ghost cells, tent including the sizable nucleoid was released which subsequently became converted to spher- through this envelope gap (Fig. 2c). At these ical ghosts (Fig. 1 and 2). Confirming earlier higher incubation temperatures a vesiculation observations (14), spheroplasts were never ob- of the ghost cell envelopes was observed concurserved either during the lysozyme treatment or rent with the appearance of spherical ghosts after dilution of the osmotic sensitive cells with (Fig. 2g and e). Simultaneously, the ghost cells the detergent mixture. started to aggregate while the lysate became The envelope structure of the rod-shaped highly viscous, indicating nucleoid unfolding. ghost cells was dependent on the lysis temperaAlthough the sequence of cytological changes ture. At 0°C convoluted envelopes were ob- during lysis was invariant, the time ofoccurrence served (Fig. 2f), whereas at higher incubation temperatures (20 to 25°C) the envelopes were smooth and revealed a single gap, frequently located either near the cell equator or at the pole (Fig. 2c and d). Since serial sections did not show additional interruptions in the continuity of the ghost envelope, the entire cell con-
of the respective changes in cell morphology depended on several parameters, including lysis temperature (Table 2, Fig. 3) and salt concentration (Fig. 4). Temperature dependence of cell lysis. Measured on a time course basis, the decrease in absorbance of the lysate after detergent ad-
NUCLEOID RELEASE FROM E. COLI
VOL. 133, 1978
TABLE 2. Time course of gross morphological changes and decrease in absorbance during cell lysis as a function of the incubation temperature Absorb- Reversal Rod-shaped ghost cells Spherical ance eofplasTemp crease molysis (O) (660 rum) (P) (t¶5(tE tSG;j s) _ O_.5 _ ( 5tAI.5 [8]) (tp05 [8]) [SG) 5 a(t [s]) [_ tS(;
881
z
0 J9
(RGhstG)R
0
263 (4)
b
IL
54 _d
138 360 Nonec 318 480 None 180 450 None 25 284 (19) 52 60 180 180 170 90 _ a to.5 values indicate, respectively, the time interval required after detergent addition to obtain a 50% decrease of the original value (tAo.5 and tpo.5) or to reach half the maximum value (tRsGo.5 and tSGo.5)bAverage values with the number of observations indicated in parentheses. c No spherical ghosts were observed at this incubation temperature. d No value available because the morphological change occurred too fast to be measured accurately.
0
2
4
6
8 0
2
4
6
-_
8
TIME (min )
FIG. 4. Effect of high salt concentration on the conversion of plasmolyzed cells to spherical ghosts. Plasmolyzed cells (sucrose, 20%o [wt/vol]) were incubated with lysozyme (800 lg/ml) and sodium citrate (10 mM), respectively, at 0°C (A and C) and 25°C (B and D). To reaction mixtures C and D, NaCl at a I M final concentration was added. At 1-min time intervals, 0.3-ml samples were diluted twofold in distilled water and immediately fixed with 2% formaldehyde before microscopic observation. Symbols: *,
nonplasmolyzed, rod-shaped cells; E, spheroplasts; 0, rod-shaped ghost cells; O, spherical ghost cells.
tion temperature was raised from 0 to 25°C, and a maximum number of rod-shaped ghosts was obtained 7 and 2 min, respectively, after addition of detergents (Table 2, Fig. 3). At the higher incubation temperatures the number of rod0. shaped ghosts decreased beyond its maximum, and a proportional number of spherical ghosts in the lysate (Fig. 3B). This observa. _ A _ _ _~ A A S_appeared _ 0 tion suggests that the spherical ghosts originated )B from rod-shaped ghost cells and did not arise from the lysis of spheroplasts, which were never observed in the lysate. Consequently, incubation z of the lysate beyond the appearance of rodshaped ghost cells is not necessary for nucleoid release and should be avoided because of the _ _-_nonspecific trapping of the DNA in vesiculating envelope fragments (Fig. 2g). 2 0 10 4 6 8 Cell lysis at high ionic strength. The use TIME (min) of a high salt concentration (1 M NaCl) for FIG. 3. Effect of the incubation temperature ofcell stabilization of the released nucleoids was found lysis on ithe decrease in number of rod-shaped nonto have unfavorable consequences with respect plasmoly,zed cells (A) and the appearance of rodto a fast and gentle isolation at low temperature shaped ((O) and spherical (5) ghost cells. (A) Incuof a fragile cel component. bation at 0O'C; (B) incubation at 250(C. A first consequence of this high-ionic-strength dition was observed to be exponential. Therefore environment is illustrated in Fig. 4, which shows the senssitivity of cells to detergent lysis could the effect of 1 M NaCl on the activity of lysobe charsacterized by the time required to reduce zyme measured over the temperature range northe abs(Drbance of the lytic mixture to half its mally used. At high salt concentration the rate of conversion of plasmolyzed cells to spherical original value (tA.5). This parameter and the ghosts was considerably lower, especially at correspcrnding one for reversal of plasmolysis higher incubation temperatures (cf. Fig. 4B and (tPO.5) Wras essentially temperature independent (Table '2). However, despite considerable fluc- D). A further analysis revealed that at high salt tuations3, the rate of formation of rod-shaped concentration the fraction of spheroplasts was ghosts i]ncreased considerably when the incuba- considerably reduced parallel with the appearA
z
0
4
0
0.
>
w
C-)
4
B
0
0
w
U
B
882
MATERMAN AND VAN GOOL
ance of a high percentage of rod-shaped ghost cells (cf. Fig. 4A and B with C and D). To overcome this inhibitory effect on lysozyme ac-
tivity, excessive amounts of this enzyme have been used ranging from final concentrations of 0.8 up to 2.0 mg/ml (3, 12). However, cell lysis became strongly inhibited when cells were incubated with increasing concentrations of lysozyme in the presence of a chelating agent (Fig. 5). Although the extent of cell lysis may not have been appropriately reflected by the decrease in turbidity of the incubation mixture, parallel microscopic observations revealed that rapid and extensive cell aggregation occurred at a lysozyme concentration of 800 ,ug/ml, preventing cell lysis almost completely. When, according to Kornberg et al. (6), 1 M NaCl was replaced by 5 mM spermidine, an immediate and complete cell lysis was observed upon incubation with the same detergent mixture. This suggests that a preincubation of plasmolyzed, exponentially grown cells for 30 s with lysozyme and citrate was sufficient to render all the cells susceptible to detergent lysis. Consequently, a second major inhibitory effect of the high salt concentration on cell lysis was postu-
E 0 0
(0
LU
uJ
5
10
15
J. BACTERIOL.
lated at the level of the detergent action. The lysis of lysozyme-citrate-treated cells by the detergent mixture was considerably slower in the presence of 1 M NaCl over a temperature range extending from 0 to 25°C (Fig. 6). DISCUSSION The examination of morphological changes during cell lysis directly illustrates that the undisturbed release of nucleoids can only result from a compromise between two extremes. On the one hand, genome release may be restrained by a convoluted, unappropriately disrupted cell envelope as it occurs in cell lysis at 00C (Fig. 2f), whereas at the other extreme the nucleoid may unfold and bind nonspecifically to vesiculating envelopes after a prolonged incubation of the lysate at higher temperatures (Fig. 2g). To avoid these extremes, cell lysis should be carried out in correspondence with two essential requirements for an efficient release of folded nucleoids. First, cell lysis should provide the possibility of an undisturbed release of the sizable nucleoid through the cell envelope. Second, the fragile DNA molecule must be protected by a fast and gentle lysis at low temperature in the presence of 1 M NaCl as a counterionic charge (4, 12). These requirements, however, are difficult to reconcile since the high salt concentration was shown to inhibit the lytic effect of both lysozyme (Fig. 4) and the detergent mixture (Fig. 6). Attempts to improve lysis efficiency using higher lysozyme concentrations seemed to be unfavorable (Fig. 5). Cell lysis has to proceed at higher incubation temperatures (20 to 25°C) to obtain a fast and undisturbed release of nu-
20
z co
4 2K
2.8
B 2.4
0 m
(0
0
4
2.0
CD 1,6
1.2
m
0
co
1,2 0.8
0,4
0
10
20
30
40
TIME (min)
FIG. 5. Effect of lysozyme concentration on osmotic sensitivity. Exponential cells were suspended in TEA buffer (10 mM, pH 8.2) at n = 6 x 107/ml. After addition of sodium citrate (10 mM final concentration) and lysozyme the absorbance of the incubation mixture was measured as a function of time. (A) Incubation at 25°C; (B) incubation at 0°C. Symbols: 0, final concentration of lysozyme, 800 ug/ml; E, 400 ,g/ml; A, 200 pg/ml; x, 100ug/ml; 0, 25 pg/ml.
0
L4
12 8 TIME (mn)
16
FIG. 6. Effect of high salt concentration on the detergent lysis of lysozyme-treated, plasmolyzed cells. Plasmolyzed cells were incubated with lysozyme (200 pg/ml) and sodium citrate (10 mM). After 30 s the incubation mixture was diluted twofold with a detergent mixture containing 1% Brij-58 and 0.4% deoxycholate. Symbols: *, 0°C; A, 25°C; 0, 0°C, 1 M NaCl; A, 25°C, 1 M NaCl.
VOL. 133, 1978
NUCLEOID RELEASE FROM E. COLI
cleoids through a single gap in the cell envelope (Fig. 2 and 3). Since unlike the effect of detergents (2), lysozyme activity is temperature dependent, this gap presumably results from a selective interaction of lysozyme with zones of enhanced murein synthesis in the cell wall (13). However, prolonged incubation of the lysate at high temperature results in a partial unfolding of the released nucleoids (9) and the trapping of vesiculating cell envelope fragments within nucleoids (Fig. 2g). In addition, extensive detergent exposure could lead to a denaturation of proteins, contributing to stabilization of the folded nucleoid in situ (10). It is therefore suggested that a microscopic control on the formation of rod-shaped ghosts should occur shortly after addition of the detergents.
bacterial nucleoids. J. Mol. Biol. 96:217-237. 4. Godson, G. N. 1967. A technique of rapid lysis for the preparation of Escherichia coli polyribosomes. Methods Enzymol. 12:503-516. 5. Korch, C., S. Ovreb0, and K. Kleppe. 1976. Envelopeassociated folded chromosomes from Escherichia coli: variations under different physiological conditions. J. Bacteriol. 127:904-916. 6. Kornberg, T., A. Lockwood, and A. Worcel. 1974. Replication of the Escherichia coli chromosome with a soluble enzyme system. Proc. Natl. Acad. Sci. U.S.A. 71:3189-3193. 7. Meyer, M., M. A. De Jong, C. L. Woldringh, and N. Nanninga. 1976. Factors affecting the release of folded chromosomes from Escherichia coli. Eur. J. Biochem. 63:469-475. 8. Pettijohn, D. E. 1976. Prokaryotic DNA in nucleoid structure. CRC Crit. Rev. Biochem. 4:175-202. 9. Pettijohn, D. E., and R. Hecht. 1973. RNA molecules bound to the folded bacterial genome stabilize DNA folds and segregate domains of supercoiling. Cold Spring Harbor Symp. Quant. Biol. 38:31-41. 10. Rouviere-Yaniv, J., and F. Gros. 1975. Characterization of a novel, low-molecular-weight DNA-binding protein from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:3428-3432. 11. Ryter, A., E. Kellenberger, 0. Birch-Andersen, and 0. Maal0e. 1958. Etude au microscope electronique de plasmas contenant de l'acide desoxyribonucleique. Z. Naturforsch. Teil B 13:597-605. 12. Stonington, 0. G., and D. E. Pettijohn. 1971. The folded genome of Escherichia coli isolated in a protein DNA-RNA complex. Proc. Natl. Acad. Sci. U.S.A. 68:6-9. 13. Trentini, W. C., and R. G. E. Murray. 1975. Ultrastructural effect of lysozyme on the cell wall of Caryophanon latum. Can. J. Microbiol. 21:164-172. 14. Voss, J. G. 1964. Lysozyme lysis of gram-negative bacteria without production of spheroplasts. J. Gen. Microbiol. 35:313-317. 15. Worcel, A., and E. Burgi. 1974. Properties of a membrane-attached form of the folded chromosome of Escherichia coli. J. Mol. Biol. 82:91-105. 16. Zussman, D. R., A. Carbonell, and J. Y. Haga. 1973. Nucleoid condensation and cell division in Escherichia coli MX74T2 ts52 after inhibition of protein synthesis. J. Bacteriol. 115:1167-1178.
ACKNOWLEDGMENTS We are grateful to N. Nanninga of the Laboratory for Electron Microscopy, University of Amsterdam, for the use of electron microscope facilities and to M. J. Heuts for critical reading of the manuscript. Special thanks are due to C. L. Woldringh for his profound interest in this study and his direct guidance of E. M. while carrying out the electron microscopic part of this work. This work was supported by the Belgian Nationaal Fonds voor Wetenschappelijk Onderzoek (A.V.G.), by the Bilateraal Cultereel Akkoord Belgie-Nederland (E.M.), and partly by a doctorandus-scholarship from the Onderzoeksfonds K.U.Leuven (E.M.).
LITERATURE CITED 1. Delius, H., and A. Worcel. 1974. Electronmicroscopic visualization of the folded chromosome of Escherichia coli. J. Mol. Biol. 82:107-109. 2. Filip, C., G. Fletcher, J. L. Wulff, and C. F. Earhart. 1973. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol. 115:717-722. 3. Giorno, R., R. Stamato, B. Lydersen, and D. Pettijohn. 1975. Transcription in vitro of DNA in isolated
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