ANALYTICAL BIOCHEMISTRY 99, 454--457 (1979)
Extraction of Polysomes from Escherichia coli without Use of Detergent MARIE-PIERRE D E B E T H U N E , JACQUELINE MARCHAL, AND A L A I N J. COZZONE
Department of Molecular Biology, University of Lyon, Villeurbanne 69621, France Received February 20, 1979 A technique of extracting Escherichia coli polysomes in the absence of detergent is presented. It is based on the conversion of cells to spheroplasts and their opening by osmotic shock combined with freeze-thaw treatment.
Various techniques have been used to open Escherichia coli cells in view of polysome extraction. A first type of techniques involves vigorous treatments such as grinding of bacteria with abrasives or ultrasonic oscillation or use of French pressure cell, and therefore results in an extensive destruction of polysomes (1,2). Another type of technique, more commonly used, involves dissolution of the cell wall with detergents after it has been weakened by freezing and thawing or by incubation with lysozyme and ethylenediaminetetraacetate or by penicillin treatment (3-8). The latter procedures yield, in general, a high proportion of undegraded polysomes. However, because of the utilization of detergents during preparation, polysomes thus obtained may not be suitable for further in vitro studies e.g., enzymatic assays, even though neutral detergents appear to be less harmful than ionic detergents (6,8). We now describe a new technique of extracting polysomes from E. coli K-12 that avoids the use of detergent of any kind. It is based on the conversion of cells to spheroplasts (5-7) and opening of these altered cells by osmotic shock and freezethaw treatment. A quantitative and qualitative comparison with a technique previously reported (5) making use of detergents is thereafter presented.
0003- 2697/79/160454-04502.00/0 Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved.
MATERIALS AND METHODS
The auxotrophic strain CP78 (rel A +, arg-, leu-, thr-, his-, vit.Ba-) ofE. coli K-12 was used throughout. Bacteria were grown under forced aeration at 37°C in a minimal medium (10) supplemented with the four essential amino acids (50/zg/ml each) and the vitamin (5/zg/ml). In a typical experiment, 90 ml of an exponentially growing culture (approx. 2 × 10s cells/ml) was poured over crushed ice. Cells were harvested by centrifugation and resuspended in 9 ml of a sucrose-buffer solution containing 0.5 M ribonuclease-free sucrose (Mann Research Laboratories), 0.016 M Tris-HC1 at pH 8.1, and 0.05 M KC1. Spheroplasts were then prepared as previously described (11). Briefly, the cell suspension was treated with 1.1 ml of freshly dissolved lysozyme solution (10 mg/ml in sucrose-buffer solution; Boehringer-Mannheim GmbH) and 0.22 ml of 10% EDTA at pH 8.0. After stirring for 10 min, 0.22 ml of 1 M MgCI2 was added to stop lysozyme action, and spheroplasts were centrifuged. They were then subjected to osmotic shock by resuspension in 0.5 ml of a buffer solution containing 0.01 M Tris-HCl at pH 7.8, 0.05 M NH4C1, and 0.01 M MgC12, and treated with 2/zg ofribonuclease-free deoxyribonuclease
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E. coli POLYSOME EXTRACTION WITHOUT DETERGENT
(Sigma Chemical Co.). After 10 min, the suspension was quickly frozen in a CO2acetone mixture then slowly thawed at room temperature for about 30 min. This freezethaw cycle was sometimes performed one more time (see Results). The cellular lysate thus obtained was finally clarified by lowspeed centrifugation and further analyzed by zonal sedimentation. In other comparative experiments, spheroplasts were instead suspended in 0.5 ml of a lysing medium prepared in 0.01 M TrisHC1 at pH 7.8, 0.05 M NH4C1, and 0.01 M MgCI2, and containing either 0.5% Brij 58 detergent (polyoxyethylene [20] cetylether; Arias Chemical Ind.) or 0.5% sodium deoxycholate, or both. After l0 min, lysates were clarified by low-speed centrifugation then analyzed. Zonal sedimentation analysis of the various preparations was carried out by loading 0.2 to 0.3 ml of clarified lysate onto 15 to 40% linear sucrose gradients (11) and centrifuging for 150 rain at 4°C in a Beckman SW41 Ti rotor at 39,000 rpm. Gradients were then pumped through the continuous-flow cell of a Beckman Model 25 recording spectrophotometer which monitored the absorbance at 260 nm, and 0.3- to 0.4-ml fractions were collected when necessary.
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Long-term labeling of RNA was achieved, in some experiments, by growing cells for two to three generations in the presence of [5-3H]uracil (0.4 /xCi and 6.5 /zg/ml; from the French C.E.A.). The amount of radioactivity incorporated into nucleic acids was determined after precipitation by 5% trichloroacetic acid at 4°C. The amount of total ribosomes recovered (ribosomal subunits + monosomes + polysomes) was determined from the absorbance at 260 nm of the relevant fractions collected after sucrose gradient centrifugation (15 A~60 units corresponding to 1 mg of ribosomes). The proportion of polysomes in the total ribosomal material taken as 100% was calculated from either the absorbance or the amount of incorporated radioactivity in all ribosomal populations heavier than monosomes. Similar values were obtained in both kinds of calculation. In all experiments here reported, polysomes were purposely prepared in the absence of chloramphenicol even though this drug is known to inhibit ribosome translocation and, consequently, to reduce polysome degradation during isolation (5,7). Our aim was, indeed, to avoid any treatment that might interfere with the functional properties of polysome preparations.
TABLE 1 EXTRACTION OF RIBOSOMES FROM SPHEROPLASTS UNDER VARIOUS TREATMENTSa Treatment
Total ribosomes (mg) Proportion of polysomes (%)
Control
Freezingthawing (one cycle)
Freezingthawing (two cycles)
Brij 58
Deoxycholate
Brij-58 + deoxycholate
0.25 60
0.39 62
0.38 59
0.44 58
0.68 58
0.66 62
a Spheroplasts were prepared from exponentially growing cells (approx. 101° cells) and subjected to osmotic shock (control). They were further treated either by freezing-thawing for one or two cycles, or by neutral and/or ionic detergents as indicated. The amount of total ribosomes recovered was determined from the absorbance at 260 nm of the relevant fractions after sucrose gradient centrifugation. The proportion of polysomes in ribosomes was calculated as described under Materials and Methods. Average values from three to six experiments are expressed.
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DE BETHUNE, MARCHAL, AND COZZONE
RESULTS AND DISCUSSION As shown in Table 1, whenE, coli spheroplasts are merely subjected to osmotic shock, a relatively low amount of total ribosomes is extracted. This amount is substantially increased (by more than 50%) if spheroplasts are further treated by freezing-thawing. No significant difference is observed between one or two cycles of such treatment. By analyzing, in parallel, the effects of detergents on the lysis of spheroplasts, it appears that the use of neutral Brij 58 alone improves only slightly the yield of ribosome extraction as compared to freezing-thawing. In contrast, the presence in the lysing medium of sodium deoxycholate, either alone or in addition to Brij 58 as used by Flessel et al. (5), results in the extraction of a much higher amount of total ribosomes since an increase by 70 to 75% can be measured. Therefore, from a quantitative point of view, the technique of freezing-thawing here presented is somehow less efficient than the techniques making use of the combination of neutral and ionic detergents to promote the solubilization of membranes. However, in either type of technique, the apparent structural state of polysomes as analyzed by zonal sedimentation, seems to be the same. Indeed, nearly identical patterns are obtained after centrifugation through linear sucrose gradients
(a) E
0.4
'~
0.3
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2
3
0.2
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1
(b)
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0.2
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FIG. 1. Zonal sedimentation of ribosomal material extracted from untreated or detergent-treated spheroplasts. Lysates were prepared from spheroplasts either treated with Brij 58 and sodium deoxycholate (a) or frozen and thawed for 1 cycle (b). They were analyzed by centrifugation through 15-40% sucrose gradients as indicated under Materials and Methods. In each pattern, the position of ribosomal populations from monosomes to tetrasomes is indicated by numbers, respectively, from 1 to 4.
TABLE 2 P R O P O R T I O N O F V A R I O U S CLASSES OF POLYSOMES I S O L A T E D FROM U N T R E A T E D OR D E T E R G E N T - T R E A T E D S P H E R O P L A S T S a
Relative percentage of polysomes Treatment
Disomes
Trisomes
Tetrasomes
>Tetrasomes
Detergent No detergent
22.3 21.5
18.6 20.2
16.7 17.4
42.4 40.9
a Spheroplasts were prepared from radioactively labeled exponential cells and either subjected to one cycle of freezing-thawing (no detergent) or treated with Brij 58 and sodium deoxycholate (detergent). Lysates were centrifuged through sucrose gradients and the proportion of various classes of polysomes in the total polysomal material taken as 100% was then determined from the amount of radioactivity present in the relevant fractions. Average values from four experiments are expressed.
E. coli POLYSOME EXTRACTION WITHOUT DETERGENT
(Fig. 1). In addition, the proportion of polysomes in total ribosomes is very similar, whatever the technique of extraction, since it varies only from 58 to 62% (Table 1). Moreover, the precise determination of the percentage of various classes of polysomes leads to equivalent results (Table 2), thus indicating that no difference in the maintenance of polysomal structures during isolation seems to exist. This is particularly true in the case of heavier polysomes which are known yet to be readily converted to smaller populations whenever degradation occurs. The original procedure here described for isolating bacterial polysomes offers several advantages. Its main interest lies in the fact that it permits one to obtain preparations which can be qualitatively more appropriate for further in vitro studies than other techniques involving the use of detergents. Also, it is gentle, simple and reproducible, and it allows the isolation ofpolysomes in a largely undegraded state which represents a significant improvement over other techniques that also avoid detergent treatment (1,2). Finally, its applicability is apparently not limited to the type of strain used in these experiments. In particular, by strictly applying this procedure to strain CP79 (tel A-, arg-, leu-, thr-, his-, vit. B1-) orE. coli which has been
457
reported to have a cell envelope different from that of strain CP78 (12), similar results have been obtained. ACKNOWLEDGMENTS This work was supported by grants from the Centre National de la Recherche Scientifique (AI 03.0085) and the Drl~gation Grnrrale ~t la Recherche Scientifique et Technique (MRM 79.7.0152).
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