Chapter
12
Methods for Protoplast Formation in Escherichid coli RICHARD L. WEISS’ The Biological Laboratories, Harvard University, Cambridge, Massachusetts
Introduction . . . . . General Methods . . Appearance of Protoplasts . . Discussion. , A. Lysozyme Concentration . B. Temperature and Ionic Strength C. Osmotic Conditions . V. Summary and Conclusion . . References . . .
I. 11. 111. IV.
. .
. . . . . . .
. . .
.
.
. . . . .
. .
. . . . . . . . .
. . . . . . . . .
. . . . . . .
. .
. . . . .
. . . .
. . .
141 142 142 . 145 , 1 4 5 . 146 . 146 . 146 . 147
I. Introduction The investigation of bacterial membrane structure and function has relied on the isolation of membranes from osmotically sensitive forms, protoplasts and spheroplasts. In this chapter “protoplast” refers to a grampositive organism after complete digestion of the cell wall; “spheroplast” describes gram-negative forms surrounded by or retaining part of the outer membrane. The outer membrane of gram-negative bacteria is difficult to remove from spheroplasts. It either surrounds the cell (Kaback, 1971) or peels back, exposing the cell surface membrane (Birdsell and Cota-Robles, 1967). In both cases the outer membrane remains attached to the cell. A method was recently developed for complete removal of the outer membrane from gram-negative cells, and this method is described here. These protoplasts can be obtained from log-phase and stationary cultures of Escherichia coli by enzymic and chemical alteration of the cell wall. This alteration includes rapid cell wall lysis wherein the outer membrane deI
Present address: Department of Botany, San Diego State University, San Diego, California. 141
142
RICHARD L. WEISS
taches from the cell body, forming small vesicles. This detachment proceeds without restraint until the cell is bound only by the cytoplasmic or inner membrane.
11. General Methods Protoplasts of E . coli ML30 are formed by the following procedure. Cells grown in glucose mineral salts (Fiil and Branton, 1969) without iron at 37°C to the logarithmic (A,,, = 0.9) phase of growth are centrifuged at 10,000 g for 5 minutes. The cells are washed twice in 10 mMtris-HC1 buffer, pH 8, at 23°C. [Tris (hydroxymethyl) aminomethane can be obtained from Schwartz-Mann, Orangeburg, N.Y.] The pellet is resuspended in a solution containing 20% (w/w) sucrose and 0.1 M tris-HC1 (pH 8) which ispipetted directly into the centrifuge tube. The product of a culture's volume and its measured absorbance at 450 nm is calculated to determine the total absorbance units (AU) contained in the culture (Osborn et al., 1972). The cells are suspended in 1 ml of buffer per 10 AU. For example, in our experiments a culture with a volume of 500 ml and an absorbance of 0.9 was resuspended in 45 ml of buffer at 37°C. Cells are then transferred into a small flask, and the temperature is adjusted to 37°C. The cells are stirred with a magnetic stirrer, and within 1 minute 2.25 ml of lysozyme is added from a 2 mg/ml stock solution in distilled water to a final concentration of 100 pglml. The temperature is adjusted to 37"C, and stirring is continued for the next 12 minutes at 37°C. The cell suspension is diluted [I part ethylenediaminetetraacetic acid (EDTA) per 10 parts cells] with0.1 MK,EDTA prewarmed to 37°C. Continuous stirring and slow dilution over 2.5 minutes prevents lysis of cells. Na,EDTA may be used instead. After adding EDTA the temperature is adjusted to 37°C. More than 99% of the cells become spherical within 10 minutes and can be observed by phase microscopy.
111. Appearance of Protoplasts Samples for electron microscope examination are fixed at 4°C in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7, postfixed in 1% osmium tetroxide in 30 mM Verona1 buffer containing 0.5 mM CaCl,, pH 6.1, dehydrated in graded acetone, and embedded in Spurr's (1969) resin. The cell envelope of fixed and sectioned cells is shown in Fig. 1. The
FIG. 1. Control cells. The cell envelope consists of the outer membrane (om), the peptidoglycan layer (r), and the inner or cell membrane (im). FIG. 2. Protoplasts of E. coli. The cell surface is limited by the inner membrane (im); the arrow indicates a wall fragment.
144
RICHARD L. WEISS
features illustrated are the outer membrane, the peptidoglycan layer, and the inner membrane. A consistent feature of the strain depicted here, but not of other strains, is the presence of small, clear areas within the cytoplasm. This feature is observed both before and after protoplasts are formed and is unique to this strain. A field of cells followinglysozyme-EDTA treatment is presented in Fig. 2. In the protoplasts illustrated here the cell wall has been completely removed from 95% of the cells, forming small outer membrane fragments (Fig. 3, inset a). The separatio'n of such fragments is illustrated in Fig. 3, inset b. Here one fragment is partially attached to
FIG. 3. Protoplast of E. coli. The inner membrane (im) forms the outer surface of the cell. Outer membrane fragments that have separated from the cell are shown in inset a. An outer membrane fragment in the process of separating from the cell is indicated by the arrow in inset b. The marker bar also applies to the insets.
12.
PROTOPLASTS OF E. COLI
145
the cell (arrow), whereas the other has separated from it. When the cell surface is observed at higher magnification, as in Fig. 3, the inner membrane can be seen to form the outermost surface boundary, thus confirming cell wall removal. Contaminating cell wall fragments appear on some cells (Fig. 2, arrow). Protoplasts such as these can be centrifuged and resuspended without extensive lysis. Thus one may reasonably expect that membranes can be prepared from such cells and used for further biochemical or structural analysis.
IV. Discussion Since it has been reported in the literature that gram-negative organisms yield spheroplasts, it is important t o consider why this procedure yields protoplasts. Three mechanisms by which this could occur have already been suggested (Weiss, 1976). They are: (1) the enzyme/substrate ratio may be optimal; (2) the constant temperature or ionic conditions may ensure more complete cell wall lysis; and (3) the mild osmotic changes during dilution with EDTA may open up the cell wall. A consideration of the above proposed mechanisms has led us to suggest ways to adapt this method to organisms other than E. coli ML30. The modifications we consider most significant are presented in the following discussion.
A. Lysozyme Concentration It is well established that lysozyme treatment brings about spheroplast formation in E. coli (Kaback, 1971). Concentrations well below the amount used here are apparently sufficient (Birdsell and Cota-Robles, 1967) to peel back the outer membrane with appropriate osmotic treatment. However, it is critically important to have a concentration below that which aggregates the bacteria. While some variation can be tolerated, both minimum ( < 20 pg/ml) and excess (< 500 pglml) concentrations should be avoided, since they inhibit the efficiency of protoplast formation. When applying this technique to other strains of E. coli, it may be necessary to determine the optimum concentration of lysozyme experimentally; for any given organism this concentration may vary with growth conditions. As an example, an organism may respond well to 100 pgIml in log-phase growth, but require 250 pglml under starvation conditions. However, it should be noted that the conditions described here have been used successfully with several ML
146
RICHARD L . WEISS
strains, and it is expected that this procedure will work with other strains such as K12 and B.
B. Temperature and Ionic Strength It is much less likely that changes in the constant-temperature regime or ionic strength can improve this method when difficulty with protoplast formation is encountered. In this regard however, a general principle is that, when the concentration of lysozyme is increased, the ionic strength of the buffer should be decreased.
C. Osmotic Conditions The removal of the cell wall depends not only on enzyme activity, but also on the access of the enzyme to the substrate. Since lysozyme is sufficiently active over a wide range of concentrations (Birdsell and Cota-Robles, 1967; Kaback, 1971;Osborn et af., 1972), it seems reasonable to postulate that the osmotic stress attained with the present method allows more complete access of lysozyme to the substrate, perhaps as a result of the egress of loosely bound enzymes. If it is assumed that the release of such enzymes takes place through a break in the outer membrane, the osmotic imbalance would force out the enzymes, creating one or many discontinuities in this layer. Thus, during the general application of this method to gram-negative organisms, it may be advantageous to increase the osmotic stress. Fortunately, a simple approach t o determine the experimental requirements is available; it includes increased dilution which may be introduced when adding EDTA to the cells. The direct effect of this and other modifications is rapidly monitored by phase microscopy. An advantage of this method is that morphological changes as well as adherent outer membrane coils of spheroplasts can be visualized. Following this approach more elaborate methods such as electron microscopy and chemical analysis will be required to demonstrate cell wall removal.
V.
Summary and Conclusion
Cells of E . cofi ML30 have been converted into protoplasts using lysozyme-EDTA. This technique has several possible applications for this and other sensitive strains. One of these is the separation of protoplasts from the outer membrane fragments by differential centrifugation. This should
12.
PROTOPLASTS OF E. COLI
147
allow physiological studies or further manipulation that hopefully will increase the utility of E. coli in studies on the structure and function of bacterial membranes. ACKNOWLEDGMENT
The work described here was supported by a grant from the American Cancer Society (PF-876).
REFERENCES Birdsell, D. C., and Cota-Robles, E. H . (1967). J . Bacteriol. 93,427-437. Fiil, A,, and Branton, D. (1969). J . Bacteriol. 98, 1320-1327. Kaback, H. R. (1971). In “Methods in Enzymology” (W. B. Jakoby, ed.),Vol. 22, pp. 84-120. Academic Press, New York. Osborn, M. J., Gander, J. E., Parisi, E., and Carson, J. (1972). J . Biol. Chem. 247,3962-3972. Spurr, A. R. (1969). J . Ultrastruct. Res. 26, 3 1 4 3 . Weiss, R. L. (1976). J . Bacteriol. 128, 668-670.
12
Methods for Protoplast Formation in Escherichid coli RICHARD L. WEISS’ The Biological Laboratories, Harvard University, Cambridge, Massachusetts
Introduction . . . . . General Methods . . Appearance of Protoplasts . . Discussion. , A. Lysozyme Concentration . B. Temperature and Ionic Strength C. Osmotic Conditions . V. Summary and Conclusion . . References . . .
I. 11. 111. IV.
. .
. . . . . . .
. . .
.
.
. . . . .
. .
. . . . . . . . .
. . . . . . . . .
. . . . . . .
. .
. . . . .
. . . .
. . .
141 142 142 . 145 , 1 4 5 . 146 . 146 . 146 . 147
I. Introduction The investigation of bacterial membrane structure and function has relied on the isolation of membranes from osmotically sensitive forms, protoplasts and spheroplasts. In this chapter “protoplast” refers to a grampositive organism after complete digestion of the cell wall; “spheroplast” describes gram-negative forms surrounded by or retaining part of the outer membrane. The outer membrane of gram-negative bacteria is difficult to remove from spheroplasts. It either surrounds the cell (Kaback, 1971) or peels back, exposing the cell surface membrane (Birdsell and Cota-Robles, 1967). In both cases the outer membrane remains attached to the cell. A method was recently developed for complete removal of the outer membrane from gram-negative cells, and this method is described here. These protoplasts can be obtained from log-phase and stationary cultures of Escherichia coli by enzymic and chemical alteration of the cell wall. This alteration includes rapid cell wall lysis wherein the outer membrane deI
Present address: Department of Botany, San Diego State University, San Diego, California. 141
142
RICHARD L. WEISS
taches from the cell body, forming small vesicles. This detachment proceeds without restraint until the cell is bound only by the cytoplasmic or inner membrane.
11. General Methods Protoplasts of E . coli ML30 are formed by the following procedure. Cells grown in glucose mineral salts (Fiil and Branton, 1969) without iron at 37°C to the logarithmic (A,,, = 0.9) phase of growth are centrifuged at 10,000 g for 5 minutes. The cells are washed twice in 10 mMtris-HC1 buffer, pH 8, at 23°C. [Tris (hydroxymethyl) aminomethane can be obtained from Schwartz-Mann, Orangeburg, N.Y.] The pellet is resuspended in a solution containing 20% (w/w) sucrose and 0.1 M tris-HC1 (pH 8) which ispipetted directly into the centrifuge tube. The product of a culture's volume and its measured absorbance at 450 nm is calculated to determine the total absorbance units (AU) contained in the culture (Osborn et al., 1972). The cells are suspended in 1 ml of buffer per 10 AU. For example, in our experiments a culture with a volume of 500 ml and an absorbance of 0.9 was resuspended in 45 ml of buffer at 37°C. Cells are then transferred into a small flask, and the temperature is adjusted to 37°C. The cells are stirred with a magnetic stirrer, and within 1 minute 2.25 ml of lysozyme is added from a 2 mg/ml stock solution in distilled water to a final concentration of 100 pglml. The temperature is adjusted to 37"C, and stirring is continued for the next 12 minutes at 37°C. The cell suspension is diluted [I part ethylenediaminetetraacetic acid (EDTA) per 10 parts cells] with0.1 MK,EDTA prewarmed to 37°C. Continuous stirring and slow dilution over 2.5 minutes prevents lysis of cells. Na,EDTA may be used instead. After adding EDTA the temperature is adjusted to 37°C. More than 99% of the cells become spherical within 10 minutes and can be observed by phase microscopy.
111. Appearance of Protoplasts Samples for electron microscope examination are fixed at 4°C in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7, postfixed in 1% osmium tetroxide in 30 mM Verona1 buffer containing 0.5 mM CaCl,, pH 6.1, dehydrated in graded acetone, and embedded in Spurr's (1969) resin. The cell envelope of fixed and sectioned cells is shown in Fig. 1. The
FIG. 1. Control cells. The cell envelope consists of the outer membrane (om), the peptidoglycan layer (r), and the inner or cell membrane (im). FIG. 2. Protoplasts of E. coli. The cell surface is limited by the inner membrane (im); the arrow indicates a wall fragment.
144
RICHARD L. WEISS
features illustrated are the outer membrane, the peptidoglycan layer, and the inner membrane. A consistent feature of the strain depicted here, but not of other strains, is the presence of small, clear areas within the cytoplasm. This feature is observed both before and after protoplasts are formed and is unique to this strain. A field of cells followinglysozyme-EDTA treatment is presented in Fig. 2. In the protoplasts illustrated here the cell wall has been completely removed from 95% of the cells, forming small outer membrane fragments (Fig. 3, inset a). The separatio'n of such fragments is illustrated in Fig. 3, inset b. Here one fragment is partially attached to
FIG. 3. Protoplast of E. coli. The inner membrane (im) forms the outer surface of the cell. Outer membrane fragments that have separated from the cell are shown in inset a. An outer membrane fragment in the process of separating from the cell is indicated by the arrow in inset b. The marker bar also applies to the insets.
12.
PROTOPLASTS OF E. COLI
145
the cell (arrow), whereas the other has separated from it. When the cell surface is observed at higher magnification, as in Fig. 3, the inner membrane can be seen to form the outermost surface boundary, thus confirming cell wall removal. Contaminating cell wall fragments appear on some cells (Fig. 2, arrow). Protoplasts such as these can be centrifuged and resuspended without extensive lysis. Thus one may reasonably expect that membranes can be prepared from such cells and used for further biochemical or structural analysis.
IV. Discussion Since it has been reported in the literature that gram-negative organisms yield spheroplasts, it is important t o consider why this procedure yields protoplasts. Three mechanisms by which this could occur have already been suggested (Weiss, 1976). They are: (1) the enzyme/substrate ratio may be optimal; (2) the constant temperature or ionic conditions may ensure more complete cell wall lysis; and (3) the mild osmotic changes during dilution with EDTA may open up the cell wall. A consideration of the above proposed mechanisms has led us to suggest ways to adapt this method to organisms other than E. coli ML30. The modifications we consider most significant are presented in the following discussion.
A. Lysozyme Concentration It is well established that lysozyme treatment brings about spheroplast formation in E. coli (Kaback, 1971). Concentrations well below the amount used here are apparently sufficient (Birdsell and Cota-Robles, 1967) to peel back the outer membrane with appropriate osmotic treatment. However, it is critically important to have a concentration below that which aggregates the bacteria. While some variation can be tolerated, both minimum ( < 20 pg/ml) and excess (< 500 pglml) concentrations should be avoided, since they inhibit the efficiency of protoplast formation. When applying this technique to other strains of E. coli, it may be necessary to determine the optimum concentration of lysozyme experimentally; for any given organism this concentration may vary with growth conditions. As an example, an organism may respond well to 100 pgIml in log-phase growth, but require 250 pglml under starvation conditions. However, it should be noted that the conditions described here have been used successfully with several ML
146
RICHARD L . WEISS
strains, and it is expected that this procedure will work with other strains such as K12 and B.
B. Temperature and Ionic Strength It is much less likely that changes in the constant-temperature regime or ionic strength can improve this method when difficulty with protoplast formation is encountered. In this regard however, a general principle is that, when the concentration of lysozyme is increased, the ionic strength of the buffer should be decreased.
C. Osmotic Conditions The removal of the cell wall depends not only on enzyme activity, but also on the access of the enzyme to the substrate. Since lysozyme is sufficiently active over a wide range of concentrations (Birdsell and Cota-Robles, 1967; Kaback, 1971;Osborn et af., 1972), it seems reasonable to postulate that the osmotic stress attained with the present method allows more complete access of lysozyme to the substrate, perhaps as a result of the egress of loosely bound enzymes. If it is assumed that the release of such enzymes takes place through a break in the outer membrane, the osmotic imbalance would force out the enzymes, creating one or many discontinuities in this layer. Thus, during the general application of this method to gram-negative organisms, it may be advantageous to increase the osmotic stress. Fortunately, a simple approach t o determine the experimental requirements is available; it includes increased dilution which may be introduced when adding EDTA to the cells. The direct effect of this and other modifications is rapidly monitored by phase microscopy. An advantage of this method is that morphological changes as well as adherent outer membrane coils of spheroplasts can be visualized. Following this approach more elaborate methods such as electron microscopy and chemical analysis will be required to demonstrate cell wall removal.
V.
Summary and Conclusion
Cells of E . cofi ML30 have been converted into protoplasts using lysozyme-EDTA. This technique has several possible applications for this and other sensitive strains. One of these is the separation of protoplasts from the outer membrane fragments by differential centrifugation. This should
12.
PROTOPLASTS OF E. COLI
147
allow physiological studies or further manipulation that hopefully will increase the utility of E. coli in studies on the structure and function of bacterial membranes. ACKNOWLEDGMENT
The work described here was supported by a grant from the American Cancer Society (PF-876).
REFERENCES Birdsell, D. C., and Cota-Robles, E. H . (1967). J . Bacteriol. 93,427-437. Fiil, A,, and Branton, D. (1969). J . Bacteriol. 98, 1320-1327. Kaback, H. R. (1971). In “Methods in Enzymology” (W. B. Jakoby, ed.),Vol. 22, pp. 84-120. Academic Press, New York. Osborn, M. J., Gander, J. E., Parisi, E., and Carson, J. (1972). J . Biol. Chem. 247,3962-3972. Spurr, A. R. (1969). J . Ultrastruct. Res. 26, 3 1 4 3 . Weiss, R. L. (1976). J . Bacteriol. 128, 668-670.
