JOURNAL OF BACTERIOLOGY, Sept. 1977, p. 815-820 Copyright 0D 1977 American Society for Microbiology
Vol. 131, No. 3 Printed in U.S.A.
Intracellular Localization of the Superoxide Dismutases of Escherichia coli: a Reevaluation LARRY BRITTON AND IRWIN FRIDOVICH* Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received for publication 2 May 1977
All of the superoxide dismutase isozymes of Escherichia coli have been shown to occur in the cell matrix, and none have been found in the periplasm. This was the case with both E. coli B and E. coli K-12, whether grown on a low phosphate medium or on a Trypticase soy-yeast extract medium. Alkaline phosphatase was used as a marker of the periplasm; adenosine deaminase and glucose 6-phosphate dehydrogenase were used as matrix markers, and consistent results were obtained by osmotic shock, spheroplast formation, and use of a diazonium salt that penetrates the periplasm but cannot cross the plasma membrane. A previous report that the iron-containing superoxide dismutase of E. coli is a periplasmic enzyme is now seen to have been in error.
The superoxide radical (021 is a common intermediate in the biological reduction of oxygen, and the superoxide dismutases (SOD), which catalytically scavenge this radical, are important defense components against the toxicity of oxygen. Some of the work supporting these assertions has been reviewed (3, 11-13). In the case of several microorganisms, increased levels of SOD correlate with increased resistance toward the toxicity of oxygen and the oxygen enhancement of streptonigrin toxicity (16-18, 20, 28, 32, 34). Polyacrylamide gel electrophoresis of crude extracts of Escherichia coli reveals three distinct superoxide dismutases (20). One of these contains manganese (MnSOD) (26); another contains iron (FeSOD) (33); and the third (New SOD) appears to be a hybrid composed of one subunit from each of the other two enzymes (H. Dougherty, Merck, Sharp and Dohme, Rahway, N. J.; personal communication). E. coli grown under rigorously anaerobic conditions contain only FeSOD, and exposure to oxygen induces the synthesis of MnSOD and of New SOD (17, 20). MnSOD and FeSOD have been assigned distinct subcellular localizations and physiological functions. Thus it was reported (19) that osmotic shock of E. coli occasioned a preferential release of FeSOD but not of MnSOD, and it was therefore concluded that FeSOD is a periplasmic enzyme, whereas MnSOD is localized in the cell matrix. This distinctive localization was further correlated with discrete functions, in that increased levels of FeSOD were correlated with increased resistance against exogenous 02-, whereas increased MnSOD correlated with resistance towards endogenous 2-
(19). A growing body of work implicates 2- as an important agent of the microbicidal action of human polymorphonuclear leukocytes (1, 5, 6, 10, 25, 31), and this lends increased significance to a periplasmic SOD capable of protecting bacteria against exogenous 02- This importance led us to reexamine the data base for the conclusion that FeSOD was a periplasmic enzyme (19). Since that data base seemed weak, in retrospect, the entire question was reexamined. We now report experimental results indicating that FeSOD is not, in fact, localized in the periplasmic space of E. coli. MATERIALS AND METHODS
Bacterial strains and media. E. coli K-12 his thi (ATCC 23794) and E. coli B B12-, the strain used in previous SOD localization studies (19), were grown on either TSY or low phosphate medium. The TSY medium contained 3% Trypticase soy broth supplemented with 0.5% yeast extract (Baltimore Biological Laboratories, Cockeysville, Md.). The low phosphate medium described by Neu and Heppel (29) consisted of a tris(hydroxymethyl)aminomethane (Tris)-hydrochloride-buffered minimal medium with 0.5% glucose, 0.5% Difco agar-peptone, and either 150 ,ug of histidine plus 30 jig of thiamine per ml for E. coli K-12 or 1 ,ug of B12 per ml for E. coli B. Incubations were at 37°C with shaking at 200 rpm on a rotary platform shaker. Osmotic shocking. Cultures were grown to late log phase and washed in 0.01 M Tris-hydrochloride, pH 8.0. Cells were suspended in 20% sucrose-0.03 M Tris-hydrochloride, pH 8.0, at a ratio of 1 g of wet cells to 80 ml of buffer. Sodium ethylenediaminetetraacetate (EDTA) was then added to a final concentration of 0.001 M. After 10 min of incubation, cells were centrifuged, and the pellet was rapidly suspended in cold distilled water (80 ml/g [wet weight]
of cells). After 10 min of incubation at 0 to 4°C, the suspension was centrifuged, and the supernatant or shock fluid was saved for assay. Since enzyme activities were low in shock fluids, these fractions were concentrated 5- to 10-fold in an Amicon ultrafiltration cell, with a PM-10 membrane, prior to assay. The shocked cells were suspended in 0.01 M Tris, pH 8.0, and were disrupted by sonic oscillation for 10 min with a Branson model W185 sonifier, at an output of 70 W. During sonic oscillation the cell suspension was kept at a low temperature by immersion in an ice-salt bath and by applying sonifier power intermittently. Clearing of the cell suspension indicated complete disruption of cells. Lysozyme spheroplast formation. Cells were grown in low phosphate medium and harvested in log phase. After washing in 0.01 M Tris-hydrochloride, pH 8.0, cells were suspended 1:80 (wt/vol) in 20% sucrose-0.03 M Tris-hydrochloride, pH 8.0, followed by the addition of sodium EDTA to 0.001 M. Egg-white lysozyme (Sigma Chemical Co., St. Louis, Mo.) was added to 10 jug/ml, and spheroplast formation was monitored by following the decrease in absorbance at 600 nm (AmN) when samples were diluted 10-fold in distilled water. After 12 min, MgS04 was added to 0.01 M to stabilize the spheroplasts. The spheroplasts were separated from the "spheroplast medium" by centrifugation and then osmotically lysed in cold distilled water (80 mg/g [wet weight] of cells). After centrifugation, the supernatant fluid from the lysed spheroplasts was collected, and the pellet was resuspended in 0.01 M Tris and disrupted by sonic oscillation for 5 min. All fractions were dialyzed 24 h against several volumes of 0.01 M Tris-hydrochloride, pH 8.0. The spheroplast medium and the supernatant from osmotically lysed spheroplasts were concentrated 5to 10-fold in an Amicon apparatus, with a PM-10 membrane, prior to assaying for enzyme activities. Localization with diazo-NDS. Diazonium reagent was prepared from 7-amino-1,3-naphthalene-
disulfonic acid (NDS) (Eastman Organic Chemicals) according to Pardee and Watanabe (30). Late log phase cells were washed with 0.05 M Tris-hydrochloride, pH 8.0-5 mM MgCl2 and were resuspended in the same buffer to give a cell concentration of approximately 1010 cells per ml. Half ofthis suspension was used as untreated control. Diazo-NDS, 1 ml per 10 ml of bacterial suspension (5 mM), was added to the remaining portion and incubated at 256C with gentle agitation. After 60 min the reaction was quenched by the addition of imidazole to 0.1 M. Control and treated cells were washed twice in 0.05 M Tris-5 mM Mgl2 and suspended in 0.05 M Tris. Extracts were prepared by sonic oscillation for 3 min followed by centrifugation at 20,000 x g for 15 min to remove cell debris. To determine if selected enzymes could be inhibited by diazo-NDS, cell-free extracts of untreated controls were treated with 2.5 mM diazoNDS for 20 min. The reactions were quenched by the addition of imidazole to 0.1 M, and unreacted diazoNDS was removed by gel filtration through a Sepha-
dex G-25 column. Electrophoresis and asays. Unless otherwise indicated, electrophoresis was performed on 10%
J. BACTZIUOL. acrylamide gels by the method of Davis (7). Alkaline phosphatase activity was localized by soaking gels in 0.033 M Tris-hydrochloride buffer, pH 9.5, containing 0.025 M sodium a-naphthyl-phosphate (Sigma) and 1 mg of Fast Red TR (Sigma). SOD activity on acrylamide gels was detected by the photochemical methods previously described (2). After scanning gels at 560 nm with a Gilford gel scanner, activities of SOD isoenzymes were estimated from the areas under the corresponding absorbance troughs. Methods for the assay of SOD (27), alkaline phosphatase (14), adenosine deaminase (4), and glucose 6-phosphate dehydrogenase (9) have been previously described. Protein was estimated by the biuret method (15).
RESULTS Osmotic shock. Exposure of gram-negative bacteria to an osmotic shock, after treatment with EDTA, results in substantial release of enzymes from the periplasmic space, without significant release of enzymes from the matrix space (22, 23). Indee4, release from the cell by osmotic shock or by spheroplast formation constitutes an operational definition for periplasmic enzymes (23). E. coli K-12 and B were osmotically shocked after growth on TSY or on a low phosphate medium, and shock fluid and shocked cells were assayed for the various isozymes of SOD as described above. Alkaline phosphatase was assayed as a marker enzyme for the periplasmic space, and adenosine deaminase was similarly assayed as a matrix marker (Table 1). TSY medium is a high phosphate medium, and alkaline phosphatase, the periplasmic marker enzyme, is only induced in low phosphate media. For that reason there are no values for the alkaline phosphatase activities of fractions of TSY-grown cells in Table 1. It is clear that osmotic shock did not occasion preferential release of any of the SOD isoenzymes, which behaved very much like adenosine deaminase in that they were largely retained by the shocked cells. With both E. coli K-12 and B, grown in low phosphate medium, virtually complete release of alkaline phosphatase in the shock fluid occurred. None of the SOD isoenzymes behaves like a periplasmic enzyme when examined by the criterion of release upon osmotic shocking. Spheroplast formation. A periplasmic enzyme could be retained by the cell during osmotic shock if it bound tightly to components of the cell surface external to the plasma membrane. This possibility was examined by the formation of spheroplasts. Sensitivity to osmotic lysis was used as a criterion of spheroplast formation. Spheroplast formation, performed as described above, was essentially complete within 2 min after lysozyme addition.
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LOCALIZATION OF E. COLI SUPEROXIDE DISMUTASES
This was indicated by a 14-fold decrease in turbidity (Am*) upon 10-fold dilution with water. Activities released from the cells during spheroplast formation and those released only upon subsequent lysis of the spheroplasts were assayed (Table 2). Alkaline phosphatase was again used as a marker of the periplasmic space, while glucose 6-phosphate dehydrogenase was used as a marker of the matrix (22). As expected, alkaline phosphatase was largely released during spheroplast formation and glucose 6-phosphate dehydrogenase was largely re-
tained. All of the SOD isoenzymes behaved like matrix enzymes in that they were retained during spheroplast formation. Attack by diazotized NDS. Diazonium salts have been used as group-specific reagents in studies aimed at exposing the essentiality of specific amino acid residues in enzymes. These reagents couple primarily with histidine and tyrosine residues (24). Diazo-NDS appears to be unable to penetrate the plasma membrane,
probably by virtue of the negative charges on its sulfonate groups; but it readily gains access
TABLz 1. Release of enzymes by osmotic shock ofE. coli B and K-12 cells Activity of enzymes Fraction and growth conditions
TSY-grown E. coli K-12 Shock fluid Sonic extract of shocked cells
TSY-grown E. coli B Shock fluid Sonic extract of shocked cells
Low phosphate-grown E. coli K-12 Shock fluid Sonic extract of shocked cells
Low phosphate-grown E. coli B 14 97.8 13.4 15.7 Shock fluid 12.7 10.4 86 2.2 86.6 84.3 87.3 89.6 Sonic extract of shocked cells a Activities are expressed as percentage ofthe total activity of shock fluid plus extracts of shocked cells. b APase, Alkaline phosphatase. c -, Alkaline phosphatase was not induced in high-phosphate TSY medium.
TABLz 2. Release of enzymes after spheroplwst formation of E. coli B and K-12 Activity of enzymes"
12.4 94.8 10.9 10.7 11.5 Supernatant, after removal of spheroplasts 4.5 64 67.1 64.4 63.1 Supernatant, osmotically lysed spheroplasts 23.6 0.8 22.0 26.2 24.1 Sonic extract of pellet from lysed spheroplasts a Activities are expressed as percentage of the total activity of the three fractions. b APase, Alkaline phosphatase. c G6PDH, Glucose 6-phosphate dehydrogenase.
E. coli K-12
Supernatant, after removal of spheroplasts Supernatant, osmotically lysed spheroplasts Sonic extract of pellet from lysed spheroplasts E. coli B
BRI'ITON AND FRIDOVICH
to the periplasmic space. This reagent is therefore useful in identifying proteins that are external, or are attached, to the outer surface of the plasma membrane (30). It has been used to prove the surface location of the (3-galactoside permease of E. coli (30), the sulfate-binding protein of Salmonella typhimurium (30), and the a-galactosidase of Pseudomonas atlantica (8). Diazo-NDS inactivated the SOD activity of cell-free extracts of E. coli but had trivial effect on the activity of whole cells. Diazo-NDS was similarly without effect on the glucose 6-phosphate dehydrogenase of the matrix of whole cells but readily inactivated this enzyme in free solution (Table 3). The SOD in whole cells, like the glucose 6-phosphate dehydrogenase, was inaccessible to this diazo reagent, and thus behaved like a matrix enzyme. Alkaline phosphatase is known not to be inactivated by diazo-NDS (30). We did, however, find that this reagent coupled with the alkaline phosphatase of whole cells, as evidenced by a change in its electrophoretic mobility. Figure 1 presents the electrophoretic mobilities of the alkaline phosphatase of untreated and of diazoNDS-treated cells, as a function of gel concentration. It is apparent that the periplasmic alkaline phosphatase of whole cells was accessible to diazo-NDS and that its interaction with this reagent changed its net charge rather than its hydrodynamic size (21), as would be expected for a simple coupling between the enzyme and this anionic reagent. The data in Fig. 1 were obtained withE. coli K-12, but identical
results were obtained with E. coli B. The data in Table 3 show that SOD in free solution was inactivated from 69 to 91% by diazo-NDS, depending on the strain ofcells and the conditions of growth. Since FeSOD constitutes between 40 and 60% of the total SOD of these cells, this degree of inactivation must have involved at least some of the FeSOD. Nevertheless, one could argue that FeSOD, even in free solution, was relatively resistant to diazo-NDS compared with the other SOD iso.0
6 e 10 Gel Concentration (%)
FIG. 1. Effect of treatment of whole cells of E. coli K-12 with diazo-NDS on the electrophoretic mobility ofalkaline phosphatase. Samples of cell-free extracts, containing 0.040 mg ofprotein, were applied to polyacrylamide gels ofgraded porosity. After electrophoresis and staining for alkaline phosphatase, log of the mobility, relative to the marker dye, was graphed as a function of gel density. (A) Extracts taken from untreated whole cells; (0) extracts from whole cells treated for 1 h with 5 mM diazo-NDS as described in the text.
TABLz 3. Effect of diazo-NDS treatment of whole cells on enzyme activity
Sample lSOD (U/mg)
TSY-grown E. coli K-12 Whole cells without NDS Whole cells + 5 mM NDS for 60 min Cell-free extract + 2.5 mM NDS for 20 min
23.2 21.1 2.27
0 9 91
0.089 0.087 0
0 2 100
TSY-grown E. coli B Whole cells without NDS Whole cells + 5 mM NDS for 60 min Cell-free extract + 2.5 mM NDS for 20 min
25.0 24.2 7.19
0 3 71
0.109 0.095 0
0 13 100
Low phosphate-grown E. coli K-12 Whole cells without NDS Whole cells + 5 mM NDS for 60 min Cell-free extract + 2.5 mM NDS for 20 min
30.3 28.3 9.43
0 7 69
0.062 0.059 0
0 5 100
Low phosphate-grown E. coli B Whole cells without NDS Whole cells + 5 mM NDS for 60 min Cell-free extract + 25 mM NDS for 20 min a G6PDH, Glucose 6-phosphate dehydrogenase.
39.5 38.2 9.61
0 3 76
0.042 0.028 0
0 33 100
LOCALIZATION OF E. COLI SUPEROXIDE DISMUTASES
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zymes, and, hence, the failure ofthis reagent to greatly inactivate the net SOD of whole cells did not really demonstrate that FeSOD was a matrix enzyme. To examine this objection, the electrophoretic properties of the SOD of whole cells, before and after treatment with this reagent. Figure 2 presents the results in the form of densitometric scans of the gels after activity staining for SOD. Line 1 reflects the electrophoretic mobility of the residual SOD activity of cell-free extracts after treatment with diazoNDS. It is apparent that the enzyme in free solution, although only partially inactivated, had been completely derivatized by this reagent. In contrast, line 2 represents the pattern of SOD bands exhibited both by untreated and by diazo-NDS-treated cells. It is apparent that the diazo reagent, although capable of reacting with all of the SOD isozymes in free solution, was unable to gain access to any of the SOD isozymes in whole cells. The results shown in Fig. 2 were obtained with E. coli B, but identical results were obtained with E. coli K-12. Since the diazo reagent did gain access to the periplasmic space, as evidenced by its effect on alkaline phosphatase, we must conclude that all of the SOD isozymes are matrix enzymes.
DISCUSSION The results obtained by osmotic shocking, by apheroplast formation, and through use of a reagent selective for ectoenzymes consistently indicate that all of the SOD isozymes of E. coli are matrix enzymes. The previous report that FeSOD is a periplasmic enzyme (19) seems certainly to have been in error. Although it is difficult to discern the source of that error, its probable cause was the small net release of SOD seen upon osmotic shocking coupled with the uncertainties of estimating relative quantities of isozymes by densitometry of stained polyacrylamide gels. Thus the earlier report of selective release of FeSOD by osmotic shock was based upon a net release of only 6% of the total cell SOD. We are pleased now to have several lines of strong evidence to clarify this point. The story previously seemed beautifully self-consistent. Thus FeSOD was periplasmic in localization and specialized to deal with exogenous 02- in function. We now know that FeSOD is not a periplasmic enzyme and must proceed to reexamine its physiological function vis-a-vis the other SOD isozymes. ACKNOWLEDGMENTS This work was supported by research grant GM-10287 from the National Institute of General Medical Sciences and DAHC-0474-G-0194 from the United States Army Research Office. L. Britton is a postdoctoral fellow of the National Institute of Environmental and Health Sciences and is supported by training grant 5 T 32-ES07002.
4 5 Gel Distance (cm)
FIG. 2. Effects of diazo-NDS on the electrophoretic mobility ofE. coli B SOD. Samples were applied to polyacrylamide gels, and, after electrophoresis and staining for SOD activity, the gels were scanned at 560 nm. Gel density is here plotted as a function of linear distance from the cathodic end of the gel. Since the activity stain used leaves achromatic zones at the positions bearing SOD activity, absorbance troughs mark the positions of SOD species. Line I was obtained from a cell-free extract of E. coli B, which had been treated as an extract with 2.5 mM diazo-NDS for 20 min. Line 2 was obtained with cell-free extracts of untreated E. coli B or with cell-free extracts of E. coli B that had been treated, as whole cells, with 5 mM diazo-NDS for 1 h.
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