T H E JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 269, No. 41, Issue of October 14, pp. 25310-25314, 1994 Printed in U.S.A.

Escherichia coli Expresses a Copper- and Zinc-containing Superoxide Dismutase*

(Received for publication, April 22, 1994, and in revised form, July 11, 1994)

Ludmil T. BenovS and Irwin Fridovichg From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

A mutant of Escherichia coli, unable to produce man- ganese- or iron-containing superoxide dismutase (SOD), was found to contain modest levels of an SOD that was judged to be a copper- and zinc-containing SOD on the basis of inhibition by cyanide and inactivation by either H,O, or diethyldithiocarbamate. Moreover, the diethyl- dithiocarbamate-inactivated enzyme could be reacti- vated with Cu(II), and this reconstituted enzyme, like the native enzyme, was unaffected by EDTA and was inhibited by cyanide. This enzyme was, furthermore, se- lectively released by osmotic shock, in keeping with a periplasmic localization, and it was strongly induced during aerobic growth. This enzyme was also present in the SOD-competent parental strain. Failure to detect it previously can be attributed to its periplasmic localiza- tion, thermal lability, sensitivity to pH, and to its rela- tive paucity. It will now be interesting to explore the phenotypic consequences imposed by the absence of this SOD.

Bacteria have generally been found to contain Fe.SOD’ and/or Mn.SOD; whereas CuZn.SOD has been considered to be characteristic of the cytosol of eukaryotic cells. A few species of bacteria have, however, been found to contain CuZn.SOD. These include Photobacter leiognathi (1, 2) Pseudomonas diminuta and Pseudomonas maltophila (31, Caulobacter cres- centis (41, Brucella abortus ( 5 ) and other species of Brucella (6), and several species of Haemophilus (7). The C. crescentis gene (sodC) coding for CuZnBOD was cloned and sequenced and a 22-residue leader sequence was identified (8). This suggested that this enzyme might be periplasmic and this supposition was subsequently affirmed (9).

Mutants with insertional defects in the bacterial CuZn.SOD gene have been prepared, in attempts to learn the function of this enzyme. In the case of B. abortus the CuZn.SOD-null ap- peared less able to survive in mice, than was the parental strain, suggesting that the CuZn.SOD might be a pathogenicity factor (10). In free-living C. crescentis the CuZn.SOD-null did not exhibit defects in growth rate, motility, plating efficiency, or sensitivity toward paraquat; but it was more sensitive toward

* This work was supported by research grants from the Council for Tobacco Research-U.S.A., Inc., Johnson & Johnson Focused Giving, and Eukarion Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

# Recipient of Fellowship 1 F05 TWO4975 from the Fogarty Interna- tional Center. Permanent address: Medical University, Dept. of Bio- physics, 11 Armeiska St., BU-6000 Stara Zagora, Bulgaria.

6 To whom all correspondence should be addressed. Tel.: 919-684-

iron-containing SOD; Mn.SOD, manganese-containing SOD; CuZn. The abbreviations used are: SOD, superoxide dismutase(s1; FeSOD,

SOD, copper- and zinc-containing SOD; DDC, diethyldithiocarbamate; PAGE, polyacrylamide gel electrophoresis.

5122; Fax: 919-684-8885.

citrate, and this citrate effect was neutralized by Ca(I1) and Mg(I1) (11). Since Ca(I1) and Mg(I1) bind to the negatively charged lipopolysaccharide in the outer leaflet of the outer membrane, the CuZn4OD may serve to protect some outer membrane function against defects which are exposed when citrate extracts Ca(I1) and Mg(I1) from the cell surface.

We now report that E. coli produce a CuZn.SOD, which is inducible by dioxygen, and which also appears to be periplas- mic. Some of the properties of this enzyme, which explain why i t has never previously been noticed in this well studied orga- nism, are also reported.

MATERIALS AND METHODS The strains of E. coli used in this work are JI132, which bears

insertional defects in the genes coding for both Mn.SOD and Fe.SOD, and AB1157, which is the parental strain (12). Media were prepared with tap water, except where the use of deionized water is specified. LB medium contained 10 g of Bacto-Tryptone, 5 g of yeast extract, and 10 g of NaCl per liter, and the pH was adjusted to 7.4 with &HPO,. Minimal medium contained minimal A salts (13) with 0.2% glucose, 150 mg/liter pantothenate, 50 mg/liter thiamine, and either 0.5 mM of all 20 lz-amino acids or 0.2% casamino acids at pH 7.4. Cultures were grown at 37 “C, and cells were harvested by centrifugation, washed in 50 II~M potassium phosphate at pH 7.5, and were then lysed by either sonica- tion (31, freezing-thawing (141, the French press, or by osmotic shock (15), as specified. Extracts were dialyzed for 5 h against cold 50 mM potassium phosphate at pH 7.5 prior to assay. Anaerobic growth, when desired, was performed in a Coy chamber under 90% N, + 10% Hz.

SOD activity was measured with xanthine oxidase as the source of 0; and with either cytochrome c (16), nitro blue tetrazolium (171, or epinephrine (16) serving as indicating scavengers of 0;. Native PAGE was done at 4 “C and at pH 7.5, and gels were stained for protein and for SOD activity (17).

Reagents-Bacto-Tryptone, yeast extract, and casamino acids were obtained from Difco. Acrylamide and bisacrylamide were from Bio-Rad. Bovine CuZn.SOD was supplied by Diagnostic Data Inc. Xanthine ox- idase isolated by the procedure of Waud et al. (18) was kindly provided by Dr. K. V. Rajagopalan. All other compounds were reagent grade from commercial sources.

RESULTS SOD Activity in the SOD-null Stra in41132 was thought to

be completely devoid of SOD activity. We noted, however, that extracts of this strain, grown aerobically to late log phase in LB medium, contained approximately 2% as much activity as was present in comparable extracts of the parental AB1157 strain. This SOD activity in the extracts of 51132 was detectable in all of the assays used. It was, moreover, thermolabile and non- dialyzable, indicating that i t reflected an enzyme. Control ex- periments established that the extract of JI132 did not influ- ence the rate of the xanthine oxidase reaction followed in terms of accumulation of urate at 295 nm.

CuZn.SODs are inhibitable by cyanide, whereas Mn4ODs and Fe.SODs are not (19, 20). By this criterion the SOD in JI132 is a CuZn.SOD. Thus, as shown in Fig. 1, it was inhibited by 2.0 mM CN-. Another compound which can be used to dis- tinguish CuZn.SOD from Mn4OD and Fe6OD is diethyldi-

25310

CuZn.SOD in E. coli 25311

0.15 -

- 0 In

5 0.10 - ? EJ 0

4 n L

0.05 - 0 -2

0.00 ! , 1

0 2 4 6 8

Time (min)

FIG. 1. CN- inhibition of the SOD activity in extracts of E. coli JI132. The reaction mixture contained 1.0 mM xanthine, 8.3 1" cyto- chrome c, 0.1 mM EDTA, and 50 mM potassium phosphate in 3.0 ml and at 25 "C. The reaction was started by adding xanthine oxidase to 2.0 x

M. The sample was 50 pl of a dialyzed extract of E. coli JI132 which had been grown overnight in LB medium and then disrupted with the French press. CN- was added as indicated.

thiocarbamate (21, 22). As shown in Fig. 2 the SOD activity in an extract of JI132 was strongly inhibited by this hydrophobic chelating agent; whereas the activity in an extract of the pa- rental strain, which was overwhelmingly Mn.SOD plus Fe.SOD, was not detectably inhibited. Measurements in the presence of diethyldithiocarbamate were done with nitro blue tetrazolium in the place of cytochrome c; because this chelating agent can directly reduce the cytochrome, but not the tetrazo- lium.

A third method for distinguishing CuZn-SODS from Mn.SODs and Fe.SODs is based upon the effects of chloroform plus ethanol. This Tsuchihashi (23) procedure does not inacti- vate CuZn+OD (16), but does inactivate Mn.SOD and Fe.SOD (24). Treatment of an extract of aerobically grown JI132 with chloroform plus ethanol did not diminish its SOD activity. Un- der the same conditions an extract of the parental strain (AB1157) lost 75% of its activity. Indeed, since this treatment did denature and precipitate other proteins in the extract of the SOD-null strain, it increased the specific activity of SOD in the supernatant obtained by centrifugation following the Tsuchi- hashi treatment.

Reactivation of the DDC-inactivated Enzyme-DDC inacti- vates CuZn4OD by extracting Cu(I1) from the enzyme. If the SOD in JI132 was indeed a CuZn.SOD, then it should be pos- sible to restore its activity with Cu(I1) following, or accompa- nying, removal of the DDC by dialysis. Extract of JI132 was treated with 2 mM DDC for 15 min at 25 "C, during which time its activity fell from 8.5 unitdm1 to -1.0 unitiml. The DDC- treated extract was then dialyzed for 48 h against cold 10 mM phosphate a t pH 7.5. There was no regain of activity during this dialysis. However, subsequent dialysis against 15 CuC1, in 1.0 mM Tris at pH 7.5 restored over 90% of the original activity. Comparable dialyses against FeC1, or MnCl, restored only 10-15% of the original activity, and this could reflect the presence of some Cu(I1) in the iron and manganese salts used.

All of these results indicate that the SOD activity in extracts of JI132 was due to a CuZn-SOD. Variation in the level of this activity as a function of the growth cycle was examined. As shown in Fig. 3 the SOD activity increased during the early part of the growth cycle and then declined gradually during the late stationary phase. Aliquots were taken for 10,000-fold dilu-

8o 1

'pi 20

1 - o n . B . n . l . r . ? . J

0 1 2 3 4 5 6

DDC (mM)

in extracts of E. coli JI132. E. coli JI132 (line 1 ) and AB1157 (line 2 ) FIG. 2. Diethyldithiocarbamate inhibition of the SOD activity

were grown overnight, extracted, and dialyzed as in Fig. 1. The SOD activity per ml of these extracts was initially very discordant. For the purpose of this experiment they were brought into the same range by using ultrafiltration to concentrate the extract of JI132 by a factor of -15-fold, while diluting the extract of AB1157 by -50-fold. Aliquots (300 pl) of the extracts were incubated for 5 min at 25 "C with the indicated concentrations of DDC prior to assay with 1.7 x M nitro blue tetrazolium serving as the indicating scavenger for O,, in place of cytochrome c.

3 1 - 30

- Cd E

-20 .g

n

. e - 0

- 10

0 Y .

2 0 40 60 I "

80

Incubation (hours)

FIG. 3. Variation of SOD activity during the growth cycle of JI132. An overnight culture of JI132 in LB medium was used as the innoculum. Aliquots (100 pl) of this culture were diluted into 20 ml of fresh LB medium in 100-ml flasks; which were then shaken at 220 rpm at 37 "C. At intervals samples were taken for measurement of Aswnm (line 1 ) and for extraction and assay, using the cytochrome method (line 2) . Extraction was by osmotic shock.

tion and plating on LB agar at 24, 48, and 72 h. The 24-h sample yielded several hundred colonies per plate and these were entirely uniform in appearance, indicating the absence of contaminants. The 48- and 72-h samples yielded no colonies at all. This indicates that the JI132 rapidly lost viability during the stationary phase under aerobic conditions. This phenotypic deficit must be considered to be another consequence of the paucity of SOD activity in this strain and it also explains the loss of SOD activity seen during the stationary phase.

The SOD activity in JI132 was increased by aerobic growth. Thus, extracts of anaerobically grown JI132 were prepared by osmotic shock and were found to contain 1.1 units of SOD/mg of protein. Comparable extracts of aerobically grown JI132 had a

25312 CuZn6'OD in E. coli

specific activity 32-fold higher. It should be noted that extracts which were prepared by osmotic shock contain much less pro- tein than those prepared by cell lysis with the French press and are thus selectively enriched in the periplasmic SOD.

CuZn.SOD in the Parental Strain-Given that the level of activity due to the CuZn-SOD in the JI132 strain was only 2% as great as the total SOD activity in AB1157; it should be difficult to detect against the background of Mn.SOD and Fe4OD present in the SOD-competent strain. This was the case. CuZn.SODs have been purified from C. crescentis (4) and from I? leiognathi (2) and a modification of these procedures was used to enrich CuZn.SOD from E. coli AB1157.

The SOD-competent strain was grown overnight in aerobic LB medium and was washed once with 50 mM potassium phos- phate a t pH 7.5. The cell paste (6.8 g) was suspended in 6.8 ml of this buffer and was passed through the French press at 20,000 p.s.i. The lysate was diluted with 30.0 ml of the phos- phate buffer and was clarified by centrifugation. The superna- tant solution (40 ml) was diluted with an equal volume of buffer, and solid (NH,),SO, was added to 50% of saturation. After 75 min of stirring at 23 "C the precipitate was removed by centrifugation, and (NH,),SO, was added to 85% of saturation, with stirring for 75 min. The precipitated protein was collected by centrifugation, was taken up in 5 ml of H,O, and was dia- lyzed for 20 h at 4 "C against 4.0-liter batches of buffer the first of which was 50 mM phosphate, pH 7.5, while the second was 20 mM at the same pH, and the third was 15 mM at pH 8.4. The dialyzed sample was applied to a 3.0 x 20-cm column of DEAE- cellulose (Whatman DE-52) which had been equilibrated with 15 mM phosphate a t pH 8.4. Elution with this buffer at a flow rate of 120 ml/h, while 4.0-ml fractions were collected, gave the results shown in Fig. 4. E-acing 1 in Panel A shows two well separated peaks of SOD activity, while Panel B shows that only one of these was inhibited by 2.0 mM CN-. I t is thus clear that the CuZn.SOD, first detected in the sodA sodB strain (JI1321, was also present in the parental strain (AB1157).

Why the E. coli CuZn.SOD Has Been Evasive Heretofore- Detection of the activity due to the CuZn3OD in JI132 was critically dependent upon the conditions of lysis. Thus, when the French press was used, dense suspensions (cell paste:buffer = 1:l) yielded active extracts, whereas moderately more dilute suspensions (paste:buffer < 1:2) did not. Lysis by freezing and thawing, or by ultrasonication, also failed to produce active extracts. On the other hand lysis by agitation with 0.2-mm glass beads in the Bead Beater, or by osmotic shock (15) did yield active extracts. These results are summarized in Table I . Since sonication or passage of relatively dilute suspensions of cells through the French press are the most commonly used methods of lysis of E. coli, it is not surprising that the CuZn-SOD has not been noticed heretofore in the SOD-null strain. In SOD-competent strains the great excess of Mn6OD and Fe.SOD over CuZn.SOD is another confounding factor.

There are additional reasons as well. Thus the E. coli CuZn4OD is very thermolabile and lost activity when exposed to temperatures in the range 40-55 "C, as shown in Fig. 5. The mammalian CuZn.SODs are, in contrast, quite resistant to- ward thermal inactivation (25). Further, the mammalian CuZn.SOD exhibits essentially constant activity over the pH range 5-9.5 (26); whereas the E. coli CuZn.SOD exhibits an optimum at pH 6.8 and progressively less activity as the pH is raised. This is shown in Fig. 6 (line 1). Because the rate of reaction of 0, with cytochrome c falls as the pH is raised (27) and because the xanthine oxidase-cytochrome c assay depends upon competition between SOD and cytochrome c for the flux of O,, this assay becomes progressively more sensitive toward SOD as the pH is raised. This is the explanation for the appar-

100 1 r600

0 100 200 ml of effluent

B

300

- 600

- 500

- 400

-300

- 200

- 100

0

B

0 1" ,

1

0 100 200 300 0

ml of emuent

AB1167. The dialyzed 5685% (NH,),SO, fraction was chromato- FIG. 4. Column chromatography of SOD activities from

graphed over DE-52 as described in the text, and the eluate was assayed for protein (trace 2) and for SOD activity by the cytochrome c method (trace 1 ). In Panel A SOD activity was measured in the absence of CN-, while in Panel B 2.0 mM CN- was present.

Effect of method of cytolysis on release of CuZnSOD TABLE I

from E. coli 51132

Method SOD

French press (bufferxells > 2:l) French press (bufferxells = 1:l)' French press (buffer:cells = l : l )*

Freezindthawing Sonication

Bead beater Osmotic shock

unitslmg

0 0.9

0 1.6

0 5.4 9.8

a Cells grown in LB prepared with deionized water. Cells grown in LB prepared with tap water.

ent increase in activity of the bovine CuZn4OD with increas- ing pH (line 1). A correct representation of the effect of pH on the E. coli CuZneSOD can be obtained by normalizing its ac- tivity, at each pH, to that of the bovine enzyme and that result is shown by line 3. This loss of catalytic activity as the pH is raised in the range 7-9 explains why activity-stained (17), gel electropherograms, usually at pH 8.7 (28), might also fail t o reveal the presence of a CuZn4OD in extracts of E. coli.

Detection of E, coli CuZn.SOD on Electropherograms-Initial attempts to visualize the E. coli CuZn.SOD on native PAGE gels by use of the photochemical nitro blue tetrazolium reduction method (17) were not successful. However, when the pH of both the gel and ofthe buffer reservoirs was adjusted to 7.5 and when care was taken to avoid resistive warming of the gels, the CuZn.SOD could be seen. Thus, lane A in Fig. 7 exhibits the

CuZnSOD in E. coli 25313

10 20 30 4 0 5 0 6 0 Temperature ( C )

FIG. 5. Thermal inactivation of SOD in extracts of JI132. E. coli JI132 was extracted by osmotic shock and then dialyzed against 50 mM phosphate buffer at pH 7.5. Aliquots of the dialyzed extract (200 pl) were sealed into small tubes and were then incubated for 15 min over a range of temperatures. The tubes were then chilled, opened and assayed for residual SOD activity.

0

5 6 7 X 9 10 I 1

pH

FIG. 6. Effect of pH on the SOD activity. Bovine erythrocyte CuZn.SOD (line 1 ) and the CuZn.SOD fromJI132 (line 2) were assayed in 50 mM potassium phosphate + 0.1 mM EDTA over a range of pH by the xanthine oxidasdcytochrome c method. Line 3 represents the ratio of the activity of the E. coli enzyme to that of the bovine enzyme.

achromatic zone due to the CuZn4OD in an extract of the JI132 strain; while lane B shows that this activity was inhibited by 2.0 mM cyanide. Lanes C and D show that the parental AB1157 also shows a band of CN--sensitive SOD activity whose anodic mo- bility matched that of the activity in JI132. The band of activity which was sensitive to CN- could also be eliminated by soaking the gel in 2.0 mM DDC for 15 min prior to activity staining.

DISCUSSION When a bacterial CuZn.SOD was first described (1,2) it was

in F! leiognathi, which lives as a symbiont in the luminescent gland of the pony fish. Since CuZn.SOD was at that t ime thought to be unique to eukaryotes it seemed reasonable to propose that its presence in F! leiognathi was the result of a gene transfer from the host fish (29-31). This hypothesis did not stand up under closer scrutiny (32, 33). Subsequent detec- tion of CuZn.SOD in a handful of other bacteria (3-7), includ- ing free-living species such as C. crescentis, effectively elimi-

El i i l

* I

,' .

ii !

A B C 0 1

FIG. 7. Detection of the E. coli CuZnaSOD on gel electrophero- grams. Partially purified extracu of 51132 (gels A and H I and of AB1157 (ge ls C and D ) were subjected to polyacrylamide gr.1 elrctrn- phoresis at pH 7.6, followed by staining or SOD activity. Gel H was soaked in 2 mM CN- for 15 min before being rinsrd and stained; while gel D was similarly treated with 2 my DDC prior to activity staining.

nated gene transfer from eukaryotes as an explanation for the or iginal of this bacter ia l enzyme. I t never the less has re- mained the dominant view that CuZn6OD is only rarely found in prokaryotes.

Given the presence of CuZn.SOD in E. coli, it now seems likely t h a t CuZn.SOD is a common attribute of prokaryotes; or at least of Gram-negative bacteria. It may well he that in other bacteria, as is the case in E. coli, that the CuZn4OD will always be a minor fraction of the total complement of SODS present and that i t will be localized in the periplasm. It will now be interesting to explore the physiological function of the periplasmic CuZn4OD in E. coli; which could he done by ex- amining a mutant of JI132 which had a defect in the CuZn.SOD gene. One also wonders whether the prokaryotic and eukaryotic CuZn.SODs represent descent from a common ancestral SOD or, alternatively, convergent evolution from in- dependent sources. The regulation of the CuZn.SOD, as illus- trated by its relative abundance in aerobic, hut not in anaerobic cultures, also requires exploration.

I t i s now clear that the sodA sodB strains of E. coli, produced to date, are not truly SOD-nulls; since they retain a CuZn.SOD. The oxygen-dependent phenotypic deficits of the sodA sodR strain (14, 34) thus reflect a deficiency of SOD, rather than a complete lack of this activity and these phenotypic deficits may be exacerbated in sodA sodB sodC strains yet to be produced. Preparation of quantities of the E. coli CuZn.SOD suitable for metal analyses and other physicochemical characterization is being pursued.

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