Vol. 138, No. 1
JOURNAL
OF BACTERIOLOGY, Apr. 1979, p. 139-145 0021-9193/79/04-0139/07$02.00/0
Superoxide Dismutase and 02 Lethality in Bacteroides fragilis CHRISTOPHER T. PRIVALLE AND EUGENE M. GREGORY* Department of Biochemistry and Nutrition, College of Agriculture and Life Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received for publication 6 December 1978
Exposure of midlog Bacteroides fragilis (VPI 2393) to 2% 02-98% N2 caused a three- to fivefold increase in superoxide dismutase specific activity within the cells. The increase in specific activity was completed within 90 min after exposure to oxygen and was dependent upon protein synthesis. Cells containing the higher superoxide dismutase level were more resistant to the effects of 5 atm of oxygen tension than were cells containing the lower level of superoxide dismutase but were equally resistant to 5 atm of nitrogen tension. Similar results were observed upon comparing viability experiments with B. fragilis and B. vulgatus. Superoxide dismutase activity in sonic extracts of B. fragilis was rapidly inactivated by exposure to 5 mM H202 and was inhibited by 1 mM NaN3 but not 5 mM NaCN. The inhibition pattern is identical to the pattern demonstrated for the purified iron-containing enzyme from Escherichia coli B and suggests that the superoxide dismutase in B. fragilis is an iron enzyme.
The reduction of triplet oxygen by a single electron generates the superoxide radical. The radical can disproportionate spontaneously to form hydrogen peroxide and singlet 02 and can interact with iron chelates and hydrogen peroxide in a catalytic cycle to generate hydroxyl radical (OH.). The disproportionation products and the superoxide radical itself have been shown to damage a number of cellular processes. Aerobic and aerotolerant bacteria have evolved ferric and manganic superoxide dismutases (EC 1.15.1.1) to diminish steady-state levels of superoxide radical. The superoxide dismutases catalyze disproportionation of the superoxide radical to hydrogen peroxide and ground-state 02, curtailing singlet O2 production, and remove 02 from the catalytic cycle with iron chelates and H202, diminishing OH - production (9). A direct correlation between superoxide dismutase level and resistance to oxygen lethality has been reported for a number of procaryotes and eucaryotes (11-13). These data and the attendant hypothesis have been reviewed (8, 9). Anaerobic bacteria were thought to be devoid of superoxide dismutase and, lacking that defense, were quite sensitive to oxygen-dependent lethality (22). A degree of aerotolerance variation was documented by Loesche (20) for a number of anaerobes based on the ability of the cells to grow under a defined 02 concentration. It now appears as if that variation in aerotolerance might be explained, in part, by the presence of superoxide dismutase. We have shown that 23
of 28 Bacteroides strains, several Clostridia, anaerobic cocci, and anaerobic nonspore-forming rods possessed superoxide dismutase activity (14). Tally et al. (24) and Carlsson et al. (6) have reported superoxide dismutase levels in anaerobes isolated from clinical specimens and have suggested, using a test system different from that we report herein, that there exists a correlation between superoxide dismutase level and aerotolerance. Generally, those comparisons were made among genera or species of anaerobes. The purpose of this communication is to present evidence that (i) the superoxide dismutase level in Bacteroides fragilis is inducible by exposing the organism to 02, (ii) induced levels of superoxide dismutase protect the cells from hyperbaric 02 lethality, and (iii) the major inducible form of superoxide dismutase appears to be the ferric-enzyme form.
MATERIALS AND METHODS A 1% inoculum of B. fragilis (VPI 2393) was transferred under 02-free CO2 from chopped-meat medium (17) into a prereduced medium containing 1.0% peptone, 1.0% yeast extract, and 1.0% glucose supplemented with 7.7 ,uM hemin and 2.2 ,uM- vitamin Ki, and was incubated at 37°C for 5.5 h. Bacterial growth was monitored turbidimetrically at 660 nm by the method of Koch (19). Cells were harvested by centrifugation, washed with 50 mM potassium phosphate plus 1 mM EDTA (pH 7.8), and sonically disrupted with cooling with a Branson model W185D soniflier operated at a power output of 65 W into the microtip. Cell debris was removed by centrifugation at 35,000 139
140
PRIVALLE AND GREGORY
x g for 15 min. All manipulations in preparing the crude cell extract were performed aerobically. Protein content of the crude extract was determined spectrophotometrically by absorbance at 280 and 260 nm, assuming that a 1 mg/ml solution of protein in a 1-cm cuvette would give an absorbance of 1.0 (26). Catalase was assayed by the method of Beers and Sizer (3). Superoxide dismutase was assayed by its ability to inhibit the 02-mediated reduction of ferricytochrome c as described by McCord and Fridovich (21). Malate dehydrogenase (10) and lactate dehydrogenase (4) were assayed by previously reported methods. Disc gel electrophoresis was performed in 7.5 or 10% acrylamide gels (7) with a 5 mM Tris-39 mM glycine buffer at pH 8.3 at a constant current of 2.5 mA per gel until the bromophenol blue tracking dye had swept through most of the gel. Each gel received 300,ug of crude cell extract and approximately 40 mg of sucrose. Superoxide dismutase activity was localized on gels by the method of Beauchamp and Fridovich (2). Superoxide dismutase isozymes or relative amounts of the major band were estimated from areas under densitometric scans of polyacrylamide gels which had been stained for superoxide dismutase activity. A Schoeffel model SD 3000 spectrodensitometer was used for scanning the gels at 560 nm. Treatment of the crude cell extract with H202 was used to determine which of the superoxide dismutase activity bands were inactivated. Final concentrations of inhibitors were 5.0 mM H202 and 1.0 mM KCN used in pretreatment of extracts in the activity gels. The presence of KCN was to suppress any residual catalase activity which would have diminished the effective concentration of H202. The effects of KCN (1 and 5 mM) and NaN3 (1 mM) on superoxide dismutase activity were tested by the addition of these reagents to the assay mixture immediately prior to the addition of the crude cell extract. At the concentrations used, azide neither inhibited the oxidation of xanthine by xanthine oxidase nor inhibited the reduction of cytochrome c by 02'. The slight inhibitory effect of sodium cyanide on xanthine oxidase activity was corrected in the superoxide dismutase activity calculation. The effect of H202 was tested by the addition to the standard assay mix of a small aliquot of enzyme which had been incubated at 22°C with a final concentration of 5.0 mM H202 and 1.0 mM KCN. Corrections for any inhibitory effects of these reagents on the standard assay system were made by addition of these reagents in the absence of crude cell extract. Final concentration of H202 in the assay mixture was 83 pM. The strains of bacteria, obtained from the VPI Anaerobe Laboratory Culture Collection, were B. fragilis (VPI 2393 from H. Berens, no. 12, Institut Pasteur, Lille, France), B. fragilis (VPI 2553 -* ATCC 25285), B. ditasonis (VPI 4243, ATCC 8503), and B. vulgatus (VPI 4245, ATCC 8482). For oxygen toxicity testing, B. fragilis (VPI 2393) was grown in peptone-yeast-glucose broth supplemented with 7.7 pM hemin and 2.2 ,uM vitamin K1 (17). One milliliter each was removed from the control and induced cultures, and a series of 10-fold dilutions was made for each into VPI salt dilution blanks (17). A 0.1-ml volume of appropriate dilutions was plated
J. BACTrERIOL. onto brain heart infusion (BHI) plates supplemented with 7.7 uM hemin and 2.2 uM vitamin K1. Plates were exposed to hyperbaric 02 or N2 at 37°C in a stainlesssteel pressure vessel. Compressions were carried out over a 2- to 3-min period, whereas decompressions were carried out gradually over a 4- to 5-min period to minimize formation of bubbles in the agar plates. A final pressure of 5 ATA (atmospheres absolute) of 100% 02 was used in the oxygen lethality studies whereas 5 ATA of 100% N2 was used in the control experiments. Other pressure conditions used are described in the text. Anaerobic incubation of the plates was carried out in a GasPak jar at 37°C (BD & Co., Cockeysville, Md.) for 72 to 96 h. Materials. Rifampin, chloramphenicol, cytochrome c (type III), xanthine, hemin, vitamin K,, Nitro Blue Tetrazolium, and riboflavin were purchased from Sigma Chemical Co. Acrylamide and bisacrylamide were Aldrich Gold Label products. Hydrogen peroxide (30%), sodium azide, and potassium cyanide were products of J. T. Baker Chemicals. All other chemicals were reagent grade. Bacterial cultures were obtained from the VPI Anaerobe Laboratory Collection.
RESULTS Induction of superoxide dismutase in B. frag4ilis (VPI 2393). Exposure of midlog B. fragilis culture growing in the peptone-yeast extract-glucose medium to 2% 02-98% N2 gassing for 2 h led to an average three- to fivefold increase in total superoxide dismutase activity when compared with control midlog cultures held under anaerobiosis for that same time period (Table 1). Culture turbidity measured by absorbance at 660 nm increased from approximately 0.7 to 1.2 in the control and to 1.0 in the 02-treated culture. Lactate dehydrogenase activity increased whereas malate dehydrogenase activity remained constant during oxygenation of the cell culture. Catalase activity varied from no detectable activity to approximately 1.2 U/mg in both control and 02-exposed cultures. Inhibition of the superoxide-mediated cytochrome c reduction was totally abolished by boiling the crude cell extract for several minutes. The time course of the superoxide dismutase activity increase is shown in Fig. 1. Initially, superoxide dismutase activity in the culture at midlog anaerobic state was 0.4 U/mg, and the specific activity increased within 15 min upon exposure to 2% 02 (Fig. 1). Activity increase was complete after 90 min of 2% 02 exposure, with the superoxide dismutase level being 3.7 U/mg. Superoxide dismutase levels in the control culture held under anaerobiosis increased only 20% during the 2-h experiment. Addition of chloramphenicol (0.5 mg/ml) or rifampin (1 pAg/ml) (27) prevented the 02-dependent increase in superoxide dismutase specific activity. Chloramphenicol completely inhibited increase in culture turbid-
141
SUPEROXIDE DISMUTASE IN B. FRAGILIS
VOL. 138, 1979
TABLE 1. Enzyme activity levels in B. fragilis (VPI 2393) Sp act (U/mg)" Conditions
Superoxide dismutase
Malate dehydro-
Catalase
Lactate dehydrogenase
0.2 (8) 0.05 ± 0.02 (6) 0.3 (6) 1.5 ± 0.6 (11)b Anaerobic 0.5 (8) 0.15 ± 0.05 (6) 0.3 (6) 4.9 ± 1.5 (11) 2% 02sparged a Malate dehydrogenase, lactate dehydrogenase, and catalase activities are reported as international units per milligram of protein. Superoxide dismutase activity units are those defined by McCord and Fridovich (21). b The numbers in parentheses are the number of determinations.
0
n
0 -6
C)
IE40 0
2.0+
arate experiment, B. fragilis were grown to midlog in peptone-yeast-glucose medium and inhibited by the addition of chloramphenicol (0.5 mg/ml), and the system was then sparged with 2% 02 for 2 h. Cells treated in that manner had not induced superoxide dismutase nor were they any more resistant to 02 lethality than were
la41 TC
2 cl.v (f)
.0
100 u
zu
40
Minutes
60 02
80
100
120
60
Exposure
FIG. 1. Time course of superoxide dismutase induction in B. fragilis (VPI 2393). B. fragilis (VPI 2393) was grown to midlogphase at 37°C in 800 ml of hemin and prereduced BHI broth containing 7.7 2.2 puM vitamin K1. A mixture of 2% 02-98% N2 was introduced into the culture via a sterile sintered glass sparger. The culture was maintained at 37°C during this treatment. As a function of time, 50-ml aliquots were removed from the culture, and the superoxide dismutase specific activity was determined for the crude extract of the cells therein. Specific activity for the cells exposed to 2% 02 (0) is shown as a function of time. ity during the 2-h experiment, but culture tur-
bidity increased -10% in the rifampin-treated test system.
Superoxide dismutase protection against hyperbaric 02 lethality in B. fraglis (VPI 2393). Exposure of B. fragilis on BHI agar plate surface to 5 ATA of 02 pressure resulted in a decrease in colony-forming units in both the uninduced, anaerobic sample (0.6 U of superoxide dismutase per mg) and in the 02-treated induced sample (4.9 U of superoxide dismutase per mg). However, the rates of cell viability loss were markedly different for the two test systems. Only 2% of initial viable colony-forming units remained after 4 h of exposure to hyperbaric 02 in the uninduced cells (Fig. 2). Under the same conditions, 15% of initial viable colonies re-
o
0
20
0
. 10
20 Hours
3.0
4.0
02 E oposure
FIG. 2. Rates of hyperoxic cell kill in induced and noninduced B. fragilis (VPI 2393) cells. Midlog cultures of B. fragilis (VPI 2393) containing 0.6 U of superoxide dismutase per mg (0) or 4.9 U of superoxide dismutase per mg (A) were appropriately diluted into prereduced dilution blanks, and 0.1 ml of the 10i, 10-, iO6¶ i0- dilutions were plated onto the surface of BHI agar supplemented with 7.7 zi and 2.2 p.M vitamin K,. The plated cells were exposed to 5 ATA of 02 at 37°C in a stainless-steel pressure vessel for 1 to 4 h. Plates were removed from the pressure vessel and incubated anaerobically at 37°C for 72 to 96 h. Control experiments with 5 ATA of N2 replacingr the 02 were performed. The data are expressed as percent viable colonies in the 02-treated mained (Fig. 2) in the 02-induced cells. If N2 samples compared with control values for the N2 test. replaced 02, no measurable loss of cell viability The N2 control values for the uninduced cultures was observed in either cell sample, implicating (0) and superoxide dismutase-induced cultures (A) oxygen as the toxic agent and not the mechanics are shown. These N2 test values were calculated on of pressurization and depressurization. In a sep- the basis of zero time cell count being 1tXS%.
142
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PRIVALLE AND GREGORY
uninduced cells. Since it is known that anaerobic prereduced medium may generate toxic metabolites, among them H202 (5), sterile BHI agar plates were first exposed to 5 ATA of oxygen, and then B. fragilis was diluted and plated onto the preexposed plates, as well as being plated onto BHI agar plates which had not been preexposed to hyperbaric oxygen. There was no difference in recovery of viable colony-forming units between unexposed or hyperbarically preexposed plates. The observed differential in cell viability loss was affected, at most, minimally due to toxic metabolites generated upon oxidation of the plating medium. Superoxide dismutase levels and oxygen toxicity in B vulgatus (VPI 4245) and B fiagilis (VPI 2553). B. vulgatus (VPI 4245) contained 0.5 U of superoxide dismutase per mg, no detectable catalase activity, and was relatively sensitive to hyperbaric oxygen. After 2 h of exposure to 5 ATA of 02 on BHI agar surfaces, 104 colony-forming units of original culture per ml was rendered nonviable (Fig. 3). Exposure of the cells to hyperbaric nitrogen or exposure to 1 ATA of 02 for that same time period resulted in only modest loss of cell viability (Fig. 3). B. fragilis (VPI 2553) contained 4.2 U of superoxide dismutase per mg at midlog, was devoid of detectable catalase activity, and was more resistant to hyperbaric oxygen toxicity than either B. vulgatus or B. fragilis (VPI 2393). Thus, exposure of midlog cells to 5 ATA of 02 pressure for 5 h (Fig. 4) resulted in no detectable loss in cell viability. Exposure of the cell to 10 ATA of 02 resulted in 2.5 decades of loss of viable colonyforming units in 2 h, and exposure to 15 ATA of 02 led to 3.5 decades of loss in numbers of colony-forming units in 2 h (Fig. 4). Inhibition of superoxide dismutase in Bacteroides species. Superoxide dismutase activity in each of the Bacteroides species tested was insensitive to inhibition by a final concentration of 1 mM or 5 mM potassium cyanide in the assay system. Each of the four strains or species tested was inhibited by the addition of sodium azide to a final concentration of 1 mM in the assay mixture. The inhibition levels ranged from 75% for B. vulgatus to 93% for B. distasonis (Table 2). The manganic superoxide dismutase from Escherichia coli was unaffected by either sodium cyanide or sodium azide (Table 2) (18). Upon incubation of bacterial crude cell extracts with 5 mM H202 at 220C, the superoxide dismutase activity diminished in a pseudo-firstorder manner with a half-life of 2.3 to 4.0 min. The crude cell extracts were diluted to contain approximately 30 U of superoxide dismutase activity in the 1-ml incubation mxture. The mix-
7 0
06 s
5
0
3
2
4
5
Hours
FIG. 3. Effects of hyperbaric oxygen on B. vulgatus (VPI 4245). Midlog cultures of B. vulgatus (VPI 4245) were diluted andplated onto BHI agar supplemented with 7.7/tMhemin and 2.2 AM vitamin Ki as described in Fig. 2. The cells were then exposed at 37°C to 5 ATA of 02 (0), I ATA of 02 (A), or 5 ATA of N2 (A). Plates were removed from the vessel as a fiunction of time for incubation anaerobicaly. Viabk cells, enumerated as colonies, were determined, and the data are presented as viable colonies per milliliter of culture based on viable cell density at zero time.
ture was also 1 mM in KCN to inhibit catalase activity. The data in Fig. 5 are comparisons of inactivation rates by 5 mM H202 of B. fragilis
(VPI 2393, open squares), B. distasonis (VPI 4243, triangles), and the manganic superoxide dismutase from E. coli (open circles). The halflives derived from these inactivation studies are shown in Table 2. Electrophoretic characterization of superoxide dismutase from Bacteroides species. Histochemical examination of 10% polyacrylamide gels for superoxide dismutase activity revealed that in each extract, except that of B. distasonis (VPI 4243), there were major and minor achromatic activity bands. The more rapidly migrating band (relative mobility, 0.55 to 0.63) in each case was the minor band. The major band, accounting for -80% of the superoxide dismutase activity, migrated at a mobility of 0.33 to 0.45 with respect to the tracking dye. Figure 6 shows the densitometric measurement of extracts of B. fragilis (VPI 2393) electrophoretically separated on 10% acrylamide gels and stained for superoxide dismutase activity. The upper gel scan is that of crude extract with no further treatment whereas the lower scan results
SUPEROXIDE DISMUTASE IN B. FRAGILIS
VOL. 138, 1979
both the major and minor activity bands. The manganic superoxide dismutase from E. coli was stable for at least 12 h under these conditions.
9_ 8
>7 I
>1
o 6 _J
5
0
DISCUSSION Exposure of mnidlog B. fragilis (VPI 2393) to 2% 02 results in a three- to fivefold induction of superoxide dismutase levels. The induction re\ quires de novo protein synthesis since inhibitors of protein synthesis also inhibit induction of \superoxide dismutase. The oxygen-induced cells are more resistant to the bactericidal effect of hyperbaric oxygen than are the noninduced cells. The cell kill observed appears to be due to the effects of 02 and not mechanical disruption of the cells due to abrupt pressure changes or to 2
3
100
4
510
E
Hours
TABLE 2. Characteristics of Bacteroides superoxide dismutase inhibition Sp act (U/mg)
B. fragilis (VPI 1.5 2393) B. fragilis (VPI 4.2 2553) B. distasonis 3.2 (VPI 4243) B. vulgatus (VPI 1.1 4245) E. coli Mn 3,300k
Half-life in Inhibition 5 mm by I1mm (%) azide H202 (min)
Mn SOD
40
' 20
0
4iB.frogilis (2393)
o
\ \
> '
Relative
mobility in 10% acrylamide gels
4.0
78
0.33", 0.55
3.8
83
0.39*, 0.63
3.7
93
0.37
2.3
75
0.45*, 0.61
Stable
0
NDc
superoxide dismutase Asterisk denotes major band. Reference 29. ND, Not determined.
coli
70
FIG. 4. Oxygen toxicity studies with B. fragilis (VPI 2553). Midlog culures of B. fragilis (VPI 2553) were diluted and plated as described (Fig. 2 and 3) and exposed to 5 (0), 10 (0), or 15 (A) ATA of 02 at 37°C. Plates were removed from the hyperbaric chamber and incubated anaerobically. Viable cells per milliliter of initial culture were determined. Nitrogen controls were performed for each pressure, and no loss in cell viability was observed in the control cultures. Results are shown as viable colonies per milliliter of culture based on viable cell density at zero time.
Organism
143
0
4
8
12
16
20
MINUTES
FIG. 5. Effects ofH202 upon superoxide dismutase in B. fragilis (VPI2393). Aliquots of crude cell extract of B. fragilis (VPI 2393) containing 30 U of superoxide dismutase activity were diluted in a final volume of 1 ml of 50 mM sodium phosphate, I mM EDTA (pH 7.8), 5 mM H20, and 1 mM KCN. Samples were if the extract were preincubated with 5 mM withdrawn as a function of incubation time at 22°C assayed for superoxide dismutase activity (0). H202 and 1 mM KCN for 1 h prior to electro- and treatments of crude cell extract of B. distaSimilar phoresis. The minor, rapidly migrating band is sonis (VPI 4243, A) and pure E. coli B manganic apparently unaffected by the H202 under these superoxide dismutase (0) are shown as log activity but the of the form is conditions, activity major as a function of time. Half-lives for the inactivation abolished. Incubation of the extracts under these of the two H202-sensitive species were derived from conditions for 12 h resulted in inactivation of these first-order plots.
144
PRIVALLE AND GREGORY
J. BACTERIOL.
siderably resistant to 5 ATA of 02, and was killed slowly by 10 ATA of 02 (see Fig. 3). Fifteen ATA of 02 was required to rapidly render those cells nonviable. B. fragilis (VPI 2393) was susceptible to 5 ATA of 02 tension even with prior induction of superoxide dismutase. Although the data suggest that there does exist a correlation between superoxide dismutase content and oxygen tolerance, precise tolerance estimates must be based upon studies with the individual species, provided that levels of superoxide dismuTOP
CONTROL
H2°O TREATED DYE
.53
.34
FIG. 6. Effects of H202 upon B. fragiLlis (VPI 2393) superoxide dismutase: densitometric anialysis. Crude cell extracts of B. fragilis (VPI 2393) w ,ere incubated for I h at 22°C in the presence of 5 mA H202 and 1 mM KCN. Three hundred micrograms of the crude extract was added to tubes containing ,40 mg of sucrose and then transferred to 10% acrylamide gels. Similarly, crude extract which had A iot been pretreated was placed onto gels. Electrop horetic separation was performed at 4°C until the bromophenol blue tracking dye had migrated throuAgh 75% of the gel length, and the gels were removed a?nd stained for superoxide dismutase activity. Areas o f achromaticity were determined by scanning the ge I at 560 nm in a Schoeffel densitometer. A recorder wcis attached to the densitometer to allow apositive reco coincident with achromaticity in the g4rel The upper bands in scan depicts superoxide dismutase acti untreated crude cell extract, whereas tihe lower scan is from gels after separation of the H202--treated crude
ft
vlity
cell
extract.
tase can be manipulated in the test system.
We recently demonstrated (28) that the catalase level in a number of Bacteroides strains varied widely depending upon the amount of hemin added to the medium and whether the hemin was added prior to sterilization of the medium or was added aseptically to the cool sterile medium. Thus, in the differential oxygen kill studies (Fig. 2), catalase activity was not detectable in 4 of the 11 repetitions of the experiment. In all 11 of the experiments, there was a consistent differential in superoxide dismutase activity. The rates of cell kill were not measurably affected by the presence or absence of catalase activity. Under the experimental conditions utilized for the results shown in Fig. 3 and 4, catalase activity was not detected in cell extracts of either B. fragilis (VPI 2553) or B. vulgatus (VPI 4245). The data thus suggest that, at least in the species tested, variation in catalase activity had no marked effect. Superoxide dismutases have been differentiated into three classes of metalloenzymes, based upon analytical data obtained with the enzyme isolated from various sources (21, 25, 29). Those clases are: cupro-zinc, manganic, and ferric superoxide dismutases. Differentiation of those enzymes in crude extracts is possible due to differential inhibition or inactivation of the
various forms of superoxide dismutase. Thus, toxic metabolites generated in the growth medium on hyperbaric oxygenation. T'hese results parallel observations with aerobic arid aerotolerant bacteria which suggest a role foir superoxide dismutase in oxygen tolerance. Oxyg,en inhibited growth of this strain of B. fragilis at a level of 2% when the cells were exposed on algar surfaces, but it is clearly shown by the data ti hat the toxic effect of oxygen requires exposurEa to several atmospheres of 02. Moreover, the ratte ofoxygenmediated cell kill appears to be deLpendent on the species tested. Thus, B. vulgatuws (VPI 4245) containing 0.5 U of superoxide dismLutase activity per mg was killed more rapidly by 5 ATA of 02 than were the uninduced B. ft -agilis (VPI 2393) containing comparable supero)xide dismutase activity. B. fragilis (VPI 2553}) contained 4.2 U of superoxide dismutase per nng, was con-
cupro-zinc enzymes are inhibited by NaCN (15) whereas manganic and ferric enzymes are not; low levels of azide inhibit the ferric enzyme but not manganic or cupro-zinc enzymes (23); hydrogen peroxide irreversibly inactivates the cuprozinc (16) and ferric enzymes (1) but not the manganic form. B. fragilis (VPI 2393) contains two electrophoretically distinct superoxide dismutases. The slower migrating form, which increases in intensity upon oxygen exposure, is rapidly inactivated by 5 mM H202 and is inhibited by 1 mM sodium azide, but not by 1 or 5 mM KCN. These observations suggest that the major form of superoxide dismutase is an iron-containing enzyme. The minor, more rapidly migrating form of the enzyme is inactivated by 5 mM H202 but over a 12-h period. Reaction of the ferric superoxide
SUPEROXIDE DISMUTASE IN B. FRAGILIS
VOL. 138, 1979
dismutase and H202 must be at least second order so the low levels of the minor band would considerably slow the inactivation. Even under the conditions required to inactivate the minor band, manganic superoxide dismutase is stable, suggesting that the minor band is also an iron metalloprotein. We observed similar results with another strain of B. fragilis (VPI 2553) and with B. vulgatus (VPI 4245) and B. distasonis (VPI 4243). Iron-containing superoxide dismutases have been characterized from the anaerobes Chlorobium thiosulfatophilum and Chromatum vinosum as weil as the aerotolerant E. coli B. E. coli B contains a ferric superoxide dismutase which may be constitutive and a manganic superoxide dismutase which is induced upon exposure to 02. In B. fragilis, the iron enzyme is apparently inducible upon exposure of the cells to low 02 levels. The role of superoxide dismutase as an 02 scavenger has been documented in defined chemical systems and in complex organisms. The logical argument based upon those data suggests that aerotolerance in Bacteroides species is based in part upon the presence of superoxide dismutase. Although it is not known how certain anaerobes acquired this enzymatic activity, superoxide dismutase appears to function in protecting the B. fragilis from the lethal action of oxygen. The genus Bacteroides is most frequently isolated from soft tissue infections, does not form spores, and must migrate from the reservoir (most commonly the intestinal tract) to the infection site. It is plausible to consider that superoxide dismutase offers some protection to brief exposure to 02 during that transit. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grant AI15250 from the National Institute of Allergy and Infectious Disease, Young Investigator grant HL19609 from National Heart, Lung and Blood Institute, and Hatch Project 6122540.
LITERATURE CITED 1. Asada, K., K. Yoshikawa, M.-A. Takahashi, Y.
2. 3.
4.
5.
Maeda, and K. Enmanji. 1975. Superoxide dismutase from a blue-green algae, Plectonema boryanum. J. Biol. Chem. 250:2801-2807. Beauchamp, C. O., and I. Fridovich. 1971. Superoxide dismutase: improved assay and an assay applicable to acrylamide gels. Anal. Biochem. 44:276-287. Beers, R. F., Jr., and I. W. Sizer. 1952. A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. Biol. Chem. 195:133140. Bergmeyer, H. U., and E. Bernt. 1974. Assay of lactate dehydrogenase activity, p. 574-578. In H. U. Bergmeyer (ed.), Methods of biochemical analysis, vol. 2. Academic Press Inc., New York. Carlsson, J., G. Nyberg, and J. Wreth6n. 1978. Hydrogen peroxide and superoxide radical formation in anaerobic broth media exposed to atmospheric oxygen. Appl. Environ. Microbiol. 36:223-229.
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6. Carl8son, J., J. Wrethen, and G. Beckman. 1977. Superoxide dismutase in Bacteroides fragilis and related Bacteroides species. J. Clin. Microbiol. 6:280-284. 7. Davis, B. J. 1964. Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121:404-427. 8. Fridovich, L 1975. Superoxide dismutases. Annu. Rev. Biochem. 44:147-159. 9. Fridovich, L 1978. The biology of oxygen radicals. Science 201:875-80. 10. Gregory, E. M. 1975. Chemical modification of bovine heart mitochondrial malate dehydrogenase. J. Biol. Chem. 250:5470-5474. 11. Gregory, E. M., and I. Fridovich. 1973. Induction of superoxide dismutase by molecular oxygen. J. Bacteriol. 114:543-548. 12. Gregory, E. M., and I. Fridovich. 1973. Oxygen toxicity and the superoxide dismutase. J. Bacteriol. 114:11931197. 13. Gregory, E. M., S. A. Goscin, and L. Fridovich. 1974. Superoxide dismutase and oxygen toxicity in a eukaryote. J. Bacteriol. 117:456-460. 14. Gregory, E. M., W. E. C. Moore, and L. V. Holdeman. 1978. Superoxide dismutase in anaerobes: survey. Appl. Environ. Microbiol. 35:988-991. 15. Hoffner, P. H., and J. E. Coleman. 1973. Cu(II)-carbon bonding in cyanide complexes of copper enzymes. J. Biol. Chem. 28:6626-6629. 16. Hodgson, E. K., and I. Fridovich. 1975. The interaction of bovine erythrocyte superoxide dismutase with hydrogen peroxide. Inactivation of the enzyme. Biochemistry 14:5294-5299. 17. Holdeman, L. V., and W. E. C. Moore (ed.). 1975. Anaerobe laboratory manual, 3rd ed. Anaerobe Laboratory, Virginia Polytechnic Institute and State University, Blacksburg, Va. 18. Keele, B. B., Jr., J. M. McCord, and I. Fridovich. 1970. Superoxide dismutase from Escherichia coli B. A new manganese containing enzyme. J. Biol. Chem. 245: 6176-6181. 19. Koch, A. L. 1970. Turbidity measurements of bacterial cultures in some available commercial instruments. Anal. Biochem. 38:252-259. 20. Loesche, W. J. 1969. Oxygen sensitivity of various anaerobic bacteria. Appl. Microbiol. 18:723-727. 21. McCord, J. M., and I. Fridovich. 1969. Superoxide dismutase: an enzymatic function for erythrocuprein. J. Biol. Chem. 244:6049-6055. 22. McCord, J. M., B. B. Keele, Jr., and I. Fridovich. 1971. An enzyme based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc. Natl. Acad. Sci. U.S.A. 68:1024-1027. 23. Misra, H. P., and I. Fridovich. 1978. Inhibition of superoxide dismutase by azide. Arch. Biochem. Biophys. 189:317-322. 24. Tally, F. P., B. R. Goldin, N. V. Jacobus, and S. L. Gorbach. 1977. Superoxide dismutase in anaerobic bacteria of clinical significance. Infect. Immun. 16:2025. 25. Vance, P. G., B. B. Keele, Jr., and K. V. Rajagopalan. 1972. Superoxide dismutase from Streptococcus mutans. Isolation and characterization of two forms of the enzyme. J. Biol. Chem. 247:4782-4786. 26. Warburg, O., and W. Christian. 1941. Isolierung und Kristalisation des Garung ferments Enolase. Biochem. Z. 310:384421. 27. Wehrli, W., F. Knusel, K. Schmid, and M. Staehelin. 1968. Interaction of rifamycin with bacterial RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 61:667-673. 28. Wilkins, T. D., D. L. Wagner, B. J. Veltri, Jr., and E. M. Gregory. 1978. Factors affecting production of catalase by Bacteroides. J. Clin. Microbiol. 8:553-557. 29. Yost, F. J., and I. Fridovich. 1973. An iron-containing superoxide dismutase from Escherichia coli. J. Biol. Chem. 248:4905-4908.
JOURNAL
OF BACTERIOLOGY, Apr. 1979, p. 139-145 0021-9193/79/04-0139/07$02.00/0
Superoxide Dismutase and 02 Lethality in Bacteroides fragilis CHRISTOPHER T. PRIVALLE AND EUGENE M. GREGORY* Department of Biochemistry and Nutrition, College of Agriculture and Life Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 Received for publication 6 December 1978
Exposure of midlog Bacteroides fragilis (VPI 2393) to 2% 02-98% N2 caused a three- to fivefold increase in superoxide dismutase specific activity within the cells. The increase in specific activity was completed within 90 min after exposure to oxygen and was dependent upon protein synthesis. Cells containing the higher superoxide dismutase level were more resistant to the effects of 5 atm of oxygen tension than were cells containing the lower level of superoxide dismutase but were equally resistant to 5 atm of nitrogen tension. Similar results were observed upon comparing viability experiments with B. fragilis and B. vulgatus. Superoxide dismutase activity in sonic extracts of B. fragilis was rapidly inactivated by exposure to 5 mM H202 and was inhibited by 1 mM NaN3 but not 5 mM NaCN. The inhibition pattern is identical to the pattern demonstrated for the purified iron-containing enzyme from Escherichia coli B and suggests that the superoxide dismutase in B. fragilis is an iron enzyme.
The reduction of triplet oxygen by a single electron generates the superoxide radical. The radical can disproportionate spontaneously to form hydrogen peroxide and singlet 02 and can interact with iron chelates and hydrogen peroxide in a catalytic cycle to generate hydroxyl radical (OH.). The disproportionation products and the superoxide radical itself have been shown to damage a number of cellular processes. Aerobic and aerotolerant bacteria have evolved ferric and manganic superoxide dismutases (EC 1.15.1.1) to diminish steady-state levels of superoxide radical. The superoxide dismutases catalyze disproportionation of the superoxide radical to hydrogen peroxide and ground-state 02, curtailing singlet O2 production, and remove 02 from the catalytic cycle with iron chelates and H202, diminishing OH - production (9). A direct correlation between superoxide dismutase level and resistance to oxygen lethality has been reported for a number of procaryotes and eucaryotes (11-13). These data and the attendant hypothesis have been reviewed (8, 9). Anaerobic bacteria were thought to be devoid of superoxide dismutase and, lacking that defense, were quite sensitive to oxygen-dependent lethality (22). A degree of aerotolerance variation was documented by Loesche (20) for a number of anaerobes based on the ability of the cells to grow under a defined 02 concentration. It now appears as if that variation in aerotolerance might be explained, in part, by the presence of superoxide dismutase. We have shown that 23
of 28 Bacteroides strains, several Clostridia, anaerobic cocci, and anaerobic nonspore-forming rods possessed superoxide dismutase activity (14). Tally et al. (24) and Carlsson et al. (6) have reported superoxide dismutase levels in anaerobes isolated from clinical specimens and have suggested, using a test system different from that we report herein, that there exists a correlation between superoxide dismutase level and aerotolerance. Generally, those comparisons were made among genera or species of anaerobes. The purpose of this communication is to present evidence that (i) the superoxide dismutase level in Bacteroides fragilis is inducible by exposing the organism to 02, (ii) induced levels of superoxide dismutase protect the cells from hyperbaric 02 lethality, and (iii) the major inducible form of superoxide dismutase appears to be the ferric-enzyme form.
MATERIALS AND METHODS A 1% inoculum of B. fragilis (VPI 2393) was transferred under 02-free CO2 from chopped-meat medium (17) into a prereduced medium containing 1.0% peptone, 1.0% yeast extract, and 1.0% glucose supplemented with 7.7 ,uM hemin and 2.2 ,uM- vitamin Ki, and was incubated at 37°C for 5.5 h. Bacterial growth was monitored turbidimetrically at 660 nm by the method of Koch (19). Cells were harvested by centrifugation, washed with 50 mM potassium phosphate plus 1 mM EDTA (pH 7.8), and sonically disrupted with cooling with a Branson model W185D soniflier operated at a power output of 65 W into the microtip. Cell debris was removed by centrifugation at 35,000 139
140
PRIVALLE AND GREGORY
x g for 15 min. All manipulations in preparing the crude cell extract were performed aerobically. Protein content of the crude extract was determined spectrophotometrically by absorbance at 280 and 260 nm, assuming that a 1 mg/ml solution of protein in a 1-cm cuvette would give an absorbance of 1.0 (26). Catalase was assayed by the method of Beers and Sizer (3). Superoxide dismutase was assayed by its ability to inhibit the 02-mediated reduction of ferricytochrome c as described by McCord and Fridovich (21). Malate dehydrogenase (10) and lactate dehydrogenase (4) were assayed by previously reported methods. Disc gel electrophoresis was performed in 7.5 or 10% acrylamide gels (7) with a 5 mM Tris-39 mM glycine buffer at pH 8.3 at a constant current of 2.5 mA per gel until the bromophenol blue tracking dye had swept through most of the gel. Each gel received 300,ug of crude cell extract and approximately 40 mg of sucrose. Superoxide dismutase activity was localized on gels by the method of Beauchamp and Fridovich (2). Superoxide dismutase isozymes or relative amounts of the major band were estimated from areas under densitometric scans of polyacrylamide gels which had been stained for superoxide dismutase activity. A Schoeffel model SD 3000 spectrodensitometer was used for scanning the gels at 560 nm. Treatment of the crude cell extract with H202 was used to determine which of the superoxide dismutase activity bands were inactivated. Final concentrations of inhibitors were 5.0 mM H202 and 1.0 mM KCN used in pretreatment of extracts in the activity gels. The presence of KCN was to suppress any residual catalase activity which would have diminished the effective concentration of H202. The effects of KCN (1 and 5 mM) and NaN3 (1 mM) on superoxide dismutase activity were tested by the addition of these reagents to the assay mixture immediately prior to the addition of the crude cell extract. At the concentrations used, azide neither inhibited the oxidation of xanthine by xanthine oxidase nor inhibited the reduction of cytochrome c by 02'. The slight inhibitory effect of sodium cyanide on xanthine oxidase activity was corrected in the superoxide dismutase activity calculation. The effect of H202 was tested by the addition to the standard assay mix of a small aliquot of enzyme which had been incubated at 22°C with a final concentration of 5.0 mM H202 and 1.0 mM KCN. Corrections for any inhibitory effects of these reagents on the standard assay system were made by addition of these reagents in the absence of crude cell extract. Final concentration of H202 in the assay mixture was 83 pM. The strains of bacteria, obtained from the VPI Anaerobe Laboratory Culture Collection, were B. fragilis (VPI 2393 from H. Berens, no. 12, Institut Pasteur, Lille, France), B. fragilis (VPI 2553 -* ATCC 25285), B. ditasonis (VPI 4243, ATCC 8503), and B. vulgatus (VPI 4245, ATCC 8482). For oxygen toxicity testing, B. fragilis (VPI 2393) was grown in peptone-yeast-glucose broth supplemented with 7.7 pM hemin and 2.2 ,uM vitamin K1 (17). One milliliter each was removed from the control and induced cultures, and a series of 10-fold dilutions was made for each into VPI salt dilution blanks (17). A 0.1-ml volume of appropriate dilutions was plated
J. BACTrERIOL. onto brain heart infusion (BHI) plates supplemented with 7.7 uM hemin and 2.2 uM vitamin K1. Plates were exposed to hyperbaric 02 or N2 at 37°C in a stainlesssteel pressure vessel. Compressions were carried out over a 2- to 3-min period, whereas decompressions were carried out gradually over a 4- to 5-min period to minimize formation of bubbles in the agar plates. A final pressure of 5 ATA (atmospheres absolute) of 100% 02 was used in the oxygen lethality studies whereas 5 ATA of 100% N2 was used in the control experiments. Other pressure conditions used are described in the text. Anaerobic incubation of the plates was carried out in a GasPak jar at 37°C (BD & Co., Cockeysville, Md.) for 72 to 96 h. Materials. Rifampin, chloramphenicol, cytochrome c (type III), xanthine, hemin, vitamin K,, Nitro Blue Tetrazolium, and riboflavin were purchased from Sigma Chemical Co. Acrylamide and bisacrylamide were Aldrich Gold Label products. Hydrogen peroxide (30%), sodium azide, and potassium cyanide were products of J. T. Baker Chemicals. All other chemicals were reagent grade. Bacterial cultures were obtained from the VPI Anaerobe Laboratory Collection.
RESULTS Induction of superoxide dismutase in B. frag4ilis (VPI 2393). Exposure of midlog B. fragilis culture growing in the peptone-yeast extract-glucose medium to 2% 02-98% N2 gassing for 2 h led to an average three- to fivefold increase in total superoxide dismutase activity when compared with control midlog cultures held under anaerobiosis for that same time period (Table 1). Culture turbidity measured by absorbance at 660 nm increased from approximately 0.7 to 1.2 in the control and to 1.0 in the 02-treated culture. Lactate dehydrogenase activity increased whereas malate dehydrogenase activity remained constant during oxygenation of the cell culture. Catalase activity varied from no detectable activity to approximately 1.2 U/mg in both control and 02-exposed cultures. Inhibition of the superoxide-mediated cytochrome c reduction was totally abolished by boiling the crude cell extract for several minutes. The time course of the superoxide dismutase activity increase is shown in Fig. 1. Initially, superoxide dismutase activity in the culture at midlog anaerobic state was 0.4 U/mg, and the specific activity increased within 15 min upon exposure to 2% 02 (Fig. 1). Activity increase was complete after 90 min of 2% 02 exposure, with the superoxide dismutase level being 3.7 U/mg. Superoxide dismutase levels in the control culture held under anaerobiosis increased only 20% during the 2-h experiment. Addition of chloramphenicol (0.5 mg/ml) or rifampin (1 pAg/ml) (27) prevented the 02-dependent increase in superoxide dismutase specific activity. Chloramphenicol completely inhibited increase in culture turbid-
141
SUPEROXIDE DISMUTASE IN B. FRAGILIS
VOL. 138, 1979
TABLE 1. Enzyme activity levels in B. fragilis (VPI 2393) Sp act (U/mg)" Conditions
Superoxide dismutase
Malate dehydro-
Catalase
Lactate dehydrogenase
0.2 (8) 0.05 ± 0.02 (6) 0.3 (6) 1.5 ± 0.6 (11)b Anaerobic 0.5 (8) 0.15 ± 0.05 (6) 0.3 (6) 4.9 ± 1.5 (11) 2% 02sparged a Malate dehydrogenase, lactate dehydrogenase, and catalase activities are reported as international units per milligram of protein. Superoxide dismutase activity units are those defined by McCord and Fridovich (21). b The numbers in parentheses are the number of determinations.
0
n
0 -6
C)
IE40 0
2.0+
arate experiment, B. fragilis were grown to midlog in peptone-yeast-glucose medium and inhibited by the addition of chloramphenicol (0.5 mg/ml), and the system was then sparged with 2% 02 for 2 h. Cells treated in that manner had not induced superoxide dismutase nor were they any more resistant to 02 lethality than were
la41 TC
2 cl.v (f)
.0
100 u
zu
40
Minutes
60 02
80
100
120
60
Exposure
FIG. 1. Time course of superoxide dismutase induction in B. fragilis (VPI 2393). B. fragilis (VPI 2393) was grown to midlogphase at 37°C in 800 ml of hemin and prereduced BHI broth containing 7.7 2.2 puM vitamin K1. A mixture of 2% 02-98% N2 was introduced into the culture via a sterile sintered glass sparger. The culture was maintained at 37°C during this treatment. As a function of time, 50-ml aliquots were removed from the culture, and the superoxide dismutase specific activity was determined for the crude extract of the cells therein. Specific activity for the cells exposed to 2% 02 (0) is shown as a function of time. ity during the 2-h experiment, but culture tur-
bidity increased -10% in the rifampin-treated test system.
Superoxide dismutase protection against hyperbaric 02 lethality in B. fraglis (VPI 2393). Exposure of B. fragilis on BHI agar plate surface to 5 ATA of 02 pressure resulted in a decrease in colony-forming units in both the uninduced, anaerobic sample (0.6 U of superoxide dismutase per mg) and in the 02-treated induced sample (4.9 U of superoxide dismutase per mg). However, the rates of cell viability loss were markedly different for the two test systems. Only 2% of initial viable colony-forming units remained after 4 h of exposure to hyperbaric 02 in the uninduced cells (Fig. 2). Under the same conditions, 15% of initial viable colonies re-
o
0
20
0
. 10
20 Hours
3.0
4.0
02 E oposure
FIG. 2. Rates of hyperoxic cell kill in induced and noninduced B. fragilis (VPI 2393) cells. Midlog cultures of B. fragilis (VPI 2393) containing 0.6 U of superoxide dismutase per mg (0) or 4.9 U of superoxide dismutase per mg (A) were appropriately diluted into prereduced dilution blanks, and 0.1 ml of the 10i, 10-, iO6¶ i0- dilutions were plated onto the surface of BHI agar supplemented with 7.7 zi and 2.2 p.M vitamin K,. The plated cells were exposed to 5 ATA of 02 at 37°C in a stainless-steel pressure vessel for 1 to 4 h. Plates were removed from the pressure vessel and incubated anaerobically at 37°C for 72 to 96 h. Control experiments with 5 ATA of N2 replacingr the 02 were performed. The data are expressed as percent viable colonies in the 02-treated mained (Fig. 2) in the 02-induced cells. If N2 samples compared with control values for the N2 test. replaced 02, no measurable loss of cell viability The N2 control values for the uninduced cultures was observed in either cell sample, implicating (0) and superoxide dismutase-induced cultures (A) oxygen as the toxic agent and not the mechanics are shown. These N2 test values were calculated on of pressurization and depressurization. In a sep- the basis of zero time cell count being 1tXS%.
142
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PRIVALLE AND GREGORY
uninduced cells. Since it is known that anaerobic prereduced medium may generate toxic metabolites, among them H202 (5), sterile BHI agar plates were first exposed to 5 ATA of oxygen, and then B. fragilis was diluted and plated onto the preexposed plates, as well as being plated onto BHI agar plates which had not been preexposed to hyperbaric oxygen. There was no difference in recovery of viable colony-forming units between unexposed or hyperbarically preexposed plates. The observed differential in cell viability loss was affected, at most, minimally due to toxic metabolites generated upon oxidation of the plating medium. Superoxide dismutase levels and oxygen toxicity in B vulgatus (VPI 4245) and B fiagilis (VPI 2553). B. vulgatus (VPI 4245) contained 0.5 U of superoxide dismutase per mg, no detectable catalase activity, and was relatively sensitive to hyperbaric oxygen. After 2 h of exposure to 5 ATA of 02 on BHI agar surfaces, 104 colony-forming units of original culture per ml was rendered nonviable (Fig. 3). Exposure of the cells to hyperbaric nitrogen or exposure to 1 ATA of 02 for that same time period resulted in only modest loss of cell viability (Fig. 3). B. fragilis (VPI 2553) contained 4.2 U of superoxide dismutase per mg at midlog, was devoid of detectable catalase activity, and was more resistant to hyperbaric oxygen toxicity than either B. vulgatus or B. fragilis (VPI 2393). Thus, exposure of midlog cells to 5 ATA of 02 pressure for 5 h (Fig. 4) resulted in no detectable loss in cell viability. Exposure of the cell to 10 ATA of 02 resulted in 2.5 decades of loss of viable colonyforming units in 2 h, and exposure to 15 ATA of 02 led to 3.5 decades of loss in numbers of colony-forming units in 2 h (Fig. 4). Inhibition of superoxide dismutase in Bacteroides species. Superoxide dismutase activity in each of the Bacteroides species tested was insensitive to inhibition by a final concentration of 1 mM or 5 mM potassium cyanide in the assay system. Each of the four strains or species tested was inhibited by the addition of sodium azide to a final concentration of 1 mM in the assay mixture. The inhibition levels ranged from 75% for B. vulgatus to 93% for B. distasonis (Table 2). The manganic superoxide dismutase from Escherichia coli was unaffected by either sodium cyanide or sodium azide (Table 2) (18). Upon incubation of bacterial crude cell extracts with 5 mM H202 at 220C, the superoxide dismutase activity diminished in a pseudo-firstorder manner with a half-life of 2.3 to 4.0 min. The crude cell extracts were diluted to contain approximately 30 U of superoxide dismutase activity in the 1-ml incubation mxture. The mix-
7 0
06 s
5
0
3
2
4
5
Hours
FIG. 3. Effects of hyperbaric oxygen on B. vulgatus (VPI 4245). Midlog cultures of B. vulgatus (VPI 4245) were diluted andplated onto BHI agar supplemented with 7.7/tMhemin and 2.2 AM vitamin Ki as described in Fig. 2. The cells were then exposed at 37°C to 5 ATA of 02 (0), I ATA of 02 (A), or 5 ATA of N2 (A). Plates were removed from the vessel as a fiunction of time for incubation anaerobicaly. Viabk cells, enumerated as colonies, were determined, and the data are presented as viable colonies per milliliter of culture based on viable cell density at zero time.
ture was also 1 mM in KCN to inhibit catalase activity. The data in Fig. 5 are comparisons of inactivation rates by 5 mM H202 of B. fragilis
(VPI 2393, open squares), B. distasonis (VPI 4243, triangles), and the manganic superoxide dismutase from E. coli (open circles). The halflives derived from these inactivation studies are shown in Table 2. Electrophoretic characterization of superoxide dismutase from Bacteroides species. Histochemical examination of 10% polyacrylamide gels for superoxide dismutase activity revealed that in each extract, except that of B. distasonis (VPI 4243), there were major and minor achromatic activity bands. The more rapidly migrating band (relative mobility, 0.55 to 0.63) in each case was the minor band. The major band, accounting for -80% of the superoxide dismutase activity, migrated at a mobility of 0.33 to 0.45 with respect to the tracking dye. Figure 6 shows the densitometric measurement of extracts of B. fragilis (VPI 2393) electrophoretically separated on 10% acrylamide gels and stained for superoxide dismutase activity. The upper gel scan is that of crude extract with no further treatment whereas the lower scan results
SUPEROXIDE DISMUTASE IN B. FRAGILIS
VOL. 138, 1979
both the major and minor activity bands. The manganic superoxide dismutase from E. coli was stable for at least 12 h under these conditions.
9_ 8
>7 I
>1
o 6 _J
5
0
DISCUSSION Exposure of mnidlog B. fragilis (VPI 2393) to 2% 02 results in a three- to fivefold induction of superoxide dismutase levels. The induction re\ quires de novo protein synthesis since inhibitors of protein synthesis also inhibit induction of \superoxide dismutase. The oxygen-induced cells are more resistant to the bactericidal effect of hyperbaric oxygen than are the noninduced cells. The cell kill observed appears to be due to the effects of 02 and not mechanical disruption of the cells due to abrupt pressure changes or to 2
3
100
4
510
E
Hours
TABLE 2. Characteristics of Bacteroides superoxide dismutase inhibition Sp act (U/mg)
B. fragilis (VPI 1.5 2393) B. fragilis (VPI 4.2 2553) B. distasonis 3.2 (VPI 4243) B. vulgatus (VPI 1.1 4245) E. coli Mn 3,300k
Half-life in Inhibition 5 mm by I1mm (%) azide H202 (min)
Mn SOD
40
' 20
0
4iB.frogilis (2393)
o
\ \
> '
Relative
mobility in 10% acrylamide gels
4.0
78
0.33", 0.55
3.8
83
0.39*, 0.63
3.7
93
0.37
2.3
75
0.45*, 0.61
Stable
0
NDc
superoxide dismutase Asterisk denotes major band. Reference 29. ND, Not determined.
coli
70
FIG. 4. Oxygen toxicity studies with B. fragilis (VPI 2553). Midlog culures of B. fragilis (VPI 2553) were diluted and plated as described (Fig. 2 and 3) and exposed to 5 (0), 10 (0), or 15 (A) ATA of 02 at 37°C. Plates were removed from the hyperbaric chamber and incubated anaerobically. Viable cells per milliliter of initial culture were determined. Nitrogen controls were performed for each pressure, and no loss in cell viability was observed in the control cultures. Results are shown as viable colonies per milliliter of culture based on viable cell density at zero time.
Organism
143
0
4
8
12
16
20
MINUTES
FIG. 5. Effects ofH202 upon superoxide dismutase in B. fragilis (VPI2393). Aliquots of crude cell extract of B. fragilis (VPI 2393) containing 30 U of superoxide dismutase activity were diluted in a final volume of 1 ml of 50 mM sodium phosphate, I mM EDTA (pH 7.8), 5 mM H20, and 1 mM KCN. Samples were if the extract were preincubated with 5 mM withdrawn as a function of incubation time at 22°C assayed for superoxide dismutase activity (0). H202 and 1 mM KCN for 1 h prior to electro- and treatments of crude cell extract of B. distaSimilar phoresis. The minor, rapidly migrating band is sonis (VPI 4243, A) and pure E. coli B manganic apparently unaffected by the H202 under these superoxide dismutase (0) are shown as log activity but the of the form is conditions, activity major as a function of time. Half-lives for the inactivation abolished. Incubation of the extracts under these of the two H202-sensitive species were derived from conditions for 12 h resulted in inactivation of these first-order plots.
144
PRIVALLE AND GREGORY
J. BACTERIOL.
siderably resistant to 5 ATA of 02, and was killed slowly by 10 ATA of 02 (see Fig. 3). Fifteen ATA of 02 was required to rapidly render those cells nonviable. B. fragilis (VPI 2393) was susceptible to 5 ATA of 02 tension even with prior induction of superoxide dismutase. Although the data suggest that there does exist a correlation between superoxide dismutase content and oxygen tolerance, precise tolerance estimates must be based upon studies with the individual species, provided that levels of superoxide dismuTOP
CONTROL
H2°O TREATED DYE
.53
.34
FIG. 6. Effects of H202 upon B. fragiLlis (VPI 2393) superoxide dismutase: densitometric anialysis. Crude cell extracts of B. fragilis (VPI 2393) w ,ere incubated for I h at 22°C in the presence of 5 mA H202 and 1 mM KCN. Three hundred micrograms of the crude extract was added to tubes containing ,40 mg of sucrose and then transferred to 10% acrylamide gels. Similarly, crude extract which had A iot been pretreated was placed onto gels. Electrop horetic separation was performed at 4°C until the bromophenol blue tracking dye had migrated throuAgh 75% of the gel length, and the gels were removed a?nd stained for superoxide dismutase activity. Areas o f achromaticity were determined by scanning the ge I at 560 nm in a Schoeffel densitometer. A recorder wcis attached to the densitometer to allow apositive reco coincident with achromaticity in the g4rel The upper bands in scan depicts superoxide dismutase acti untreated crude cell extract, whereas tihe lower scan is from gels after separation of the H202--treated crude
ft
vlity
cell
extract.
tase can be manipulated in the test system.
We recently demonstrated (28) that the catalase level in a number of Bacteroides strains varied widely depending upon the amount of hemin added to the medium and whether the hemin was added prior to sterilization of the medium or was added aseptically to the cool sterile medium. Thus, in the differential oxygen kill studies (Fig. 2), catalase activity was not detectable in 4 of the 11 repetitions of the experiment. In all 11 of the experiments, there was a consistent differential in superoxide dismutase activity. The rates of cell kill were not measurably affected by the presence or absence of catalase activity. Under the experimental conditions utilized for the results shown in Fig. 3 and 4, catalase activity was not detected in cell extracts of either B. fragilis (VPI 2553) or B. vulgatus (VPI 4245). The data thus suggest that, at least in the species tested, variation in catalase activity had no marked effect. Superoxide dismutases have been differentiated into three classes of metalloenzymes, based upon analytical data obtained with the enzyme isolated from various sources (21, 25, 29). Those clases are: cupro-zinc, manganic, and ferric superoxide dismutases. Differentiation of those enzymes in crude extracts is possible due to differential inhibition or inactivation of the
various forms of superoxide dismutase. Thus, toxic metabolites generated in the growth medium on hyperbaric oxygenation. T'hese results parallel observations with aerobic arid aerotolerant bacteria which suggest a role foir superoxide dismutase in oxygen tolerance. Oxyg,en inhibited growth of this strain of B. fragilis at a level of 2% when the cells were exposed on algar surfaces, but it is clearly shown by the data ti hat the toxic effect of oxygen requires exposurEa to several atmospheres of 02. Moreover, the ratte ofoxygenmediated cell kill appears to be deLpendent on the species tested. Thus, B. vulgatuws (VPI 4245) containing 0.5 U of superoxide dismLutase activity per mg was killed more rapidly by 5 ATA of 02 than were the uninduced B. ft -agilis (VPI 2393) containing comparable supero)xide dismutase activity. B. fragilis (VPI 2553}) contained 4.2 U of superoxide dismutase per nng, was con-
cupro-zinc enzymes are inhibited by NaCN (15) whereas manganic and ferric enzymes are not; low levels of azide inhibit the ferric enzyme but not manganic or cupro-zinc enzymes (23); hydrogen peroxide irreversibly inactivates the cuprozinc (16) and ferric enzymes (1) but not the manganic form. B. fragilis (VPI 2393) contains two electrophoretically distinct superoxide dismutases. The slower migrating form, which increases in intensity upon oxygen exposure, is rapidly inactivated by 5 mM H202 and is inhibited by 1 mM sodium azide, but not by 1 or 5 mM KCN. These observations suggest that the major form of superoxide dismutase is an iron-containing enzyme. The minor, more rapidly migrating form of the enzyme is inactivated by 5 mM H202 but over a 12-h period. Reaction of the ferric superoxide
SUPEROXIDE DISMUTASE IN B. FRAGILIS
VOL. 138, 1979
dismutase and H202 must be at least second order so the low levels of the minor band would considerably slow the inactivation. Even under the conditions required to inactivate the minor band, manganic superoxide dismutase is stable, suggesting that the minor band is also an iron metalloprotein. We observed similar results with another strain of B. fragilis (VPI 2553) and with B. vulgatus (VPI 4245) and B. distasonis (VPI 4243). Iron-containing superoxide dismutases have been characterized from the anaerobes Chlorobium thiosulfatophilum and Chromatum vinosum as weil as the aerotolerant E. coli B. E. coli B contains a ferric superoxide dismutase which may be constitutive and a manganic superoxide dismutase which is induced upon exposure to 02. In B. fragilis, the iron enzyme is apparently inducible upon exposure of the cells to low 02 levels. The role of superoxide dismutase as an 02 scavenger has been documented in defined chemical systems and in complex organisms. The logical argument based upon those data suggests that aerotolerance in Bacteroides species is based in part upon the presence of superoxide dismutase. Although it is not known how certain anaerobes acquired this enzymatic activity, superoxide dismutase appears to function in protecting the B. fragilis from the lethal action of oxygen. The genus Bacteroides is most frequently isolated from soft tissue infections, does not form spores, and must migrate from the reservoir (most commonly the intestinal tract) to the infection site. It is plausible to consider that superoxide dismutase offers some protection to brief exposure to 02 during that transit. ACKNOWLEDGMENTS This work was supported in part by Public Health Service grant AI15250 from the National Institute of Allergy and Infectious Disease, Young Investigator grant HL19609 from National Heart, Lung and Blood Institute, and Hatch Project 6122540.
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