fourfold increase in SOD activity in the aerobic cells, suggesting that oxygen acts as an inducer for SOD in the vibrios as has beenreported for E. coli. Inone strain ... aerobic respiration which, in phagocytic cells, ... levels are increased as a result of a shift from an anaerobic .... vigorous aeration (Table 2), as described in Ma-.
Vol. 134, No. 2
JOURNAL OF BACTERIOLOGY, May 1978, p. 375-380
0021-9193/78/00134-0375$02.00/0 Copyright 0 1978 American Society for Microbiology
Printed in U.S.A.
Superoxide Dismutase and Catalase Levels in Halophilic Vibrios OTIS P. DAILY,* ROBERT M. DEBELL, AND SAM W. JOSEPH Department of Microbiology, Naval Medical Research Institute, Bethesda, Maryland 20014 Received for publication 31 October 1977
Superoxide dismutase (SOD) and catalase (CAT) levels were determined for several aerobically grown halophilic vibrios and compared with those found in aerobically grown Escherichia coli K-12. The SOD levels ranged from 25 to 103.6 U/mg of protein for the vibrios compared with 44.6 U/mg of protein for E. coli. The CAT levels ranged from 2.1 to 32.1 U/mg of protein. Electrophoretic analysis of cell extracts revealed that the halophilic vibrios tested possessed only one detectable SOD enzyme, except one strain which possessed two distinct enzymes, as compared with the three SOD enzymes in aerobically grown E. coli K-12. A comparison of anaerobically and aerobically grown vibrios revealed a three- to fourfold increase in SOD activity in the aerobic cells, suggesting that oxygen acts as an inducer for SOD in the vibrios as has been reported for E. coli. In one strain, Vibrio parahaemolyticus 27519, both SOD enzymes were observed in low levels in anaerobic and at higher levels in aerobically grown cells as compared with only one SOD enzyme in anaerobically grown E. coli. This suggests that differences in SOD regulation occur between the two genera. Our results indicate that halophilic vibrios possess SOD, which could enhance virulence by allowing the organisms to survive in oxygenated environments.
Vibrioparahaemolyticus and other halophilic vibrios have been implicated with increasing frequency in gastro- and extraintestinal infections of humans after seafood ingestion or contact with marine and coastal environments in many parts of the world, including the United States (2, 12, 17, 24). Although the precise nature of their marine habitat is not known, these bacteria are presumably exposed to an anaerobic environment in the intestines of humans and to aerobic conditions when they invade the mucosa and other tissues (6, 11) and are exposed to the action of leukocytes (1, 19, 25). Superoxide radicals and hydrogen peroxide are toxic products of mammalian and bacterial aerobic respiration which, in phagocytic cells, are believed to play a role in the killing of ingested bacteria (1, 10, 18, 19, 25). Certain microorganisms are protected from the oxygen toxicity of phagocytic cells by their ability to synthesize superoxide dismutase (SOD) and catalase (CAT) (1, 10, 13, 14, 19, 25). It was of interest to determine whether pathogenic halophilic vibrios have these two enzymes and whether the levels are increased as a result of a shift from an anaerobic to aerobic growth environment. This study demonstrates that several related halophilic vibrios possess one or more SOD enzymes and that exposure to higher oxygen tensions results in increased SOD levels in these organisms.
MATERIALS AND METHODS The following organisms were used in this study: V. parahaenolyticus strains ATCC 27519, ATCC 17802, and DJ1242A, a human isolate from Indonesia; V. alginolyticus DJ7608, an enteric isolate from Indonesia; two lactose-positive Vibrio species, A8694 and A1402, both of which are extraintestinal human isolates obtained from R. Weaver of the Center for Disease Control, Atlanta, Ga.; V. cholerae 569B, a vaccine strain obtained from L. T. Callahan, formerly of our laboratory, and Escherichia coli K-12 (ATCC 23716). Stock cultures were maintained on brain heart infusion (BHI) agar (Baltimore Biological Laboratories) supplemented to contain 2.0% NaCl for all strains except E. coli and V. cholerae, which were maintained on the standard BHI agar. Aerobic growth conditions for broth cultures were achieved by oscillation of broth cultures in 500-ml baffled Erlenmeyer flasks on a rotary platform shaker at 250 rpm at 37°C. Anaerobic growth conditions were established by transfer of freshly sterilized broth into an anaerobic glove box (Germ Free Laboratories, Inc., Miami, Fla.) containing an atmosphere of 85% N2, 10% CO2, and 5% H2. Palladium catalysts were used to continually maintain anaerobiosis. Resazurin solutions were indicators of anaerobic conditions. Freshly sterilized broth was allowed to equilibrate with the anaerobic environment for 72 h before use for batch cultures. Anaerobic batch cultures were prepared in the following manner: aerobic cultures (in 10-ml aliquots) were transferred into the anaerobic glove box, equilibrated for several hours, subcultured to anaerobic broth, and incubated overnight at 37°C. This process 375
DAILY, DEBELL, AND JOSEPH
was repeated for five successive transfers; the last subculture was then used to inoculate batch cultures for anaerobic growth. Incubation was at 37°C without shaking. Aerobic cell extracts of E. coli K-12 and V. cholerae were prepared from 200-ml batch cultures grown in 1% peptone (Difco). This medium was supplemented to contain 2% NaCl for the halophiles. Anaerobic E. coli K-12 extracts were prepared from cultures grown in peptone containing 1% glucose. Anaerobic vibrio extracts, due to poor growth in peptone, were prepared from cultures grown in BHI broth supplemented to contain 1% glucose and 2% NaCl. Two hundred milliliters of log-phase cells was harvested at 4°C by centrifugation for 10 min at 7,000 x g and washed once with 0.05 M potassium phosphate (pH 7.8) containing 1 mM ethylenediaminetetraacetic acid. The washed cells were resuspended in 10 ml of buffer and disrupted at 4°C for 3 min with a Branson Sonifier (Branson Sonic Power Co., Danbury, Conn.) operated at maximum output (150 W, 20 kHz). Cell debris was removed by centrifugation at 30,000 x g for 30 min, and the cell extracts were stored at -70°C until used. Protein concentrations of the cell extracts were estimated by the method of Lowry et al. (20). SOD assays were performed by using the xanthinexanthine oxidase superoxide radical generating system coupled with the nitroblue tetrazolium indicator (3, 22). All measurements were made with a Beckman model 26-S spectrophotometer at 25°C. One unit of SOD is defined as the amount of enzyme that effected a 50% inhibition of the rate of nitroblue tetrazolium reduction by the xanthine-xanthine oxidase reaction (3, 22). Catalase assays were performed spectrophotometrically according to the method of Beers and Sizer (5). Polyacrylamide disc gel electrophoresis (PAGE) of cell extracts was performed on 7.5% gels by the method of Davis (8). Polymerization of the acrylamide gels was accomplished by the addition of 0.028% final ammonium persulfate concentration. Cell extracts were placed on the gels in sucrose, and electrophoresis was performed at 4 mA/tube until the bromophenol blue marker dye had swept through most of the 100-mmlong tubes. SOD activity was localized by the method of Beauchamp and Fridovich (3). Gels stained for SOD activity were scanned with an Acta III spectrophotometer equipped with a gel-scanning accessory (Beckman Instruments, Inc., Palo Alto, Calif.) at 550 nm. Rf values of SOD enzymes were calculated from the gel scan tracings. In the SOD induction experiments, relative amounts of SOD enzymes were estimated from areas under the peaks of scan tracings. Cell extracts of V. parahaemolyticus ATCC 27519, Vibrio sp. A8694, and, for comparison, E. coli K-12, all of which were grown under anaerobic and aerobic conditions, were prepared by splitting the batch cultures into two aliquots; one aliquot was transferred to aerobic growth conditions at 37°C, while the other remained under anaerobic conditions. Cell extracts were prepared at the time of the transfer and at 1, 3, and 6 h after the transfer. Care was taken to assure that protein synthesis was terminated with the addition of 25 ftg of chloramphenicol (Ames Co., Elkhart, Ind.) per ml of culture before the cultures were re-
moved from their growth environments. Cell extracts were assayed for SOD activity and the presence of one or more SOD enzymes, and their respective levels were determined by subjecting the cell extracts to PAGE.
RESULTS Levels of SOD and CAT in vibrios. Table 1 summarizes the results of SOD and CAT determinations on aerobically grown strains. Most of the vibrios possessed SOD levels slightly lower (range 25.0 to 39.4 U/mg of protein) than those of E. coli K-12 (44.6) except for V. alginolyticus 7608, which had a level 2.3 times greater than that of E. coli K-12. Although two vibrios (27519 and A1402) possessed CAT activities similar to those of E. coli K-12 (30.1), the other vibrios possessed only about 50% of this activity; one strain, 1242A, had the unusually low level of 2.1, or about 7% of that of E. coli. SOD enzymes. Densitometric scans of PAGE patterns of extracts from late-log-phase, aerobically grown cells, stained for SOD activity, are shown in Fig. 1. The Rf value for each SOD enzyme is listed adjacent to the appropriate scan. V. parahaemolyticus 27519 appeared to have one major SOD enzyme (Rf = 0.81) and one minor enzyme (Rf= 0.72), whereas the other vibrios appeared to have only one detectable SOD enzyme. E. coli K-12 displayed three SOD enzymes (Rf values of 0.29, 0.42, and 0.54) as previously reported (16). All of the vibrio SOD enzymes migrated at considerably faster rates than the three E. coli SOD enzymes. Induction of SOD. To determine whether increased oxygen tensions might alter the levels of vibrio SOD enzymes in a manner similar to alterations reported for E. coli (13, 16), we comTABLE 1. SOD and CAT levels in several vibrios and E. coli K-12 Level (ug/ml of
V. parahaemolyticus 27519 V. parahaemolyticus 17802 V. parahaemolyticus 1242A V. alginolyticus 7608 Vibrio sp. A8694 Vibrio sp. A1402
12.9 2.1 28.7 0.9 29.2 13.8 2.1 V. cholerae 569B 44.6 30.1 1.5 E. coli K-12 a Cel extracts were prepared from late-log-phase
aerobically grown cells.
VOL. 134, 1978
SOD LEVELS IN HALOPHILIC VIBRIOS DYE
10.81 B 0.85
pared the levels of SOD in log-phase V. parahaemolyticus 27519, Vibrio sp. A8694, and E. coli K-12 cells grown for varying lengths of time under conditions ofstrict anaerobiosis and under vigorous aeration (Table 2), as described in Materials and Methods. V. parahaemolyticus 27519 log-phase cells possessed a low level of SOD activity, 6.8 U/mg of protein, when grown under strict anaerobic conditions (Fig. 2A). Transfer of these cells to conditions of vigorous aeration resulted in a sudden increase in SOD activity to 22.4 U/mg of protein within 1 h and 29.8 U/mg of protein
after 6 h. The anaerobic controls did not show any increase in activity. The SOD activities of Vibrio sp. A8694 grown under anaerobic and aerobic conditions are
shown in Fig. 2B. An increase in SOD activity from 10.2 to 29.2 U/mg of protein occurred within the first hour of aerobic incubations and was maintained at this level, or higher, for 6 h. The anaerobic controls did not show any increase in activity. Figure 2C shows the results of a similar induction experiment with E. coli K-12. A somewhat less rapid overall increase in SOD activity, from 17 U/mg of protein at to to 51.4 U/mg of protein at 6 h, was observed for the aerobically grown cells. In contrast to the vibrios, the anaerobic controls showed an increase in activity from 17 to 36.5 U/mg of protein in 6 h. The low level of SOD activity in the anaerobic TABLE 2. Relative amounts of SOD enzymes present in anaerobically and aerobically grown cells Relative amt of
RI of SOD
SODa AnaerAeroobic cells
V. parahaemolyticus 27519
Vibrio sp. A8694
E. coli K-12 *ao
42 CENTIMETERS FIG. 1. Densitometric scan tracings (absorbance at 550 nm) ofpolyacrylamide disc gels (7.5%) stained for SOD activity. (A) V. parahaemolyticus ATCC 27519; (B) V. parahaemolyticus ATCC 17802; (C) V. parahaemolyticus DJ1242A; (D) V. alginolyticus DJ7608; (E) Vibrio sp. A8694; (F) Vibrio sp. A1402; (G) V. cholerae 569B; (H) E. coli K-12. The top of the gel is represented on the left-side of the tracing, and the marker dye (bromophenol blue) is on the right.
0.54 1.00 1.30 0.42 0 0.44 0.31 0 1.88 a Estimated from the area under the corresponding peaks from the gel scans. The SOD with an Rf of 0.54 in anaerobic E. coli K-12 was given a relative value of 1.0, aU other SOD enzymes were compared with this one for relative activities.
Approximately 150 to 200 pg of cell extract protein applied to each gel, and electrophoresis was carried out at 4 mA/tube. The Rf values of each SOD enzyme are listed adjacent to each peak.
-~ extracts was not due to possible induction of , _ ~~ SOD during cell extract preparation, since 25 iLg of chloramphenicol per ml of culture, representing at least an eightfold increase over the mini_-_ _- o' mal inhibitory concentrations of any of the 20 - /I strains utilized, was added 20 min before removal of all cultures from the anaerobic glove box. In addition, all subsequent manipulations were carried out at ice bath temperatures. Figure 3 shows the scans of SOD electrophoI0 retic patterns of cell extracts prepared in the induction experiments. A comparison of the rel41 ative amounts of each SOD enzyme present in the scans was made by determining the area under each SOD peak, with a relative value of E 4 1.0 assigned to the constitutive SOD enzyme (Rf 6 5 12 3 60 = 0.54) in the anaerobically grown E. coli K-12 cells. Although this method does not consider possible reaction rate differences between SODs of the same or different strains, it is of value for determining which enzymes are induced by aer40 ation. In V. parahaemolyticus 27519, both SOD / " enzymes were present in anaerobically grown 0 cells and at increased levels (2.65-fold higher) in aerobically grown cells, but in the same relative proportions as those of the anaerobic cells. Vi20 brio sp. A8694 cells possessed only one detectable SOD enzyme when grown anaerobically. This enzyme was present at a 1.7-fold-higher level in aerobic cells. In contrast to the vibrios, I I I I I I E. coli K-12 contained a constitutive SOD en4 6 5 3 2 zyme (Rf= 0.54) when grown anaerobically and 60 three SOD enzymes when grown aerobically. The constitutive enzyme, although comprising 0 100% of the anaerobic relative SOD activity, did . not show a significant increase in activity upon transfer to aerobic conditions. The other two 40 enzymes (Rf = 0.42 and 0.31), although undetected in anaerobic cells, comprised 65% of the relative SOD activity in aerobic celLs.
DAILY, DEBELL, AND JOSEPH
TIME (HOURS) FIG. 2. Induction of SOD. Anaerobically grown (3
h) log-ph4ase batch cultures were split into two ali-
quots; onte was transferred from strict anaerobic growth to conditions of maximum aeration, and the other continued to be incubated anaerobically. E. coli K-12 cells were grown in peptone containing 1% glucose. The vibrios were grown in BHI supplemented to contain 1% glucose and 2% NaCI Extracts were prepared and tested for SOD activity at the tunes indicated, with to representing the time of transfer
Superoxide radicals (021 and hydrogen peroxide (H202) are extremely reactive products of aerobic metabolism that can cause cellular damage and are believed to play an important role in the phenomenon of oxygen toxicity (14, 19). SOD and CAT have been postulated to function as enzymes in a survival mechanism and possibly as virulence factors for pathogens (7, 21, 23). To establish whether these enzymes are important for the survival of halophilic vibrios, we from anaerobic to aerobic growth. Symbols: (0) Aerobically grown cels; (@) anaerobicaly grown cells. (A) V. parahaemolyticus 27519; (B) Vibrio sp. A8694; (C) E. coli K-12.
SOD LEVELS IN HALOPHILIC VIBRIOS
VOL. 134, 1978 DYE
FIG. 3. Densitometric scan tracings (absorbance at 550 nm) ofpolyacrylamide disc gels (7.5%) stained for SOD activity. 200 pg of cell extract protein from cells grown for 6 h under anaerobic or aerobic conditions was subjected to electrophoresis at 4 mA/tube until the bromophenol blue marker dye had swept through most of the tube. The gels were stained for SOD and scanned as described in Materials and Methods. (A) V. parahaemolyticus 27519 anaerobic and (B) aerobic; (C) Vibrio sp. A8694 anaerobic and (D) aerobic; (E) E. coli K-12 anaerobic and (F) aerobic.
first determined SOD and CAT levels in latelog-phase, aerobically grown cells. SOD levels in five different species of vibrios were remarkably similar to each other and to that of E. coli K-12.
Only one strain, V. alginolyticus 7608, possessed unusually high SOD levels. Although we have no direct explanation for this phenomenon, we have noted that this strain synthesizes higher amounts of other enzymes, such as ,8-lactamases, than the other vibrios (9). CAT activities were variable, and one strain, 1242A, had unusually low levels. The SOD-toCAT ratio was consistent, with a range of 0.9 to 2.1, for all but two strains. This suggests that the balance of SOD and CAT may be closely regulated in the halophilic vibrios and in E. coli. The two exceptions, strains 1242A and 7608, possessed considerably higher ratios. Electrophoretic characteristics of SOD enzymes have been used successfully by several investigators to determine the numbers of different SOD enzymes present in various cell types (4, 15, 16). Using similar techniques, we have shown that only one SOD enzyme is detectable in most vibrios, with the exception of V. parahaemolyticus 27519, which possesses two distinct SOD enzymes. The numbers of SOD enzymes and their Rf values for aerobically grown halophilic vibrios and E. coli K-12 revealed dissimilarities between the two genera. Three distinct SOD enzymes have been described for E. coli K-12. One enzyme is constitutive in that it is present in similar levels in cells grown in either anaerobic or aerobic environments, whereas the other two enzymes appear only during conditions of aerobic growth (13, 14). In the present study we have examined the halophilic vibrios for the presence of SOD in environments of low and high oxygen tensions. The SOD enzymes of the halophilic vibrios are present in low levels under anaerobic conditions and appear at higher levels in response to exposure to aerobic environments. These observations with halophilic vibrios, in contrast to those for E. coli SOD enzymes, suggest that SOD regulatory differences occur between the two genera. In addition, we have confirmed the existence of a third distinct SOD enzyme recently reported by Hassan and Fridovich (16). In conclusion, SOD activity is present in low levels in anaerobically grown halophilic vibrios and is induced to higher levels in response to increased oxygen tensions. This inductive process may be helpful in allowing vibrios in aerobic tissues to survive long enough to reduce the environment and create foci of infections. Although we have described differences in electrophoretic migration patterns and possibly regulation between SOD enzymes of the halophilic vibrios and those of E. coli, it is likely that SOD functions as a survival enzyme in halophilic vibrios in a manner similar to that reported for E. coli (14, 16, 19).
DAILY, DEBELL, AND JOSEPH
ACKNOWLEDGMENTS This work was supported by the Naval Medical Research and Development Command, Department of the Navy, Research Work Unit no. M0099PNOO1.6020, ZF51524009.0057, and MR0000101.1157. We thank Emilio Weiss for his critical review and discussion of this manuscript, Nathan Murray and Lois Gadd for their expert technical astance, and Adele Buterbaugh and Donita Marconi for typing the manuscript.
J. BACTERIOL. 11. Fernandes, P. B., and H. L Smith, Jr. 1977. The effect of anerobiosis and bile salts on the growth and toxin
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