JOURNAL OF BACTERIOLOGY, May 1992, p. 3386-3391
Vol. 174, No. 10
0021-9193/92/103386-06$02.00/0 Copyright ©) 1992, American Society for Microbiology
Physiological Functions of Hydroperoxidases in Rhodobacter capsulatus AYALA HOCHMAN,1* ANTONIO FIGUEREDO,2 AND JUDY D. WALL3
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel1; Centro de Investigaciones Multidisciplinarias, Universidad Nacional de Asuncion, Asuncion, Paraguay2; and Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 652113 Received 15 July 1991/Accepted 10 March 1992
Metabolic activation of molecular oxygen very often results in the production of hydrogen peroxide, which has been shown to be deleterious to most cellular components (12, 29). The potential damage is especially significant in cells faced with stresses characteristic of special conditions. For example, photosynthetic organisms are challenged by the photodynamic reactions that enhance partial reduction of 02 (12); in nitrogen-free medium, diazotrophs are vulnerable because of the extreme lability to oxygen of the nitrogen fixation system (35); and elevated steady-state concentrations of toxic oxygen products play a significant role in aging of cells and tissues (15, 16, 35). Hydrogen peroxide is metabolized by three different types of hydroperoxidases: catalase, peroxidase, and catalase-peroxidase. The typical catalases, which catalyze the dismutation of H202 to 02 and H20, have been isolated from animals, plants, and microorganisms (7, 10). It is generally accepted that the major physiological role of the typical catalase is protection of the cells against the damaging effect of hydrogen peroxide (7). Peroxidase, which catalyzes oxidation of H202 by a large variety of substrates, also may function in detoxifying hydrogen peroxide as well as in various cellular activities of biosynthesis and degradation (12). The catalase-peroxidases, which were only recently classified as a distinct group of enzymes (19, 33), have been identified in various genera of microorganisms. These include some strains of the following bacteria: Escherichia coli (8, 33), Rhodobacter capsulatus (19), Klebsiella pneumoniae (18), Chromatium vinosum (33), Salmonella typhimunum (27), a Vitreoscilla sp. (1), an alkalophilic Bacillus sp. (43), and a Campylobacter sp. (16a). A catalase-peroxidase was also reported recently in one eukaryotic microorganism, the plant pathogenic fungus Septoria tritici (25). The catalase-peroxidases are unique in that they catalyze both catalase and peroxidase activities at significant rates; most significantly, the enzymes can oxidize the pyridine nucleotides NADH and NADPH (18) and cy-
*
tochrome c (unpublished data). These enzymes share biochemical and physicochemical properties with both catalases and peroxidases. Like the typical catalases they are tetramers, with a combined molecular mass of about 240,000 Da, and they have hydrophobic properties exhibited by their binding to phenyl-Sepharose. Unlike typical catalases and similar to peroxidases, the catalase-peroxidases are inactivated by ethanol-chloroform and are not inhibited by 3-amino-1,2,4-triazole, they have a sharp dependence of their activity on the pH, their apparent half-maximal activity is at the millimolar range, and their heme iron can be reduced by dithionite (18, 19). The unique properties of the catalase-peroxidases raise the question of whether these enzymes were tailored to perform specific physiological functions or whether they are just evolutionary relics of an ancestral hydroperoxidase. The aim of this work was to elucidate the physiological function of the catalase-peroxidase. For this purpose we selected the bacterium R. capsulatus, which synthesizes catalase-peroxidase as the only enzyme with catalase activity. Because R. capsulatus is a photosynthetic, facultatively anaerobic bacterium and is capable of fixing molecular nitrogen, it is most suitable as a test organism for studying oxidative damage and protection mechanisms. We generated a mutant lacking the catalase-peroxidase activity, and we investigated the effect of absence of the enzyme. We studied various functions of the bacterium that are potentially affected by oxygen metabolites: growth rate, viability, nitrogenase activity, pigment synthesis, and mutation rate. We found that the catalase-peroxidase-deficient mutant behaved similarly to the wild type in all these parameters, except that viable counts of its aerobic cultures declined much faster after reaching the stationary phase.
MATERIALS AND METHODS Organisms and growth conditions. R. capsulatus Jl (42) and the mutant AH18 (see below) were grown at 30°C on RCV medium (40) supplemented with 0.002% thiamine hy-
Corresponding author. Electronic mail address: ayala@taunos. 3386
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Rhiodobacter capsulatus Jl has two hydroperoxidases: a catalase-peroxidase and a peroxidase. A mutant strain, AH18, that had no catalase-peroxidase was isolated. The growth rate under aerobic and photosynthetic conditions, respiration, superoxide dismutase and peroxidase activities, and pigment content of the mutant were similar to those of the wild type. AH18 was more susceptible to killing and to inhibition of nitrogenase by H202 but not by molecular oxygen. The incidences of spontaneous mutations were similar in both strains. Viable counts in aerobic but not anaerobic cultures of AH18 started to decline as soon as the cultures reached the stationary phase, and the rate of cell death was much higher in AH18 than in the wild type. It is inferred that the peroxidase provides protection against H202 in log-phase cells and that the catalase-peroxidase provides protection under the oxidative conditions that prevail in aging cultures. This protective function might be related to the dual activity of the latter as a catalase and a peroxidase or to its capacity to oxidize NADH, NADPH, and cytochrome c.
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ments. Assays of enzyme activities. For assaying enzyme activities, the cells were collected by centrifugation at 12,000 x g for 15 min and washed once with 20 mM potassium phosphate (pH 6.8) containing 5 mM EDTA (phosphate buffer). Crude extracts were prepared by passing the cells through a French pressure cell as described previously (19). Catalase activity was measured with an oxygen electrode, and peroxidase was assayed spectrophotometrically as described previously (19). The difference in the pH profiles between the catalase-peroxidase and the peroxidase (see Fig. 2) enabled us to estimate the activities of these two enzymes in the presence of each other. Potassium phosphate (100 mM) adjusted to various pHs was used for the H+ dependence assays, and the pH of the reaction mixture was measured inside the reaction vessel immediately after the assay. Superoxide dismutase activity was estimated by the cy-
FIG. 1. Native gel electrophoresis of crude extracts from R. capsulatus stained for hydroperoxidase activities. Lanes: A and B, Jl stained for catalase and peroxidase activities, respectively; A, 0.7 U of catalase; B, 0.09 U of peroxidase; C, AH18, 0.09 U of peroxidase, stained for this activity. Experimental conditions were as described in Materials and Methods.
tochrome c inhibition method (4), and respiration was measured by monitoring the oxygen consumption of whole cells with a Clark-type oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, Ohio). The effect of oxygen on nitrogenase activity was assayed in vivo by the acetylene reduction method described by Hochman and Burris (17). Other methods. Bacteriochlorophyll and carotenoids were extracted from the cells in an acetone-methanol mixture (7:2, vol/vol), and the concentrations were calculated by using extinction coefficients of 76 mM-' cm-1 at 770 nm and 128 mM cm-1 at 484 nm, respectively (9). Protein was determined by the Coomassie blue method (6) with bovine serum albumin as a standard. For total cell protein measurements, the pelleted cells were boiled for 20 min in 1 N NaOH and the centrifuged supernatant was used for the assay. Electrophoretic separations were performed on 6% (wt/vol) polyacrylamide native gels and stained for catalase and peroxidase activities as described before (18, 19). To determine the incidence of spontaneous mutations, several dilutions of a logarithmic culture were plated on YPS plates containing
puromycin (200 ,ug/ml), streptomycin (250 mg/ml), or erythromycin (50 ,ug/m) and the colony counts were compared with those of plates without antibiotics. RESULTS
Figure 1 shows gel electrophoresis of crude extracts from the wild-type Jl and the mutant AH18 stained for catalase and peroxidase activities. The wild type, Jl, had one band with catalase activity (lane A) and two bands that stained for peroxidase activity (lane B); one of the peroxidase bands, the slower-moving one, coincided with the catalase activity and represented catalase-peroxidase. These findings showed that R. capsulatus has two hydroperoxidases: the catalaseperoxidase described previously (19) and a peroxidase. In crude extracts from mutant AH18, only the peroxidase band could be seen (lane 1C). No catalase activity was detected in the mutant on gels or in enzyme assays in bacterial extracts, showing that the mutation affected only the catalase-peroxidase. Preliminary characterization of the peroxidase showed that it could use both diaminobenzidine and o-dianisidine as electron donors, but, unlike the catalase-peroxidase, neither NADH nor NADPH could serve as a substrate. The pH optimum of the enzyme was 6.9 (Fig. 2). Characterization of
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drochloride or on YPS medium (0.3% [wt/vol] yeast extract, 0.3% [wt/vol] Bacto-Peptone [Difco], 1 mM CaCl2, 1 mM MgSO4). E. coli strains were grown in LC (10 g of BactoTryptone [Difco], 5 g of yeast extract, and 5 g of NaCl per liter) as indicated below. Photosynthetic anaerobic cultures were incubated in screw-cap test tubes filled to the top with medium. Aerobic growth was accomplished in 100-ml cultures in 2-liter Erlenmeyer flasks on a rotary shaker with shaking at 250 rpm. Growth of the bacteria was monitored spectrophotometrically at 660 nm. Generation of a catalase-peroxidase-negative mutant. A mutant lacking catalase-peroxidase was isolated from among kanamycin-resistant colonies resulting from the introduction of Tn5 on a suicide plasmid, pSUP2021 (39). (Although at the time, pRK2013 [14] was mistakenly used as a helper plasmid, this was later shown to be unnecessary for mutagenesis). Anaerobically grown R. capsulatus cells were conjugated with LC-grown E. coli donors on the surface of YPS solidified medium in a ratio of approximately 20:1. Stationary-phase cultures of R. capsulatus were mixed with the E. coli donor, which had been diluted 1:10 from an overnight culture and grown for an additional 2 h at 37°C with slow shaking. Three aliquots (0.04 ml) of the mating mixture were spotted onto the surface of petri plates containing ca. 25 ml of solidified YPS, and the liquid was allowed to soak into the surface. After 5 h of incubation at 30°C, the cells were spread on the plates with 0.2 ml of buffer (10 mM Tris-HCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM NaCl). Three milliliters of the same buffer containing 0.8% (wt/vol) agar was added as an overlayer. As soon as the top layer of molten agar was solidified, 0.5 ml of a mixture of rifampin and kanamycin (final concentrations of 80 and 25 ,ug/ml, respectively) was put under the bottom agar. The plates were incubated anaerobically in the light until colonies appeared. These colonies were replica plated with toothpicks onto two identical RCV plates containing kanamycin and rifampin and incubated under aerobic conditions at 30°C. For the identification of colonies suspected of lacking catalase-peroxidase activity, one plate of each pair was flooded with 1 M H202. Colonies that failed to produce bubbles (02) were selected from the twin plates, grown in liquid medium, and assayed for catalase activity in crude extracts. One of these colonies, designated AH18, which had no detectable activity of the catalase-peroxidase, was restreaked for single-colony purification. Chromosomal DNA was prepared by the minipreparation method (5), digested with EcoRI, and probed with the internal HindIII fragment of Tn5. Southern blotting revealed a single hybridization band of approximately 10 kb (data not shown). AH18 was used in all subsequent experi-
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pH FIG. 2. pH profile of the peroxidase activity in crude extracts from AH18 (S). Cells were grown aerobically and crude extracts were prepared as described in Materials and Methods. The pH profile of the peroxidase activity of the catalase-peroxidase ---) is taken from reference 20 and is given for comparison.
oxygen-related functions in Jl and AH18 (Table 1) showed that catalase activity could be detected only in the wild type, whether grown by respiration or photosynthesis. The respiration rates and superoxide dismutase and peroxidase activities were similar for both strains and were higher in aerobic cultures than in anaerobic cultures. Pigment synthesis was shown to be regulated by oxygen, which causes inhibition of both chlorophyll and carotenoid biosynthesis. The levels of these pigments were similar in the wild type and the catalaseperoxidase-negative mutant grown aerobically in the dark or anaerobically in the light (Table 1). For studying the possible function of the catalase-peroxidase in protecting R. capsulatus against damage by oxygen, we compared the effects of H202 and molecular oxygen on various functions of logarithmic-phase cultures. The mutant AH18 was more susceptible than the wild type to killing by H202 (Fig. 3). For anaerobic cultures, the median lethal doses were 0.1 and 0.34 mM for AH18 and Ji, respectively; for aerobic cultures, the median lethal doses were 1.2 and 57 mM, respectively. However, molecular oxygen did not seem
H202
(mM)
FIG. 3. Effect of H202 on the viability of anaerobic cultures of Jl (U) and AH18 (A) and aerobic cultures of Jl (0) and AH18 (*). Cells were incubated in fresh medium containing 2 ,ug of tetracycline (previously shown to prevent changes in the cellular levels of hydroperoxidases) per ml with the indicated initial concentrations of H202. After 10 min of incubation at 30°C, samples were diluted into saline solution containing 2 ,ug of bovine catalase (to remove residual H202) per ml and plated on YPS plates for viable counts. Controls for the determination of 100% were run by the same methods but without hydrogen peroxide; they contained 1 x 10' to 1.2 x 108 cells per ml. The datum points are the averages of three to four measurements. to have an inhibitory effect on the catalase-peroxidasenegative mutant, since its generation time when grown either aerobically or anaerobically was identical to that of the wild type (Table 1). Since nitrogen fixation is considered to be one of the most oxygen-sensitive biological activities (35), we studied the effect of lack of the catalase-peroxidase on the inhibition of nitrogenase by oxygen. Figure 4 shows a comparison of the inhibition by H202 of nitrogenase activity in vivo in the two strains of R. capsulatus. Fifty percent inhibition of acetylene reduction in the wild type was achieved with 8.9 x 10' M H202 and was lower for the catalase-peroxidase-negative mutant (2.8 x 10-5 M H202).
TABLE 1. 02-related cell activities in wild-type R. capsulatus Jl and the catalase-peroxidase-negative mutant AH18C Enzyme activity
c Growth t Stra
conditions
C
adu
pCratal!dasee
Peroxidasef
Superoxide
peroxidaseedsuae
Ji
Aerobic Anaerobic
210 + 13 19.6 ± 1.4
0.42 0.14
+ t
0.02 0.02
84.4 30.2
AH18
Aerobic Anaerobic
ND ND
0.45 0.16
t t
0.08 0.03
81.6 ± 5.2 27.7 ± 1.9
+ t
4.9 2.5
Superoxide
dismutase 10 mM
+
N3f
Respiration
Chlorophyll
Carotenoid
contentd
Generation time (h)
rateb
content'
69.1 + 3.2 26.5 ± 1.6
3,834
+ 120 1,707 ± 114
33.6 + 2.1 1.7 ± 0.2
19.7 + 1 2.8 ± 0.2
3.0 ± 0.2 3.3 ± 0.4
61.7 26.4
3,854 - 150 1,770 + 210
31.1 t 6 1.8 ± 0.2
18.6 ± 4 2.6 ± 0.3
3.1 t 0.3 3.5 ± 0.4
t t
4 2
a Catalase, peroxidase, and superoxide dismutase activities were assayed in crude extracts; respiration rates and chlorophyll and carotenoid contents were assayed in whole cells. Cultures in the mid-log phase were used. Experimental conditions were as described in Materials and Methods. b Nanomoles of 02 decomposed per minute per milligram of protein. c Micrograms per milligram of protein. d Nanomoles per milligram of protein. e Micromoles of H202 decomposed per minute per milligram of protein. ND, not detected. f Increase in units of optical density per minute per milligram of protein. g Units per milligram of protein.
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mM
FIG. 4. Effect of H202 on acetylene reduction activity in whole cells of R. capsulatus Jl (0) and AH18 (A). Samples (1 ml) of logarithmic cultures of R. capsulatus were incubated photosynthetically in 25-ml bottles filled with 10% (vol/vol) acetylene in argon and the indicated initial concentrations of H202. The rate of ethylene production during a 10-min incubation was determined as the nitrogenase activity. The results are calculated in comparison to controls, which were run under the same conditions without H202 and for which the rate of production was 210 nmol of C2H2 per h per ml. The datum points are the averages of three to four measurements.
However, nitrogenase activity in vivo in both strains was still equally sensitive to 02 inactivation (Fig. 5); 0.75 mM dissolved 02 was required for 50% inhibition. Oxygen has been shown to damage DNA, resulting in mutations (41). To determine the possible participation of catalase-peroxidase in protection of the DNA against damage by oxygen, we studied the effect of oxygen on the mutation rate in R. capsulatus. The incidences of spontaneous mutations leading to resistance to the antibiotics puromycin, streptomycin, and erythromycin were similar in the wild type and the mutant AH18 grown aerobically or anaerobically (data not shown).
0
10
20
30 TIME
40
50 (hours)
60
70
FIG. 6. Effect of aging on survival of R. capsulatus Jl (0) and AH18 (El) in aerobic cultures. Cells were grown in RCV medium. Samples of 100 ml in 2-liter Erlenmeyer flasks were shaken at 250 rpm at 300C. Aliquots were removed at the indicated time points and plated on YPS plates for determination of viable counts.
The results described so far indicate that the enzyme catalase-peroxidase can contribute to the protection of logphase R. capsulatus against H202, but this function is manifest only when hydrogen peroxide concentrations in vivo are higher than those present in cells grown in air (37). There are indications in the literature that aged cells are more vulnerable to oxidative damage, both because their metabolic activities produce more deleterious oxygen products and because they have a lower potential for protection (15, 16, 34). In Fig. 6 the effects of aging on aerobic cultures of Jl and AH18 are compared. The viable counts in the catalase-peroxidase-negative strain started to decline as soon as the culture reached the stationary phase, and the rate of cell death was much higher in AH18 than in the wild type. Cells of Jl and AH18 grown under anaerobic conditions had comparable patterns of loss of viability (data not shown). DISCUSSION
O
1
2
1°2l Jilm
3
4
FIG. 5. Effect of dissolved 02 concentrations on acetylene reduction in whole cells of R. capsulatus AH18 (-). Cells were grown anaerobically in the light with glutamate as the sole nitrogen source. Experimental conditions were as described in Materials and Methods; the control rate was 195 nmol of C2H2 per h per ml. The results for the wild type (--- -) were taken from reference 18.
Physiological and biochemical studies of H202 metabolism in microorganisms have shown that it is catalyzed by multiple enzyme forms that are subject to intricate modes of regulation. Our data show that R. capsulatus synthesizes two enzymes that potentially participate in the protection against H202: a catalase-peroxidase (19) and a peroxidase. In order to assess the specific function of each of these enzymes, we generated a mutant strain, AH18, that was deficient in the catalase-peroxidase activity. AH18 reacted to molecular oxygen in a manner similar to that of the wild type, as reflected by its generation time under aerobic conditions, pigment content, mutation rate, and in vivo nitrogen fixation capacity. Hydrogen peroxide, on the other hand, had a more deleterious effect on the mutant, as shown by a lower viability and inhibition of nitrogen fixation. A relationship between viability in the presence of H202 and the functioning of the catalase-peroxidase was inferred from the finding that aerobic cultures of the wild type, which had 10 times higher activity of the enzyme, were much less sensitive than anaerobic cells to H202. However, the H202 concentrations used to distinguish between the mutant and
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stresses (21). A putative sigma factor, katf, was shown to be a major switch that controls this starvation-mediated response (22, 31). The proteins controlled by katF endowed the cells with increased protection against oxidative stress (21) and include the catalase HPII. It is concluded that starved bacteria are subjected to oxidative stress and that H202-scavenging activity is crucial for survival of the cells. In E. coli this activity is furnished by HPII, whereas in R. capsulatus it is furnished by the catalase-peroxidase, which is its only enzyme with catalase activity. Furthermore, the catalase-peroxidase can also function via its peroxidatic activity by catalyzing biosynthesis or degradation activities (11), which are part of the cellular adaptation to starvation conditions, or by oxidizing NADH, NADPH (18), or cytochrome c, thereby lowering the cell's reducing activity, which might be responsible for the generation of H202. ACKNOWLEDGMENTS This research was partially supported by grant 1-105-260.3/88 from the German-Israeli Foundation for Scientific Research and Development to Ayala Hochman and by Department of Energy grant DE-FG02-87ER13713 and the Missouri Soybean Merchandising Council grant 040 to Judy D. Wall. We thank Tara Wickman for the Southern analysis. REFERENCES 1. Abrams, J. J., and D. A. Webster. 1990. Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla. Arch. Biochem. Biophys. 279:54-59. 2. Abril, N., and C. Pueyo. 1990. Mutagenesis in Escherichia coli lacking catalase. Environ. Mol. Mutagen. 15:184-189. 3. Alonso-Moraga, A., A. Bocanegra, J. M. Torres, J. Lopez-Barea, and C. Pueyo. 1987. Glutathione status and sensitivity to GSHreacting compounds of Escherichia coli strain deficient in glutathione metabolism and/or catalase activity. Mol. Cell. Biochem. 73:61-68. 4. Asada, K., M. Takahashi, and M. Nagate. 1974. Assay and inhibition of spinach superoxide dismutase. Agric. Biol. Chem. 38:471-473. 5. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 6. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 7. Chance, B., H. Sies, and A. Boveris. 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527-605. 8. Claiborne, A., and I. Fridovich. 1979. Purification of o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities. J. Biol. Chem. 254:4245-4252. 9. Clayton, R. K. 1966. Spectroscopic analysis of bacteriochlorophylls in vitro and in vivo. Photochem. Photobiol. 5:669-688. 10. Deisseroth, A., and A. L. Dounce. 1970. Catalase: physical and chemical properties, mechanism of catalysis, and physiological role. Physiol. Rev. 50:319-374. 11. Dunford, H. B., and J. S. Stillman. 1976. On the function and mechanism of action of peroxidases. Coord. Chem. Rev. 19: 187-251. 12. Elstner, E. F. 1982. Oxygen activation and oxygen toxicity. Annu. Rev. Plant Physiol. 33:73-96. 13. Farr, S. B., D. Touati, and T. Kogoma. 1988. Effects of oxygen stress on membrane functions in Escherichia coli: role of HPI catalase. J. Bacteriol. 170:1837-1842. 14. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76:1648-1652. 15. Harman, D. 1956. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11:298-300.
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the wild type were significantly higher than those formed intracellularly in actively growing cells (estimated in E. coli to be below 10-8 M [37]). As the following examples show, studies of the physiological functions of other bacterial catalases also reveal that they do not have a crucial function in actively growing aerobic cultures, unless the cells are exposed to externally added hydrogen peroxide. Similar to catalase-deficient mutants of R. capsulatus, catalase-deficient mutants of S. typhimurium grew under aerobic conditions as well as the wild type, but they had a slightly longer lag period (24). Since growth was initiated by diluting stationary-phase cultures and was monitored spectrophotometrically, the longer lag period of the mutants might simply reflect lower numbers of viable cells in the inoculum, which in turn indicate enhanced stationary-phase death of the mutants, similar to the case of AH18. E. coli strains that had no detectable catalase activity were shown to be comparable to the wild type with respect to growth rate and extent of growth (26), but they were more sensitive to lethal effects of hydrogen peroxide (2, 32). The increase in glutathione oxidation in a catalase-deficient E. coli mutant (3) indicated that, in the absence of catalase, the cells could switch to utilizing glutathione as an antioxidant. Unlike the R. capsulatus mutants, the E. coli null mutants appeared to have an increased spontaneous mutation rate (26). In a different study, however, the increased mutation rate in a superoxide dismutase-negative strain was not exacerbated in a hydroperoxidase-negative background (36). In contrast to the results with R. capsulatus, studies in E. coli showed that there was no difference between catalase-deficient mutants and the parental strain in the loss of viability during storage at room temperature for periods up to 2 weeks (26). However, a critical comparison cannot be made, because there was no description of the method used for viability determination and quantitation. There are only a few reports in the literature that suggest a distinct function for catalase-peroxidases. E. coli synthesizes two different enzymes with catalase activity, a catalase-peroxidase, designated HPI (8), and an atypical catalase, HPII (28), which are regulated differently. The level of HPI increases in response to external H202, and HPII has a 10- to 20-fold-higher activity in the stationary phase (26). It was suggested that HPII functions in nongrowing cells, whereas HPI has a general antioxidant role in log-phase cells, with a specific effect on membrane structure and function (13, 23). The aerobic bacterium Vitreoscilla sp. has one enzyme with catalase activity, characterized as a catalase-peroxidase, and it was suggested that its specific role was to scavenge the peroxide generated by its bacterial hemoglobin (1). The only significant difference found in our studies between the wild-type R. capsulatus and the catalase-peroxidase-deficient mutant was the enhanced death of the mutant cells after reaching the stationary phase under aerobic conditions. We infer from the similarity in growth rates that the catalase-peroxidase has a minor function in log-phase cells. Therefore, in actively growing R. capsulatus cells it is possible that the main protection against oxidative damage is conferred by the peroxidase. Alternatively, the damage repair systems may be efficient enough to prevent manifestation of oxidative stress in the log phase until the culture reaches the stationary phase, when the catalase-peroxidase has a crucial function. Stationary-phase bacteria are starved or stressed cultures. Starved bacteria undergo various changes that are crucial for their survival (20, 30). In E. coli it includes increased expression of over 40 proteins (38), some of which confer on the bacterium resistance to other
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30. Matin, A., E. A. Auger, P. H. Blum, and J. E. Schultz. 1989. Genetic basis of starvation survival in nondifferentiating bacteria. Annu. Rev. Microbiol. 43:293-316. 31. McCann, M. P., J. P. Kidwell, and A. Matin. 1991. The putative sigma factor katF has a central role in development of starvation-mediated general resistance in Escherichia coli. J. Bacteriol. 173:4188-4194. 32. Meir, E., and E. Yagil. 1984. Catalase-negative mutants of Escherichia coli. Curr. Microbiol. 11:13-18. 33. Nadler, V., I. Goldberg, and A. Hochman. 1986. Comparative study of bacterial catalases. Biochim. Biophys. Acta 882:234241. 34. Rao, G., E. Xia, and A. Richerdson. 1990. Effect of age on the expression of antioxidant enzymes in male Fischer F344 rats. Mech. Ageing Dev. 53:49-60. 35. Robson, R. L., and J. R. Postgate. 1980. Oxygen and hydrogen in biological nitrogen fixation. Annu. Rev. Microbiol. 34:183207. 36. Schellhorn, H. E., and H. M. Hassan. 1988. Response of hydroperoxidase and superoxide dismutase deficient mutants of Escherichia coli K-12 to oxidative stress. Can. J. Microbiol. 34:1171-1176. 37. Schellhorn, H. E., S. Pou, C. Moody, and H. M. Hassan. 1989. An electron spin resonance study of oxyradical generation in superoxide dismutase- and catalase-deficient mutants of Escherichia coli K-12. Arch. Biochem. Biophys. 271:323-331. 38. Schultz, J. E., and A. Matin. 1991. Molecular and functional characterization of a carbon starvation gene of Escherichia coli. J. Mol. Biol. 218:129-140. 39. Simon, R., U. Pfiefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1:784791. 40. Takakuwa, S., J. M. Odom, and J. D. Wall. 1983. Hydrogen uptake deficient mutants of Rhodopseudomonas capsulata. Arch. Microbiol. 136:20-25. 41. Vuillaume, M. 1987. Reduced oxygen species, mutation, induction and cancer initiation. Mutat. Res. 186:43-72. 42. Wall, J. D., and K. Braddock. 1984. Mapping of Rhodopseudomonas capsulata nif genes. J. Bacteriol. 158:404-410. 43. Yumoto, I., Y. Fukumori, and T. Yamanaka. 1990. Purification and characterization of catalase from the facultative alkalophilic Bacillus. J. Biochem. 108:583-587.
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16. Harman, D. 1981. The aging process. Proc. Natl. Acad. Sci. USA 78:7124-7128. 16a.Hochman, A. Unpublished data. 17. Hochman, A., and R. H. Bums. 1981. Effect of oxygen on acetylene reduction by photosynthetic bacteria. J. Bacteriol. 147:492-499. 18. Hochman, A., and I. Goldberg. 1991. Purification and characterization of a catalase-peroxidase and a typical catalase from the bacterium Klebsiella pneumoniae. Biochim. Biophys. Acta 1077:299-307. 19. Hochman, A., and A. Shemesh. 1987. Purification and characterization of a catalase-peroxidase from the photosynthetic bacterium Rhodopseudomonas capsulata. J. Biol. Chem. 262: 6871-6876. 20. Hood, M. A., J. B. Guckert, D. C. White, and F. Deck. 1986. Effect of nutrient deprivation on lipid, carbohydrate, DNA, RNA, and protein levels in Vibno cholerae. Appl. Environ. Microbiol. 52:788-793. 21. Jenkins, D. E., J. E. Schultz, and A. Matin. 1988. Starvationinduced cross protection against heat or H202 challenge in Escherichia coli. J. Bacteriol. 170:3910-3914. 22. Lange, R., and R. Henge-Aronis. 1991. Identification of a central regulator of stationary-phase gene expression in Escherichia coli. Mol. Microbiol. 5:49-59. 23. Leven, S., A. Heimberger, and A. Eisenstark. 1990. Catalase HPI influences membrane permeability in Escherichia coli following near-UV stress. Biochem. Biophys. Res. Commun. 171:1224-1228. 24. Levine, S. A. 1977. Isolation and characterization of catalase deficient mutants of Salmonella typhimurium. Mol. Gen. Genet. 150:205-209. 25. Levy, E., Z. Eyal, and A. Hochman. Arch. Biochem. Biophys., in press. 26. Loewen, P. C. 1984. Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity. J. Bacteriol. 157:622-626. 27. Loewen, P. C., and G. V. Stauffer. 1990. Nucleotide sequence of katG of Salmonella typhimurium LT2 and characterization of its product, hydroperoxidase I. Mol. Gen. Genet. 224:147-151. 28. Loewen, P. C., and J. Switala. 1986. Purification and characterization of catalase HPII from Escherichia coli K12. Biochem. Cell Biol. 64:638-646. 29. Malmstrom, B. G. 1982. Enzymology of oxygen. Annu. Rev. Biochem. 51:21-59.
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0021-9193/92/103386-06$02.00/0 Copyright ©) 1992, American Society for Microbiology
Physiological Functions of Hydroperoxidases in Rhodobacter capsulatus AYALA HOCHMAN,1* ANTONIO FIGUEREDO,2 AND JUDY D. WALL3
Department of Biochemistry, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel1; Centro de Investigaciones Multidisciplinarias, Universidad Nacional de Asuncion, Asuncion, Paraguay2; and Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 652113 Received 15 July 1991/Accepted 10 March 1992
Metabolic activation of molecular oxygen very often results in the production of hydrogen peroxide, which has been shown to be deleterious to most cellular components (12, 29). The potential damage is especially significant in cells faced with stresses characteristic of special conditions. For example, photosynthetic organisms are challenged by the photodynamic reactions that enhance partial reduction of 02 (12); in nitrogen-free medium, diazotrophs are vulnerable because of the extreme lability to oxygen of the nitrogen fixation system (35); and elevated steady-state concentrations of toxic oxygen products play a significant role in aging of cells and tissues (15, 16, 35). Hydrogen peroxide is metabolized by three different types of hydroperoxidases: catalase, peroxidase, and catalase-peroxidase. The typical catalases, which catalyze the dismutation of H202 to 02 and H20, have been isolated from animals, plants, and microorganisms (7, 10). It is generally accepted that the major physiological role of the typical catalase is protection of the cells against the damaging effect of hydrogen peroxide (7). Peroxidase, which catalyzes oxidation of H202 by a large variety of substrates, also may function in detoxifying hydrogen peroxide as well as in various cellular activities of biosynthesis and degradation (12). The catalase-peroxidases, which were only recently classified as a distinct group of enzymes (19, 33), have been identified in various genera of microorganisms. These include some strains of the following bacteria: Escherichia coli (8, 33), Rhodobacter capsulatus (19), Klebsiella pneumoniae (18), Chromatium vinosum (33), Salmonella typhimunum (27), a Vitreoscilla sp. (1), an alkalophilic Bacillus sp. (43), and a Campylobacter sp. (16a). A catalase-peroxidase was also reported recently in one eukaryotic microorganism, the plant pathogenic fungus Septoria tritici (25). The catalase-peroxidases are unique in that they catalyze both catalase and peroxidase activities at significant rates; most significantly, the enzymes can oxidize the pyridine nucleotides NADH and NADPH (18) and cy-
*
tochrome c (unpublished data). These enzymes share biochemical and physicochemical properties with both catalases and peroxidases. Like the typical catalases they are tetramers, with a combined molecular mass of about 240,000 Da, and they have hydrophobic properties exhibited by their binding to phenyl-Sepharose. Unlike typical catalases and similar to peroxidases, the catalase-peroxidases are inactivated by ethanol-chloroform and are not inhibited by 3-amino-1,2,4-triazole, they have a sharp dependence of their activity on the pH, their apparent half-maximal activity is at the millimolar range, and their heme iron can be reduced by dithionite (18, 19). The unique properties of the catalase-peroxidases raise the question of whether these enzymes were tailored to perform specific physiological functions or whether they are just evolutionary relics of an ancestral hydroperoxidase. The aim of this work was to elucidate the physiological function of the catalase-peroxidase. For this purpose we selected the bacterium R. capsulatus, which synthesizes catalase-peroxidase as the only enzyme with catalase activity. Because R. capsulatus is a photosynthetic, facultatively anaerobic bacterium and is capable of fixing molecular nitrogen, it is most suitable as a test organism for studying oxidative damage and protection mechanisms. We generated a mutant lacking the catalase-peroxidase activity, and we investigated the effect of absence of the enzyme. We studied various functions of the bacterium that are potentially affected by oxygen metabolites: growth rate, viability, nitrogenase activity, pigment synthesis, and mutation rate. We found that the catalase-peroxidase-deficient mutant behaved similarly to the wild type in all these parameters, except that viable counts of its aerobic cultures declined much faster after reaching the stationary phase.
MATERIALS AND METHODS Organisms and growth conditions. R. capsulatus Jl (42) and the mutant AH18 (see below) were grown at 30°C on RCV medium (40) supplemented with 0.002% thiamine hy-
Corresponding author. Electronic mail address: ayala@taunos. 3386
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Rhiodobacter capsulatus Jl has two hydroperoxidases: a catalase-peroxidase and a peroxidase. A mutant strain, AH18, that had no catalase-peroxidase was isolated. The growth rate under aerobic and photosynthetic conditions, respiration, superoxide dismutase and peroxidase activities, and pigment content of the mutant were similar to those of the wild type. AH18 was more susceptible to killing and to inhibition of nitrogenase by H202 but not by molecular oxygen. The incidences of spontaneous mutations were similar in both strains. Viable counts in aerobic but not anaerobic cultures of AH18 started to decline as soon as the cultures reached the stationary phase, and the rate of cell death was much higher in AH18 than in the wild type. It is inferred that the peroxidase provides protection against H202 in log-phase cells and that the catalase-peroxidase provides protection under the oxidative conditions that prevail in aging cultures. This protective function might be related to the dual activity of the latter as a catalase and a peroxidase or to its capacity to oxidize NADH, NADPH, and cytochrome c.
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ments. Assays of enzyme activities. For assaying enzyme activities, the cells were collected by centrifugation at 12,000 x g for 15 min and washed once with 20 mM potassium phosphate (pH 6.8) containing 5 mM EDTA (phosphate buffer). Crude extracts were prepared by passing the cells through a French pressure cell as described previously (19). Catalase activity was measured with an oxygen electrode, and peroxidase was assayed spectrophotometrically as described previously (19). The difference in the pH profiles between the catalase-peroxidase and the peroxidase (see Fig. 2) enabled us to estimate the activities of these two enzymes in the presence of each other. Potassium phosphate (100 mM) adjusted to various pHs was used for the H+ dependence assays, and the pH of the reaction mixture was measured inside the reaction vessel immediately after the assay. Superoxide dismutase activity was estimated by the cy-
FIG. 1. Native gel electrophoresis of crude extracts from R. capsulatus stained for hydroperoxidase activities. Lanes: A and B, Jl stained for catalase and peroxidase activities, respectively; A, 0.7 U of catalase; B, 0.09 U of peroxidase; C, AH18, 0.09 U of peroxidase, stained for this activity. Experimental conditions were as described in Materials and Methods.
tochrome c inhibition method (4), and respiration was measured by monitoring the oxygen consumption of whole cells with a Clark-type oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, Ohio). The effect of oxygen on nitrogenase activity was assayed in vivo by the acetylene reduction method described by Hochman and Burris (17). Other methods. Bacteriochlorophyll and carotenoids were extracted from the cells in an acetone-methanol mixture (7:2, vol/vol), and the concentrations were calculated by using extinction coefficients of 76 mM-' cm-1 at 770 nm and 128 mM cm-1 at 484 nm, respectively (9). Protein was determined by the Coomassie blue method (6) with bovine serum albumin as a standard. For total cell protein measurements, the pelleted cells were boiled for 20 min in 1 N NaOH and the centrifuged supernatant was used for the assay. Electrophoretic separations were performed on 6% (wt/vol) polyacrylamide native gels and stained for catalase and peroxidase activities as described before (18, 19). To determine the incidence of spontaneous mutations, several dilutions of a logarithmic culture were plated on YPS plates containing
puromycin (200 ,ug/ml), streptomycin (250 mg/ml), or erythromycin (50 ,ug/m) and the colony counts were compared with those of plates without antibiotics. RESULTS
Figure 1 shows gel electrophoresis of crude extracts from the wild-type Jl and the mutant AH18 stained for catalase and peroxidase activities. The wild type, Jl, had one band with catalase activity (lane A) and two bands that stained for peroxidase activity (lane B); one of the peroxidase bands, the slower-moving one, coincided with the catalase activity and represented catalase-peroxidase. These findings showed that R. capsulatus has two hydroperoxidases: the catalaseperoxidase described previously (19) and a peroxidase. In crude extracts from mutant AH18, only the peroxidase band could be seen (lane 1C). No catalase activity was detected in the mutant on gels or in enzyme assays in bacterial extracts, showing that the mutation affected only the catalase-peroxidase. Preliminary characterization of the peroxidase showed that it could use both diaminobenzidine and o-dianisidine as electron donors, but, unlike the catalase-peroxidase, neither NADH nor NADPH could serve as a substrate. The pH optimum of the enzyme was 6.9 (Fig. 2). Characterization of
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drochloride or on YPS medium (0.3% [wt/vol] yeast extract, 0.3% [wt/vol] Bacto-Peptone [Difco], 1 mM CaCl2, 1 mM MgSO4). E. coli strains were grown in LC (10 g of BactoTryptone [Difco], 5 g of yeast extract, and 5 g of NaCl per liter) as indicated below. Photosynthetic anaerobic cultures were incubated in screw-cap test tubes filled to the top with medium. Aerobic growth was accomplished in 100-ml cultures in 2-liter Erlenmeyer flasks on a rotary shaker with shaking at 250 rpm. Growth of the bacteria was monitored spectrophotometrically at 660 nm. Generation of a catalase-peroxidase-negative mutant. A mutant lacking catalase-peroxidase was isolated from among kanamycin-resistant colonies resulting from the introduction of Tn5 on a suicide plasmid, pSUP2021 (39). (Although at the time, pRK2013 [14] was mistakenly used as a helper plasmid, this was later shown to be unnecessary for mutagenesis). Anaerobically grown R. capsulatus cells were conjugated with LC-grown E. coli donors on the surface of YPS solidified medium in a ratio of approximately 20:1. Stationary-phase cultures of R. capsulatus were mixed with the E. coli donor, which had been diluted 1:10 from an overnight culture and grown for an additional 2 h at 37°C with slow shaking. Three aliquots (0.04 ml) of the mating mixture were spotted onto the surface of petri plates containing ca. 25 ml of solidified YPS, and the liquid was allowed to soak into the surface. After 5 h of incubation at 30°C, the cells were spread on the plates with 0.2 ml of buffer (10 mM Tris-HCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM NaCl). Three milliliters of the same buffer containing 0.8% (wt/vol) agar was added as an overlayer. As soon as the top layer of molten agar was solidified, 0.5 ml of a mixture of rifampin and kanamycin (final concentrations of 80 and 25 ,ug/ml, respectively) was put under the bottom agar. The plates were incubated anaerobically in the light until colonies appeared. These colonies were replica plated with toothpicks onto two identical RCV plates containing kanamycin and rifampin and incubated under aerobic conditions at 30°C. For the identification of colonies suspected of lacking catalase-peroxidase activity, one plate of each pair was flooded with 1 M H202. Colonies that failed to produce bubbles (02) were selected from the twin plates, grown in liquid medium, and assayed for catalase activity in crude extracts. One of these colonies, designated AH18, which had no detectable activity of the catalase-peroxidase, was restreaked for single-colony purification. Chromosomal DNA was prepared by the minipreparation method (5), digested with EcoRI, and probed with the internal HindIII fragment of Tn5. Southern blotting revealed a single hybridization band of approximately 10 kb (data not shown). AH18 was used in all subsequent experi-
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pH FIG. 2. pH profile of the peroxidase activity in crude extracts from AH18 (S). Cells were grown aerobically and crude extracts were prepared as described in Materials and Methods. The pH profile of the peroxidase activity of the catalase-peroxidase ---) is taken from reference 20 and is given for comparison.
oxygen-related functions in Jl and AH18 (Table 1) showed that catalase activity could be detected only in the wild type, whether grown by respiration or photosynthesis. The respiration rates and superoxide dismutase and peroxidase activities were similar for both strains and were higher in aerobic cultures than in anaerobic cultures. Pigment synthesis was shown to be regulated by oxygen, which causes inhibition of both chlorophyll and carotenoid biosynthesis. The levels of these pigments were similar in the wild type and the catalaseperoxidase-negative mutant grown aerobically in the dark or anaerobically in the light (Table 1). For studying the possible function of the catalase-peroxidase in protecting R. capsulatus against damage by oxygen, we compared the effects of H202 and molecular oxygen on various functions of logarithmic-phase cultures. The mutant AH18 was more susceptible than the wild type to killing by H202 (Fig. 3). For anaerobic cultures, the median lethal doses were 0.1 and 0.34 mM for AH18 and Ji, respectively; for aerobic cultures, the median lethal doses were 1.2 and 57 mM, respectively. However, molecular oxygen did not seem
H202
(mM)
FIG. 3. Effect of H202 on the viability of anaerobic cultures of Jl (U) and AH18 (A) and aerobic cultures of Jl (0) and AH18 (*). Cells were incubated in fresh medium containing 2 ,ug of tetracycline (previously shown to prevent changes in the cellular levels of hydroperoxidases) per ml with the indicated initial concentrations of H202. After 10 min of incubation at 30°C, samples were diluted into saline solution containing 2 ,ug of bovine catalase (to remove residual H202) per ml and plated on YPS plates for viable counts. Controls for the determination of 100% were run by the same methods but without hydrogen peroxide; they contained 1 x 10' to 1.2 x 108 cells per ml. The datum points are the averages of three to four measurements. to have an inhibitory effect on the catalase-peroxidasenegative mutant, since its generation time when grown either aerobically or anaerobically was identical to that of the wild type (Table 1). Since nitrogen fixation is considered to be one of the most oxygen-sensitive biological activities (35), we studied the effect of lack of the catalase-peroxidase on the inhibition of nitrogenase by oxygen. Figure 4 shows a comparison of the inhibition by H202 of nitrogenase activity in vivo in the two strains of R. capsulatus. Fifty percent inhibition of acetylene reduction in the wild type was achieved with 8.9 x 10' M H202 and was lower for the catalase-peroxidase-negative mutant (2.8 x 10-5 M H202).
TABLE 1. 02-related cell activities in wild-type R. capsulatus Jl and the catalase-peroxidase-negative mutant AH18C Enzyme activity
c Growth t Stra
conditions
C
adu
pCratal!dasee
Peroxidasef
Superoxide
peroxidaseedsuae
Ji
Aerobic Anaerobic
210 + 13 19.6 ± 1.4
0.42 0.14
+ t
0.02 0.02
84.4 30.2
AH18
Aerobic Anaerobic
ND ND
0.45 0.16
t t
0.08 0.03
81.6 ± 5.2 27.7 ± 1.9
+ t
4.9 2.5
Superoxide
dismutase 10 mM
+
N3f
Respiration
Chlorophyll
Carotenoid
contentd
Generation time (h)
rateb
content'
69.1 + 3.2 26.5 ± 1.6
3,834
+ 120 1,707 ± 114
33.6 + 2.1 1.7 ± 0.2
19.7 + 1 2.8 ± 0.2
3.0 ± 0.2 3.3 ± 0.4
61.7 26.4
3,854 - 150 1,770 + 210
31.1 t 6 1.8 ± 0.2
18.6 ± 4 2.6 ± 0.3
3.1 t 0.3 3.5 ± 0.4
t t
4 2
a Catalase, peroxidase, and superoxide dismutase activities were assayed in crude extracts; respiration rates and chlorophyll and carotenoid contents were assayed in whole cells. Cultures in the mid-log phase were used. Experimental conditions were as described in Materials and Methods. b Nanomoles of 02 decomposed per minute per milligram of protein. c Micrograms per milligram of protein. d Nanomoles per milligram of protein. e Micromoles of H202 decomposed per minute per milligram of protein. ND, not detected. f Increase in units of optical density per minute per milligram of protein. g Units per milligram of protein.
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cc C6
LU
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mM
FIG. 4. Effect of H202 on acetylene reduction activity in whole cells of R. capsulatus Jl (0) and AH18 (A). Samples (1 ml) of logarithmic cultures of R. capsulatus were incubated photosynthetically in 25-ml bottles filled with 10% (vol/vol) acetylene in argon and the indicated initial concentrations of H202. The rate of ethylene production during a 10-min incubation was determined as the nitrogenase activity. The results are calculated in comparison to controls, which were run under the same conditions without H202 and for which the rate of production was 210 nmol of C2H2 per h per ml. The datum points are the averages of three to four measurements.
However, nitrogenase activity in vivo in both strains was still equally sensitive to 02 inactivation (Fig. 5); 0.75 mM dissolved 02 was required for 50% inhibition. Oxygen has been shown to damage DNA, resulting in mutations (41). To determine the possible participation of catalase-peroxidase in protection of the DNA against damage by oxygen, we studied the effect of oxygen on the mutation rate in R. capsulatus. The incidences of spontaneous mutations leading to resistance to the antibiotics puromycin, streptomycin, and erythromycin were similar in the wild type and the mutant AH18 grown aerobically or anaerobically (data not shown).
0
10
20
30 TIME
40
50 (hours)
60
70
FIG. 6. Effect of aging on survival of R. capsulatus Jl (0) and AH18 (El) in aerobic cultures. Cells were grown in RCV medium. Samples of 100 ml in 2-liter Erlenmeyer flasks were shaken at 250 rpm at 300C. Aliquots were removed at the indicated time points and plated on YPS plates for determination of viable counts.
The results described so far indicate that the enzyme catalase-peroxidase can contribute to the protection of logphase R. capsulatus against H202, but this function is manifest only when hydrogen peroxide concentrations in vivo are higher than those present in cells grown in air (37). There are indications in the literature that aged cells are more vulnerable to oxidative damage, both because their metabolic activities produce more deleterious oxygen products and because they have a lower potential for protection (15, 16, 34). In Fig. 6 the effects of aging on aerobic cultures of Jl and AH18 are compared. The viable counts in the catalase-peroxidase-negative strain started to decline as soon as the culture reached the stationary phase, and the rate of cell death was much higher in AH18 than in the wild type. Cells of Jl and AH18 grown under anaerobic conditions had comparable patterns of loss of viability (data not shown). DISCUSSION
O
1
2
1°2l Jilm
3
4
FIG. 5. Effect of dissolved 02 concentrations on acetylene reduction in whole cells of R. capsulatus AH18 (-). Cells were grown anaerobically in the light with glutamate as the sole nitrogen source. Experimental conditions were as described in Materials and Methods; the control rate was 195 nmol of C2H2 per h per ml. The results for the wild type (--- -) were taken from reference 18.
Physiological and biochemical studies of H202 metabolism in microorganisms have shown that it is catalyzed by multiple enzyme forms that are subject to intricate modes of regulation. Our data show that R. capsulatus synthesizes two enzymes that potentially participate in the protection against H202: a catalase-peroxidase (19) and a peroxidase. In order to assess the specific function of each of these enzymes, we generated a mutant strain, AH18, that was deficient in the catalase-peroxidase activity. AH18 reacted to molecular oxygen in a manner similar to that of the wild type, as reflected by its generation time under aerobic conditions, pigment content, mutation rate, and in vivo nitrogen fixation capacity. Hydrogen peroxide, on the other hand, had a more deleterious effect on the mutant, as shown by a lower viability and inhibition of nitrogen fixation. A relationship between viability in the presence of H202 and the functioning of the catalase-peroxidase was inferred from the finding that aerobic cultures of the wild type, which had 10 times higher activity of the enzyme, were much less sensitive than anaerobic cells to H202. However, the H202 concentrations used to distinguish between the mutant and
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stresses (21). A putative sigma factor, katf, was shown to be a major switch that controls this starvation-mediated response (22, 31). The proteins controlled by katF endowed the cells with increased protection against oxidative stress (21) and include the catalase HPII. It is concluded that starved bacteria are subjected to oxidative stress and that H202-scavenging activity is crucial for survival of the cells. In E. coli this activity is furnished by HPII, whereas in R. capsulatus it is furnished by the catalase-peroxidase, which is its only enzyme with catalase activity. Furthermore, the catalase-peroxidase can also function via its peroxidatic activity by catalyzing biosynthesis or degradation activities (11), which are part of the cellular adaptation to starvation conditions, or by oxidizing NADH, NADPH (18), or cytochrome c, thereby lowering the cell's reducing activity, which might be responsible for the generation of H202. ACKNOWLEDGMENTS This research was partially supported by grant 1-105-260.3/88 from the German-Israeli Foundation for Scientific Research and Development to Ayala Hochman and by Department of Energy grant DE-FG02-87ER13713 and the Missouri Soybean Merchandising Council grant 040 to Judy D. Wall. We thank Tara Wickman for the Southern analysis. REFERENCES 1. Abrams, J. J., and D. A. Webster. 1990. Purification, partial characterization, and possible role of catalase in the bacterium Vitreoscilla. Arch. Biochem. Biophys. 279:54-59. 2. Abril, N., and C. Pueyo. 1990. Mutagenesis in Escherichia coli lacking catalase. Environ. Mol. Mutagen. 15:184-189. 3. Alonso-Moraga, A., A. Bocanegra, J. M. Torres, J. Lopez-Barea, and C. Pueyo. 1987. Glutathione status and sensitivity to GSHreacting compounds of Escherichia coli strain deficient in glutathione metabolism and/or catalase activity. Mol. Cell. Biochem. 73:61-68. 4. Asada, K., M. Takahashi, and M. Nagate. 1974. Assay and inhibition of spinach superoxide dismutase. Agric. Biol. Chem. 38:471-473. 5. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1989. Current protocols in molecular biology. John Wiley & Sons, Inc., New York. 6. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. 7. Chance, B., H. Sies, and A. Boveris. 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527-605. 8. Claiborne, A., and I. Fridovich. 1979. Purification of o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities. J. Biol. Chem. 254:4245-4252. 9. Clayton, R. K. 1966. Spectroscopic analysis of bacteriochlorophylls in vitro and in vivo. Photochem. Photobiol. 5:669-688. 10. Deisseroth, A., and A. L. Dounce. 1970. Catalase: physical and chemical properties, mechanism of catalysis, and physiological role. Physiol. Rev. 50:319-374. 11. Dunford, H. B., and J. S. Stillman. 1976. On the function and mechanism of action of peroxidases. Coord. Chem. Rev. 19: 187-251. 12. Elstner, E. F. 1982. Oxygen activation and oxygen toxicity. Annu. Rev. Plant Physiol. 33:73-96. 13. Farr, S. B., D. Touati, and T. Kogoma. 1988. Effects of oxygen stress on membrane functions in Escherichia coli: role of HPI catalase. J. Bacteriol. 170:1837-1842. 14. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76:1648-1652. 15. Harman, D. 1956. Aging: a theory based on free radical and radiation chemistry. J. Gerontol. 11:298-300.
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the wild type were significantly higher than those formed intracellularly in actively growing cells (estimated in E. coli to be below 10-8 M [37]). As the following examples show, studies of the physiological functions of other bacterial catalases also reveal that they do not have a crucial function in actively growing aerobic cultures, unless the cells are exposed to externally added hydrogen peroxide. Similar to catalase-deficient mutants of R. capsulatus, catalase-deficient mutants of S. typhimurium grew under aerobic conditions as well as the wild type, but they had a slightly longer lag period (24). Since growth was initiated by diluting stationary-phase cultures and was monitored spectrophotometrically, the longer lag period of the mutants might simply reflect lower numbers of viable cells in the inoculum, which in turn indicate enhanced stationary-phase death of the mutants, similar to the case of AH18. E. coli strains that had no detectable catalase activity were shown to be comparable to the wild type with respect to growth rate and extent of growth (26), but they were more sensitive to lethal effects of hydrogen peroxide (2, 32). The increase in glutathione oxidation in a catalase-deficient E. coli mutant (3) indicated that, in the absence of catalase, the cells could switch to utilizing glutathione as an antioxidant. Unlike the R. capsulatus mutants, the E. coli null mutants appeared to have an increased spontaneous mutation rate (26). In a different study, however, the increased mutation rate in a superoxide dismutase-negative strain was not exacerbated in a hydroperoxidase-negative background (36). In contrast to the results with R. capsulatus, studies in E. coli showed that there was no difference between catalase-deficient mutants and the parental strain in the loss of viability during storage at room temperature for periods up to 2 weeks (26). However, a critical comparison cannot be made, because there was no description of the method used for viability determination and quantitation. There are only a few reports in the literature that suggest a distinct function for catalase-peroxidases. E. coli synthesizes two different enzymes with catalase activity, a catalase-peroxidase, designated HPI (8), and an atypical catalase, HPII (28), which are regulated differently. The level of HPI increases in response to external H202, and HPII has a 10- to 20-fold-higher activity in the stationary phase (26). It was suggested that HPII functions in nongrowing cells, whereas HPI has a general antioxidant role in log-phase cells, with a specific effect on membrane structure and function (13, 23). The aerobic bacterium Vitreoscilla sp. has one enzyme with catalase activity, characterized as a catalase-peroxidase, and it was suggested that its specific role was to scavenge the peroxide generated by its bacterial hemoglobin (1). The only significant difference found in our studies between the wild-type R. capsulatus and the catalase-peroxidase-deficient mutant was the enhanced death of the mutant cells after reaching the stationary phase under aerobic conditions. We infer from the similarity in growth rates that the catalase-peroxidase has a minor function in log-phase cells. Therefore, in actively growing R. capsulatus cells it is possible that the main protection against oxidative damage is conferred by the peroxidase. Alternatively, the damage repair systems may be efficient enough to prevent manifestation of oxidative stress in the log phase until the culture reaches the stationary phase, when the catalase-peroxidase has a crucial function. Stationary-phase bacteria are starved or stressed cultures. Starved bacteria undergo various changes that are crucial for their survival (20, 30). In E. coli it includes increased expression of over 40 proteins (38), some of which confer on the bacterium resistance to other
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