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Superoxide Dismutase in Some Obligately Anaerobic Bacteria

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Superoxide Dismutase in Some Obligately Anaerobic Bacteria CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Volume 5 0, number 3 FEBS LETTERS February 1975 SUPEROXIDE DISMUTASE IN SOME OBLIGATELY ANAEROBIC BACTERIA Joan HEWITT and J. G. MORRIS Department of Botany & Microbiology, School of Biological Sciences, The University College of Wales, Aberystwyth SY23 3DA, Wales, U.K. Received 7 December 1974 1. Introduction collection, and all other organisms were obtained from the National Collection of Industrial Bacteria, Substantial protection against oxygen toxicity is Aberdeen, Scotland. The E. coli strains were grown afforded to aerobic and facultatively anaerobic organisms aerobically with shaking at 37°C in glucose (1:/o) by their possession of superoxide dismutase [ 1,2]. This nutrient broth (Oxoid Ltd., London, England) and enzyme was reportedly not present in obligately anaero- all other organisms were grown anaerobically in bic bacteria [3], i.e. species which exhibit sensitivity to batch culture, Chlorobium thiosulfatophilum NCIB oxygen at 0.2 atm or less [ 11. Thus it appeared that the 8346 with illumination at 30°C in CCT medium [5], distinction between the obligate and facultative anaerobe, Chromatium sp. NCIB 8348 with illumination at 30°C formerly a somewhat arbitrary line drawn on a spectrum in the medium of Eymers and Wassink [6] supple- of aerotolerance, could be more satisfactorily defined mented with 0.05% yeast extract, Clostridium species in biochemical terms, viz.: the obligate anaerobe is de- at their optimum growth temperatures in Reinforced void of superoxide dismutase, the facultative (aeroto- Clostridial Medium (Oxoid Ltd.), Desulfotomaculum lerant) anaerobe contains this enzyme. nigrificans NCIB 8395 at 55°C in BET1 broth [7] and In this communication we report that possession Desulfovibrio desulfuricans NCIB 8307 at 30°C in of superoxide dismutase is not restricted to organisms medium C of Postgate [8]. The cultures were harvested capable of growth in air. We have found superoxide in the late exponential phase by centrifuging at 12 000 dismutase in moderately high specific activity in X g for 20 min at 4’C. After washing in 0.05 M Chlorobium thiosulfatophilum and Clostridium per- potassium phosphate buffer pH 7.0, the cells were fringens, and have partially purified the cyanide-insen- resuspended in a small volume of this buffer and dis- sitive enzyme of the latter organism. Lesser activities rupted by passage at 20 000 psi through a chilled of the enzyme were detected in Chromatium sp., French pressure cell (Aminco, Silver Spring, Md., Desulfotomaculum nigrificans, Desulfovibrio desul- U.S.A.). Cell debris was removed by centrifugation furicans and some other species of Clostridium. at 26 000 X g for 30 min at 4°C. Protein was measured by the method of Lowry et al. [9]. Polyacrylamide gel disc electrophoresis of cell extracts was performed by 2. Experimental the method of Davis [lo], protein bands being located by staining with Coomassie Blue [ 1 l] and superoxide Strains of Clostridium butyricum were kindly dismutase activity being located on duplicate gels by provided by Mr J. Wolf, University of Leeds, England, the method of Beauchamp and Fridovich [ 121. Super- Clostridium pasteurianum ATCC 6013 was supplied oxide dismutase activity in crude and partially purified by Mrs Winifred Ego, University of Hawaii, U.S.A. cell extracts was measured spectrophotometrically by and Clostridium perfringens type A NCIB 11105 was the method of McCord and Fridovich [ 131. This assay a local isolate. Escherichia coli B and E. coli K-l 2 was complicated by the substantial soluble cytochrome mutant ABl157 [4] were from our laboratory c reductase activity present in many of the extracts. North-Holland Publishing Company - Amsterdam 315 Volume 50, number 3 k’EBS LETTERS I’cbruarv 1975 This could be removed by prolonged dialysis, but it Table 1 was found that the superoxide dismutase activity di- Qualitative demonstration of superoxide dismutase activity in extracts of some anaerobic bacteria” rectly measurable after dialysis, equalled that indirect- -~--___~_~_._.-.___ ly assayable in the crude, undialysed extract by making Organism Superoxid; allowance for the ‘background’ cytochrome c reduction dismutrsc‘ L (measured in the absence of the superoxide generating system of xanthine plus xanthine oxidase EC 1.2.3.2). Photosynthetic anaerobes: NCIB 8346 One unit of superoxide dismutase activity is defined as Chlorobium thiosulfatophilum + Chromatium sp. NCIB 8348 + that quantity of enzyme which would cause 50% inhi- Sulphate reducers: bition of the rate of cytochrome c reduction by 0; Desulfotomaculum nigrifcans NUB 8395 + generated by xanthine oxidase, though in the assay, Desulfovibrio desulfuricans NCIB 8307 + stoichiometry was often not maintained at levels of Fermentative anaerobes: inhibition greater than 40%. The superoxide dismutase Clostridium acetobutylicum NCIB 6445 t of Cl. perfringens NCIB 1110.5 was purified 15fold by Clostridium acetobutylicum NCIB 8049 t a procedure (to be published elsewhere) involving Clostridium acetobutylicum NCIB 8052 t precipitation in the 50 to 70% saturated ammonium Clostridium beijerinckii NCIB 9362 t NCIB 506 sulphate fraction of the cell extract, followed by gel Clostridium bifermentans + Clostridium butyricum SA I t filtration through Sephadex G-75 and chromatography Clostridium butyricum SA II - on DEAE-cellulose. Cytochrome c and enzymes were Clostridium butyricum CNRZ 528 t purchased from Sigma (London) Chemical Co., DEAE- Clostridium butyricum CNRZ 531 _ cellulose from Whatman Biochemicals Ltd., Maidstone, Clostridium pasteurianum ATCC 6013 + type A NCIB 11105 t England, Sephadex G-75 from Pharmacia (Great Britain) Clostridium perfringens Clostridium sporogenes NCIB 532 t Ltd., London, and reagents for polyacrylamide gel ____- electrophoresis from Eastman-Kodak, Rochester, N.Y., Organisms were grown, and extracts prepared, as describ- U.S.A. ed in Experimental. Crude cell extract (2 mg protein) was subjected to polyacrylamide gel electrophoresis and the band(s) of superoxide dismutase activity were visualised by the method of Beauchamp & Fridovich [ 121. 3. Results Presence (t) of superoxide dismutase is contrasted with absence of sufficient activity of enzyme to be detectable A preliminary survey of the distribution of superoxide in this assay (-). dismutase in several obligately anaerobic bacteria, was carried out by visual inspection of polyacrylamide disc gel electrophoretograms made using samples of crude cell Spectrophotometric assay of superoxide dismutase extracts, and treated to reveal the presence of bands activity in crude cell extracts (2.0) enabled a com- of superoxide dismutase activity [ 121. The enzyme parison to be made between the specific activities of was present in several of these organisms (table l), the enzyme in various anaerobes with those found in Chlorobium thiosulfatophilum NCIB 8346 and Clostri- aerobically grown strains of .F. coli (table 2). Though dium perfringens NCIB 11105 yielding particularly the enzyme was present in surprisingly high specific prominent bands of superoxide dismutase activity in activity in Chl. thiosulfatophilum NCIB 8346 and this assay. Some activity was also discernible in a num- Cl. perfringens NCIB 11105, in those other anaerobes ber of other species, including Cl. acetobutylicum and possessing a superoxide dismutase, this was present CI. pasteurianum which McCord et al. [3] had reported at only 1 to 6% of the specific activity found in to be devoid of the enzyme. Several other Clostridium aerobic E. coli. species contained either no superoxide dismutase activ- The partly purified preparation of superoxide dis- ity, or less than could be detected by this procedure, mutase from Cl. perfringens (2.0) migrated as two the findings being somewhat unpredictable in that major protein bands on polyacrylamide gel electroph- different strains of the same species could differ in oresis, the enzyme activity coinciding with the greater their contents of the enzyme (e.g. CE. butyricum). of these bands (fig. 1). The nature and properties of 316 Volume 50, number 3 FEBS LETTERS lebruary 1975 Table 2 this clostridial superoxide dismutase, which was not Specific activities of superoxide dismutase in crude extracts of inhibited by 1 V3 M cyanide, are now being studied. (a) aerobically grown E. coli and (b) several anaerobic bacteria Organism Specific activity 4. Discussion of superoxide dismutase (units/ Though an organism can evidently contain quite mg of protein)” - substantial superoxide dismutase activity and yet be incapable of growth in air, this does not mean that Escherichia coli B 44.0 Escherichia coli K-l 2, mutant AB 1157 36.8 the enzyme renders it no significant service. Even obligate anaerobes display a spectrum of oxygen Chlorobium thiosulfatophilum NCIB 8346 14.0 Chromutium sp. NCIB 8348 0.6 sensitivity ranging from those bacteria for which Desttlfotomaculum nigrificans NCIB 8395 2.6 oxygen is apparently bactericidal at very low con- Desulfovibrio desulfurican:r NCIB 8307 0.6 centrations to those which can tolerate limited expo- sure to air, and for which atmospheric oxygen may. Clostridium pastcurianum ATCC 6013 0.5 Clostridium perfringens NCIB 11 105 15.6 in the short term, be reversibly bacteriostatic rather _____-.___ than bactericidal [ 141. This range of oxygen sensi- a Superoxide dismutase activity was measured spectro- tivity is displayed within the genus Clostridium; Cl. photometrically in undialysed, crude
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