Dec 5, 1989 - Mn superoxide dismutase and is, consequently, very sensitive to oxidative ..... 7219. 3. Hassan, H. M. & Fridovich, I. (1977) J. Biol. Chem. 252,.
Proc. Nati. Acad. Sci. USA Vol. 87, pp. 2608-2612, April 1990 Biochemistry
Cloned manganese superoxide dismutase reduces oxidative stress in Escherichia coli and Anacystis nidulans (shuttle vectors/paraquat/activated oxygen/photobleaching)
MARGARET Y. GRUBER*t, BERNARD R. GLICK*, AND JOHN E. THOMPSON*: *Department of Biology, University of Waterloo, Waterloo, ON, N2L 3G1 Canada; and tDepartment of Horticultural Science, University of Guelph, Guelph, ON, N1G 2W1 Canada
Communicated by P. K. Stumpf, December 5, 1989 (received for review August 7, 1989)
tase as well as the more H202-resistant Mn superoxide dismutase are present (11). In the present study, the prospect that increased tolerance to oxidative stress can be achieved by genetic manipulation of Mn superoxide dismutase was examined by subcloning the E. coli Mn superoxide dismutase gene into an E. coli-A. nidulans shuttle vector and introducing this new vector into both E. coli and A. nidulans. The resulting transformants were tested for resistance to oxidative stress induced by exposure to paraquat.
The Mn superoxide dismutase gene of EschABSTRACT erichia cofi was subcloned into the E. col-Anacystis nidulans shuttle vector pSG111 to make the plasmid pMYG1. Transformation of E. coil HB101 with pMYG1 resulted in a 6-fold increase in superoxide dismutase activity. There was also induction of Mn superoxide dismutase in the transformants upon exposure to paraquat, as evidenced by dramatically increased levels of the Mn superoxide dismutase polypeptide in cytoplasmic extracts and a 16-fold further increase in superoxide dismutase activity. As well, the E. coil transformants showed resistance to paraquat-mediated inhibition of growth. Anacystis nidulans, a cyanobacterium that has no detectable Mn superoxide dismutase and is, consequently, very sensitive to oxidative stress, was also transformed with pMYG1. The transformants had detectable levels of Mn superoxide dismutase protein and showed resistance to paraquat-mediated inhibition of growth and photobleaching of pigments. Paraquat is known to promote formation of the superoxide radical anion, O2*, and thus the data have been interpreted as indicating that the cloned Mn superoxide dismutase provides protection in both E. coli and A. nidulans against damage attributable to °2..
MATERIALS AND METHODS Culture Conditions and Plasmid Construction. Anacystis nidulans SPC (the R2 strain cured of pANS; ref. 12) was obtained from N. Straus (Department of Botany, University of Toronto) and was grown at 300C in BG11 medium with 100 gE-m-2's-1 [1 einstein (E) = 1 mol of photons] continuous cool white illumination (13). E. coli HB101 was grown at 300C in YT medium (14). The plasmid pDT1-5, which contains the E. coli Mn superoxide dismutase gene (15), was obtained from D. Touati (Institut Jacques Monod, Centre National de la Recherche Scientifique, Universitd de Paris), and the E. coli-A. nidulans shuttle vector pSG111 (16) was provided by L. Sherman (Division of Biological Sciences, University of Missouri). Plasmid pMYG1 was constructed by ligating pSG111, which had been completely digested with EcoRI and partially digested with BamHI, with an EcoRI-BamHI fragment from pDT1-5 that contained the E. coli Mn superoxide dismutase gene (15) and transforming E. coli HB101 with the ligation mixture. pMYG1 was selected as an ampicillin-resistant, chloramphenicol-sensitive colony that showed increased superoxide dismutase activity. By contrast, both E. coli and A. nidulans transformants containing pSG111 (16) were ampicillin-resistant and chloramphenicol-resistant. E. coli HB101 was transformed with pMYG1 or pSG111 as described by Maniatis et al. (17), and A. nidulans was transformed with the same plasmids according to Gendel et al. (12). Plasmid DNA was isolated from E. coli according to Maniatis et al. (17) and from A. nidulans as described by Daniell et al. (18). Restriction fragment analyses were carried out as described
Escherichia coli exhibits symptoms of oxidative stress when exposed to redox cycling agents such as paraquat (1) and certain photoactivated dyes (2). Protection against oxidative stress has been correlated with the presence of Mn superoxide dismutase. For example, expression of the Mn superoxide dismutase gene in E. coli is induced during exposure to redox cycling agents, whereas the gene for Fe superoxide dismutase is expressed constitutively (3). Indeed, levels of the Fe superoxide dismutase protein decline during oxidative stress (4), and the enzyme is inactivated by H202 (5). The protective role attributed to Mn superoxide dismutase in the event of oxidative stress is further substantiated by the finding that the mutation rate under normal aerobic conditions for a Mn superoxide dismutase-minus mutant of E. coli was 9-fold higher than that for the corresponding wild type or for an Fe superoxide dismutase-minus mutant (6). Cyanobacteria experience photooxidative stress when exposed to high light intensities (7, 8) or paraquat (9). Manifestations of these stresses include a reduction in growth rate and pigment photobleaching. Photooxidative injury in cyanobacteria is intensified when synthesis of superoxide dismutase and catalase is inhibited, suggesting that these enzymes provide protection against the effects of oxidative stress (8, 10). Some cyanobacteria, such as Anacystis nidulans, are particularly sensitive to photooxidative stress because they possess only Fe superoxide dismutase. This sensitivity is reduced in other cyanobacteria when Fe superoxide dismu-
(17).
Hybridization. Restriction enzyme-digested DNA was fractionated by agarose gel electrophoresis, and the fragments were denatured and electroblotted onto Biotrans nylon membranes (ICN) for 4.5 hr at 4°C in 25 mM sodium phosphate (pH 6.5) (19). The membranes were air-dried and baked at 80°C. The blots were hybridized (17) with a 1.1kilobase Ava I DNA fragment from plasmid pDT1-5 containing the E. coli Mn superoxide dismutase gene (20). The DNA
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tPresent address: Agriculture Canada Research Station, 107 Science Crescent, Saskatoon, SK, S7N OX2 Canada.
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Biochemistry: Gruber et al. probe was radiolabeled with [a-32PldCTP by using a random oligonucleotide-primed labeling kit from Pharmacia (21). Cell-Free Synthesis of Plasmid-Encoded Proteins. Protein synthesis in an E. coli C600 cell-free system (22) was programmed with plasmid DNA. The reaction mixtures (50 pl) contained, in addition to buffer, salts, tRNAs, an ATPgenerating system, and E. coli C600 cell-free extract (12.5 ,ug/IAI), 7 ACi of [35Slmethionine (800 Ci/mmol; 1 Ci = 37 GBq) and 0.5-3.0 Ag of plasmid DNA. After 40 min of reaction at 370C, protein was precipitated with cold 5% trichloroacetic acid and analyzed by SDS/PAGE in a 10-20%o gradient polyacrylamide gel with a discontinuous buffer system (23). Protein was measured according to Bradford
(24).
Cell Fractionation. Cells (200 ml) of control E. coli cultures or cultures grown in the presence of paraquat (0.1-8 mM) were sedimented by centrifugation at 8000 X g, resuspended in 1 ml of 10 mM potassium phosphate (pH 7.0), and lysed by sonication on ice (five 20-s bursts with 1-min intervening cooling periods with a Branson Sonifier cell disrupter). The lysate was centrifuged for 30 min at 4°C in an Eppendorf centrifuge. The supernatant was assayed for superoxide dismutase activity (25), and the membrane and supernatant fractions were analyzed by SDS/PAGE in 10-20o gradient gels (23). A. nidulans cells were plated onto nylon membranes (diameter, 100 mm) and grown on solid BG11 medium [containing ampicillin (1 ,g/ml) if the cells had been transformed with pMYG1 or pSG111] for 4 days at 30°C in 100 ,E m 2s-1 light. The filters were then transferred either to fresh solid BG11 medium or to solid BG11 medium containing 10, 100, or 1000 nM paraquat and the cells were grown at 30°C for 2 days in 100 ,E m 2-s- light. Cells scraped from six to eight filters were resuspended in 2 ml of lysozyme (1 mg/ml) in 10 mM potassium phosphate (pH 7.0), lysed by sonication on ice as for E. coli, and centrifuged for 30 min at 4°C in an Eppendorf centrifuge. The supernatant was analyzed by SDS/PAGE in a 10-20% gradient gel (23) or assayed for superoxide dismutase activity (25).
RESULTS The E. coli gene for Mn superoxide dismutase was introduced into both E. coli and A. nidulans in the E. coli-A. nidulans shuttle vector pMYG1. Plasmid pMYG1 was constructed by ligating the E. coli Mn superoxide dismutase gene from pDT1-5 into the shuttle vector pSG111. Published maps of pDT1-5 (15) and pSG111 (16) together with EcoRI, BamHI, and Xho I restriction fragment analyses of pMYG1, pDT1-5, and pSG111 and the results of cell-free translations of pMYG1 fragments were used to deduce a restriction endonuclease map for pMYG1 (Fig. 1). EcoRI restriction fragment patterns for pMYG1 isolated from E. coli HB101/pMYG1 and A. nidulans SPC/pMYG1 were identical, indicating that the plasmid is not subject to rearrangement in A. nidulans. The isolated plasmids also hybridized strongly to the E. coli Mn superoxide dismutase gene probe. Furthermore, pMYG1 from either E. coli HB101/pMYG1 or A. nidulans SPC/ pMYG1 and the plasmid pDT1-5 directed the synthesis of the 20.5-kDa Mn superoxide dismutase polypeptide when used to program protein synthesis in an E. coli cell-free system (Fig. 2). pMYG1 also programmed the synthesis of 43-lactamase (29.5 kDa) but not the 21.3-kDa chloramphenicol acetyltransferase polypeptide encoded by the parent shuttle vector pSG111 (Fig. 2). The 27.4-kDa f-lactamase cleavage product was also detectable in some gels (Fig. 2B). Superoxide dismutase specific activity was 6- to 7-fold higher in E. coli HB101/pMYG1, which contained the cloned Mn superoxide dismutase gene, than in HB101/pSG111. When cells of either transformant were exposed to paraquat
Proc. Natl. Acad. Sci. USA 87 (1990) Pvull
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BamH}
PvuII
EcoRlI
BomHI HindIl
FIG. 1. Restriction endonuclease map of pMYG1. Thick line, A. nidulans plasmid DNA; thin line, E. coli plasmid DNA; gray bar, E. coli chromosomal DNA containing the Mn superoxide dismutase (MnSOD) gene; Amp', ampicillin-resistance gene; Ori E, E. coli replication origin; Ori A, A. nidulans replication origin; kb, kilobases.
(0.5-5.0 mM), the activity of the enzyme rose 10- to 100-fold, although HB101/pMYG1 cells always displayed a higher level of superoxide dismutase activity than HB101/pSG111 cells (Table 1). SDS/PAGE analysis of cytoplasmic proteins confirmed that the increased superoxide dismutase activity upon exposure to paraquat reflected induction of Mn superoxide dismutase. The 20.5-kDa Mn superoxide dismutase polypeptide was induced in both E. coli HB101/pSG111 and HB101/pMYG1 during a 9-hr exposure to 0.5 mM paraquat (Fig. 3, lanes 2, 4, 6, and 8), and for HB101/pMYG1 became the major cytoplasmic protein as a result of the paraquat treatment (Fig. 3, lane 8). By contrast, Mn superoxide dismutase was not induced in A. nidulans SPC/pMYG1 during treatment with paraquat. The 20.5-kDa polypeptide corresponding to Mn superoxide dismutase was not detectB
A
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FIG. 2. Autoradiograms showing cell-free expression of pMYG1. (A) pMYG1 isolated from E. coli. Lane 1, 50 lug of cell-free extract, 2 .g of pSG111; lane 2, 50 Itg of cell-free extract, 2 j.g of pMYG1 isolated from E. coli; lane 3, 50 pug of cell-free extract, 2 ,ug of pDT1-5; lane 4, 50 ,ug of cell-free extract, no DNA. (B) pMYG1 isolated from A. nidulans. Lane 1, 67 jig of cell-free extract, no DNA; lane 2, 33 Ag of cell-free extract, 2 pug of pSG111; lane 3, 33 J.g of cell-free extract, 2 j.g of pDT1-5; lane 4, 67 ug of cell-free extract,
