NOTE

Azotobacter chroococcum does not contain sodAor its gene product Mn-superoxide dismutase

Jane M. Caldwell and Hosni M. Hassan

Abstract: Azotobacter chroococcum and Azotobacter vinelandii grown in Burk medium with 1% mannitol (BM) or inBM supplemented with 2.2 mg/mL ammonium acetate (BM+N) were found to have only iron-containing and CuZn-containing superoxide dismutase. Furthermore, genomic DNA from A. chroococcum and A. vinelandii were subjected topolymerase chain reaction analysis using sodA- and sodB-specific primers and yielded only a sodB product. These re-sults dispute the assertion by Buchanan and Lees (Can. J. Microbiol. 26: 441–447, 1980) that A. chroococcum containsMn-superoxide dismutase.

Key words: FeSOD, Cu-ZnSOD, MnSOD, Azotobacter chroococcum, Azotobacter vinelandii.

Caldwell and HassanRésumé : Cultivés dans du milieu Burk contenant 1 % de mannitol (BM) ou dans du milieu BM additionné de2,2 mg/mL d’acétate d’ammonium (BM+N), l’Azotobacter chroococcum et l’Azotobacter vinelandii ne possèdentseulement que la superoxyde dismutase contenant du fer ou celle contenant du CuZn. D’autre part l’ADN génomique aété soumis à une analyse réaction en chaîne de la polymérase en utilisant les amorces spécifiques sodA- et sodB- etseul un composé sodB a été obtenu. Ces résultats sont en désaccord avec Buchanan et Lees (Can. J. Microbiol. 26:441–447, 1980) affirmant que l’A. chroococcum contient une Mn-superoxyde dismutase.

Mots clés : FeSOD, Cu-ZnSOD, MnSOD, Azotobacter chroococcum, Azotobacter vinelandii.

[Traduit par la Rédaction] 187

As one of the major cellular defenses against oxidativedamage, superoxide dismutases (SODs) convert superoxideanions (O2

–) to molecular oxygen and hydrogen peroxide(Fridovich 1975). SODs, isolated from a wide range of or-ganisms, fall into three classes, depending on the metalfound in their active center: manganese, iron, or copper-zinc(Hassan 1989). In 1969, McCord and Fridovich were thefirst to describe the activity of an enzyme now known ascopper-zinc superoxide dismutase (Cu-ZnSOD). Two othermetallo-enzymes were quickly discovered, one containingmanganese (MnSOD) (Keele et al. 1970) and the other con-taining iron (FeSOD) (Yost and Fridovich 1973). Inprokaryotes, Cu-ZnSODs are found in the periplasm ofgram-negative organisms (Benov et al. 1995), whileMnSODs and FeSODs are found in the cytosol (Britton andFridovich 1977).

MnSODs and FeSODs have significant amino acid se-quence (Steinman 1978) and structural (Carlioz et al. 1988)homology, suggesting a common ancestral protein. However,

in Escherichia coli, MnSODs and FeSODs areimmunologically distinct from each other (Schiavone andHassan 1988).

Azotobacter chroococcum and Azotobacter vinelandii aregram-negative, aerobic, nitrogen-fixing soil bacteria that haveextremely high respiration rates. Azotobacter species are ubiq-uitous in neutral to alkaline soils, with A. chroococcum beingthe most abundant species isolated (Hill and Sawers 2000).Nitrogen fixation is accomplished by the enzyme nitrogenase,which reduces dinitrogen to ammonia, but paradoxically, thisenzyme is extremely sensitive to oxygen in Azotobacter spe-cies. High respiration rates together with conformational pro-tection of the enzyme are thought to allow nitrogen fixation toproceed in an aerobic environment (Hill and Sawers 2000).Reduction of O2 by Azotobacter species occurs at such a highrate that large amounts of superoxide radicals are produced(Jurtshuk et al. 1984). Yet, little is known about AzotobacterSODs.

Buchanan and Lees (1980) reported that A. chroococcumcontained MnSOD. In 1995, Genovese et al. reported aperiplasmic Cu-ZnSOD and a cytoplasmic FeSOD in A.vinelandii. Qurollo et al. (2001) confirmed the presence ofFeSOD and Cu-ZnSOD in A. vinelandii and cloned and se-quenced the gene for FeSOD (sodB). In an attempt to re-solve this difference in the distribution of MnSODs amongthese two strains of Azotobacter, we examined the possibilitythat MnSOD or its gene sodA is present in A. vinelandii orA. chroococcum.

Can. J. Microbiol. 48: 183–187 (2002) DOI: 10.1139/W02-003 © 2002 NRC Canada

183

Received 10 October 2001. Revision received 7 December2001. Accepted 10 December 2001. Published on the NRCResearch Press Web site at http://cjm.nrc.ca on 3 March 2002.

J.M. Caldwell and H.M. Hassan.1 Department ofMicrobiology, North Carolina State University, Raleigh, NC27695-7615, U.S.A.

1Corresponding author (e-mail: hosni_hassan@ncsu.edu).

Azotobacter vinelandii(strain CA; Bush and Wilson1959) and A. chroococcum(ATCC 7493) cultures weregrown aerobically at 30°C with shaking at 150 rpm inBurk’s nitrogen-free media (Strandberg and Wilson 1968),containing 1% mannitol (BM) or in nitrogen-supplementedmedia, by adding ammonium acetate (2.2 g/L) to BM toyield BM+N. Solid media were prepared by adding 2% agarto liquid BM or BM+N media.Escherichia coli(GC4468)was grown in Luria–Bertani media at 37°C at 200 rpm. Cul-ture samples were collected at the late logarithmic or sta-tionary phases of growth.Azotobacter chroococcuminBM+N were grown for 25 h to a final OD600 of ca. 3.7. Cul-tures were then spun at 23 240 ×g, and pellets were frozenovernight and used to prepare dialyzed cell-free extract(CFE). Briefly, the pellet was resuspended in 0.05 M phos-phate buffer plus 0.1 mM EDTA, pH 7.8 (KPi–EDTA buffer)and sonicated at 60 A (Heat Systems – Ultrasonics Inc. CellDisrupter W370) for five 45-s bursts. Samples were placedon ice for 15 s between bursts. Sonicated samples wereplaced in 6.4 mm dialysis tubing (BioDesign, Inc. Carmel,N.Y.) and dialyzed overnight with three changes of KPi–EDTA buffer.

Protein concentration in dialyzed CFE was assayed ac-

cording to Lowry et al. (1951) using bovine serum albuminas standard. Total SOD was assayed by the cytochromecmethod (McCord and Fridovich 1969). SOD isoenzymeswere separated by electrophoresis on 10% polyacrylamidegels (Davis 1964) and visualized using a SOD activity stain(Beauchamp and Fridovich 1971). By adding cyanide or hy-drogen peroxide to the reagents used to develop the gels,one can differentiate between the three classes of SOD(Beauchamp and Fridovich 1971; Asada et al. 1975). UnlikeCu-ZnSODs, FeSODs and MnSODs are resistant to cyanide.Therefore, the use of cyanide has been a convenient tool fordistinguishing between the two families: FeSODs andMnSODs versus Cu-ZnSODs (Hassan 1989). Hydrogen per-oxide irreversibly inactivates FeSODs, but has no effect onMnSODs (Asada et al. 1975). Thus, the gels were soaked inthe nitro blue tetrazolium (NBT) stain containing either1 mM NaCN or 5 mM hydrogen peroxide, prior to exposureto light. Activity gels revealed thatA. chroococcum, like A.vinelandii, contained FeSOD and Cu-ZnSOD, but notMnSOD, when grown to late logarithmic or stationary phaseunder either nitrogen-fixing or non-nitrogen-fixing condi-tions. Figures 1A and 1B show SOD activity bands forA.chroococcumcells grown under non-nitrogen-fixing and ni-

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Fig. 1. Identification of the types of SOD inAzotobacter chroococcum. (A) Cells were grown in BM+N to stationary phase, and cell-free extracts (300µg protein/lane) were subjected to 10% nondenaturing polyacrylamide gel electrophoresis and stained for SOD activ-ity. Gels were subjected to different inhibitors added to the NBT staining solutions. 1, no addition; 2, 1 mM NaCN; 3, 5 mM H2O2.NaCN inhibits Cu-ZnSOD, while H2O2 inhibits FeSOD. (B) Same as in (A) except the cells were grown in BM.

trogen-fixing conditions, respectively. Similar results (datanot shown) were found with A. vinelandii as previously re-ported (Genovese et al. 1995; Qurollo et al. 2001). These re-sults dispute the assertion of Buchanan and Lees (1980) thatA. chroococcum contains MnSOD. Next, we examined thepossible presence of sodA in these two strains ofAzotobacter.

Extraction of bacterial genomic DNA was performed us-ing the Qiagen DNeasy kit. BioEdit software (Hall 1999)(available at www.mbio.ncsu.edu/BioEdit/BioEdit.html) wasused to align DNA sequences and to design the differentprimers for the amplification of genomic sodA and sodB.Polymerase chain reactions (PCR) were performed usingthree different pairs of the following primers:

Sodita A5 (5′-GACAAGAAAACCGTA-3′), forwardSodita A3 (5′-ATAATCGGGAAGCCG-3′), reverseSod B5 (5′-TGGAACCAYACHTTCTACTGG-3′), forwardSod B3 (5′-GACRTCRMMGGTCAGCAGCGG-3′), re-

verse

Poyart A5 (5′-CCITAYICITAYGAYGCIYTIGARCC–3′),forward

Poyart A3 (5′-ARRTARTAIGCRTGYTCCCAIACRTC-3′),reverse

The GenBank database nucleotide sequences of the sodAgene of E. coli (M94879) were aligned against sodB genesfrom two strains of E. coli (AB009855; AB026684) and twostrains of A. vinelandii (AB025798; AF077373) to find the“gaps” between the sodA and sodB nucleotide sequences(data not shown). The Sodita A primers were designed usingthose gaps to bind the sodA, but exclude the sodB gene dur-ing PCR. The GenBank database nucleotide sequences ofthe sodB genes from Photobacterium leiognathi (AB050790;AB050791), Photobacterium phosphoreum (AB050790;AB050791), two E. coli (AB009855; AB026684), two A.vinelandii (AB025798; AF077373), and Pseudomonasaeruginosa (L25675) were aligned (data not shown), andtwo highly conserved stretches of nucleotides were chosenas templates for design of Sod B primers. The Sodita A andPoyart A primers amplified a 295-bp and 480-bp internal re-gion of sodA, respectively. The Sod B primers amplified a250-bp internal region of sodB. The Sod B primers delineatea segment that represents ca. 50% of the sodB gene and doesnot bind sodA. In PCR studies using sodA and sodBplasmids, Sodita A and Sod B primers were shown to reactexclusively with their intended gene (data not shown).

Amplification of E. coli (strain GC4468), A. vinelandii,and A. chroococcum genomic DNAs was accomplished us-ing reagents from a Qiagen Taq DNA polymerase kit. DNAamplification was performed in a final volume of 50 µL con-taining 500 ng of genomic DNA, 0.5 µM of each primer,200 µM of each dNTP, and 2.5 U of Taq DNA polymerase in1× amplification buffer (TrisCl, KCl, (NH4)2S04, 1.5 mMMgCl2, pH 8.7). The PCR mixture for Sodita A and Sod Bprimers was subjected to a denaturation step (4 min at95°C), followed by 30 cycles of amplification (30 s of dena-turation at 95°C, 30 s of annealing at 45°C, 30 s of elonga-tion at 72°C), and final elongation (7 min at 72°C) followedby a 4°C temperature hold. A Icycler thermal cycler(BioRad, Hercules, Calif.) was employed for the above pro-tocol.

The primers Poyart A5 and A3 correspond to d1 and d2reported by Poyart et al. (1995) that were used as universalprimers for sodA from gram-positive bacteria (Poyart et al.1998). The same PCR mixtures used with Sodita A and SodB primers were used with Poyart primers, but the protocolreported by Poyart et al. (1995) was used for amplification.In short, a denaturation step (3 min at 95°C) was followedby 35 cycles of amplification (2 min of annealing at 37°C,90 s of elongation at 72°C, and 30 s of denaturation at 95°C)and a final annealing (4 min at 37°C) and elongation(12 min at 72°C) followed by a 4°C temperature hold. APerkin Elmer thermal cycler was employed for the Poyartprimers because of the lower annealing temperatures thatcould not be accommodated in the Icycler. PCR productswere run on 1.2% agarose gels and imaged with GelDoc2000 (BioRad).

PCR assays indicate that there is no sodA gene in either A.choococcum or A. vinelandii, only sodB (Fig. 2). Both setsof Sod A primers, Sodita A (Fig. 2A, lanes 4 and 6) andPoyart A (Fig. 2B, lanes 4–7), failed to produce any PCR

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Caldwell and Hassan 185

Fig. 2. PCR products of three different bacterial genomic DNAs.(A) Using sodA- or sodB-specific primers. Genomic DNAs fromEscherichia coli (lanes 2 and 3), Azotobacter vinelandii (lanes 4and 5), and Azotobacter chroococcum (lanes 6 and 7) were pre-pared and used in PCR reactions using sodA-specific primers(even-numbered lanes) or sodB-specific primers (odd-numberedlanes) to test for PCR products. Molecular weight standards arein lanes 1 and 8. (B) Using Poyart’s sodA primers. Conditionsare the same as in (A) except that lane 8 is a PCR blank andlanes 1 and 9 are molecular weight standards. E.C., E. coli;A.V., A. vinelandii; A.C., A. chroococcum.

products with either of the Azotobacter strains. Yet, thesesame primers produced a single expected band for sodA inE. coli (GC4468) (Fig. 2A, lane 2; Fig. 2B, lanes 2 and 3).These results, combined with the SOD activity gel data(Fig. 1) and Qurollo et al. (2001), clearly suggest that A.chroococcum and A. vinelandii do not contain the sodAgene.

In the course of this study, a 240-base partial sequence ofsodB from A. chroococcum was determined (Iowa State Se-quencing Facility) and was submitted to GenBank underAY055761. This partial sequence was translated, and the re-sulting partial amino acid sequence was compared with otherSodB proteins (Fig. 3), using the NCBI blast search. Thepartial SodB sequence from A. chroococcum was found tohave a 93% identity with A. vinelandii (AF077373), a 91%identity with P. putida (U64798) and P. aeruginosa(NC_002516), a 62% identity with E. coli (AB009855), anda 66% identity with Salmonella typhimurium (AE008762)SodB. These results are consistent with phylogenetic studies(Loveless et al. 1999) showing Azotobacter to be closely re-lated to the fluorescent pseudomonads.

Acknowledgements

We wish to thank Dr. Paul Bishop and Telisa Loveless forsupplying the Azotobacter strains and Dr. Steve Bowen, Dr.Jason Andrus, Alan House, and Tim Dean for technical as-sistance.

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The author has requested enhancement of the downloaded file. All in-text references underlined in blue are linked to publications on ResearchGate.The author has requested enhancement of the downloaded file. All in-text references underlined in blue are linked to publications on ResearchGate.