1. NOTE
Azotobacter chroococcum does not contain sodA
or 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 in
BM 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 to
polymerase 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 contains
Mn-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 de
2,2 mg/mL d’acétate d’ammonium (BM+N), l’Azotobacter chroococcum et l’Azotobacter vinelandii ne possèdent
seulement que la superoxyde dismutase contenant du fer ou celle contenant du CuZn. D’autre part l’ADN génomique a
été soumis à une analyse réaction en chaîne de la polymérase en utilisant les amorces spécifiques sodA- et sodB- et
seul 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 oxidative
damage, superoxide dismutases (SODs) convert superoxide
anions (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 metal
found in their active center: manganese, iron, or copper-zinc
(Hassan 1989). In 1969, McCord and Fridovich were the
first to describe the activity of an enzyme now known as
copper-zinc superoxide dismutase (Cu-ZnSOD). Two other
metallo-enzymes were quickly discovered, one containing
manganese (MnSOD) (Keele et al. 1970) and the other con-
taining iron (FeSOD) (Yost and Fridovich 1973). In
prokaryotes, Cu-ZnSODs are found in the periplasm of
gram-negative organisms (Benov et al. 1995), while
MnSODs and FeSODs are found in the cytosol (Britton and
Fridovich 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 are
immunologically distinct from each other (Schiavone and
Hassan 1988).
Azotobacter chroococcum and Azotobacter vinelandii are
gram-negative, aerobic, nitrogen-fixing soil bacteria that have
extremely high respiration rates. Azotobacter species are ubiq-
uitous in neutral to alkaline soils, with A. chroococcum being
the most abundant species isolated (Hill and Sawers 2000).
Nitrogen fixation is accomplished by the enzyme nitrogenase,
which reduces dinitrogen to ammonia, but paradoxically, this
enzyme 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 to
proceed in an aerobic environment (Hill and Sawers 2000).
Reduction of O2 by Azotobacter species occurs at such a high
rate that large amounts of superoxide radicals are produced
(Jurtshuk et al. 1984). Yet, little is known about Azotobacter
SODs.
Buchanan and Lees (1980) reported that A. chroococcum
contained MnSOD. In 1995, Genovese et al. reported a
periplasmic Cu-ZnSOD and a cytoplasmic FeSOD in A.
vinelandii. Qurollo et al. (2001) confirmed the presence of
FeSOD 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 among
these two strains of Azotobacter, we examined the possibility
that MnSOD or its gene sodA is present in A. vinelandii or
A. chroococcum.
Can. J. Microbiol. 48: 183–187 (2002) DOI: 10.1139/W02-003 © 2002 NRC Canada
183
Received 10 October 2001. Revision received 7 December
2001. Accepted 10 December 2001. Published on the NRC
Research Press Web site at http://cjm.nrc.ca on 3 March 2002.
J.M. Caldwell and H.M. Hassan.1
Department of
Microbiology, North Carolina State University, Raleigh, NC
27695-7615, U.S.A.
1
Corresponding author (e-mail: hosni_hassan@ncsu.edu).

2. Azotobacter vinelandii (strain CA; Bush and Wilson
1959) and A. chroococcum (ATCC 7493) cultures were
grown aerobically at 30°C with shaking at 150 rpm in
Burk’s nitrogen-free media (Strandberg and Wilson 1968),
containing 1% mannitol (BM) or in nitrogen-supplemented
media, by adding ammonium acetate (2.2 g/L) to BM to
yield BM+N. Solid media were prepared by adding 2% agar
to 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 chroococcum in
BM+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 frozen
overnight 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. Cell
Disrupter W370) for five 45-s bursts. Samples were placed
on ice for 15 s between bursts. Sonicated samples were
placed 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 albumin
as standard. Total SOD was assayed by the cytochrome c
method (McCord and Fridovich 1969). SOD isoenzymes
were separated by electrophoresis on 10% polyacrylamide
gels (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). Unlike
Cu-ZnSODs, FeSODs and MnSODs are resistant to cyanide.
Therefore, the use of cyanide has been a convenient tool for
distinguishing between the two families: FeSODs and
MnSODs versus Cu-ZnSODs (Hassan 1989). Hydrogen per-
oxide irreversibly inactivates FeSODs, but has no effect on
MnSODs (Asada et al. 1975). Thus, the gels were soaked in
the nitro blue tetrazolium (NBT) stain containing either
1 mM NaCN or 5 mM hydrogen peroxide, prior to exposure
to light. Activity gels revealed that A. chroococcum, like A.
vinelandii, contained FeSOD and Cu-ZnSOD, but not
MnSOD, when grown to late logarithmic or stationary phase
under either nitrogen-fixing or non-nitrogen-fixing condi-
tions. Figures 1A and 1B show SOD activity bands for A.
chroococcum cells grown under non-nitrogen-fixing and ni-
© 2002 NRC Canada
184 Can. J. Microbiol. Vol. 48, 2002
Fig. 1. Identification of the types of SOD in Azotobacter chroococcum. (A) Cells were grown in BM+N to stationary phase, and cell-
free extracts (300 µg 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.

3. trogen-fixing conditions, respectively. Similar results (data
not 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) that
A. chroococcum contains MnSOD. Next, we examined the
possible presence of sodA in these two strains of
Azotobacter.
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) was
used to align DNA sequences and to design the different
primers for the amplification of genomic sodA and sodB.
Polymerase chain reactions (PCR) were performed using
three different pairs of the following primers:
Sodita A5 (5′-GACAAGAAAACCGTA-3′), forward
Sodita A3 (5′-ATAATCGGGAAGCCG-3′), reverse
Sod B5 (5′-TGGAACCAYACHTTCTACTGG-3′), forward
Sod 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 sodA
gene of E. coli (M94879) were aligned against sodB genes
from two strains of E. coli (AB009855; AB026684) and two
strains 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 using
those gaps to bind the sodA, but exclude the sodB gene dur-
ing PCR. The GenBank database nucleotide sequences of
the sodB genes from Photobacterium leiognathi (AB050790;
AB050791), Photobacterium phosphoreum (AB050790;
AB050791), two E. coli (AB009855; AB026684), two A.
vinelandii (AB025798; AF077373), and Pseudomonas
aeruginosa (L25675) were aligned (data not shown), and
two highly conserved stretches of nucleotides were chosen
as templates for design of Sod B primers. The Sodita A and
Poyart A primers amplified a 295-bp and 480-bp internal re-
gion of sodA, respectively. The Sod B primers amplified a
250-bp internal region of sodB. The Sod B primers delineate
a segment that represents ca. 50% of the sodB gene and does
not bind sodA. In PCR studies using sodA and sodB
plasmids, Sodita A and Sod B primers were shown to react
exclusively 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. DNA
amplification 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 in
1× amplification buffer (TrisCl, KCl, (NH4)2S04, 1.5 mM
MgCl2, pH 8.7). The PCR mixture for Sodita A and Sod B
primers was subjected to a denaturation step (4 min at
95°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) followed
by 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 d2
reported by Poyart et al. (1995) that were used as universal
primers for sodA from gram-positive bacteria (Poyart et al.
1998). The same PCR mixtures used with Sodita A and Sod
B primers were used with Poyart primers, but the protocol
reported by Poyart et al. (1995) was used for amplification.
In short, a denaturation step (3 min at 95°C) was followed
by 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. A
Perkin Elmer thermal cycler was employed for the Poyart
primers because of the lower annealing temperatures that
could not be accommodated in the Icycler. PCR products
were run on 1.2% agarose gels and imaged with GelDoc
2000 (BioRad).
PCR assays indicate that there is no sodA gene in either A.
choococcum or A. vinelandii, only sodB (Fig. 2). Both sets
of Sod A primers, Sodita A (Fig. 2A, lanes 4 and 6) and
Poyart A (Fig. 2B, lanes 4–7), failed to produce any PCR
© 2002 NRC Canada
Caldwell and Hassan 185
Fig. 2. PCR products of three different bacterial genomic DNAs.
(A) Using sodA- or sodB-specific primers. Genomic DNAs from
Escherichia coli (lanes 2 and 3), Azotobacter vinelandii (lanes 4
and 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-numbered
lanes) to test for PCR products. Molecular weight standards are
in lanes 1 and 8. (B) Using Poyart’s sodA primers. Conditions
are the same as in (A) except that lane 8 is a PCR blank and
lanes 1 and 9 are molecular weight standards. E.C., E. coli;
A.V., A. vinelandii; A.C., A. chroococcum.

4. products with either of the Azotobacter strains. Yet, these
same primers produced a single expected band for sodA in
E. 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 sodA
gene.
In the course of this study, a 240-base partial sequence of
sodB from A. chroococcum was determined (Iowa State Se-
quencing Facility) and was submitted to GenBank under
AY055761. This partial sequence was translated, and the re-
sulting partial amino acid sequence was compared with other
SodB proteins (Fig. 3), using the NCBI blast search. The
partial SodB sequence from A. chroococcum was found to
have 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), and
a 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 for
supplying the Azotobacter strains and Dr. Steve Bowen, Dr.
Jason Andrus, Alan House, and Tim Dean for technical as-
sistance.
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Caldwell and Hassan 187
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