1. business and industrial
2. agriculture and forestry
3. farms and ranches

The EMBO Journal vol.5 no.3 pp.623-630, 1986
Isolation of superoxide dismutase mutants in Escherichia coli: is
superoxide dismutase necessary for aerobic life?
Antoine Carlioz and Danide Touati
Institut Jacques Monod, CNRS, Universite Paris 7, 2 Place Jussieu, 75251
Paris Cedex 05, France
Communicated by M.Radman
Mu transposons carrying the chloramphenicol resistance
marker have been inserted into the cloned Escherichia coli
genes sodA and sodB coding for manganese superoxide
dismutase (MnSOD) and iron superoxide dismutase (FeSOD)
respectively, creating mutations and gene fusions. The
mutated sod4 or sodB genes were introduced into the bacterial
chromosome by allelic exchange. The resulting mutants were
shown to lack the corresponding SOD by activity measurements and immunoblot analysis. AerobicaDly, in rich medium,
the absence of FeSOD or MnSOD had no major effect on
growth or sensitivity to the superoxide generator, paraquat.
In minimal medium aerobic growth was not affected, but the
sensitivity to paraquat was increased, especially in the sodA
mutant. A sodA sodB double mutant completely devoid of
SOD was also obtained. It was able to grow aerobically in
rich medium, its catalase level was unaffected and it was
highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose
medium. Growth could be restored by removing oxygen, by
providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that
the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.
Key words: conditional aerobic survival/E. coli mutants/oxygen
toxicity/superoxide dismutase
Introduction
Oxygen toxicity has been reported in various species (Gottlieb,
1971; Wolfe and De Vries, 1975). It has been shown to be
mediated by products resulting from univalent reduction of
molecular oxygen, including the superoxide radical (O°2),
hydrogen peroxide (H202) and the hydroxyl radical (OH-)
(Fridovich, 1983; Halliwell and Gutteridge, 1984). Superoxide
dismutase (SOD), which catalyzes the dismutation of superoxide radicals, thus appears as the first line of defence against oxygen toxicity. A wide variety of deleterious effects produced by
exposure to O' are diminished or eliminated by SOD (Fridovich,
1983). Therefore, SOD, found in almost all organisms exposed
to oxygen, might be expected to be essential for aerobic life
(Fridovich, 1978).
Escherichia coli, like many Gram-negative bacteria, contains
two SODs: an iron superoxide dismutase (FeSOD, Yost and
Fridovich, 1973) and a manganese superoxide dismutase
(MnSOD, Keele et al., 1970). No specific cellular localization
of these isoenzymes has been demonstrated and there is no
evidence that they have different biological roles. However, their
expression in the cell is different. FeSOD is synthesized
anaerobically and aerobically at a constant rate (Hassan and
©) IRL Press Limited, Oxford, England
Fridovich, 1977a), whereas MnSOD is not synthesized
anaerobically and is induced by exposure to oxygen (Hassan and
Fridovich, 1977b) or O' (Hassan and Fridovich, 1979).
We have previously described the cloning of the E. coli structural genes for MnSOD (sodA; Touati, 1983) and FeSOD (sodB;
Sakamoto and Touati, 1984). We report here the isolation of sod4
and sodB mutations on these plasmids, the transfer of the mutations to the E. coli chromosome and the physiological
characterization of the resulting mutants lacking MnSOD, FeSOD
or both enzymes. Preliminary results have been reported
elsewhere (Touati and Carlioz, 1986).
Results
Construction of sodA and sodB mutants
Insertions of Mu transposons in the sodA and sodB genes. Mutations were made in the sodA and sodB genes by insertion of Mu
transposons. MudIIPR13 was inserted into pDT1-5, a plasmid
which carries the sodA gene and overproduces MnSOD about
8-fold. MudIIPR3 was inserted into pHSI-7, a plasmid which
carries the sodB gene and overproduces FeSOD about 15- to
20-fold. Both plasmids carry the bla gene, which confers
resistance to ampicillin.
The procedure for insertion into plasmids was as described by
Castilho et al. (1984). Strain MC4100 caryring a Mucts prophage
and either MudIIPR3 or MudIIPR13 was rendered recA (see
Table I) to minimize the formation of plasmid multimers before
transformation at 30°C with pDT1-5 or pHSl-7. Mu transposition and phage growth were induced at 42°C, and the resulting
lysate was used to transduce strain M8820Mu to AprCmr, thus
selecting for plasmids carrying a Mud transposon. AprCmr
clones were further scored for protein fusion phenotypes: Lac+
or Kmr. Among the various classes (CmrApr, CmrAprLac+,
CmrAprKmr), clones carrying insertions into sod genes were
detected as those in which the SOD level had returned to the
wild-type SOD level (by immunoprecipitation assays on crude
extracts, confirmed by SOD assays).
The characterization was completed by establishment of a
restriction map. The insertion sites were compared to the location of the sod genes, as determined by subcloning (Figure la
and b). Subcloning and fusions showed that the middle of the
sodA gene is at the PstI site on the EcoRI-BamHI fragment carried by pDT1-5, with a transcription direction from EcoRI
towards BamHI, and the beginning of the sodB gene is between
the BamHI and EcoRI sites on the DNA fragment carried by
pHS1-7, at about 0.6 kb from the EcoRI site, with transcription
from BamHI towards EcoRI.
The studies described below were done starting from
pDTl-54)(sodA-lacZ)49 (Figure la) and pHSl-74)(sodB-kan)1-A2
(Figure lb). Use of other insertions gave similar results (not
shown). pDT1-54(sodA-lacZ)49 confers an AprCmrLac+
phenotype. It carries the complete MudI 1PR13 transposon and
a fusion which puts the ,B-galactosidase gene expression under
sodA control (in preparation). pHSI-7cI(sodB-kan)1-A2 confers
an AprKmr phenotype. Kanamycin resistance is expressed under
623
A.Carlioz and D.Touati
(a )
SOD
10 kb
overproduction
hicu
4
-C
.0
la
.9
0
pp
pp
P
B
I
I
f-
.1
~
zB
E
E
1
d
Ci
-
pST1-0
-I-
pST1-5
none
pDT1-5
p DT1t6
none
pDT1-8
-
E
B
E
B
--
B
- --
A
Mu
Z
LB
ya
none
f(sodA-lacZ49
none
pDT15 (sodA::MudPRI3J
none
I (sodA_IacZ )60
none
pDT15
B
E
pDl1JO
pDT15
\Mu%
lac
(b )
SOD
overproduction
5 kb
-1
*4
sod B
r EfeAPt
r
EjP2
r2rP.
p
Ef P2
3 p
p
B
I
Mu
ir-
B
Ejf_
Ek!:.2..
EUS
p___
I
Iant Mu
_;
Ika
C
S.
a
I
_
III"
Ev
pHS 1L8
F
p
5P
p
pHS14
p
p
N
--_
_,
PHS 1L6
-F
El
kan- EjC._
I JrV2
cat kan
t
-0-
pHS 1L2
none
pHS 1-3
none
pHS-17J9 (sod B.kan)l
none
:|B
-
p~~
c
a
pHS IJ
1.7 t(sodB-kan:)l2
none
pHS I 7(sodB:: MUdBR3)i
none
p HS
3
Fig. 1. Restriction map of plasmids carrying sod genes and insertions into sod genes. (a) Plasmids carrying the sodA gene; (b) plasmids carrying the sodB
pHC79
gene. Restriction endonuclease cleavage sites are indicated as follows: EcoRI, E; BamHI, B; PstI, P; Pvul, P2; HpaI, H; ClaI, C. Symbols:
Mu transposon DNA;
bacterial DNA. SOD was measured in crude extracts; overproduction was at least 4-fold above the total SOD
DNA;
activity in wild-type. The location of the pstl-O restriction map of cpxA, pfJA, tpi and glpK genes was deduced from the data of Albin and Silverman (1984),
Shimosaka et al. (1982) and Conrad et al. (1984), and from complementation tests for rha, plA and tpi genes.
,
,
624
E. colb SOD mutants: conditional aerobic survival
Table I. E. coli K12 bacterial strains
Bacterial
strain
Relevant characteristics
Reference
MC4100
QC756
QC720
QC759
QC721
M8820 Mu
TS214
HVCI
QC748
CGS5263
GC4468
QC772
QC773
QC774
LCB18
F- araDJ39 A(IacIPOZYA-argF)U169 rpsL thi
MC4100 MuCts, MudIIPR3
MC4100 MuCts, MudIIPR13
QC756 srl::TnJO recA
QC720 srl::TnJO recA
F- araDJ39 A(ara-leu)7697 A(proAB-argF-lacIPOZYA)XEII rpsL Mu c+
F- leu-6 thy his-i arg metBI lacYI xyl-7 rpsLJ04 polAts214
TS214 cured of ColEl
Castilho et al. (1984)
P.Ratet and F.Richaud
P.Ratet and F.Richaud
srl::TnlO recA transduction from LCB18 into QC756
srl::TnJO recA transduction from LCB18 into QC720
Castilho et al. (1984)
D.Helinski
A.Goze
Obtained by cross between HVC1 and HfrH 3000 XIII
Bachmann and Low (1980)
R.D'Ari
This work
This work
This work
Chippaux et al. (1982)
F-A(proAB-argF-1acIPOZXYA)XIII his' polAts214
HfrH 3000 XIII PO1 thi-J relAl spoTI X- A(proAB-argA-lacIPOZYA)XJII
F- Alac4169 rpsL
GC4468 4(sodA-lacZ)49 Cmr Lac+
GC4468 4(sodB-kan)1-A2 Kmr
GC4468 (b(sodA-lacZ)49 4)(sodB-kan)1-A2 Cmr Kmr
F-pyrD recA srl:TnlO rpsLicos srl+AprrecA+
sodB control; the Tn9 part of MudIIPR3 and the major part of
the sodB gene have been deleted.
Allele exchange between the mutated sod gene on the plasmid
and the wild-type chromosomal allele. This was carried out
following the rationale described by Gutterson and Koshland
(1983) using a polA strain, unable to replicate ColEl-like
replicons extrachromosomally.
A polA(Ts) strain deleted from the lactose operon was constructed (see Table I) and transformed at 30°C with
pDTl-54(sodA-lacZ)49 or pHSl-7b(sodB-kan)l-A2 selecting
AprCmr or AprKmr, respectively. Transformants were inoculated
at 30°C in LB containing antibiotics and at an OD600 of 1 shifted
to 42°C for the night to select for plasmid integrates. Overnight
cultures were then diluted 1/100 in prewarmed 42°C medium
containing antibiotics and grown to saturation before plating at
42°C on LB containing antibiotics. After purification at 420C
colonies were inoculated at 30°C without selective pressure,
grown for a few generations for resolution of integrated plasmids
and used for P1 lysates. The prototroph strain GC4468, which
is deleted for the lactose operon, was then transduced, selecting
for Cmr or Kmr at 37°C. Ampicillin-sensitive transductants
(which have lost vector sequences and carry an altered
chromosomal segment) were analysed for SOD content.
Characterization of sodA and sodB mutants
There is no MnSOD activity in the sodA mutant, and the lack
of MnSOD activity is not compensated for by an increase in
FeSOD activity, even in the presence of excess superoxide
radicals induced by paraquat as shown on electrophoretic gel
stained for SOD activity (Figure 2). Direct SOD activity
measurements (units/mg of protein) gave the same results (not
shown). Similarly there is no FeSOD in the sodB mutant, and
the lack of FeSOD activity is not compensated for by an increase
in MnSOD activity. In the presence of paraquat MnSOD is induced as in wild-type.
When cells are grown anaerobically MnSOD is not expressed. In the sodB mutant, no SOD activity was detectable under
these conditions (Figure 5a). Similar results were obtained by
immunoblotting analysis (Figure 5b), showing that no inactive
SOD antigen is present. Staining of protein subunits on Coomassie
Fig. 2. SOD activity in sod4 and sodB mutants. Cells were grown as
described in Materials and methods to an OD of 1.5 for crude extracts.
Crude extract (30 Al; about 30 1tg protein) was loaded on a non-denaturing
7% polyacrylamide gel, run under 25 mA constant current, then stained for
SOD activity. Lane a represents cultures without addition of paraquat, lanes
b and c represent cultures grown with 5 x 10-6 M and 5 x I0-5 M
paraquat, respectively, as described in Materials and methods. Symbols:
MnSOD, Mn; HySOD, Hy; FeSOD, Fe.
blue-coloured gel (Figure 5c) confirmed the disappearance of
MnSOD or FeSOD subunits.
Sensitivity of sodA and sodB mutants to paraquat
In rich medium the sodB mutant exhibits the same sensitivity to
paraquat as the wild-type strain, whereas the sodA mutant showed
slightly increased sensitivity (Figure 3). Bacterial survival curves
were similar to optical density curves for all three strains (not
shown).
625
A.Carlioz and D.Touati
6 hours
Fig. 3. Paraquat sensitivity of sodA and sodB mutants in rich medium. Paraquat was added to exponential cultures at 0,0 M; A, 5 x 10-6 M; M,
5 x 10-5 M; O, 10-4 M, V, 2.5 x 10-4 M; 0, 10-3 M (final concentration).
ODsoOnm
sad B
sod A
Fig. 4. Paraquat sensitivity of sodA and sodB mutants in glucose minimal medium. Paraquat was added to exponential cultures at 0, 0 M; 0, 10-7 M;
*, 3x10-7M; V, 10-6 M; A, 3 x 10-6 M; *, 10- M; 0, 3 x 10-5 M (final concentration).
In minimal medium, on the other hand, the mutants were more
sensitive to paraquat (Figure 4). A concentration of 10-5 M
paraquat, which had little effect on the growth of the wild-type
strain, completely inhibited growth of the sodB mutant, and in
the sodA mutant a concentration as low as 3 X 10-7 M was sufficient to inhibit growth almost completely.
Isolation of a sodA sodB double mutant
To construct a sodA sodB double mutant, the 4D (sodB-kan) I-A2
allele was P1 tunsduced into the 4c(sodA-lacZ)49 mutant. Plating
was done immediately after transduction in both aerobic and
anaerobic conditions: transductants were obtained in both conditions, they were all Cmr and after further studies were shown
to be identical.
One such presumptive sod4 sodB mutant was analysed for SOD
activity, in aerobic and anerobic growth conditions. No SOD activity could be detected either by direct SOD measurements (not
shown) or by revelation on electrophoretic gels (Figure 5a), and
no SOD protein was detectable on SDS electrophoretic gels
(Figure 5c) or by immunoblotting analysis (Figure 5b).
Physiological properties of the sodA sodB double mutant
The double mutant formed small colonies on rich medium under
626
aerobic conditions, although colonies were the same size as wildtype under anaerobic conditions. The anaerobic growth rate of
the double mutant was the same as that of wild-type (not shown),
whereas the doubling time was almost twice that of wild-type
in aerobic growth in rich medium (Figure 6a). Sensitivity to paraquat (Figure 6a) or oxygen (Figure 6b) was dramatically increased. Introduction of a plasmid overproducing MnSOD (Figure 6c)
or FeSOD (not shown) restored a normal growth rate and paraquat resistance. To determine whether lack of SOD was compensated for by an increase in catalase level, the latter was
measured and found, for all three mutants, to be the same as
for wild-type (not shown).
In Figure 7 the hydrogen peroxide sensitivity of the different
mutants is shown. The double mutant was hypersensitive to this
oxidizing agent. A challenge with 2.5 mM H202, which has little or no effect on wild-type or single mutants, gave only 1 %
survival with the sodA sodB double mutant after 20 min. At
5 mM the single mutants, especially sodA mutants, are more sensitive to H202 than the wild-type. Moreover, in sodA sodB
mutants, the surviving colonies were very heterogeneous in size.
In glucose minimal medium, the double mutant was totally
unable to grow in aerobic conditions, although growth was as
-. g.a
+
A
B
E. coh SOD mutants: conditional aerobic survival
-
-
-
-
*,,,,
s::
-
Fe
MM
Ai
,.
-
| zFt -|
B
.
(a'
Fe
A
Mn
.!.
i. ':t
-
l
+A B
li
13~1.-ttit
.,
_ aerobic
Mn
:1-
-
*'t .; ',. ,. #, . ,
S S S
F
AB
-
Mn
!tssP'' ;.0.
:-s K
B
I
C n
S
A
AB
+
A
.XA jIB jAB
j
IiiI1
A B AB
ft-I
AS
4414444
'X
1
iii'
_
nSOD af3
(c)
_.aroi
MnSOD-...
F
a
4m
SOD_-.-
qb
aerobic
----
-
Am
_.. anaerobic
..... so,
ied
...
;q-
"
PIR
l,,
$4
'f a
1
)Ia1r1i
( b)
Fig. 5. Crude extract of aerobic and anaerobic cultures of sod mutants analysed by polyacrylamide gel electrophoresis. Symbols + A, B and AB represent
respectively the wild-type strain, sodA, sodB and sodA sodB mutants; Fe and Mn represent purified FeSOD and MnSOD protein. In a, 7% non-denaturing
polyacrylamide disc gels were stained to reveal SOD activity. Last lane shows wild-type control grown in aerobic conditions run with anaerobic samples. In b
and c, proteins were analysed on a sodium dodecyl sulphate 15% polyacrylamide gel, run for 3 h under 40 mA constant current. (b) proteins were transferred
to a nitrocellulose filter which was incubated with FeSOD and MnSOD antibodies; (c) Coomassie blue stained proteins immediately after electrophoresis.
Labelling of immunoprecipitated proteins was achieved by incubation with iodinated protein A and b represents the autoradiography of immunoprecipitated
proteins. The upper band present in all crude extract lanes corresponds to a contaminating antibody present in the FeSOD antibody serum (which was raised
against partially purified FeSOD protein). This contaminant does not appear with more purified FeSOD protein.
for wild-type in anaerobic conditions. Addition of the 20 amino
acids restored growth in minimal medium (Figure 8). To determine whether all 20 amino acids were necessary, minimal media
containing 19 amino acids were used. Growth was completely
inhibited only when leucine, valine or isoleucine was lacking.
However, a medium supplemented with these three amino acids
and pantothenate was not sufficient to restore growth (Figure 8).
Various degrees of inhibition were observed in the absence of
other amino acids. Boehme et al. (1976) found that the biosynthesis of 10 amino acids was sensitive to hyperbaric oxygen; addition of these 10 amino acids, however, did not restore growth
as completely as addition of all 20 amino acids (Figure 8).
When a plasmid causing overproduction of MnSOD or FeSOD
was introduced into the double mutant, aerobic growth on
minimal medium was completely restored, as shown by plating.
Discussion
To study the role and regulation of SOD we isolated mutants
in the structural genes of E. coli SOD, using the cloned genes.
The procedure chosen to isolate mutants - insertion of Mu
transposons able to create fusions - permitted us to localize the
sod genes on restriction maps and determine their direction of
transcription, and furnished tools for improving our understanding of SOD regulatory mechanisms (in preparation).
The presence of two isoenzymes in numerous microorganisms
raised the question of why an organism produces more than one
superoxide dismutase. Different functions related to distinct
localization of the enzymes has been suggested but has not found
support to date (Britton and Fridovich, 1977). Another explanation was suggested by the finding that FeSOD is produced constitutively, even in anaerobic conditions: FeSOD would provide
a constant standby defence against oxygen toxicity, while
MnSOD, which is inducible by oxygen, would adjust the response
to the challenge (Fridovich, 1983). The physiological properties
of sodA and sodB mutants are in favor of an adaptative value
of MnSOD rather than a specialized function. Indeed, in usual
627
A.Carlioz and D.Touati
(a)
Fig. 6. Aerobic growth of the sodA sodB mutant in rich medium: (a) in the presence of paraquat; (b) in the presence of excess oxygen; (c) in the presence of
an MnSOD overproducing plasmid and paraquat. (a) Parquat was added to an exponential culture at 0, 0 M; A, 5 x 10-6 M; *, 5 x 10-5 M, OI,
10-4 M; V, 2.5 x 10-4 M (final concentration). Dotted lines represent the wild-type strain. (b) Oxygen was bubbled through exponential cultures: 0, wildtype; 0, sodA; o, sodB, *, sodA sodB mutants. Dotted lines represent cultures grown in aerobic conditions without oxygen bubbling. (c) 0, 0 M or E
5 X 10-5 M paraquat was added to an exponential culture of the so&4 sodB (pDTI-5) strain. Dotted lines represent the sodA+ sodB+ strain in the same
conditions.
Minutes
after H20, Challenge
Fig. 7. Sensitivity of sod mutants to hydrogen peroxide. Symbols: *, wild0, sodA; *, sodB; A, sodA sodB.
type;
Fig. 8. Growth of the sodA4 sodB mutant in glucose minimal media. Preculture in glucose minimal medium supplemented with the 20 amino acids
was diluted in glucose minimal medium with supplements
A, none, or
all amino acids but Leu, Ile or Val; *, all amino acids; O], all but
aromatic amino acids; V, Asp, Cys, Ile, Leu, Met, Phe, Thr, Try, Tyr
and Val; 0, Leu, Ile, Val and pantothenate.
-
628
aerobic conditions the two mutants, which have about the same
SOD activity, show no difference in growth in rich or minimal
medium. Under oxidative stress (exposure to paraquat, to H202),
however, the sodA mutant, unable to increase its SOD level,
shows increased sensitivity compared to wild-type, whereas the
sodB mutant is affected only in drastic conditions.
It is noteworthy that half of the usual SOD activity is sufficient to ensure normal aerobic growth, as seen with both mutants.
Here again the cells seem to have a margin of safety over and
above the minimal SOD activity necessary, as in the case of
anaerobes that have SOD (Hewitt and Morris, 1975; Gregory
et al., 1978).
Superoxide dismutase has been found in almost all oxygentolerant organisms, although some exceptions are known for
which no detoxification replacement mechanisms have been
demonstrated (Lynch and Cole, 1980). By isolation, in aerobic
conditions, of an E. coli mutant completely devoid of SOD, we
have shown that superoxide dismutase is not strictly necessary
for aerobic survival in this organism. However, the slow growth
of the sodA sodB mutant in rich medium and its low saturation
level suggest serious damage to the cells. Further studies are
necessary to determine whether this reflects altered metabolism
in all or only part of the population.
The controversy over direct toxicity of the superoxide radical,
based on its poor reactivity, has been lively (Fee, 1980; Sawyer
and Valentine, 1981; Fridovich, 1983). The mechanism which
generates highly reactive OH- radicals from O° and H202 has
been extensively discussed (see Halliwell and Gutteridge, 1984,
for review). Recent data on FefIm reduction by °2 (Bielski and
Cabelli, 1986) support the idea of a metal-mediated in vivo
generation of OH- radicals from H202 by °2 (a metal-catalyzed
Haber-Weiss reaction). The hypersensitivity of the sodA sodB
double mutant to H202 in rich medium, with a concomitant normal catalase level, suggests that part of the damage attributed
to °2 must be mediated by OH- radicals formed in the presence
of H202 and O°, whatever the mechanism of their formation.
The molecular nature of the lethal effects observed remains to
be determined.
On the other hand, the double mutant's inability to grow
aerobically in minimal medium, if not supplemented by the 20
amino acids, show a drastic effect of °2 (or derivatives) on
E. col SOD mutants: conditional aerobic survival
amino acid biosynthesis. A sensitivity of amino acid biosynthesis
was previously reported (Boehme et al.,
1976). Brown and Yein (1978) observed particular sensitivity
of the branched amino acids and showed that dihydroxyacid
dehydratase, a common enzyme in the biosynthetic pathway of
these three amino acids, was rapidly inactivated by exposure of
E. coli to hyperbaric oxygen. The same effect was obtained with
paraquat (Fee et al., 1980; Brown and Seither, 1983). The complete growth inhibition of the sodA sodB mutant in minimal
medium supplemented with all amino acids but leucine or valine
presumably results from the same event.
The peculiar sensitivity of branched amino acid biosynthesis
may reflect a specific role of this pathway in the detection of
oxidative stress. However, it appears that the biosynthesis of all
amino acids is affected to various degrees. Thus the inactivation
of dihydroxyacid dehydratase may simply reflect a general process of protein denaturation. tRNA modifications could also be
involved: Buck and Ames (1984) described a tRNA adenosine
residue (A37) whose modification is dependent on oxygenation.
This modification is known to play a role in the regulation of
biosynthesis of those amino acids having A37 in the corresponding tRNA (Phe, Tyr, Tyr, Ser, Leu, Cys).
All specific deleterious effects observed in sod mutants have
been shown to be directly dependent on exposure to oxygen or
O2j. Those tested were reversed by SOD. Although an E. coli
mutant devoid of SOD activity can survive aerobically, its
physiological properties described above lead to the conclusion
that it would be rapidly eliminated under conditions of oxidative
stress. It would be further handicapped during the course of
evolution by its associated amino acid auxotrophies.
Further studies with the sodA sodB mutant should permit us
to discriminate among the numerous effects which have been attributed to O2 and to progress in our knowledge of the molecular
basis of oxygen toxicity in vivo.
to hyperbaric oxygen
Materials and methods
General genetic methods (phage stocks, P1 transduction), gel electrophoresis,
standard DNA cloning procedures (DNA preparation, DNA digestion, agarose
gel electrophoresis, transformation) have been described previously (Miller, 1972;
Davis et al., 1980; Maniatis et al., 1982). Methods for handling bacteriophage
Mu transposition and infection were as described by Casadaban and Cohen (1979).
Bactena and plasmids
The E. coli K12 strains used are described in Table I. Defective Mu prophages,
MudIIPR3 (submitted) and its derivative MudIIPR13 were constructed by P.Ratet
and F.Richaud. They can transpose only if complemented by wild-type Mu; they
carry a complete cat gene from Tn9 which confers the resistance to chloramphenciol, and either the kanamycin or lactose operon, lacking the first few codons
and upstream regulatory elements. They can create gene fusions between the control
elements of the gene into which insertion occurs and the coding region of the
neomycin phosphotransferase or I3-galactosidase gene. These fusions can confer
a kanamycin-resistant or Lac' phenotype. Transposition events which do not
create active fusions can be detected by resistance to chloramphenicol. Various
plasmids are represented in Figure 1.
Growth media
Rich medium was LB and minimal medium was M63 supplemented with 0.4%
glucose and thiamin (1 Ag/mi) (Miller, 1972). Required amino acids were added
at 0.5 mM.
Growth conditions
Pre-cultures in LB or M63 glucose inoculated from single colonies were grown
at 37°C, in the presence of appropriate antibiotics when carrying resistance markers
(Cm 20 Ag/ml; Ap 500 jig/ml in liquid medium and 50 iLg/ml in solid medium;
Tc 10 jg/ml; Km 40 tsg/ml), to an OD of 1, at which point they were rapidly
chilled in a water-ice mixture and kept in the cold for a maximum of 40 h.
Cultures were inoculated from pre-cultures into the same pre-warmed medium
without antibiotics (unless otherwise specified) at an OD of about 0.02. When
used, paraquat (PQ2+) was added when the OD reached about 0.2. Cultures were
shaken (200 r.p.m. in a rotary bath) and culture volume did not exceed one-tenth
of the total Erlenmeyer volume to ensure good aeration. Growth (unless otherwise specified) was monitored by OD at 600 nm. Anaerobic conditions were obtained in a Forma Scientific anaerobic station and 1 % glucose was added to media.
Growth in oxygen was obtained by continuous bubbling of oxygen in the medium
at a pressure of one bar.
Hydrogen peroxide treatment
Cells were grown in LB medium at 37°C to an OD of 0.5 and treated with
hydrogen peroxide for 60 min. Aliquots were withdrawn, diluted in ice-cold saline
buffer and plated on LB plates at 370C to monitor cell viability.
Crude extracts and assays
Crude extracts were prepared by cycles of freezing and thawing as previously
described (Touati, 1983), except that 5 x 10-2 M phosphate bufer, pH 7.8, was
used for washing and extraction (with 0.2 mg/ml lysozyme and 10-4 M EDTA). An alternative method was disruption of the cells by ultrasonic treatment
as described by Gregory and Fridovich (1973), this method gave similar results
to the former. Crude extracts from anaerobic cultures were prepared in the
anaerobic chamber, except for centrifugation and freezing and thawing, which
were performed in sealed tubes.
SOD activity was assayed by the Beauchamp and Fridovich method (1971)
slightly modified (Maral et al., 1977): SOD isoenzymes were separated by electrophoresis on -non-denaturing polyacrylamide gels and visualized using an activity stain (Beauchamp and Fridovich, 1971). Catalase activity was determined
by spectrophotometric methods as described previously (Beers and Sizer, 1952)
and protein was estimated by the method of Lowry et al. (1951).
Immunological assays and immunoblot analysis ('Western blotting') (Burnette,
1981) were as described previously (Touati, 1983; Sakamoto and Touati, 1984).
Acknowledgements
We thank P.Ratet for the gift of Mu transposon before publication and R.D'Ari
for his friendly, enthusiastic support during this work and careful reading of the
manuscript. This research was supported by ATP no. 96 0114 from the Centre
de la Recherche Scientifique.
References
Albin,R. and Silverman,P.M. (1984) Mol. Gen. Genet., 197, 261-271.
Bachmann,B. and Low,K.B. (1980) Microbiol. Rev., 44, 1-56.
Beauchamp,B. and Fridovich,I. (1971) Anal. Biochem., 44, 276-287.
Beers,R.F.,Jr. and Sizer,I.W. (1952) J. Biol. Chem., 195, 133-140.
Bielski,B.H. and Cabelli,D.E. (1986) Fourth Internaional Conference on Superoxide and Superoxide Dismutase. Elsevier Biomedical Press, in press.
Bielski,B.H. and Cabelli,D.E. (1986) In Geelin,J. (ed.), Fourth Interational Conference on Superoxide and Superoxide Dismutase. Elsevier Biomedical Press,
in press.
Boehme,D.E., Vincent,K. and Brown,O.R. (1976) Nature, 263, 418-420.
Britton,L. and Fridovich,I. (1977) J. Bacteriol., 131, 815-820.
Brown,O.R. and Yein,F. (1978) Biochem. Biophys. Res. Commun., 85,
1219-1224.
Brown,O.R. and Seither,R.L. (1983) Fund. Appl. Toxicol., 3, 209-211.
Buck,M. and Ames,B.N. (1984) Cell, 523-531.
Burnette,W.N. (1981) Anal. Biochem., 112, 195-203.
Casadaban,M.J. and Cohen,S.N. (1979) Proc. Natl. Acad. Sci. USA, 76,
4530-4533.
Castilho,B.A., Olfson,P. and Casadaban,MJ. (1984) J. Bacteriol., 158, 488-495.
Chippaux,M., Feutrier,J., Lepelletier,M., Touati-Schwartz,D. and Pascal,M.C.
(1982) Biochem. Biophys. Res. Commun., 106, 1269-1271.
Conrad,C., Stearns,G., Prater,W., Rheiner,J. and Johnson,J. (1984) Mol. Gen.
Genet., 193, 376-378.
Davis,R.W., Botstein,D. and Roth,J.R (eds) (1980) A Manual for Genetic
Engineering. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Fee,J.A. (1980) Dev. Biochem., llB, 41-48.
Fee,J.A., Lees,A.C., Block,P.L., Gilliland,P.L. and Brown,O.R. (1980) Biochem
Int., 1, 304-311.
Fridovich,I. (1978) Photochem. Photobiol., 28, 733-741.
Fridovich,I. (1983) Annu. Rev. Pharmacol. Toxicol., 23, 239-257.
Gottlieb,S.F. (1971) Annu. Rev. Microbiol., 25, 111-152.
Gregory,E.M. and Fridovich,I. (1973) J. Bacteriol., 114, 543-548.
Gregory,E.M., Moore,W.E.C. and Holdeman,L.V. (1978) Appl. Environ.
Microbiol., 35, 988-991.
Gutterson,N.I. and Koshland,D.E.,Jr. (1983) Proc. Natl. Acad. Sci. USA, 80,
4894-4898.
Halliwell,B. and Gutteridge,J.M.C. (1984) Biochem. J., 219, 1-14.
Hassan,H.M. and Fridovich,I. (1977a) J. Biol. Chem., 52, 7667-7672.
Hassan,H.M. and Fridovich,I. (1977b) J. BacterioL, 129, 1574-1583.
Hassan,H.M. and Fridovich,I. (1979) Arch. Biochem Biophys., 196, 385-395.
629
A.Carlioz and D.Touati
Hewitt,J. and Morris,J.G. (1975) FEBS Lett., 50, 315-317.
Keele,B.B.,Jr., McCord,J.M. and Fridovich,I. (1970) J. Biol. Chem., 245,
6176-6181.
Lowry,O.H., Rosebrough,N.J., Farr,A.L. and Randall,R.J. (1951) J. Biol.
Chem., 193, 265-275.
Lynch,R.E. and Cole,B.C. (1980) Biochem. Biophys. Res. Commun., 96,
98-105.
Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning. A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Maral,J., Puget,K. and Michelson,A.M. (1977) Biochem. Biophys. Res. Commun., 77, 1525-1535.
Miller,J.H. (1972) Experiments in Molecular Genetics. Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY.
Nettleton,J.C., Bull,C., Baldwin,T.O. and Fee,J.A. (1984) Proc. Natl. Acad.
Sci. USA, 81, 4970-4973.
Sakamoto,H. and Touati,D. (1984) J. Bacteriol., 159, 418-420.
Sawyer,D.T. and Valentine,J.S. (1981) Acc. Chem. Res., 14, 393-400.
Shimosaka,M., Fukuda,Y., Murata,K. and Kimura,A. (1982) J. Bacteriol., 152,
98-103.
Touati,D. (1983) J. Bacteriol., 155, 1078-1085.
Touati,D. and Carlioz,A. (1986) In Geelin,J. (ed.), Fourth International Conference on Superoxide and Superoxide Dismutase. Elsevier Biomedical Press,
in press.
Wolfe,W.G. and De Vries,W.C. (1975) Annu. Rev. Med., 26, 203-217.
Yost,G.J. and Fridovich,I. (1973) J. Biol. Chem., 248, 4905-4908.
Received on 6 December 1985
630