Critical protective role of bacterial superoxide dismutase in Rhizobium–legume symbiosis

Molecular Microbiology (2000) 38(4), 750±759

Critical protective role of bacterial superoxidedismutase in Rhizobium±legume symbiosis

Renata Santos,1 Didier HeÂrouart,2 Alain Puppo2 and

DanieÁle Touati1*1Laboratoire de GeÂneÂtique MoleÂculaire des ReÂponses

Adaptatives, Institut Jacques Monod, CNRS-UniversiteÂs

Paris 6 and Paris 7, 2 place Jussieu 75251 Paris cedex

05, France.2Laboratoire de Biologie VeÂgeÂtale et Microbiologie,

CNRS ESA 6169, UniversiteÂ de Nice-Sophia Antipolis,

06108 Nice cedex 02, France.

Summary

In nitrogen-poor soils, rhizobia elicit nodule forma-

tion on legume roots, within which they differentiate

into bacteroids that fix atmospheric nitrogen. Protec-

tion against reactive oxygen species (ROS) was

anticipated to play an important role in Rhizobium±

legume symbiosis because nitrogenase is extremely

oxygen sensitive. We deleted the sodA gene encod-

ing the sole cytoplasmic superoxide dismutase (SOD)

of Sinorhizobium meliloti. The resulting mutant, defi-

cient in superoxide dismutase, grew almost normally

and was only moderately sensitive to oxidative stress

when free living. In contrast, its symbiotic properties

in alfalfa were drastically affected. Nitrogen-fixing

ability was severely impaired. More strikingly, most

SOD-deficient bacteria did not reach the differentiation

stage of nitrogen-fixing bacteroids. The SOD-deficient

mutant nodulated poorly and displayed abnormal

infection. After release into plant cells, a large number

of bacteria failed to differentiate into bacteroids and

rapidly underwent senescence. Thus, bacterial SOD

plays a key protective role in the symbiotic process.

Introduction

Symbiosis with rhizobial bacteria enables legumes to use

atmospheric nitrogen fixed by the microsymbiont. Effect-

ive symbiosis requires a series of co-ordinated recognition

and development steps in which molecular signals are

exchanged between the two partners (reviewed by

Mylona et al., 1995; Geurts and Franssen, 1996; Long,

1996; Bladergroen and Spaink, 1998; Schultze and

Kondorosi, 1998). Bacteria respond to a plant signal

(flavonoids) by producing nod factors, which elicit nodule

organogenesis (Peters et al., 1986; Truchet et al., 1991;

Fisher and Long, 1992; Schultze and Kondorosi, 1996).

They enter the developing nodule via an infection thread

and are released into the nodule primordia cells,

surrounded by a membrane of plant origin, the peribac-

teroid membrane (PBM). Once released into the host

cytoplasm, they stop dividing and undergo differentiation

into nitrogen-fixing bacteroids. The release of the bacteria

triggers the expression of a specific set of nodule-

associated plant genes, encoding the late nodulins,

which are necessary for bacteroid differentiation and

nitrogen fixation. As nitrogen-fixing activity drops, bacter-

oids and plant nodule cells degenerate and undergo

senescence.

In the nodule, maintenance of nitrogenase activity is

subject to a delicate equilibrium. A high rate of respiration

is necessary to supply the energy demands of the

nitrogen reduction process, but oxygen and reactive

oxygen species (ROS) irreversibly inactivate the nitro-

genase complex (Buchanan, 1977; Robson and Postgate,

1980). A diffusion barrier in the cortex of nodules greatly

limits permeability to oxygen, and the necessary oxygen is

delivered by the plant oxygen carrier, leghaemoglobin

(Fischer, 1996), present exclusively in the nodule. Despite

these strategies ensuring a low free oxygen concentra-

tion, the high rate of respiration inevitably results in there

being large amounts of ROS (Halliwell and Gutteridge,

1989) in the nodule. Therefore, efficient protection against

oxidative stress is thought to be necessary for efficient

nitrogen fixation and to delay senescence. Previous

studies have suggested that bacterial superoxide dis-

mutase (SOD) plays a critical role in protecting the

nitrogen fixation process (Puppo and Rigaud, 1986).

SODs are metalloenzymes that detoxify superoxide

(O2´2), the first radical produced in the ROS cascade.

They play a key role in protection against oxidative stress.

Although O2´2 is not extremely reactive per se (it cannot

attack DNA and does not cause lipid peroxidation

directly), it inactivates [4Fe24S] cluster-containing

enzymes releasing iron, thereby increasing the intracel-

lular free iron pool (Flint et al., 1993). This favours the

Fenton reaction (H2O2 1 Fe21 ! Fe31 1 OH´ 1 OH2),

resulting in the production of the highly reactive hydroxyl

radical (OH´), which can damage any biological macro-

molecule (Liochev and Fridovich, 1994; Keyer and Imlay,

1996; McCormick et al., 1998). O2´2 also reacts with nitric

Q 2000 Blackwell Science Ltd

Accepted 6 September, 2000. *For correspondence. E-mail [email protected]; Tel. (133) 1 44 27 47 19; Fax (133) 1 44 27 76 67.

oxide (NO´) to generate the deleterious peroxynitrite

(ONOO) (Squadrito and Pryor, 1998). Thus, SODs

safeguard cells from the damage caused by O2´2 and

from indirect injury caused by OH´ and ONOO. In

bacteroids, enzymes such as the dinitrogenase reductase

of the nitrogenase complex contain oxidizable [4Fe24S]

clusters (Howard and Rees, 1994) that are potential

targets for superoxide, although no direct effect has yet

been demonstrated.

We previously isolated the sodA gene encoding the

single Sinorhizobium meliloti Rm5000 cytoplasmic SOD

(Santos et al., 1999). This enzyme was found to be an

atypical `cambialistic' type of SOD, active with manganese

or iron as cofactor. To investigate the role of SOD in

protecting against oxidative stress in the symbiotic

process, we deleted the sodA gene and assessed the

symbiotic properties of the SOD-deficient mutant in

alfalfa. We report here the protective effects of SOD in

free-living and symbiotic conditions. We present evidence

that bacterial SOD is critical for efficient nodulation and

nitrogen fixation and delays senescence. We show that

SOD effects extend far beyond protection of the nitro-

genase complex and that defects are observed in the

mutant at all steps of symbiosis, including infection,

nodulation and bacteroid differentiation.

Results

Growth impairment and sensitivity to paraquat

The growth of the sodA mutant was slightly impaired in

rich medium: doubling time was about 1.2-fold longer and

maximal growth was reduced (almost twofold lower than

wild type, Fig. 1A). Addition of MnTM-2-PyP, a SOD

mimic that penetrates the bacterial membrane (Faulkner

et al., 1994), restored growth to wild-type levels,

demonstrating that the growth defect was caused by

SOD deficiency (Fig. 1A). The mutant was more sensitive

than the wild type to paraquat, a superoxide generator.

Growth of the mutant was significantly inhibited by

100 mM paraquat, whereas 500 mM did not affect the

growth of wild type (Fig. 1A). In minimal medium, growth

rate and maximal growth were significantly reduced for

the mutant (1.8-fold and fourfold lower than wild type

respectively) (Fig. 1B). Addition of the 20 amino acids did

not restore it to wild-type levels, suggesting that O2´2

caused defects in other metabolites.

DNA damage: mutagenesis

Oxidative damage to DNA causes an increase in

spontaneous mutagenesis in SOD-deficient mutants

(Farr et al., 1986; Keyer et al., 1995). The mutation

frequency was recorded in sodA mutants as the number

of rifampicin-resistant colonies in a rifampicin-sensitive

population. The number of Rifr mutants per 108 cells was

7.23 (^ 1.78) in QC3136, the SOD-deficient strain, and

2.08 (^ 0.54) in GMI211, the SOD-proficient strain (t-test;

n � 8, P , 0.001). Thus, the frequency of spontaneous

mutation in the S. meliloti SOD mutant was 3.5 times

higher than that of the wild type, indicating DNA damage.

Sensitivity to hydrogen peroxide and nitric oxide

Another reported consequence of superoxide-mediated

Fenton chemistry in SOD-deficient strains is an increase

in sensitivity to H2O2 (Carlioz and Touati, 1986; Imlay and

Linn, 1987). Surprisingly, the S. meliloti sodA mutant

(QC3132) was more resistant than its SOD-proficient

counterpart (Rm5000) to H2O2 at a wide range of

concentrations (Fig. 2A). We suspected that this could

be caused by the induction of catalase activity by

superoxide. Measurement of total catalase activity in

rich medium revealed a fivefold higher activity in sodA

than in wild type (50.5 ^ 2.4 and 10.9 ^ 0.5 U mg21

protein respectively). Three catalases, the products of

katA, katB and katC, have been described in S. meliloti

(HeÂrouart et al., 1996; Sigaud et al., 1999). In free-living

conditions in minimal medium, the catalase genes are

expressed differentially, with KatA and KatB produced

during exponential growth and KatC production peaking in

late stationary phase. The katA gene is strongly induced

by exposure to H2O2 (HeÂrouart et al., 1996), whereas

katC is not and is instead induced by heat stress, salt

stress, ethanol and paraquat in minimal medium (Sigaud

et al., 1999). The induction of katC by paraquat suggested

that protection against H2O2 killing in sodA may result

from O2´2-induced KatC production. Catalase activities

were analysed by electrophoresis in non-denaturing

polyacrylamide gels of crude extracts isolated from the

sodA mutant and the wild type grown in rich medium

(Fig. 2B). We found that KatA activity was considerably

higher in the SOD-deficient strain than in the wild type

(Fig. 2B), whereas KatB activity was similar in the two

strains. KatC activity, which is not detected in rich medium

in the wild type, was not induced in the sodA mutant

(Fig. 2B). This strongly suggests that protection against

H2O2 in sodA resulted from katA induction. However,

whereas the katA mutant was more sensitive to H2O2 than

the wild type, the sodA katA double mutant was resistant

to 2 mM H2O2 (Fig. 2C), despite the decrease in total

catalase activity (8.9 ^ 0.4 U mg21 protein). This indi-

cates that another type of protection against H2O2, in

addition to KatA production, is triggered in the sodA

mutant.

O2´2 can react with nitric oxide (NO´) to produce the

damaging peroxynitrite (Squadrito and Pryor, 1998). We

therefore assessed the effects of NO´ donors on sodA

Role of bacterial superoxide dismutase in symbiosis 751

Q 2000 Blackwell Science Ltd, Molecular Microbiology, 38, 750±759

survival. Neither S-nitrosoglutathione nor sodium nitro-

prusside affected the viability of the sodA mutant (not

shown).

SOD is critical for efficient nodulation and nitrogen fixation

Upon infection with the sodA mutant, nodulation was

delayed and the final nodule number smaller, although the

nodules formed were the same size as those of wild-type

S. meliloti (Fig. 3, inset). The percentage of nodulated

plants and the nodule number per plant were almost

halved with the mutant strain. The number of new nodules

over 3 weeks was clearly lower for the sodA mutant than

for the wild type, although the first nodules appeared

8 days after infection for both strains (Fig. 4A).

Nitrogenase activity was assayed 4±15 weeks after

inoculation. Almost half the plants that nodulated with the

sodA strain showed no detectable acetylene reduction

and, for plants with detectable nitrogenase activity,

significantly lower levels (by a factor of three to five)

were recorded for sodA-infected nodules (Fig. 4B).

Activity was highest for both wild type and sodA after

5 weeks. It decreased after 7 weeks for sodA, but

remained high for up to 12 weeks with the wild type.

After 9 weeks, nitrogenase activity was barely detectable

in nodules infected by the mutant, and the aerial part of

the plants was reduced in size, with chlorotic leaves

indicating the onset of senescence (Fig. 3).

SOD is required for effective infection of alfalfa

The lower level of nodules observed in alfalfa roots

inoculated with the sodA mutant may result from fewer

infection events and/or a larger number of arrested

infections. As the sodA gene appeared to be expressed

constitutively in both the sodA mutant and the wild type

(not shown), a sodA±lacZ fusion was used to follow

the infection process histochemically. Three days after

Fig. 1. Growth of the S. meliloti sodA mutant in rich and minimal media.A. Effect of paraquat and of the SOD mimic MnTM-2-PyP on growth in rich medium. Wild-type (Rm5000) and sodA mutant (QC3132) strainswere cultured for 24 h in LBMC Rif medium and diluted (1:25) to an OD600 of 0.04. Growth was monitored by OD measurements. Paraquatand MnTM-2-PyP were added when OD600 was 0.2 at the times indicated by the arrows. Symbols: W, no addition; V, 0.1 mM PQ21 O,0.5 mM PQ21 B, 25 mM MnTM-2-PyP.B. Growth in minimal medium. The wild-type (Rm5000) and sodA mutant (QC3132) strains were grown overnight in M9 medium plus 0.5 mMof all amino acids (enriched M9) until OD600 reached 0.3. The cells were washed and diluted to OD � 0.03 in M9 (filled symbols) and enrichedM9 (open symbols).

752 R. Santos, D. HeÂrouart, A. Puppo and D. Touati


infection with the sodA mutant, roots displayed hair curling

and branching, indicating that the mutant strain was

recognized normally by the plant. After 5 days, the

numbers of infection threads per root in the mutant and

wild type were similar. However, after 10 days, half the

roots had no nodules, suggesting that some infection

threads began to form but aborted before elongation.

Indeed, sodA bacteria progressed differently in the

threads, and they infected the cortical cells less efficiently

(Fig. 5A±D). Numerous infection threads were not com-

pletely full, as shown by the discontinuous, blue coloration

within the thread (Fig. 5B), a pattern not observed in the

wild type (Fig. 5A). Only 20% of sodA-elicited nodules had

a healthy zone of infection, the rest being poorly colonized

(Fig. 5F and H) or empty (not shown).

Arrest of bacteroid differentiation in the sodA mutant

The discrepancy between the number of nodules elicited

by the sodA mutant and final total nitrogen fixation led us

to follow the fate of bacteria in developing nodules. In

nodules induced by S. meliloti in alfalfa, steps in bacterial

development are correlated with specific histological

zones (Vasse et al., 1990). The most distal zone (zone

I) is formed by uninfected meristematic cells. Bacteria

are released in infection zone II, in which they start to

differentiate into bacteroids. Zone III is the nitrogen

fixation zone, containing the elongated, fully differentiated

bacteroids (type 4). Zone IV is the senescent zone;

bacteroids no longer fix nitrogen, but degenerate together

with plant cells. Electron microscopy showed that, upon

infection with sodA, bacteroid differentiation was blocked

in most nodules at the level of infection zone II. In most

cases, the infection threads contained bacteria that had

lost their characteristic rod shape (Fig. 6A) and become

irregular (Fig. 6B and C). The bacteria were released into

the cytoplasm without a peribacteroid membrane and did

not differentiate (Fig. 6B). In other cases, the infection

threads aborted and the bacteria degenerated without

release (Fig. 6C). Both phenotypes were frequently seen

in the same nodule. The cytoplasm of the host cells and

the bacteria progressively degenerated, and vesicles and

ghost membranes accumulated, indicating senescence of

Fig. 2. Sensitivity of the sodA mutant to H2O2. The survivingfraction is the ratio between the number of bacteria in the sampleschallenged with H2O2 and that in untreated samples. Values aremeans of at least three independent experiments.A. W, wt (Rm5000); D, sodA (QC3132).B. Catalase activity in the wild type and sodA mutant (QC3132) innon-denaturing polyacrylamide gel (35 mg of protein).C. Surviving fraction of bacteria challenged with 2 mM H2O2.

Fig. 3. Nine-week-old inoculated alfalfa plants with the wild-typeand sodA strains. The inset shows an expanded view of thenodules.



the two partners (Fig. 6D). As soon as a few bacteroids

were halted at some stage of differentiation, complete

degeneration of the nodule was induced, particularly in

the proximal zones. In a few cases (20%), bacteroids

differentiated into nitrogen-fixing type 4 (not shown), pre-

sumably responsible for the low level of nitrogen fixation

observed (Fig. 4B).

Early senescence in sodA nodules

Autofluorescence reveals the presence of phenolic sub-

stances and phytoalexins, which accumulate in degen-

erating tissues. These substances are classical markers

of the plant response to pathogen attack (Baron and

Zambryski, 1995; Lamb and Dixon, 1997). sodA-elicited

nodules frequently displayed strong localized autofluor-

escence under blue-violet light in cells close to infection

threads and in cells close to vascular bundles (Fig. 5J).

We did not observe this phenotype in wild-type nodules

(Fig. 5I). This suggests that the arrest of sodA bacteria

Fig. 5. Bacterial progression in infection threads and nodules wasfollowed by histochemical detection of the b-galactosidase activityof a sodA±lacZ fusion.A. Wild-type infection thread.B. sodA infection thread.C. Inner cortical cell infection with wild type.D. Abortive infection with sodA mutant.E. Wild type-induced 10-day-old nodule.F. sodA-induced 10-day-old nodule.G. Wild type-infected 14-day-old nodule.H. sodA-induced 14-day-old nodule with abortive infection threads.I and J. Nodules shown in (E) and (F), respectively, observedunder UV light by fluorescence microscopy. At each time point, 15±25 roots were stained, corresponding to a final number of 84nodules observed for the wild type and 65 for the sodA mutant.Scale bars � 100 mm.

Fig. 4. Symbiotic phenotype of the sodA mutant.A. Nodulation kinetics of alfalfa inoculated with the wild type (openbars) and sodA mutant (closed bars). The nodules were counted,and a mean for 100 plants was calculated. Plants without noduleswere excluded (5% for wild type and 50% for sodA). Values aremeans ^ SE. The difference between the means was significant(P , 0.01) after 14 days (Student's t-test).B. Effect of sodA mutation (closed bars) on nitrogen fixation.Values are means ^ SE of measurements from at least six tubes(three plants each) with detectable acetylene levels. The differencebetween the means was highly significant (P , 0.001) at all timepoints except 5 weeks; P , 0.01 (Student's t-test).



before or shortly after their release from infection threads

triggers a plant defence response.

Discussion

The lack of cytoplasmic SOD affects bacterial survival

and functions differently according to the environment.

Escherichia coli, although subjected to oxygen-dependent

damage, survive relatively well in rich medium, but cannot

overcome SOD deficiency in poor medium (Carlioz and

Touati, 1986). In the interaction of pathogens with their

host, intracellular SODs contribute differentially to patho-

genicity, depending on the mode of infection of the

pathogen. The effects of SOD mutation may also differ

for a given bacterial mutant depending on the site of

infection, as for Yersinia enterocolitica serotype O8

(Roggenkamp et al., 1997). Here, we show that a lack

of cytoplasmic SOD affects free-living S. meliloti only

moderately but has drastic effects on the development of

symbiosis with alfalfa.

SOD-deficient mutants were first isolated and their

phenotypic properties extensively investigated in E. coli

(Carlioz and Touati, 1986; Farr et al., 1986; Keyer et al.,

1995; McCormick et al., 1998). The phenotype of these

mutants is therefore often used as a reference. Free-living

S. meliloti tolerate the lack of SOD better than E. coli.

Thus, S. meliloti can grow in minimal medium, although its

growth is impaired, suggesting that the O2´2-sensitive

enzyme dihydroxy acid dehydratase used in E. coli for

branched amino acid biosynthetic pathway (Flint et al.,

1993) is different in S. meliloti. The missing compound(s),

synthesized to a lesser extent or not at all in sodA and

responsible for growth impairment in minimal medium,

may be difficult to identify because of the lack of detailed

knowledge concerning metabolic pathways in S. meliloti.

Another aspect of the efficient defence of the sodA

mutant against oxidative stress is the O2´2-mediated

protection against H2O2, including the induction of

catalase. As H2O2 is a partner of the Fenton reaction,

the O2´2-induced protection against H2O2 presumably

minimizes both the amplitude and the deleterious effect of

the Fenton reaction, which is exacerbated in the presence

of O2´2. O2´2-induced protection against H2O2 has been

reported to date only for a catalase in Xanthomonas

campestris, a non-symbiotic nitrogen-fixing bacterium

(Chamnongpol et al., 1995). The sodA mutant showed

no increase in sensitivity to NO´ donors, suggesting that

sodA is well protected against NO´. These additional

defences against oxidative stress displayed by S. meliloti

in the absence of SOD in oxidative stress conditions may

reflect the adaptation of a bacterium unable to ferment,

which therefore has no other means of escaping oxidative

stress.

In contrast to what was observed in free-living condi-

tions, SOD protection was crucial for effective symbiosis

and nitrogen fixation. Based on the oxygen sensitivity of

Fig. 6. Ultrastructure of the infection zone of3-week-old nodules.A. Wild type.B±D. sodA.A. Wild-type bacteria are released frominfection threads as type 1 bacteroids. Theyare rod shaped and have a central nucleoid,DNA fibrils (asterisks) and an irregularperibacteroid membrane (PBM, arrows).Some have poly b-hydroxybutyrate granules(white granules).B. sodA bacteria within infection threads areirregularly shaped and are released into thedisorganized host cytoplasm without PBM.These bacteria differentiate no further.C. Degenerating sodA bacteria inside infectionthreads.D. Senescent cells of the infection zone ofsodA-induced nodules are filled with non-differentiating bacteria and contain largenumbers of vesicles and ghost membranes. b,bacteroids, cw, cell wall; it, infection threads;m, mitochondria; vs, vesicles and ghostmembranes. Scale bars: A, B and D, 1 mm;C, 0.5 mm.



nitrogenase, sodA mutants were predicted to show a late

developmental block in symbiosis. Consistent with impair-

ment of nitrogenase function, the few sodA bacteroids

that reached the nitrogen fixation stage rapidly lost their

ability to fix nitrogen. However, we also found early defects

in the initiation of nodulation, infection and bacteroid

development, demonstrating a critical role for SOD even

before nitrogenase is expressed.

The symbiotic phenotype of S. meliloti sodA mutants

combines features described for other mutations.

Impaired nodulation resembles the defect described for

a glnB S. meliloti mutant (ArcondeÂguy et al., 1997), and

the early senescence upon release into plant cells is

reminiscent of the bacA (Glazebrook et al., 1993) and

DAHP synthase-deficient (Jelesko et al., 1993) pheno-

types. These multiple symbiotic phenotypes suggest that

functions critical at one or more steps in symbiosis are

impaired by oxidative stress, unless they are protected by

SOD.

The lack of severe growth impairment in free-living

sodA bacteria shows that these cells were not suffering

from excessive oxidative damage before they came into

contact with the plant. We found recently (R. Santos et al.,

submitted) that alfalfa responds to S. meliloti infection

by producing superoxide and hydrogen peroxide. This

oxidative burst at early stages of nodulation may cause

damage in the sodA mutant. Although exogenous O2´2

does not cross the inner membrane, it may be partially

converted to its cell-permeating protonated form in the

acidic environment of the infection thread, increasing

endogenous oxidative stress in sodA. Moreover, NO´,

which has been detected in the roots and nodules of

Lupinus albus (Cueto et al., 1996), may be produced as

part of the oxidative burst (Delledonne et al., 1998; Durner

et al., 1998), and NO´ can enter bacterial cells. In the

sodA mutant, the high ambient level of O2´2 may trap

NO´, yielding the highly toxic peroxynitrite. However, we

observed no increase in sensitivity to NO´ generators in

free-living bacteria, indicating that S. meliloti is well

protected against NO´. Thus, it is possible that the excess

of endogenous superoxide in the sodA mutant is solely

responsible for damaging functions critical in symbiosis.

Impairment of some functions of general metabolism that

have minor effects in free-living growth conditions may

have drastic effects in symbiosis. Alternatively or addi-

tionally, specific symbiotic functions might be sensitive to

oxidative damage.

The effects of other S. meliloti proteins involved in

resistance to oxidative stress were investigated during

symbiosis, but they appeared to be less important than

those of SOD. Single mutations in the katA and katC

genes have no effect on nodulation or N2 fixation

efficiency. However, the double katA katC mutant had a

lower than normal level of nitrogenase activity despite

KatC activity being undetectable in the wild-type bacteroid

(HeÂrouart et al., 1996; Sigaud et al., 1999). Recently, the

nex1 gene, encoding a protein with a sequence similar to

those of peroxyredoxins, was isolated along with other

genes induced during symbiosis, but disruption of this

gene had no effect on nodulation and N2 fixation efficiency

(Oke and Long, 1999).

The results reported in this paper provide the first

evidence that oxidative stress, unless counteracted by

SOD, interferes at several steps in symbiosis. The molecular

nature of the related defects should now be elucidated.

This may help to answer the question as to how 20% of the

bacterial population escapes early damage and manages

to fix nitrogen, albeit poorly.

Experimental procedures

Bacterial strains and growth conditions

All the S. meliloti strains used in this study are listed inTable 1 and were derived from Rm5000 or GMI211,rifampicin-resistant (Finan et al., 1984) and Lac2 streptomy-cin-resistant (Niel et al., 1977) derivatives of wild-type SU47respectively. Luria±Bertani medium (LB: 5 g l21 yeastextract, 10 g l21 tryptone and 10 g l21 NaCl) was used toculture E. coli strains, and 2.5 mM CaCl2 and 2.5 mM MgSO4

were added for S. meliloti (LBMC). The minimal medium usedwas M9 containing 0.4% glucose (Glazebrook and Walker,1991). If appropriate, antibiotics were added to E. colicultures at a concentration of 20 mg ml21, except ampicillin,which was added at 500 mg ml21 to liquid medium and50 mg ml21 to solid medium. The concentrations of anti-biotics added to S. meliloti cultures were 200 mg ml21 forneomycin and streptomycin, 100 mg ml21 for gentamicin and20 mg ml21 for rifampicin, added as required.

S. meliloti strain construction

Molecular cloning techniques and gel electrophoresis wereperformed as described previously (Sambrook et al., 1989;Santos et al., 1999). To construct the sodA mutants, twofragments were amplified by polymerase chain reaction(PCR) from pRS41.1 (Santos et al., 1999) using pUC18polylinker primers and two sodA internal primers, 5 0-GTTTTGGATCCATTGTGGCATCTCCTCTTG-3 0 and 5 0-GAAAAGGATCCGCTCGTCCACGGCGCAAC-3 0, which carry a5 0 BamHI site (italicized). The fragments were ligated at theBamHI site creating a sodA internal deletion of 456 bp, andthe resulting fragment was inserted between the SalI±XbaIsites of pBluescript SK1. An ApaI±XbaI fragment wasisolated and inserted between the corresponding sites ofpJQ200sk (Quandt and Hynes, 1993), creating pRS56. Theinterposons VKmr and VSmr/Spr (Fellay et al., 1987) wereintroduced into the BamHI site within the sodA gene. ThesodA::VKmr disrupted gene was transferred into strainRm5000 by biparental mating using E. coli S17-1 l-pir asthe donor, and the sodA::VSmr/Spr disrupted gene wastransferred by triparental conjugation (Glazebrook andWalker, 1991) using the helper plasmid pRK600 (Finan



et al., 1986). Mutant clones obtained by gene replacementwere selected directly on plates containing rifampicin and 5%sucrose. The resulting mutants, QC3132 and QC3199, areKmr/Nmr and Smr/Spr respectively. The GMI211 sodA::VKmr

mutant (strain QC3136) was obtained by triparental matingas described above. To construct a Rm5000 sodA katAdouble mutant (strain QC5011), we transferred the katA::Tn5mutation from the MK5001 strain (HeÂrouart et al., 1996) intoQC3199 by general transduction using the éM12 phage(Glazebrook and Walker, 1991).

To construct a fusion between the sodA promoter and thelacZ gene, the PCR-amplified fragment carrying the promoterused above to construct the sodA mutant was cloned in thepBluescript SK1 polylinker EcoRI±BamHI restriction sites. AKpnI±SacI fragment was transferred to pUC18 to obtain thepromoter fragment between two BamHI sites. The promoterfragment was removed by BamHI digestion and inserted intothe BamHI site of pIJ1363 (Rossen et al., 1985), creating atranslational sodA±lacZ fusion. The orientation of the clonedpromoter fragment was confirmed by restriction analysis.pRS49 containing the sodA±lacZ fusion was transferred byconjugation to Lac2 strains GMI211 and QC3136, creatingstrains QC3138 and QC3139 respectively.

Bacterial lysates and enzyme activity assay

Free-living S. meliloti cell lysate preparation, total catalaseactivity and catalase activity detection in non-denaturingpolyacrylamide gels were performed as previously described

(Jones, 1982; Clare et al., 1984; HeÂrouart et al., 1996; Santoset al., 1999). Total catalase activity was measured in strainscultured in rich LBMC medium to an OD600 of 1.2, and valuesare means ^ SD of three independent experiments.

Mutation frequency determination

Mutation frequency was determined essentially as describedpreviously (Touati et al., 1995). The wild type, Rifs GMI211and its sodA derivative (QC3136) were grown in rich mediumuntil saturation (2 days) and spread onto LBMC platescontaining 100 mg ml21 rifampicin. Rifr mutants were scoredafter 4 days of incubation at 308C.

Sensitivity to H2O2 and NO´

Cells were grown in LBMC medium for four generations to anOD600 of 0.2. Aliquots (5 ml) were challenged with variousH2O2 and NO´ concentrations for 1 h at 308C. The reactionwas stopped by chilling and diluting and, in the case of H2O2

treatments, by adding 400 U ml21 catalase. Samples werediluted in 10 mM MgSO4, containing 400 U ml21 catalase inthe case of H2O2 treatments, and plated on LBMC plates.Colonies were counted after 4 days of incubation at 308C.

Plant assays

Medicago sativa L. var. Europe (alfalfa) was used as host

Table 1. Bacterial strains and plasmids used in this study.

Strain or plasmid Relevant characteristics Source or reference

StrainsS. meliloti

Rm5000 SU47 rif-5 Finan et al. (1984)QC3132 Rm5000 DsodA::VKm This studyQC3199 Rm5000 DsodA::VSm/Spc This studyMK5001 Rm5000 katA::Tn5 HeÂrouart et al. (1996)QC5011 Rm5000 katA::Tn5 DsodA::VSm/Spc This studyGMI211 SU47 lac Smr Niel et al. (1977)QC3136 GMI211 sodA::VKm This studyQC3138 GMI211 carrying plasmid pRS49 (sodA±lacZ) This studyQC3139 QC3136 carrying plasmid pRS49 (sodA±lacZ) This study

E. coliDH5a F2 supE44 DlacU169(f180 dLacZDM15) Laboratory stock

hsdR17(rk2mk

1) recA1 endA1 gyrA96 thi-1 relA1S17-1 l-pir l-pir lysogen of S17±1 (recA thi pro A. Sessitsch

hsdR2M1 RP4 2-Tc::Mu-Km::Tn7(Tpr/Smr)MT616 Helper strain, pro-82 thi-1 hsdR17 supE44 recA56 (pRK600) Finan et al. (1986)

PlasmidspRS41.1 pUC18 derivative carrying sodA region, Apr Santos et al. (1999)pJQ200sk Cloning vector, p15A origin, sacB1, Gmr Quandt and Hynes (1993)pHP45 VKm pBR322 derivative carrying a VKmr cassette, Apr Kmr Fellay et al. (1987)pHP45VSm/Spc pBR322 derivative carrying a VSmr/Spcr cassette, Apr Smr/Spcr Fellay et al. (1987)pRS56 pJQ200sk with sodA gene containing This study

an internal deletion and a BamHI site, Gmr

pRS50 pRS56 derivative with DsodA::VKm, Gmr Kmr This studypRS64 pRS56 derivative with DsodA::VSm/Spc, Gmr Smr/Spcr This studypRK600 ColE1 replicon with RK2 transfer region, Cmr Finan et al. (1986)pIJ1363 Broad-host-range translational lacZ fusion vector, Tcr Rossen et al. (1985)pRS49 pIJ1363 derivative with sodA-lacZ, Tcr This study



plant for testing nodulation and N2 fixation of S. melilotistrains. Surface-sterilized germinating seedlings were grownin test tubes (three seedlings per tube) on nitrogen-free agarslats. Four-day-old plants were inoculated with S. melilotistrains cultured to an OD600 of 1.0 and diluted to an OD600

of 0.3. Nitrogenase activity was determined by acetylenereduction using a gas chromatograph as described previously(HeÂrouart et al., 1996).

Microscopy studies

For electron microscopy, nodules were harvested 3, 5 and7 weeks after inoculation. At least five nodules for each agewere examined by electron microscopy. Nodules were fixedwith 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4)for 5 h at room temperature, post-fixed with 1% osmiumtetroxide for 1 h, dehydrated in a graded series of alcohol andembedded in Spurr low-viscosity embedding medium allowedto polymerize for 48 h at 608C. Ultrathin sections werestained with uranyl acetate for 20 min at 378C and withReynold's lead citrate for 5 min and were examined in aPhilips CM12 electron microscope operating at 60 kV.

For light microscopy, b-galactosidase activity in entireroots was detected as described previously (Boivin et al.,1990). Autofluorescence of phenolic compounds wasobserved by fluorescence microscopy using ultraviolet light.All observations were performed with a Zeiss Axiophotmicroscope.

Acknowledgements

We would like to thank I. Fridovich for kindly sending usMnTMPyP, A. Sessitsch and I. Oresnik for sending usstrains, Vanessa Becquet for assistance in nitrogenaseactivity measurement, and Sophie Le Panse for assistancewith electron microscopy. We are grateful to DominiqueExpert and all the members of her laboratory (INA-PG, Paris)for their generous support. R.S. received a grant fromFundacËaÄo para a CieÃncia e a Tecnologia (Portugal).

References

ArcondeÂguy, T., Huez, I., Tillard, P., Gangneux, C., de Billy,F., Gojon, A., et al. (1997) The Rhizobium meliloti PII

protein, which controls bacterial nitrogen metabolism,affects alfalfa nodule development. Genes Dev 11: 1194±1206.

Baron, C., and Zambryski, P.C. (1995) The plant response inpathogenesis, symbiosis, and wounding: variations on acommon theme? Annu Rev Genet 29: 107±129.

Bladergroen, M.R., and Spaink, H.P. (1998) Genes andsignal molecules involved in the rhizobia±leguminoseaesymbiosis. Curr Opin Plant Biol 1: 353±359.

Boivin, C., Camut, S., Malpica, C.A., Truchet, G., andRosenberg, C. (1990) Rhizobium meliloti genes encodingcatabolism of trigonelline are induced under symbioticconditions. Plant Cell 2: 1157±1170.

Buchanan, A.G. (1977) The response of Azotobacterchroococcum to oxygen. superoxide-mediated effects.Can J Microbiol 23: 1548±1553.

Carlioz, A., and Touati, D. (1986) Isolation of superoxide

dismutase mutants in Escherichia coli : is superoxidedismutase necessary for aerobic life? EMBO J 5: 623±630.

Chamnongpol, S., Vattanaviboon, P., Loprasert, S., andMongkolsuk, S. (1995) Atypical oxidative stress regulationof a Xanthomonas oryzae pv. oryzae monofunctionalcatalase. Can J Microbiol 41: 541±547.

Clare, D.A., Duong, M.N., Darr, D., Archibald, F., andFridovich, I. (1984) Effects of molecular oxygen ondetection of superoxide radical with nitroblue tetrazoliumand on activity stains for catalase. Anal Biochem 140: 532±537.

Cueto, M., Hernandez-Perera, O., Martin, R., Bentura, M.L.,Rodrigo, L., Lamas, S., and Golvano, M.P. (1996)Presence of nitric oxide synthase activity in roots andnodules of Lupinus albus. FEBS Lett 398: 159±164.

Delledonne, M., Xia, Y., Dixon, R.A., and Lamb, C. (1998)Nitric oxide functions as a signal in plant diseaseresistance. Nature 394: 585±588.

Durner, J., Wendehenne, D., and Klessig, D.F. (1998)Defense gene induction in tobacco by nitric oxide, cyclicGMP, and cyclic ADP-ribose. Proc Natl Acad Sci USA 95:10328±10333.

Farr, S.B., D'Ari, R., and Touati, D. (1986) Oxygen-dependent mutagenesis in Escherichia coli lacking super-oxide dismutase. Proc Natl Acad Sci USA 83: 8268±8272.

Faulkner, K.M., Liochev, S.I., and Fridovich, I. (1994) StableMn(III) porphyrins mimic superoxide dismutase in vitro andsubstitute for it in vivo. J Biol Chem 269: 23471±23476.

Fellay, R., Frey, J., and Krisch, H. (1987) Interposonmutagenesis of soil and water bacteria. a family of DNAfragments designed for in vitro insertional mutagenesis ofGram-negative bacteria. Gene 52: 147±154.

Finan, T.M., Hartwieg, E., LeMieux, K., Bergman, K., Walker,G.C., and Signer, E.R. (1984) General transduction inRhizobium meliloti. J Bacteriol 159: 120±124.

Finan, T.M., Kunkel, B., De Vos, G.F., and Signer, E.R.(1986) Second symbiotic megaplasmid in Rhizobiummeliloti carrying exopolysaccharide and thiamine synthesisgenes. J Bacteriol 167: 66±72.

Fischer, H.-M. (1996) Environmental regulation of rhizobialsymbiotic nitrogen fixation genes. Trends Microbiol 4: 317±320.

Fisher, R.F., and Long, S.R. (1992) Rhizobium±plant signalexchange. Nature 357: 655±660.

Flint, D.H., Tuminello, J.F., and Emptage, M.H. (1993) Theinactivation of Fe-S cluster containing hydrolyases bysuperoxide. J Biol Chem 268: 22369±22376.

Geurts, R., and Franssen, H. (1996) Signal transduction inRhizobium-induced nodule formation. Plant Physiol 112:447±453.

Glazebrook, J., and Walker, G.C. (1991) Genetic techniquesin Rhizobium meliloti. Methods Enzymol 204: 398±418.

Glazebrook, J., Ichige, A., and Walker, G.C. (1993) ARhizobium meliloti homolog of the Escherichia coli pep-tide-antibiotic transport protein SbmA is essential forbacteroid development. Genes Dev 7: 1485±1497.

Halliwell, B., and Gutteridge, J.M.C. (1989) Free Radicals inBiology and Medicine, 2nd edn. Oxford: Clarendon Press.

HeÂrouart, D., Sigaud, S., Moreau, S., Frendo, P., Touati, D.,and Puppo, A. (1996) Cloning and characterization of the



katA gene of Rhizobium meliloti encoding a hydrogenperoxide-inducible catalase. J Bacteriol 178: 6802±6809.

Howard, J.B., and Rees, D.C. (1994) Nitrogenase: anucleotide-dependent molecular switch. Annu Rev Bio-chem 63: 235±264.

Imlay, J.A., and Linn, S. (1987) Mutagenesis and stressresponses induced in Escherichia coli by hydrogenperoxide. J Bacteriol 169: 2967±2976.

Jelesko, J.G., Lara, J.C., and Leigh, J.A. (1993) Rhizobiummeliloti mutants with decreased DAPH synthase activityare sensitive to exogenous tryptophan and phenylalanineand form ineffective nodules. Mol Plant±Microbe Interact 6:135±143.

Jones, D.P. (1982) Intracellular catalase function. analysis ofthe catalytic activity by product formation in isolated livercells. Arch Biochem Biophys 214: 806±814.

Keyer, K., and Imlay, J.A. (1996) Superoxide acceleratesDNA damage by elevating free-iron levels. Proc Natl AcadSci USA 93: 13635±13640.

Keyer, K., Gort, A.S., and Imlay, J.A. (1995) Superoxide andthe production of oxidative DNA damage. J Bacteriol 177:6782±6790.

Lamb, C., and Dixon, R.A. (1997) The oxidative burst in plantdisease resistance. Annu Rev Plant Physiol Plant Mol Biol48: 251±275.

Liochev, S.I., and Fridovich, I. (1994) The role of O2´2 in theproduction of HO´: in vitro and in vivo. Free Radical BiolMed 16: 29±33.

Long, S.R. (1996) Rhizobium symbiosis: Nod factors inperspective. Plant Cell 8: 1885±1898.

McCormick, M.L., Buettner, G.R., and Britigan, B.E. (1998)Endogenous superoxide dismutase levels regulate iron-dependent hydroxyl radical formation in Escherichia coliexposed to hydrogen peroxide. J Bacteriol 180: 622±625.

Mylona, P., Pawlowski, K., and Bisseling, T. (1995) Symbioticnitrogen fixation. Plant Cell 7: 869±885.

Niel, C., Guillaume, J.B., and Bechet, M. (1977) Mise eneÂvidence de deux enzymes preÂsentant une activiteÂ b-galactosidase chez Rhizobium meliloti. Can J Microbiol 23:1178±1181.

Oke, V., and Long, S.R. (1999) Bacterial genes inducedwithin the nodule during the Rhizobium±legume symbiosis.Mol Microbiol 32: 837±849.

Peters, N.K., Frost, J.W., and Long, S.R. (1986) A plantflavone, luteolin, induces expression of Rhizobium melilotinodulation genes. Science 233: 977±980.

Puppo, A., and Rigaud, J. (1986) Superoxide dismutase: anessential role in the protection of the nitrogen fixationprocess? FEBS Lett 201: 187±189.

Quandt, J., and Hynes, M.F. (1993) Versatile suicide vectorswhich allow direct selection for gene replacement in Gram-negative bacteria. Gene 127: 15±21.

Robson, R.L., and Postgate, J.R. (1980) Oxygen andhydrogen in biological nitrogen fixation. Annu Rev Microbiol34: 183±207.

Roggenkamp, A., Bittner, T., Leitritz, L., Sing, A., andHeesemann, J. (1997) Contribution of the Mn-cofactoredsuperoxide dismutase (SodA) to the virulence of Yersiniaenterocolitica serotype O8. Infect Immun 65: 4705±4710.

Rossen, L., Shearman, C.A., Johnston, A.W.B., and Downie,J.A. (1985) The nodD gene of Rhizobium leguminosarum isautoregulatory and in the presence of plant exudateinduces the nodA,B,C genes. EMBO J 4: 3369±3373.

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989)Molecular Cloning: A Laboratory Manual, 2nd edn.Cold Spring Harbor, NY: Cold Spring Harbor LaboratoryPress.

Santos, R., Bocquet, S., Puppo, A., and Touati, D. (1999)Characterization of an atypical superoxide dismutase fromSinorhizobium meliloti. J Bacteriol 181: 4509±4516.

Schultze, M., and Kondorosi, A. (1996) The role of lipo-chitooligosaccharides in root nodule organogenesis andplant cell growth. Curr Opin Genet Dev 6: 631±638.

Schultze, M., and Kondorosi, A. (1998) Regulation of sym-biotic root nodule development. Annu Rev Genet 32: 33±57.

Sigaud, S., Becquet, V., Frendo, P., Puppo, A., and HeÂrouart,D. (1999) Differential regulation of two divergent Sinorhi-zobium meliloti genes for HPII-like catalases during free-living growth and protective role of both catalases duringsymbiosis. J Bacteriol 181: 2634±2639.

Squadrito, G.L., and Pryor, W.A. (1998) Oxidative chemistryof nitric oxide: the roles of superoxide, peroxynitrite, andcarbon dioxide. Free Radical Biol Med 25: 392±403.

Touati, D., Jacques, M., Tardat, B., Bouchard, L., andDespied, S. (1995) Lethal oxidative damage and mutagen-esis are generated by iron in Dfur mutants of Escherichiacoli. protective role of superoxide dismutase. J Bacteriol177: 2305±2314.

Truchet, G., Roche, P., Lerouge, P., Vasse, J., Camut, S., deBilly, F., et al. (1991) Sulphated lipo-oligosaccharidesignals of Rhizobium meliloti elicit root nodule organogen-esis in alfalfa. Nature 351: 670±673.

Vasse, J., de Billy, F., Camut, S., and Truchet, G. (1990)Correlation between ultrastructural differentiation of bacter-oids and nitrogen fixation in alfalfa nodules. J Bacteriol 172:4295±4306.



Documents

Critical protective role of bacterial superoxide dismutase in Rhizobium–legume symbiosis