Proc. Natl. Acad. Sci. USAVol. 92, pp. 3759-3763, April 1995Developmental Biology

Oxygen as a key developmental regulator ofRhizobium melilotiN2-fixation gene expression within the alfalfa root noduleERIC SOUPENE, MARIE FOUSSARD, PIERRE BOISTARD*, GEORGES TRUCHET, AND JACQUES BATUTLaboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes, Centre National de la Recherche Scientifique, Institut National de la RechercheAgronomique, B.P. 27, 31326 Castanet Tolosan Cedex, France

Communicated by Sydney Kustu, University of California, Berkeley, CA, January 6, 1994 (received for review November 7, 1994)

ABSTRACT The symbiotic pattern of expression of Rhi-zobium meliloti N2-fixation genes is tightly coupled with thehistological organization of the alfalfa root nodule and thus isunder developmental control. N2-fixation gene expression isinduced very sharply at a particular zone of the nodule calledinterzone II-III that precedes the zone where N2 fixation takesplace. We show here that this coupling can be disrupted,thereby resulting in ectopic expression of N2-fixation genes inthe prefixing zone II of the nodule. Uncoupling was obtainedeither by using a R. meliloti strain in which a mutationrendered N2-fixation gene expression constitutive with respectto oxygen in free-living bacterial cultures or by placing nodulesinduced by a wild-type R. meliloti strain in a microoxic environ-ment. These results implicate oxygen as a key determinant of thesymbiotic pattern of N2-fixation gene expression.

The development of N2-fixing root nodules, elicited by rhizobiaon their legume host plants, requires the concerted differen-tiation of bacterial cells and plant tissues. In nodules of theindeterminate type, such as those elicited by Rhizobium on peaand alfalfa, a succession of histologically distinct zones differ-entiates from the apical (distal) end along the longitudinal axis(see ref. 1 for a detailed description and Fig. 1). The apicalzone I corresponds to the bacteria-free nodule meristem.Below the meristem, the prefixing (infection) zone II ischaracterized by the presence of numerous infection threads,particularly in its distal part where release of bacteria into theplant cells takes place. Released bacteria, called bacteroids,proliferate and differentiate toward a N2-fixing form foundexclusively in the N2-fixing zone III. Of particular interest is theinterzone 11-III, which consists of two or three layers ofamyloplast-rich cells, which separate the prefixing (invasion)zone II from the N2-fixing zone III (1). Interzone II-III is animportant landmark in nodule differentiation. Several keysymbiotic genes, including the bacterial N2-fixation genes (nifandfix) and plant nodulins, such as leghemoglobin, are turnedon very precisely at interzone 11-111 (refs. 2-6 and this paper).There is thus a great deal of interest in identifying theregulatory signal(s) operating at interzone 11-III.02 concentration has been shown to regulate nifandfix gene

expression in liquid cultures of Rhizobium meliloti (7-9),making it a likely candidate as the symbiotic signal. Microoxicactivation of N2-fixation gene expression in R. meliloti culturesis mediated by the pair of regulatory proteins, FixL/J (8, 9).FixL is a hemoprotein kinase and phosphatase that controlsthe phosphorylation of FixJ in response to 02 concentration(10-13). Under oxygen-limiting conditions, phosphorylatedFixJ activates the transcription of the intermediate regulatorygenes nifA andfixK (13, 14) and, hence, of nif andfix regulons.In spite of the fact that 02 concentration in zone II was so farunknown, microoxy in zone III (15, 16) was an argument insupport of the role of oxygen in the symbiotic control ofnifand

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fix gene expression. By manipulating the nodule environment,we now provide direct experimental evidence that oxygen is akey determinant of the pattern of nif and fix gene expressionwithin the alfalfa nodule. In addition, the phenotype of amutant altered in the oxygen signal transduction pathwaydemonstrates that the regulatory system that activates R.meliloti nif and fix gene expression in response to oxygenlimitation in bacterial cultures is also involved in the develop-mental control of nif and fix gene expression during noduledifferentiation.

MATERIALS AND METHODSMicrobiological Techniques. The pJJ5 (9), pGMI931 (9),

and pCHK57 (7) plasmids, carrying ftxK-lacZ, fixN-lacZ, andnifA-lacZ fusions, respectively, were introduced into the wild-type or fixJD54N R. meliloti mutant strain by conjugation.3-Galactosidase activities expressed by the lacZ fusions weremeasured in aerated or microoxic cultures as described (7, 8).

Construction of the R. melilotifixJD54N Mutant. A 424-bpXba I-EcoRV fragment that contained thefixJD54N mutation(G -> A at position 2504 in the fixLJ sequence; GenBankaccession no. J03174) in a central position was purified fromthe pJMR2 plasmid (17) and cloned between the Xba I andSma I sites of pJQ200KS (18). The resulting plasmid, pMF1,was mobilized from Escherichia coli S17-1 strain (19) to R.meliloti GMI5600 lac (8) in which it cannot replicate. Cointe-gration of the pMF1 plasmid into the resident pSym plasmidwas selected using the gentamycin-resistance gene of pJQ200.Purified colonies were grown to stationary phase in richmedium in the absence of gentamycin. Cells in which resolu-tion of the cointegrate had occurred were then selected byplating on LB agar medium supplemented with 5% sucrose.The rationale of this selection is that, in the presence of 5%sucrose, the expression of the sacB gene carried by pJQ200 islethal in Gram-negative bacteria (20). Twenty gentamycin-sensitive colonies able to grow in the presence of sucrose werescreened by the PCR (21) to identify those containing thefixJD54N mutation. Following selective amplification of a440-bp fragment of fix! encompassing the region of interest,the presence of the fixJD54N mutation in the PCR productswas readily assessed by the loss of anAva II restriction site. Thestructures of the three mutants obtained in this way wereconfirmed by Southern blot analysis of the fixLJ region.

Plant Methods. Surface-sterilized germinating seedlings ofMedicago sativa cv. Gemini were aseptically transferred ontoagar slants (two seedlings per tube) with nitrogen-free Fahr-aeus medium. Three-day-old plants were inoculated with R.meliloti strains carrying nifA-, fixK-, or fixN-lacZ fusions onplasmids. When needed (see Fig. 1B), 3-week-old noduleswere subjected to a microoxic environment for 16 hr by addingmelted agar (15 g/liter) to the tube to entirely immerse the rootsystem. Three-week-old nodules were fixed with 2% glutaral-dehyde in 0.2 M sodium cacodylate (pH 7.2) (22) and sliced

*To whom reprint requests should be addressed.

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FIG. 1. Effect of the nodule gaseousenvironment on R. meliloti fixK gene ex-pression. (A) Control nodule. fixK geneexpression is triggered in bacteroids at theinterzone Il-III. (B) Nodule subjected toa microoxic environment. ThefixKgene isnow also expressed in the prefixing zoneII, both in bacteria still enclosed in infec-tion threads at the distal end of zone IIand in bacteroids. Asterisks, nodule mer-istem; arrowheads, infection threads;black stars, prefixing zone II; arrows, dis-tal border of the interzone II-III; whitestars, N2-fixing zone III. The diagramsbeside A and B indicate the approximateextent of each zone. (A, x 60; B, x125.)

into 80-gm-thick longitudinal sections. f3-Galactosidase activ-ity was monitored as described (22). Nodule sections were thencleared with sodium hypochlorite and observed by bright-fieldmicroscopy using a Vanox Olympus light microscope.

Microelectrode Experiments. A microelectrode of the typedescribed by Revsbech (23) was used because of the 02 speci-ficity and the linear response to 02 concentration. The elec-trode was calibrated at room temperature against air-saturatedwater (250 gIM 02; measured current, 360 pA) and waterdeoxygenated by extensive flushing with N2 (0 guM 02, 0 pA).Three-week-old nodules were isolated with attached rootsegments (2-3 mm long), taped in a vertical position, and

immediately impaled at their distal end. The electrode (ca.20-gm tip diameter) was advanced with a micromanipulatorby 20-gum increments and the current was recorded at eachposition. We ensured in a control experiment that the currentat a given position of the microelectrode inside the nodule wasstable over the time length of the experiment (a few minutes).Impaled nodules were fixed in 2% glutaraldehyde and cut into80-gm longitudinal sections. The sections were then observedby bright-field microscopy to visualize the wounding caused bythe penetration of the electrode tip (see Fig. 5A). Of 20nodules assayed, 3 were found in which the tip had penetratedthe nodule through the meristem as far as interzone II-IIl. All

fixJD54N

Gm

pSym, fxL fvx

I~~~pFpSym t7xL fixJD54N sacR/B Gm frxL

lt! t J2I

e pSym fixL fJxJD54N

FIG. 2. Outline of the construction of the R. meliloti fixJD54N mutant strain. This approach can be applied generally for the site-directedintroduction of unmarked point mutations into the R. meliloti genome by homologous recombination. See text for a detailed description.

A B

*

J

''' * i14I_,S,.

pSym fixL ixJ 3I~~~~~~~~~~~~~~

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El Microaerobiosis* Aerobiosis

tf 301457

11- (I 310

1487

1000 2000 3000 4000

Miller units

FIG. 3. Effect of the fixJD54N mutation on R. meliloti nifA, fixK,and fixN expression ex planta. B-Galactosidase activities expressed bythe nifA-lacZ (A), fixN-lacZ (B), and fixK-lacZ (C) fusions in aeratedor microoxic cultures of thefLxJD54N mutant strain (group 1) or of theisogenic GM15600 wild-type strain (group 2). Each bar of the histo-gram represents the average of at least five independent experiments.

three displayed the same pattern of 02 distribution as shownin Fig. 5B. As a control, some nodules were penetrated in aradial direction from the nodule surface down to zone III.

RESULTS

To examine whether 02 concentration might play a role in thecontrol of the spatial pattern of nif andfix gene expression, wehave used two complementary approaches.

In a first, physiological, approach we lowered 02 concen-tration within the nodule by immersing 3-week-old N2-fixingnodules in agar for 16 hr. Using an 02-specific microelectrode

A

*

Proc. Natl. Acad. Sci USA 92 (1995) 3761

we have verified that this treatment indeed put nodules in amicrooxic environment (data not shown). Although this treat-ment did not result in any visible changes in the histologicalorganization of the nodule (data not shown), we found that thepattern of bacterial N2-fixation gene expression was signifi-cantly modified. Whereas fixK gene expression is normallyswitched on at the interzone II-111 in nonimmersed controlnodules (Fig. 1A), ectopic expression of the fixK gene wasobserved throughout the entire prefixing zone II of nodulessubjected to the microoxic environment, both in bacteria stillenclosed in the infection threads and in bacteroids released inthe host cells (Fig. 1B). Identical observations were made fornifA and the fixK-regulated fixN gene (data not shown).

In a second, genetic, approach, we studied the effect of amutation infixJ, which suppresses oxygen responsiveness of nifandfix gene expression explanta. We have previously describeda mutant FixJ protein, FixJD54N, in which the aspartateresidue that is phosphorylated/dephosphorylated by FixL inresponse to 02 concentration has been replaced by asparagine(17). In an in vitro transcription system as well as in an invivo-reconstituted regulatory pathway in E. coli, the mutantFixJD54N protein activates nifA andfixK independently of 02concentration in a FixL-dependent process (17). Activation ofFixJD54N transcriptional activity was correlated in vitro witha FixL-promoted but oxygen-independent phosphorylation ofthe FixJD54N protein on an alternate residue (17).The mutant fixJD54N allele was introduced into R. meliloti

by a gene replacement technique that is outlined in Fig. 2. Theresulting strain clearly expressed fixK and fixN independentlyOf 02 concentration in bacterial cultures (Fig. 3). As far as nifAis concerned, a very limited oxygen-dependent regulation (ca.2-fold) was still observed in the mutant (Fig. 3A). However thiswas much smaller than that observed in the control strain(27-fold). Thus the fixJDS4N mutation essentially abolishedoxygen regulation of all three genes.We then examined the effect of the fixJD54N mutation on

the in planta expression of nif and fix genes. It should be

B

.

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'~il .. TA ; . - .--' I.,

A i,. , $ .

FIG. 4. Effect of thefixJD54N mutation onR. melilotinifA andfixKexpression inplanta. (A) Control plant inoculated with the GM15600 wild-typestrain carrying the nifA-lacZ fusion. Note the triggering of the nifA gene expression at the interzone II-III. (X75.) (B and C) Plants inoculated withthe isogenicfixJD54N mutant strain containing the nifA-lacZ fusion (B) or thefixK-lacZ fusion (C). Both genes are expressed in the distal prefixingzone II. Symbols are the same as in Fig. 1. (X65.)

A1

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C 1

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mentioned here that the R meliloti fixJD54N mutant inducednodules that were as effective in fixing N2 as nodules inducedby the wild-type strain (data not shown). Whereas nifA ex-pression in the nodules elicited by the wild-type strain wasswitched on at the interzone II-III (Fig. 44), the expression ofnifA (Fig. 4B), fixK (Fig. 4C), and fixN (data not shown) couldbe detected throughout the entire prefixing zone II in noduleselicited by the mutant strain.As the above data pointed to a key role of oxygen in

patterning N2-fixation gene expression, we attempted to di-rectly investigate 02 distribution in the distal part of the alfalfanodule. Our approach consisted of stepwise insertion of an02-sensitive microelectrode along the longitudinal axis of thenodule, from the apex toward the interzone II-III (Fig. 5A).We found that 02 concentration progressively decreased from250 ,uM, the free 02 concentration in an air-saturated solution,to the limit of detection of the microelectrode (ca. 1 j,M) whenreaching interzone II-III (Fig. 5B).

DISCUSSION

We have found that the pattern of expression of several Rmeliloti nif and fix genes within the alfalfa nodule can bemodified either by a change in the external concentration of 02or by altering the transduction pathway that relays the 02concentration signal to the bacterial transcription apparatus.We conclude that 02 concentration inside the nodule not onlydetermines the expression of bacterial N2-fixation genes, as isthe case explanta, but also is involved in the regulation of thisexpression according to a spatial program that is related to thehistological organization of the mature nodule.How oxygen is distributed along the longitudinal axis of an

indeterminate nodule was so far unknown. Using 02-specificmicroelectrodes we have now demonstrated that 02 concen-tration decreases gradually from the apex of the nodule towardinterzone II-III (Fig. SB). This pattern of 02 distributionmarkedly differs from the one observed when nodules areimpaled laterally at the level of N2-fixing zone III (i.e., alonga radial axis). Indeed, in this case, a very sudden decrease in02 concentration from 250 ,uM to 1 ,uM takes place at the levelof, or just below, the endodermis (refs. 15 and 24; data notshown). This steep decrease in 02 concentration was attributedto the presence of an 02 diffusion barrier. Clearly, thisperipheral barrier does not exist in the apical region of thealfalfa nodule and oxygen may passively diffuse from theoutside to interzone II-III. Whether interzone II-III itselfrestricts 02 diffusion remains to be determined (see below).How can our finding that oxygen is a major determinant of

N2-fixation gene expression account for the abrupt transitionin gene expression observed at the interzone 11-III? We wouldlike to put forward two possibilities. (i) 02 concentration mayundergo a dramatic decrease at the interzone II-III boundary.Such a decrease would result in the abrupt induction of nifandfix gene expression. Although not indicated by our microelec-trode measurements, whose limit in sensitivity is around 1 ,uM,this possibility would be consistent with the observation that02 concentration in the central tissues of the N2-fixing zone ofthe nodule is around 10 nM, as indicated by leghemoglobinspectroscopy experiments (16). (ii) Alternatively, nifA andfixKcould respond sharply to a moderate change in 02 concentra-tion at the interzone II-III boundary, in continuity with thegradual decrease observed across prefixing zone II. It has beenshown in animal systems that monotonic gradients of mor-phogens can determine discrete patterns of gene expression(25), even though it is not yet clear how these gradients areinterpreted. We note that the pathway leading from oxygensensing by FixL to activation of target nifA andfixK promotersby phosphorylated FixJ is a multistep process. Oxygen, uponbinding to FixL, limits its capacity for autophosphorylation,

200

~j.6L

50

0

B

0 100 200 300 400

Distance from apex, ,um

500 600

FIG. 5. 02 distribution in the distal part of a mature alfalfa nodule.Data in A and B are from the same nodule. Note that the scales forthe two panels are different. In both panels, the site of penetration ofthe electrode is indicated by a double arrowhead and the distal borderof interzone 11-111 is indicated by a large arrow. (A) Section (80 J,m)of the nodule after impalement with the microelectrode. The trace leftby the electrode is visible (single arrowheads). The small arrows markthe endodermis. (Bar = 250 ,um.) (B) 02 concentration (,uM) isindicated on the y axis as a function of distance (,um) from the apexof the nodule (x axis).

and thus to phosphorylate FixJ, and, instead, enhances itsphosphatase activity leading to FixJ-phosphate dephosphory-lation (11, 12). Phosphorylated FixJ selectively binds DNA,possibly in an oligomeric form (26). Clearly, there is thepotential in such a system for a signal generated by a smalldecrease in 02 concentration being amplified in order to leadto a major change in gene expression. In addition, microoxymay act at more than one level and, for example, induce theproduction of a plant compound that would potentiate thebacteroid response via the FixL/J system. Finally, our data donot exclude the possibility that the pattern of expression ofN2-fixation genes is not solely under oxygen control and thatanother mode of regulation superimposes to that exerted byoxygen. However, the fact that a point mutation in theoxygen-responsive regulatory protein FixJ can alter the patternof expression of N2-fixation genes during symbiosis indicatesthat perception of any putative additional regulatory signalmust be closely related to the FixLJ regulatory pathway.

N2-fixation gene expression in R. meliloti can be inducedunder free-living microoxic conditions (7-9). Two oxygen

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control points have been identified so far. One operates at thelevel of FixLJ, the two-component system that controls ex-pression of the intermediate regulatory genes nifA andfixK (8).Oxygen also regulates the activity of NifA, the transcriptionalactivator of N2-fixation genes such as nifH (reviewed in ref. 27).fixK positively controlsfixN expression and negatively controlsits own expression as well as that of nifA (9). The regulatoryeffector of FixK, if any, is not known. The FixK protein isclosely related to Fnr, the regulator of anaerobic respirationgenes in E. coli, whose activity is oxygen sensitive. The fact thatFixK lacks a cluster of cysteines that mediate oxygen sensitivityin Fnr (reviewed in ref. 27) suggested that FixK activity was notregulated by oxygen. Our data showing that fixN is expressedunder aerobic conditions in the mutant R meliloti fixJD54N(Fig. 3) demonstrate that FixK of R. meliloti is indeed notoxygen sensitive.We have shown that the fixJD54N mutation completely abol-

ished oxygen regulation of fixK and flxN, whereas a 2-foldinduction of nifA was still observed in the mutant under condi-tions of microaerobiosis. David et aL (8) and Kahn and Ditta (28)have reported an oxygen regulation of nifA, associated with thepCHK57 plasmid, in FixL- mutants ofR meliloti. Kahn and Ditta(28) have proposed that this effect could be due to a change in thepromoter structure of nifA upon microaerobic shift. This expla-nation could be relevant to our observations as well. Another,unexpected, feature of the fixJD54N mutation is that it resultedin a significant increase (4- to 6-fold) of both fixK and fixNexpression (but not of nifA) as compared with those observed inthe wild-type strain under microaerobic conditions (Fig. 3). Thereason for this is presently not understood. Our current workinghypothesis is that thefixKpromoter, when activated by the mutantFixJD54N protein, may escape negative autoregulation by theFixK protein. This would result in an increase offixK expressionsimilar to that observed in a fixK mutant (9). By contrast,activation of nifA by the FixJD54N protein would still be undernegative regulation by flxK Preliminary evidence suggests thatthe molecular mechanisms governing the negative regulation ofnifA and fixK by the FixK protein might indeed be different(unpublished data).

Interestingly, nifH symbiotic expression, although it is alsotriggered at interzone II-111 (ref. 3; unpublished data), appearsto be under tighter control than that of nifA, fixK, or fixN.Indeed, we have been unable to elicit nifH expression in theprefixing zone II of the nodule under conditions where weobserved ectopic expression of nifA, fixK, or fixN. The lack ofnifH expression in the infection zone of nodules induced by theR. melilotifixlD54N mutant may be due to the sensitivity of theNifA protein to oxygen (27). Possibly, 02 concentration inzone II of the nodule is not compatible with NifA function.However, our attempts to trigger ectopic expression of nifH bylowering the ambient 02 concentration of the nodule havebeen unsuccessful so far. Whether the symbiotic expression ofnifi is under very stringent oxygen control or whether itundergoes an additional level of control remains to be deter-mined.

We are very much indebted to Pierre Renault for his generous helpin conducting the microelectrode experiments at the Avignon Center

of the Institut National de la Recherche Agronomique. We thankMichael Hynes for providing plasmid pJQ200 prior to publication. Weare very grateful to David Barker for his constructive criticism of themanuscript. Valerie Delage and Pascale de Philip participated in theearly phases of this work. We acknowledge the support of theEuropean Commission (BIOT CT 900166C) and of the MicrobiologyProgramme of the Institut National de la Recherche Agronomique.E.S. was supported by a fellowship from the Ministere de l'Enseigne-ment Superieur et de la Recherche.

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