Vol. 172, No. 8

Rhizobium meliloti FixL Is an Oxygen Sensor and RegulatesR. meliloti nifA and fixK Genes Differently in Escherichia coli

PASCALE DE PHILIP,* JACQUES BATUT, AND PIERRE BOISTARD

Laboratoire de Biologie Moleculaire des Relations Plantes-Microorganismes, Centre National de la RechercheScientifique-Institut National de la Recherche Agronomique, 31326 Castanet-Tolosan Cedex, France

Received 22 February 1990/Accepted 11 May 1990

In Rhizobium meliloti, nifandfix genes, involved in nitrogen fixation during symbiosis with alfalfa, are underthe control of two transcriptional regulators encoded by nifA and fixK. Expression of nifA andfixK is under thecontrol of FixL/J, a two-component regulatory system. We showed, using Escherichia coli as a heterologoushost, that FixL/J controls nifA andfixK expression in response to microaerobiosis. Furthermore, expression ofthe sensor gene fixL and of the activator gene fixj under the control of two different promoters allowed us toshow that FixL mediates microaerobic induction of nifA when the level of FixJ is low and aerobic repressionof nifA when the level of FixJ is high. Similarly, activation of fixK occurred in microaerobiosis when the FixJlevel was low in the presence of FixL. In contrast to nifA, fixK expression was not affected by FixL in aeratedcultures when the level of FixJ was high. We conclude that R. meliloti FixL senses oxygen in the heterologoushost E. coli consistent with the microaerobic induction of nifA and fixK in R. meliloti and that nifA andfixKpromoters are differentially activated by FixJ in response to the oxygen signal.

Although the structure and the biochemical properties ofthe nitrogenase complex are highly conserved, a remarkablefeature of diazotrophs is that they differ with respect to thephysiological conditions under which they fix molecularnitrogen. For example, Klebsiella pneumoniae is an entericbacterium able to use atmospheric nitrogen for growth in theabsence of oxygen and of fixed nitrogen. On the other hand,bacteria of the genus Rhizobium fix nitrogen only in non-growing, microaerobic, symbiotic conditions.

In K. pneumoniae, the expression of nifgenes, needed forthe synthesis, processing, and activity of the nitrogenasecomplex, is controlled by NifA, a transcriptional activator,and NifL, which decreases NifA activity in the presence ofoxygen and fixed nitrogen. Expression of the nifLA operonitself is controlled by the level of nitrogen by a two-component regulatory system, NtrB/NtrC. At high ratios ofot-ketoglutarate to glutamine, the NtrB protein phosphory-lates NtrC, which then activates the transcription of thenifLA operon (5, 22).A NifA homolog has been identified in Rhizobium meliloti,

as well as in other rhizobia. The R. meliloti NifA proteinactivates the symbiotic transcription of a set of genes havingtheir counterpart in free-living nitrogen-fixing bacteria (1, 9,15, 21, 52). However, symbiotic expression of R. melilotinifA is uncoupled from the NtrB/NtrC system of regulation(13, 51). In asymbiotic conditions, nifA expression is oxygenregulated and can be induced in microaerobiosis (56). Fur-thermore, R. meliloti NifA transcriptional activity is inacti-vated by oxygen (16, 25).

In addition to NifA-dependent nif and fix genes, other fixgenes, NifA independent, are needed for symbiotic nitrogenfixation by R. meliloti (10, 11, 44). Furthermore, the expres-sion of some of these genes, e.g., thefixN operon, is inducedin symbiosis and in microaerobic conditions and depends onthe presence of the fixK gene (6).We have shown that symbiotic and microaerobic expres-

sion offixK and nifA in R. meliloti is controlled by a pair ofregulatory proteins, FixL and FixJ (10). Sequence data have

* Corresponding author.

led to the prediction that FixL and FixJ belong to a family oftwo-component regulatory systems, in which a pair of sensorand regulator proteins mediates an adaptative response to anenvironmental stimulus. These systems are characterized bya C-terminal conserved region among the proteins that act as

sensors and an N-terminal conserved region among theproteins that act as regulators. The FixL C terminus ishomologous over a region of 250 amino acids to differentcomponents of the sensor class (3, 10, 50). In addition, in itsN terminus, FixL has two sequences which represent poten-tial transmembrane helices as found in many sensor compo-nents of two-component regulatory systems. FixJ showssimilarity to the N terminus of the regulator class compo-nents over the first 120 amino acids (3, 10, 50). In addition,the C-terminal domain of FixJ shows homology with the Cterminus of transcriptional activators such as UhpA ofEscherichia coli (20) and DegU of Bacillus subtilis (33).Two-component systems transduce various environmen-

tal signals such as nutrient concentrations and physical andchemical parameters and are part of complex regulationsystems which control various responses, including chemo-taxis, sporulation, symbiotic nitrogen fixation, and bacterialpathogenesis. In most cases, how the sensor senses theenvironmental stimulus is unknown. However, in someinstances it has been shown that the N-terminal domain ofthe sensor protein probably senses the stimulus directly, as

examplified by VirA (61, 62) or, alternatively, the stimulus isfirst perceived by a primary environmental sensor or inter-mediate and transduced into an intracellular signal, which isthen detected by the sensor component of the two-compo-nent systems as documented for CheA (8, 42) and NtrB (32,35). The signal could then be transmitted to the C-terminaldomain of the sensor possibly by conformational alterationsin the protein structure or by a change in the state ofoligomerization (45). This conserved domain then interactswith the N-terminal conserved domain of the regulatorprotein. It has been shown that the sensor proteins NtrB,CheA, and EnvZ are protein kinases that can autophospho-rylate and transfer the phosphate group to the cognateregulator proteins NtrC, CheB/CheY, and OmpR, respec-

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TABLE 1. Bacterial strains, plasmids, phage, and transposon used

Strain, plasmid, Relevant characteistics Source or

phage, or transposon ee~ccnisreference(s)E. coliHB101 ara gal met pro xyl supE rpsL (Sm') recA hsdR hsdM 36S17-1 F- lambda- recA hsdR carrying a modified RP4 (Aps Tcs Kms) integrated in the 47

chromosomeTG1 A(lac-pro) supE thi hsdDJ (F' tra36 proA'B' lad" lacZAM15) 12, 58

R. melilotiGM15600 lac pSym2O::TnS, Smr Nmr Bleor Tcr GM1211 derivative 10GM15704 lacfixJ2.3::Tn5, Smr Nmr Bleor Tcr GMI211 derivative 10GMI5705 lacfixL2.66::TnS, Smr Nmr Bleor Tcr GMI211 derivative 10GMI346 lacfixL2.2::Tn5, Smr Nmr Bleor Tcr GMI211 derivative 7, 10

PlasmidspRK2013 Helper plasmid for mobilization of pSUP104 derivatives tra(RK2) ori(ColE1) KMr 19pDD5 pBR322 derivative, carrying a 13.6-kilobase fragment from pHB488, Apr 11pCH2 pUC19 derivative, plac-flxJ, Apr 24pML123 pSUP104 derivative, pNm, Gm' Nmr M. LabespML133 pSUP104 derivative, ptac, Nmr Gmr M. LabespDP2 pML123 derivative, pNm-fixL, Gmr This studypDP3 pML133 derivative, ptac-fixLJ, Gmr This studypDP3::lac pDP3 derivative, ptac-fixL::lac, Gmr Kmr This studypDP4 pML133 derivative, fixLJ, Gmr This studypCHK57 pGD926 derivative, pnifA-lacZ translational fusion, Tc' 13pJJ5 pIJ1363 derivative, pfixK-lacZ translational fusion, Tcr 6

PhageLambda 573 b221(att- int-) red- Oam Pam cIts857 N. Kleckner

TransposonTn5-B20 TnS carrying a promoterless lacZ gene cloned to IS50L, KMr 48

tively (26, 27, 32, 35, 40, 42). This suggests that phosphory-lation is the general mechanism of signal transfer used bythese two-component systems. The phosphorylation/dephosphorylation of the regulator proteins would changetheir DNA-binding properties or their ability to interact withother proteins or both, thereby modulating their biologicalactivities.According to the current model, which accounts for the

properties of most members of the two..component systems(3, 45), FixL could be a transmembrane sensor protein,which reacts to a change in the bacterial environment byphosphorylating the cognate activator protein FixJ, which inturn promotes the transcription of nifA and fixK genes. Inprevious work (24), it has been shown that FixJ was indeeda transcriptional activator of fixK and nifA genes. Sinceactivation of nifA and fixK expression responds to oxygen,the most likely hypothesis was that FixL would senseoxygen concentration. Furthermore, this regulation wouldhave a biological significance since the nodule in which fixand nif genes are expressed constitutes an environmentwhere the concentration of free oxygen is maintained at alow level (63).

Here, using E. coli as a heterologous host, we show that,in the presence of FixL, FixJ-mediated activation offixK andnifA promoters is sensitive to oxygen concentration, anindication that FixL senses oxygen. In addition, our resultsindicate that there is a difference between the activation ofthe expression of nifA and that offixK by FixJ, the signifi-cance of which is discussed.

MATERIALS AND METHODSDNA manipulations. Restriction enzymes, the Klenow

fragment of E. coli DNA polymerase I, and T4 DNA ligase

were obtained from New England Biolabs, Beverly, Mass.,or Boehringer Mannheim Biochemicals, Indianapolis, Ind.,and were used as recommended by the manufacturer. Re-striction endonuclease-generated DNA fragnents were sep-arated on 1% agarose gels in TAE buffer (36). Fragmentswere eluted from gels by excision and dissolution of the gelslice in Nal foilowed by purification on GeneClean beads(BiolOl, Inc., La Jolla, Calif.). Standard procedures wereused for the extraction of plasmid DNA, in vitro recombi-nation, and transformation (36).

Bacterial strains and p ds. Strains, bacteriophages,and plasmids used in this study are described in Table 1.Plasmids constructed for this study were derived from thebroad-host-range IncQ mobilizable expression vectorspML123 and pML133 (43) kindly provided by M. Labes(submitted for publication). pML123 and pML133 carry theGmR gene (aacl) and, respectively, the promoter of theNmR gene (pNm), allowing' constitutive expression of acloned fragment in E. coli and R. meliloti (Labes, submit-ted), or the tac promoter (ptac), which can be activated bythe inducer isopropyl-3-D-thiogalactoside (IPTG) in E. coli(4) and constitutively expressed in R. meliloti (Labes, sub-mitted). pDP2, which allows expression of fixL, was ob-tained by cloning a SnaBI-BalI fragment of 2,121 base pairsfrom pDD5 (11) at a SmaI site downstream of pNm inpML123. The same fragment, in the reverse orientation,gave the recombinant plasmid pDP1, which expresses fixLunder the control of its own promoter. A SnaBl-BamHIfragment of 3,963 base pairs from pDD5 was cloned, afterfilling in the ends, at a SmaI site downstream of ptac inpML133, giving pDP3, allowing expression of fixLJ fromptac, and pDP4 in the reverse orientation. In all constructs,a 1,000-base-pair SmaI vector fragment, carrying the neo-

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OXYGEN REGULATION OF nifA AND fixK BY R. MELILOTI FixL

mycin resistance gene, was deleted. A transcriptional fusion,fixL::lacZ, was constructed in pDP3 (pDP3::1ac), usingTnS-B20 (48) to measure the expression from the tac pro-moter.Media and growth conditions. Bacterial cultures were

grown at 37°C in LB medium (36) for E. coli and at 30°C inTY medium (31) for R. meliloti. M9 minimal medium wassupplemented with MgSO4 (1 mM) (36) and glucose (0.2%),thiamine (0.5 ,ug/ml), and proline (100 jig/ml, when neces-

sary) for E. coli or saccharose (0.2%) and biotin (0.5 g/ml)for R. meliloti (46). Ampicillin (Ap; 50 jig/ml), tetracycline(TC; 10 jig/ml), kanamycin (Km; 30 pug/ml), and gentamicin(Gm; 10 pug/ml for E. coli and 45 ,ug/ml for R. meliloti) wereincluded as needed to select different strains. To induce thelac and tac promoters, IPTG was added in solid medium(0.16 mM) and in liquid medium (0.2 to 2 mM). Finally,P-galactosidase activity was visualized on plates by addingXGal (5-bromo-4-chloro-3-indolyl-p-D-galactoside; 32 ,ug/ml).

Bacterial crosses. Matings were performed between recip-ient strains, donor strains, and the helper strain HB101(pRK2013) when necessary, as described previously (54).Transconjugants were selected on appropriate media.

Mutagenesis of pDP3 with TnS-B20. Phage lambda 573carrying the transposon TnS-B20 (48) was used to muta-genize E. coli S17-1 harboring plasmid pDP3. E. coli S17-1(pDP3) was grown to a density of 109 cells per ml in LBmedium containing Gm and maltose (2 g/liter) and infectedwith lambda 573::TnS-B20 at a multiplicity of infection of1.5. Following infection, cells were plated on LB agar

containing Km and Gm. Resulting mutagenized pDP3 plas-mids were then mobilized en masse into E. coli TG1 recipi-ent strain, and transconjugants carrying a transcriptional lacfusion were identified as blue colonies on minimal mediumcontaining Gm, Km, and XGal. The location and the orien-tation of the TnS-B20 insertions in pDP3 were determined byusing restriction sites carried by TnS-B20. pDP3::lac has an

insertion infixL approximately 800 base pairs downstream ofthe tac promoter.

Microaerobic induction of fixK and nifA. E. coli or R.meliloti strains carrying transcriptional and translational lacfusions were grown at 30°C in M9 minimal medium contain-ing glucose and thiamine or saccharose and biotin, respec-

tively, and the required antibiotics in well-aerated vigorouslyshaken flasks (optical density at 600 nm, 0.1 to 0.3). Cultureswere divided into two parts: 0.5 volume was left in aeratedconditions; the other half was bubbled with a 2% oxygen-

98% nitrogen gas mixture at 0.55 liter/min per flask. ,B-Galactosidase activity was measured on samples removedafter an additional growth of 4 h.

P-Galactosidase assays. Samples were centrifuged andbacterial pellets were suspended in Z buffer as describedbefore (10). Cells were permeabilized with chloroform-so-dium dodecyl sulfate, and ,-galactosidase activity was mea-

sured as described by Miller (38). The activity is expressedin nanomoles of o-nitrophenyl-p-D-galactoside hydrolyzedper minute and per optical density (600 nm) unit. Theactivities listed are the averages of the results from fourassays. In each case the individual assays gave values within20% of the average.

RESULTS

Oxygen dependence of expression offixK and nifA in E. coli.In R. meliloti, nifA and fixK genes, whose expressiondepends on fixLJ, are induced in asymbiotic conditions in

microaerobic cultures. We asked whether this modulatedresponse to oxygen concentration was mediated throughFixL and FixJ. An approach to analyze the role of thevarious components of a regulatory pathway and theirinteraction with regulatory signals is the reconstruction ofthe pathway in a genetically distant background.To study the expression of the target genes nifA and fixK,

we used the nifA-lacZ translational fusion plasmid pCHK57(13) and afixK-lacZ translational fusion plasmid, pJJ5 (6). Toexpress the fixLJ operon on a compatible expression vectorin E. coli, we cloned the entire coding region of both genesas well as a 191-base-pair upstream region downstream ofthe tac promoter of pML133 (Fig. 1). The recombinantplasmid pDP3, which expresses fixLJ under the control ofptac, as well as pDP4, which has the same fragment but inthe reverse orientation with respect to ptac, were introducedinfixL and fixJ mutant strains of R. meliloti containing pJJ5[GMI5705(pJJ5) and GM15704(pJJ5)]. As these mutantstrains are known not to express afixK-lacZ fusion (6), pDP3and pDP4 were assayed for ability to restore the expressionof thefixK-lacZ fusion. The assay was done on plates of TYmedium containing XGal. Both plasmids allowed expressionof the fixK-lacZ fusion (data not shown), an indication thatfixL and fixJ genes are expressed in R. meliloti by theseconstructions.To have a quantitative estimate of the level of expression

of the fixLJ operon under the control of ptac in variousconditions, we used a transposon-mediated transcriptionalfixL::lacZ fusion carried by pDP3 (pDP3::lac) and measured,-galactosidase activity in either aerated or microaerobiccultures of E. coli TG1(pDP3::1ac) (Table 2). In strain TG1,which carries the lac repressor gene lacIq (12, 58), the levelof expression of genes cloned downstream of plac and ptacdepends on the concentration of the inducer IPTG. Ourresults show that there was a similar induction rate offixL::lacZ expression by IPTG in both culture conditions,which indicates thatfixLJ expression is under the control ofptac whatever the aeration status of the culture. The activitymeasured in the absence of IPTG shows a residual lacZexpression, independent of the oxygen concentration, result-ing from leakiness in the negative control of the tac promoterin the absence of the inducer.pDP3, carrying fixLJ, was introduced into E. coli

TG1(pJJ5) and TG1(pCHK57). Table 3 shows the effect ofmicroaerobic conditions on the expression of fixK-lacZ andnifA-lacZ fusions in the presence of fixLJ (pDP3). In thecontrol strains, without pDP3, there is no expression ofeitherfixK or nifA in microaerobic or aerated cultures. Thesame result was obtained with strains carrying the controlplasmid pDP4 in whichfixLJ expression is not driven by ptac(data not shown). In the presence of pDP3 and in the absenceof IPTG, microaerobiosis leads to a 40-fold induction offixKexpression. However, in the presence of IPTG,fixK expres-sion is independent of the aeration status of the culture. nifAexpression, in the presence of pDP3, shows a five-foldincrease in microaerobic conditions and in the absence ofIPTG, whereas with IPTG, a 10- to 40-fold induction is seenin microaerobic conditions. The same results were obtainedwhen the fixK-lacZ fusion was recloned on the pGD926vector used for the construction of pCHK57 (13; F. Waelk-ens; personal communication).The transcription offixLJ on plasmid pDP3 was dependent

on the presence of the inducer and not on the aeration statusof the culture (Table 2). Therefore, it is likely that microaer-obic induction of_fixK and nifA is due to an oxygen-mediatedmodulation of the activity of the FixL and/or FixJ regulatory

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RUCAC BI U iII .

I I IfixL fixJ fixK

nifAnifA

,1)D1'2pCII2)PD3

T

p,.1.1J5pCHK57

500 bp

FIG. 1. Physical genetic map offixLJ, fixK, and nWfA and plasmids used. Genes are represented as rectangular boxes, the transcriptionalfusion is represented by a black triangle, and translational fusions are represented as hatched boxes. Vertical arrows mark locations oftransposon insertions used in the experiments described in this paper. Restriction sites: Ac, AccI; Ba, Ball; B, BamHI; Bg, BgIII; R, EcoRI;Sn, SnaBl; Xh, XhoI. bp, Base pairs.

proteins. Microaerobic induction was observed for bothflxKand nifA when the level of transcription offixLJ was low. Onthe other hand, when fixLJ expression was fully induced,fixK was expressed at the same high level in aerobiosis as

well as in microaerobiosis, whereas microaerobic inductionwas observed only for nifA expression.FixL is responsible for oxygen sensitivity of nifA and flxK

transcriptional activation. In previous experiments, we hadshown that, when fixJ alone was cloned downstream of thelac promoter of the multicopy pUC19 plasmid resulting inpCH2, nifA and fixK were expressed independently of theaeration status of the bacterial culture (24). Therefore, thepresent results with pDP3, carrying both fixL and flxJ andallowing microaerobic induction of the expression of fixKand nifA in E. coli, are an indication that FixL is responsiblefor the oxygen sensitivity of the regulatory system. How-ever, oxygen sensitivity of nifA and fixK expression varieddifferently with the level of transcription of fixLJ. To dis-criminate the role of each component in the activation ofbothfixK and nifA expression and to confirm that FixL is theoxygen-sensing component in the regulatory system, we

reconstituted a regulatory system in E. coli in whichjfxL andfix. were expressed under the control of two differentpromoters carried by two compatible plasmids.

TABLE 2. Expression of ptac-fixL::1acZ fusion in E. coliTG1(pDP3::lac)

IPTG concn P-Galactosidase units

(mM) Aerated culture Microaerobic culture

0 290 3130.2 5,600 6,3002 10,200 9,500

fixL was cloned downstream of the Nm constitutive pro-moter of pML123 to give the recombinant plasmid pDP2(Fig. 1). To express filxJ, we used the pUC19 derivativepCH2 (24). pCH2 and pDP2 were introduced into E. coli

TABLE 3. Oxygen-sensitive expression offixK-lacZ andnifA-lacZ fusions in E. coli in the presence offzxLJ

p-Galactosidase unitsStrain ~IPTG concnStrain IPTGcn(MM) Aerated Microaerobic

culture culture

TG1(pJJ5) 0 0 0.070.2 0.07 0.072 0.07 0.07

TG1(pML131, pJJ5) 0 0.07 0.150.2 0.15 0.152 0.07 0.15

TG1(pDP3, pJJ5) 0 1.5 600.2 70 952 130 160

TGl(pCHK57) 0 0.2 0.60.2 0.7 0.72 0.7 0.7

TG1(pML131, pCHK57) 0 0.2 0.30.2 0.7 0.62 0.7 0.5

TG1(pDP3, pCHK57) 0 1.5 80.2 6 702 1.5 70

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OXYGEN REGULATION OF nifA AND fixK BY R. MELILOTI FixL 4259

TABLE 4. Oxygen-sensitive expression offixK-lacZ andnifA-lacZ fusion in E. coli in the presence offixL and fixJ

13-Galactosidase unitsStrain IPTG concn(mM) Aerated Microaerobic

culture culture

TG1(pJJ5) 0 0 0.072 0.07 0.07

TG1(pCH2, pJJ5) 0 3 42 350 422

TG1(pDP2, pCH2, pJJ5) 0 4 332 240 271

TG1(pCHK57) 0 0.2 0.62 0.7 0.7

TG1(pCH2, pCHK57) 0 1.5 1.52 20 22

TG1(pDP2, pCH2, pCHK57) 0 1 82 1.4 54

TG1(pJJ5) and TG1(pCHK57). Table 4 shows the effects ofpDP2 and pCH2 on the expression of pJJ5 and pCHK57 inaerated and microaerobic cultures. The presence of fixJalone did not allow expression of eitherfixK or nifA in theabsence of IPTG. However, upon induction offixJ expres-sion by the addition of IPTG, fixK-lacZ and nifA-lacZfusions were expressed independently of the aeration statusof the culture. In the presence offixL on pDP2, microaerobicinduction (eightfold) of fixK and nifA expression was ob-served in the absence of IPTG. At 2 mM IPTG, whichinduces fixJ expression, fixK-lacZ fusion was expressedindependently of the aeration status of the cultures at a levelsimilar to that observed in the absence of pDP2. Induction ofthe expression offixJ led to a situation completely differentfor nifA. Upon induction offixJ expression, in microaerobi-osis, the presence of pDP2 caused a twofold increase ofnifA-lacZ expression compared with that observed in thestrain lacking fixL (pDP2). On the other hand, the presenceof pDP2 caused a sevenfold decrease of nifA-lacZ expres-sion upon fixJ induction in an aerated culture.

Therefore, FixL exerts a positive control on bothfixK andnifA expression in microaerobic conditions at low levels offixJ expression. At high levels offixJ expression, the expres-sion offixK is independent of FixL, whereas nifA expressionis contrqlled positively by FixL in microaerobic conditionsand negatively in aerated cultures.

In previous work, we described an R. meliloti TnS inser-tion mutant, fixL::TnS 2.2, whose phenotype is a reducedability for symbiotic nitrogen fixation and a constitutiveexpression of the fixK-lac fusion on pJJ5 (7, 10). As the TnSinsertion is at the distal end of fixL, fixJ could be, in thismutant, expressed from a secondary promoter created bythe TnS insertion. Furthermore, fixJ expression at a higherlevel than in the wild-type strain could explain the constitu-tive phenotype of this mutant. We introduced pDP1, whichexpresses fixL under the control of its own promoter, intothis constitutive fixL mutant [GMI346(pJJ5)] and looked atthe expression of fixK in aerated or microaerobic cultures.Results (Table 5) show that pDP1 restores oxygen control offixK expression, resulting in the same level of microaerobicinduction as observed in the wild-type strain [GM15600(pJJ5)] containing the pDP1 plasmid.

TABLE 5. Oxygen-sensitive expression offixK-lacZ fusionin R. meliloti in the presence offixL

,B-Galactosidase unitsStrain

Aerated culture Microaerobic culture

GM15600(pJJ5) 3 443GM15600(pDP1, pJJ5) 43 150

GM1346(pJJ5) 168 159GM1346(pDP1, pJJ5) 25 71

DISCUSSION

Genetic studies have led us to propose a model for thesymbiotic activation of nifandfix genes ofR. meliloti (6, 10).In this model, two sets of nif and fix genes are under thecontrol of two different regulatory genes, nifA andfixK. BothnifA andfixK are positively regulated by a pair of regulatoryproteins, FixL and FixJ, which are predicted to belong to thefamily of two-component regulatory systems. Since thegenes regulated by fixLJ can be expressed in microaerobicculture, we hypothesized that FixL was sensing oxygenconcentration. In the present work, we show that, whenboth are present, the R. meliloti FixL and FixJ proteinsallow microaerobic activation of the expression offixK andnifA in E. coli. We further confirm the role of FixL as anoxygen sensor by showing that, in an E. coli strain in whichthe level of FixJ is insufficient for activation of nifA andfixK,expression offixL results in an oxygen-sensitive expressionof nifA and fixK. Previous results had shown that, whenoverexpressed in E. coli, the R. meliloti FixJ regulatorprotein was sufficient to allow expression of the two symbi-otic genesfixK and nifA independently of the aeration statusof the culture (24). Therefore, it can be concluded that FixLis responsible for the oxygen sensitivity of nifA and fixKexpression in E. coli. In addition, the effect of FixL onoxygen regulation offixK expression has also been observedin R. meliloti strains.The fact that oxygen-dependent expression of fixK and

nifA occurs in the heterologous host E. coli allows us topredict that all elements involved in sensing the signal in R.meliloti exist or have their counterpart in E. coli. EitherFixL itself is an oxygen-sensitive protein or it interacts withcompounds quantitatively or qualitatively modified in asimilar way in both organisms in response to oxygen. Per-ception of an environmental signal by a transmembranesensor has been shown, in some cases, to involve a directinteraction, as, for example, between the Tar chemoreceptorand the aspartate attractant (57). This interaction takes placebetween the external ligand and the periplasmic domainlimited by the two transmembrane helices. Interestingly, forVirA protein of Agrobacterium tumefaciens, which appearsto be similar in structure to the chemoreceptors (62), it hasbeen shown that the putative receptor domain for acetosy-ringone is located beyond its periplasmic domain and thatthe second transmembrane region is involved in stimulusdetection (37). Thus, FixL, which shows structural similar-ities with the chemoreceptors and VirA in the N-terminaldomain, could sense the oxygen signal by its periplasmicand/or its transmembrane helix(ces). It is not known whetherFixL is a primary sensor or whether there is an intermediatebetween the oxygen signal and FixL. There are severalarguments in favor of the second hypothesis. One is that thecytoplasmic membrane is permeable to oxygen and severalregulatory proteins which respond to oxygen concentration

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are purely cytoplasmic proteins. Fnr, for example, is a

cytoplasmic regulatory protein which, in response to anaero-

biosis, activates transcription of genes involved in anaerobicrespiratory pathways of E. coli (55). Another example couldbe provided by NifA protein of R. meliloti and Bradyrhizo-bium japonicum, which are also oxygen-sensitive transcrip-tional activators (16, 25). Cysteine clusters, similar to metal-binding motifs, are present in FNR protein of E. coli andNifA protein of B. japonicum and have been shown to beneeded for the oxygen-regulated activity of both proteins(16, 53). Furthermore, NifL of K. pneumoniae, which ap-

pears to antagonize NifA-mediated activation in response tooxygen, also has a cysteine cluster (14). NifL repressingactivity is reduced by metal ion deficiency and is restored byaddition of ferrous or manganous irons (23). Sequenceanalysis offixL (10) did not reveal any such cysteine clustersor heme-binding domains, which provides a second argu-ment in favor of the involvement of an intermediate betweenthe oxygen signal and FixL.

Since FixL is a transmembrane protein, it may interactwith oxygen-sensitive compounds located in the cytoplasmicmembrane. Components of the terminal electron transportchains, such as cytochromes or ubiquinones, which are

located in the cytoplamic membrane exist in different redoxstates according to the aeration status of the bacterialculture. Changes in their structure, mediated by their redoxstate, could result in a conformational change of the FixLtransmembrane helix(ces). Those compounds, present inboth E. coli and R. meliloti, might serve as an effectorbetween the oxygen signal and FixL. The availability of E.coli mutants affected in their electron transport chain com-

ponents should facilitate the identification of the putativeprimary oxygen sensor. Interestingly, a regulator whichmediates anaerobic repression of E. coli succinodehydroge-nase and other enzymes of the aerobic metabolism has beenidentified (30). The sequence of this gene, gene arcA, pre-dicts that it codes for the regulator component of a two-component regulatory system. Mutations in a second regu-latory gene, arcB, abolish anaerobic repression, suggesting a

likely role for the ArcB product as a sensor of oxygenconcentration (28, 29). Studies with different terminal elec-tron acceptors has indicated that the ArcB/ArcA regulatorysystem is not directly sensitive to oxygen, but rather senses

the cellular redox state (30). FixL might have functionalsimilarities with the ArcB sensor.Other results of interest concern the differential microaer-

obic induction of the target genes nifA and fixK. Althoughthe regulation offixK and nifA expression depends on FixJ,fixK and nifA promoters do not show a great similarity (6).The differential expression of nifA and fixK was seen whenfixJ was overexpressed in the cell. In this condition, nifAexpression appears to be induced by microaerobiosis andrepressed in aerobiosis in the presence of FixL. By contrast,in the presence of high levels of FixJ, fixK expression isconstitutive and does not depend on the presence of FixL.

Regulation of nifA expression is consistent with the gen-eral model of regulation predicted for two-component sys-tems. It has been shown that, in the NtrB/NtrC and EnvZ/OmpR systems, NtrB and EnvZ are bifunctional enzymesendowed with kinase and phosphatase activities whosebalance regulates the level of phosphorylated cognate regu-lator in response to the environmental signal (26, 32, 40).According to this model, the phosphorylated regulator rep-resents the active form able to promote transcription of thetarget genes. By analogy, FixL could activate FixJ byphosphorylation in response to microaerobiosis and dephos-

phorylate FixJ, thereby inactivating FixJ transcriptionalactivating function on the nifA promoter in aerobiosis. Ourresults support both the activating function of FixL inmicroaerobiosis and its repressing function in aerobiosis.When FixJ concentration is low, we observed a positiveeffect of the presence of FixL in microaerobic conditionsconsistent with a FixL-mediated phosphorylation of FixJ.When FixJ concentration is high, FixL had a marked de-pressing effect on nifA expression in aerobic conditions. Thiscould be best explained by the dephosphorylation activity ofFixL. Therefore, one has to assume that phosphorylation ofFixJ was occurring in the absence of FixL when FixJconcentration was high.

Phosphorylation of the regulator protein of a two-compo-nent system by the sensor of another system has beenobserved in vitro for the CheA/CheY,CheB system, theNtrB/NtrC system (41), and the EnvZ/OmpR system (26). Ifthis also operates in vivo, it would create a "cross-talk"between various regulatory circuits and would result in thepossibility of a regulator activating its target genes in theabsence of its cognate sensor. This has indeed been ob-served when ntrC (34), ompR (18, 39, 49), and uhpA (59, 60)regulator were overexpressed. Our previous results (24), aswell as those reported here, have shown that this was alsoobserved for FixJ.Although a marked positive effect of FixLJ onfixK expres-

sion in microaerobiosis was observed when FixJ concentra-tion was low, at high FixJ concentrations there was nodecrease in fixK expression due to the presence of FixL inaerated conditions. Therefore, taking into account the re-sults obtained with nifA, which suggest that cross-activatedFixJ is dephosphorylated by FixL in aerobiosis, we proposethat nonphosphorylated FixJ is able to activate fixK whenpresent in high concentrations. Similarly to nifA activation,FixJ in low concentrations would induce fixK expressiononly when phosphorylated by its cognate sensor FixL,suggesting that phosphorylated FixJ is more active thannonphosphorylated FixJ. This proposed differential activityof the nonphosphorylated regulator protein on differentpromoters could also apply to other regulatory systems suchas EnvZ/OmpR. It has recently been suggested that non-phosphorylated OmpR would function as a transcriptionalactivator of ompC, whereas its phosphorylated form wouldactivate ompF expression (17).An alternative explanation of the negative effect of FixL

could be that FixL and FixJ form a complex when overpro-duced that inhibits access by the cross-talking E. coli pro-tein.

Nevertheless, it is possible to explain nifA and fixKdifferential expression in E. coli by supposing that phosphor-ylated FixJ activates nifA and fixK expression and that,when overproduced, nonphosphorylated FixJ is able toactivate fixK expression. In addition, the affinity of phos-phorylated FixJ and nonphosphorylated FixJ for the fixKpromoter could be different. It has recently been reportedthat phosphorylated OmpR binds to promoters of its targetgenes with greater affinity than OmpR (2). Differential aero-bic expression of nifA and fixK could alternatively be ex-

plained by assuming that FixL does not dephosphorylate allphosphorylated FixJ in aerobiosis. The remaining phosphor-ylated FixJ could have more affinity for the fixK promoterthan for the nifA promoter. The latter hypothesis is consis-tent with the observation that fixK expression is induced bymicroaerobiosis more rapidly than nifA expression in R.meliloti (N. Nouyrigat and D. Kahn, unpublished results).Phosphorylated FixJ would first titrate the fixK promoter

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OXYGEN REGULATION OF nifA AND fixK BY R. MELILOTI FixL 4261

and then activate the nifA promoter. It is relevant to notehere that pCH2 and pDP3 carry, downstream offixJ, thefixKpromoter which could be responsible for titration of phos-phorylated FixJ in the experiments in which nifA promoteractivation was studied. To test the phosphorylation hypoth-esis, directed mutagenesis on the potential phosphorylationsites of FixJ and analysis of the transcriptional activities ofFixJ mutant proteins onfixK and nifA expression are beingperformed in this laboratory.The biological significance, if any, of the differential

expression of nifA andfixK remains to be elucidated. In theheterologous system we have used here, constitutive expres-

sion offixK is observed only when the level of the activatorFixJ is high. Previous studies in this laboratory have shownthat the concentration offixLJ mRNA is low in bacteroids as

well as in bacterial cells in asymbiotic conditions (11).However, it is not impossible that this low level, detected insymbiotic conditions, represented an average value, mask-ing differences occurring between bacteroids at differentstages of differentiation. According to this hypothesis, dif-ferential expression of fixK could be needed for a fullyefficient symbiotic process. Alternatively, fixLJ expressionin bacteroids could vary in response to environmental sig-nals such as energy supply or the level of fixed nitrogen. Theresulting differential expression offixK and as a consequence

of the fixN operon relative to nif genes could provide a

means for a fine adjustment of the nitrogen-fixing apparatusin response to variations in the physiological conditions ofthe nodule.

ACKNOWLEDGMENTS

We are grateful to M. Labes and A. Piihler for providing vectorsused in this work. We thank M. David, D. Kahn, and C. Gough forhelpful comments on the manuscript. We also thank the anonymousreferee who suggested an alternative explanation for FixL negativeeffect on nifA transcription in aerobiosis.

This work was supported in part by a grant from the Commissionof the European Communities in the frame of the BiotechnologyAction program.

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