REGULAR ARTICLE

Nitrogen fixation control in Herbaspirillum seropedicae

Leda Satie Chubatsu & Rose Adele Monteiro & Emanuel Maltempi de Souza &

Marco Aurelio Schuler de Oliveira & Marshall Geoffrey Yates & Roseli Wassem &

Ana Claudia Bonatto & Luciano Fernandes Huergo &

Maria Berenice Reynaud Steffens & Liu Un Rigo & Fabio de Oliveira Pedrosa

Received: 1 February 2011 /Accepted: 2 May 2011 /Published online: 27 May 2011# Springer Science+Business Media B.V. 2011

Abstract Herbaspirillum seropedicae is a Gram-negative endophytic diazotroph that associates withimportant agricultural crops. Several studies haveshown that this organism can contribute to plantgrowth suggesting potential for use as a biofertilizer.Nitrogen fixation in H. seropedicae is highly regu-lated both at the transcriptional and post-translationallevels. Both of these regulatory levels respond to theammonium availability in the external mediumthrough a cascade of interacting proteins. Thetranscriptional regulation of the process also respondsto oxygen, which is probably directly sensed by thetranscriptional regulator NifA. Here, we reviewcurrent knowledge of the regulation of nitrogenfixation in H. seropedicae. The signal transductionprotein GlnK is a key regulator of nitrogen fixation atboth the transcriptional and post-translational levels.In vitro analysis indicates that GlnK interacts withNifA and probably modulates its activity, thereby

controlling nif expression. GlnK, together with theammonium channel protein AmtB, also participates inthe post-translational regulation of nitrogenase activityby an unidentified mechanism. This regulatory systemefficiently controls nitrogen fixation according toprevailing fixed nitrogen and oxygen levels in H.seropedicae.

Keywords Herbaspirillum seropedicae . Nitrogenfixation . Nitrogenase regulation . NifA . PII protein

Nitrogen fixation

All living organisms require nitrogen to synthesizeessential biomolecules such as proteins and nucleicacids. However, the major source of nitrogen innature, atmospheric nitrogen, is not assimilable bymost organisms. Only a small group of prokaryotescan reduce atmospheric nitrogen to ammonium, in anenergy-demanding process called nitrogen fixation.Microorganisms with this ability are called diazo-trophs. One of the most studied group of diazotrophscomprises microorganisms that live in close associa-tion with plants, and which have the potential totransfer the fixed nitrogen to the host. Endophyticdiazotrophs are able to colonize plant internal tissueswithout damaging the host.

Atmospheric N2 reduction is a reaction catalyzed bythe nitrogenase enzyme complex. The most commonform of nitrogenase is composed of two metalopro-

Plant Soil (2012) 356:197–207DOI 10.1007/s11104-011-0819-6

Responsible Editor: Euan K. James.

L. S. Chubatsu (*) : R. A. Monteiro : E. M. de Souza :M. A. S. de Oliveira :M. G. Yates : L. F. Huergo :M. B. R. Steffens : L. U. Rigo : F. O. PedrosaDepartment of Biochemistry and Molecular Biology,Universidade Federal do Paraná,CP 19046, Curitiba, PR 81531–980, Brazile-mail: chubatsu@ufpr.br

R. Wassem :A. C. BonattoDepartment of Genetics, Universidade Federal do Paraná,CP 19071, Curitiba, PR 81531–980, Brazil

teins, the iron-protein (Fe) and the molybdenum-iron(MoFe) protein (Bulen and Lecomte 1966; Hagemanand Burris 1978; Dixon and Kahn 2004). Theproduction of active nitrogenase requires the expres-sion of at least 20 genes including nitrogenasestructural and regulatory genes, and also those requiredfor the synthesis of nitrogenase cofactors (Dixon andKahn 2004). Nitrogenase requires a supply of MgATPand, as a result, nitrogen fixation is energy intensiveand has to be tightly regulated (for review, see Dixonand Kahn 2004).

Herbaspirillum seropedicae

The β-proteobacterium Herbaspirillum seropedicae isa Gram-negative diazotroph isolated from the rhizo-sphere and tissues of several plants, includingeconomically important species, such as sugarcane,maize, wheat and rice (Baldani et al. 1986; Baldani etal. 1992). This organism was first described asAzospirillum seropedicae due to several similaritieswith the genus Azospirillum. However, further anal-ysis showed that it belonged to a new genus,Herbaspirillum, and the organism was renamedHerbaspirillum seropedicae (Baldani et al. 1986).Subsequently, the diazotroph Pseudomonas rubrisu-balbicans was also reclassified and named Herbas-pirillum rubrisubalbicans (Baldani et al. 1996). Later,the genus was expanded to include two otherreclassified species, Herbaspirillum autotrophicum(syn. Aquaspirillum autotrophicum) and Herbaspir-illum huttiense (syn. Pseudomonas huttiensis) (Dingand Yokota 2004), and six new species, Herbaspir-illum frisingense (Kirchhof et al. 2001), H. lusitanum(Valverde et al. 2003), H.chlorophenolicum (Im et al.2004), H. hiltneri (Rothballer et al. 2006), H.rhizosphaerae (Jung et al. 2007) and H. aquaticum(Dobritsa et al. 2010). Recently, the species H. putei(Ding and Yokota, 2004) was reclassified as H.huttiense ssp. putei (Dobritsa et al. 2010). H.seropedicae is the best characterized of all thesespecies.

H. seropedicae is a plant endophytic diazotrophcapable of colonizing roots, stems and leaves ofits hosts without causing disease symptoms, but ithas a low survival rate in the soil (Baldani et al.1992; Olivares et al. 1996; James and Olivares1998). H. seropedicae expresses nif genes in roots,

stems and leaves of rice, sorghum, maize and wheat(Roncato-Maccari et al. 2003, James et al. 2002),and the contribution of biological nitrogen fixationby endophytic Herbaspirillum sp. was shown inrice and sugarcane by the incorporation of 15N2

(Gyaneshwar et al. 2002; Elbeltagy et al. 2001;James et al. 2002).

Different aspects of H. seropedicae nitrogenmetabolism have been studied since the isolation ofthe nifA gene (Souza et al. 1991a, b), and significantadvances in molecular analysis have been achievedfollowing the sequencing of the genome of H.seropedicae strain SmR1 (GenBank NC_014323;Pedrosa et al. 2011). Here, we review presentknowledge of the molecular mechanisms involved inregulating nif gene expression, and the maturation andcontrol of nitrogenase activity in this nitrogen-fixingplant-growth-promoting bacterium.

H. seropedicae nif genes

The nitrogen fixation (nif) genes code for proteins thatare essential for biosynthesis, maturation and assem-bly of the nitrogenase complex. In H. seropedicae,these genes are clustered in an approximately 40-kbcontiguous region with 46 orfs (Pedrosa et al. 2011;GenBank NC_014323) comprising at least sevenoperons whose expression depends on the RNApolymerase sigma factor σ54 and the transcriptionalactivator NifA (Fig. 1).

The structural genes coding for the nitrogenasecomplex are located in the nifHDKENXHsero_2847Hsero_2846fdxA operon (Klassen et al. 1999;Machado et al. 1996; Souza et al. 2010). The H.seropedicae nifHDK genes encode the nitrogenasestructural proteins: nifH encodes the iron (Fe) proteinand nifDK the molybenum-iron (MoFe) protein(Machado et al. 1996). Mutagenesis studies showedthat the nifN gene is essential for nitrogenase activityunder normal oxygen-limited growth and that nifX andHsero_2847 are required for full nitrogenase activityunder iron or molybdenum limitation (Klassen et al.1999, 2003). The products of the nifEN and nifX genesare probably involved in the synthesis of the iron-molybdenum cofactor (FeMoco) (Brigle et al. 1987;Moreno-Vivian et al. 1989; Hernandez et al. 2007), thesite of N2 reduction in the dinitrogenase protein (Reeset al. 2005).

198 Plant Soil (2012) 356:197–207

Four genes encoding ferredoxin-like proteins wereidentified within the nif cluster, and the role of two ofthese has been determined. The fdxA gene is locatedwithin the nifHDKENXHsero_2847Hsero_2846fdxAoperon, whereas fdxN is located downstream from nifB.The product of fdxN is essential for nitrogenase activitywhile the fdxA product is necessary for maximumnitrogenase activity (Souza et al. 2010) (Fig. 1).

The H. seropedicae nif genes are transcribed fromfour promoters (GenBank NC_014323) (Fig. 1).These promoters are activated specifically by theNifA protein, a bacterial enhancer binding protein(bEBP) dependent on the RNA polymerase sigmafactor, σ54. This family of proteins activates transcrip-tion by a common mechanism: the RNA polymerase-σ54 holoenzyme binds to promoter DNA forming aclosed complex which is unable to proceed totranscription. To melt DNA and form an opencomplex, the bEBP binds to an upstream activationsite relatively far (∼100–150 bases) from the promoter.A bend in DNA, which is facilitated by the IHFprotein, allows the bEBP to contact the closed complexand hydrolysis of ATP by the bEBP triggers aremodeling of the closed complex to an open complex(for a review, see Wigneshweraraj et al. 2008).

The nifHDKENXHsero_2847Hsero_2846fdxA operonis transcribed from a promoter upstream from nifH(Klassen et al. 1999; Machado et al. 1996; Souza et al.2010). One IHF- and two NifA- binding sites are locatedupstream of the promoter (Machado et al. 1996). Thetranscription level of nifH is twofold higher than that ofnifD or nifK, suggesting transcription attenuation in theoperon (Pedrosa et al. 2001).

The nifB gene of H. seropedicae, located down-stream from nifA, encodes a protein involved in thesynthesis of FeMoco. The putative nifB promoterregion contains two upstream sequences homologousto NifA-binding sites and a σ54 consensus promoter.The expression of nifB was stimulated by IHF in an E.coli background. DNA band-shift experimentsshowed that E. coli IHF could bind the nifB promoterregion, suggesting that IHF stimulates NifA activationof the nifB promoter (Rego et al. 2006).

Regulation of nif gene expression

The expression of nif genes in H. seropedicaerequires the transcriptional activator NifA, whichresponds to the levels of fixed nitrogen and oxygen.

Nif-

modE1 hesB fdxN nifB nifA 2872 2873 pC pA

Nif-

nifS2 2864 nifZ fixU 2861 2860 fdx

2856 2857 2858 2855

2867 nifZ1

2854 nifH nifK nifD pA

Nif-

Nif-

2846 2847 nifN nifE nifQ nifX modA fdxA

Nif-

-Fe Nif-

-Mo Nif-

-Mo

nifW nifV fixX modC modB fixA fixB fixC

pA

p

2827 nifS nifU 2831 hesB1 fdx 2833 trxA1

pp

Fig. 1 Structural organization of the nif and fix gene cluster in theH. seropedicae genome. Arrows indicate genes, open trianglescharacterized mutants and their phenotypes. –Fe and -Moindicates Nif negative phenotype under low iron or lowmolybdenum conditions, respectively. pA and pC indicate NifA-

dependent or NtrC-dependent promoters previously characterized,respectively. p indicates probable NifA-dependent promoterinferred by sequence analysis. Numbers indicate orfs accordingto the annotated H. seropedicae genome (GenBank accessionnumber NC_014323)

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Moreover, nifA expression is under control of the Ntrsystem, which regulates global nitrogen metabolismin bacteria.

The Ntr system in H. seropedicae

The Ntr system modulates nitrogen metabolism inresponse to the quality of the nitrogen source andthe cellular requirements for nitrogen assimilation.This system includes several proteins allowingexpression or repression of genes required for themobilization of nitrogen sources under conditionsof ammonium ion limitation (for a review, seeMerrick and Edwards 1995). In H. seropedicae, asin other proteobacteria, the Ntr system is composedof uridylyltransferase (GlnD) (encoded by glnD), thesignal transduction proteins of the PII family GlnBand GlnK (encoded by glnB and glnK), the putativeammonium channel AmtB (encoded by amtB),glutamine synthetase (GS) (encoded by glnA),adenylyltransferase (GlnE) (encoded by glnE), andthe two-component regulatory system NtrB-NtrC(encoded by ntrB and ntrC).

InH. seropedicae, the expression of the glnAntrBntrCoperon is regulated by two promoters, glnAp1 (σ70-dependent) and glnAp2 (σ54-dependent) (Schwab etal. 2007). Expression analysis showed that NtrC isrequired to activate the glnA promoter under con-ditions of nitrogen (NH4

+) limitation. However,under conditions of nitrogen excess glnA expressionis probably regulated by a putative σ70 promoter(Persuhn et al. 2000; Schwab et al. 2007). There aretwo NtrC-binding sites in the glnAntrBC promoterregion, and under ammonium limitation, NtrCinhibits transcription from the putative σ70 promoter,probably by binding to these specific overlappingsites (Schwab et al. 2007). Moreover, mutations inthe ntrB and ntrC genes of H. seropedicae affect notonly the synthesis but also the activity of GS. Thenon-adenylylated (active) GS concentration waslower in both these mutants, irrespective of the fixednitrogen level (Persuhn et al. 2000). H. seropedicaentrC and ntrB mutant strains grow as well as thewild-type strain SmR1 in the presence of NH4Cl,glutamate or glutamine as nitrogen sources, but failto grow on either nitrate or N2, indicating that theNtrC/NtrB system is involved in nitrate assimilationas well as nif gene expression in H. seropedicae.

Similar to other proteobacteria, H. seropedicaehas two PII proteins, GlnB and GlnK, that share78% amino acid sequence identity (Benelli et al.1997; Noindorf et al. 2006). The glnB operon ismonocistronic and expressed constitutively (Benelliet al. 1997). The genes nlmA, glnK and amtBconstitute an operon which is activated by NtrC toincrease transcription under nitrogen-limiting con-ditions (Noindorf et al. 2006). The nlmA geneencodes a probable periplasmic protein of unknownfunction which is found in the glnKamtB operon inseveral organisms belonging to the α-, β- and γ-proteobacteria (Noindorf et al. 2006; Huergo et al.2010). Co-expression of nlmA with glnK and amtBsuggests that it may form part of the Ntr system.Mutations in glnB or glnK do not affect theexpression of NtrC-dependent genes, suggestingthat GlnK and GlnB are interchangeable whenregulating phosphorylation of NtrC by NtrB in H.seropedicae (Noindorf et al. 2011).

Both H. seropedicae GlnB and GlnK are reversiblyuridylylated by the bifunctional GlnD enzyme (Benelliet al. 2001; Bonatto et al. 2007) in a process thatrequires the effectors ATP and 2-oxoglutarate andwhich is inhibited by glutamine (Bonatto et al. 2007).

The structural model of H. seropedicae GlnB(Benelli et al. 2002) shows that it is very similar toE. coli PII proteins (Carr et al. 1996; Xu et al. 1998).H. seropedicae GlnB contains a C-terminal 310-helixthat is not present in E. coli GlnB, but a similar motifoccurs in the E. coli GlnK protein (Benelli et al.2002). Substitutions in the T-loop and C-terminus ofH. seropedicae GlnB resulted in different responses tohigh ammonium concentrations compared to the wild-type protein, suggesting that these regions areinvolved in nitrogen signaling (Bonatto et al. 2005).The findings described above suggest that the Ntrsystem of H. seropedicae is similar to that describedfor E. coli and for other diazotrophs (for reviews, seeNinfa et al. 2000; Arcondéguy et al. 2001).

Regulation of H. seropedicae nifA expression

Transcription of H. seropedicae nifA is σ54-dependentand is activated by NtrC under nitrogen-limitingconditions (Souza et al. 1999, 2000). This is similarto the regulation of nifLA expression in the γ-proteobacterium Klebsiella pneumoniae, which is also

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activated by NtrC (Minchin et al. 1988). The H.seropedicae nifA promoter is complex, containing twoputative NtrC sites, three putative NifA upstreamactivator sequences and a single IHF (integration hostfactor) binding site (Souza et al. 1991a). Theexpression of nifA is primarily dependent on NtrC,and while NifA is not essential for its own transcrip-tion, it is required for maximal promoter activity(Wassem et al. 2002). Since NifA activity is oxygen-sensitive, the expression of the nifA promoter undernitrogen-limiting conditions is 50% higher under lowoxygen tensions than in air (Wassem et al. 2002).

In vitro experiments showed that activation of thenifA promoter by NtrC was stimulated by the DNA-bending protein IHF (Wassem et al. 2000). Incontrast, in vitro and in vivo experiments showedthat NifA auto-activation of the nifA promoter isreduced by IHF, preventing NifA over-expressionunder nitrogen-limiting conditions (Wassem et al.2000; 2002). NifA and IHF do not compete forbinding to the nifA promoter, indicating that IHFbinding does not prevent NifA interaction with theDNA, but induces changes in DNA structure thatconsequently modulate NifA-mediated activation(Wassem et al. 2000; 2002).

Regulation of H. seropedicae NifA activity

There are two different mechanisms whereby NifAactivity can be regulated (reviewed by Dixon andKahn 2004). In γ-proteobacteria and Azoarcus spp.,NifA is regulated by the anti-activator NifL, whichsenses fixed nitrogen and oxygen levels and inhibitsNifA activity towards protein–protein interaction(Martinez-Argudo et al. 2004). In α-proteobacteriaand in H. seropedicae, there is no NifL, and NifA isdirectly regulated by environmental signals (Dixonand Kahn 2004). PII proteins are involved intransduction of fixed nitrogen availability to NifAin both mechanisms either by interaction with NifLor with NifA (Little et al. 2002; Arcondéguy et al.1999; Araújo et al. 2004; Drepper et al. 2003;Noindorf et al. 2011).

As in most σ54-dependent activators, the H.seropedicae NifA protein contains three domains(Souza et al. 1991a). The central domain is respon-sible for interaction with the RNA polymeraseholoenzyme containing σ54 (Fischer 1994). The C-

terminal domain has a conserved helix-turn-helixmotif required for DNA binding (Monteiro et al.2003b). The N-terminal region contains a regulatoryGAF domain that has relatively low sequencesimilarity with other NifA proteins. Current evidencesuggests that this domain mediates inhibition of NifAactivity in response to the level of fixed nitrogen,through interactions with PII proteins (Souza et al.1999; Monteiro et al. 1999a, b). The central and C-terminal domains of NifA are separated by aninterdomain linker (IDL linker).

The activity of the H. seropedicae NifA protein isregulated in response to both oxygen and fixednitrogen (Souza et al. 1999). The NifA protein isdirectly inactivated in response to increased levels ofoxygen (Souza et al. 1999). The mechanism ofregulation of NifA activity by oxygen is not fullyunderstood, but it probably involves a putative motifcomprised of conserved cysteine residues locatedwithin both the central domain and the IDL linker.This conserved cysteine motif has been proposed tobe part of a metal-binding site acting as a sensor foroxygen (Souza et al. 1991a). Souza and co-workers(1999) showed that the activity of NifA is iron-dependent, indicating that the cysteine motif could bea binding-site for a Fe-S cluster. Rosconi et al. (2006)showed a decrease in nifH::lacZ expression in H.seropedicae strains grown under iron-depleted con-ditions, reinforcing the suggestion of the Fe require-ment for NifA activity. Site-directed mutagenesisshowed that each cysteine of the conserved motif isessential for protein activity (Oliveira et al. 2009).Furthermore, the UV-vis absorption spectrum of H.seropedicae NifA protein purified under anaerobicconditions showed an absorption peak at 415 nm,which is typical of proteins containing an iron–sulfurcluster (Monteiro, unpublished results). These resultsare in agreement with the hypothesis that the cysteinemotif may form an iron–sulfur cluster involved in theoxygen-sensing function of the NifA protein. Thismodel of oxygen regulation has also been proposedfor α-proteobacteria azospirilla and rhizobia (Fischer1994; Zhang et al. 2006). However, this cysteinemotif is not necessary for the DNA-binding activity ofNifA, since variant proteins with substitutions in thismotif, purified under aerobic conditions, were stillable to bind to the nifB promoter in vitro with asimilar affinity to that of aerobically purified wild-type protein (Oliveira et al. 2009). The results indicate

Plant Soil (2012) 356:197–207 201

that other aspects of transcriptional activation, ratherthan DNA-binding, are oxygen sensitive.

The Fnr protein (Fumarate and Nitrate reductionregulatory protein) also seems to be involved in theO2 sensitivity of NifA. Fnr, an oxygen-sensitiveprotein, is the major regulator controlling the physi-ological switch between aerobic and anaerobic growthconditions in E. coli (Gunsalus and Park 1994).Monteiro et al. (2003a) showed that the NifA proteinof H. seropedicae is inactive and more susceptible todegradation in an E. coli fnr mutant. H. seropedicaehas three fnr-like genes (Bueno et al., unpublishedresults), which are currently being analyzed.

In the α-proteobacteria Azospirillum brasilense,Rhodospirillum rubrum and Rhodobacter capsulatus,the GlnB protein mediates the control of NifA activityin response to fixed nitrogen (Zhang et al. 2000;Drepper et al. 2003; de Zamaroczy et al. 1993). In A.brasilense, nitrogen regulation of NifA activityappears to be mediated by the interaction of GlnBwith the N-terminal GAF domain (Arsene et al. 1999;Chen et al. 2005), whereas in H. seropedicae suchregulation involves the GlnK protein rather than GlnB(Noindorf et al. 2011). Consequently, nitrogenaseactivity is impaired in H. seropedicae glnK mutantseven though the expression of nifA is similar to thewild-type. The wild-type phenotype was restored byplasmids carrying glnK or a fragment expressing atruncated form of NifA, lacking the amino-terminalGAF domain (Noindorf et al. 2011). Monteiro et al.(1999a, b) and Souza et al. (1999) have shown that, inthe absence of the amino-terminal GAF-containingdomain, NifA activity is not controlled in response tothe level of fixed nitrogen. The wild-type phenotypeof a glnK mutant was also partially restored by aplasmid expressing the GlnB protein of H. seropedi-cae, suggesting that, when expressed in higherconcentration, GlnB is also capable of activatingNifA. These results indicate that, under physiologicalconditions, GlnK rather than GlnB controls NifAactivity. Limited proteolysis assays indicate that GlnKinteracts with the N-terminal GAF domain of NifA(Monteiro and Oliveira, unpublished results). Thisinteraction seems to occur regardless of the uridyly-lation state of GlnK, suggesting that a GlnK–NifAcomplex is formed irrespective of whether or notGlnK is post-translationally modified. These resultssuggest the following mechanism for nif expressioncontrol in H. seropedicae. Under nitrogen-excess

conditions, constitutively expressed GlnB is noturidylylated, allowing NtrB to keep NtrC in itsinactive form. As a result, expression of nifA andnlmAglnKamtB is repressed (Fig. 2). On the otherhand, under nitrogen-limiting conditions, synthesis ofGlnK occurs as the result of activation of the NtrC-dependent nlmAglnKamtB promoter, and GlnK israpidly uridylylated by GlnD. Under these condi-tions, GlnK (possibly in its uridylylated form)interacts with N-terminal GAF domain of NifA,releasing the inhibitory interaction of the GAFdomain with the catalytic central domain, leadingto activation of NifA and expression of other nifgenes. Conversely, when the fixed nitrogen concen-tration rises, GlnK is rapidly deuridylylated, forminga GlnK-NifA complex in which NifA is inactive.Thus, NifA is active in the GlnK–UMP–NifAcomplex, while in the GlnK–NifA complex, thenon-modified form of GlnK inhibits NifA activity.This mechanism would enable a rapid regulation ofnif gene expression in response to changes in theextracellular ammonium availability.

Post-translational control of nitrogenase in H.seropedicae

Nitrogen fixation is an energy-demanding process,which, as mentioned above, is controlled both at thelevel of nif transcription and by regulating the activityof nitrogenase itself. This post-translational regulationis exerted in response to both fixed nitrogen and theenergy charge leading to reversible inhibition ofnitrogenase activity, a process known as nitrogenase“switch-off”. A rapid increase in extracellular ammo-nium concentration results in inhibition of nitrogenaseactivity. When the added ammonium is consumed, theactivity of nitrogenase is restored without de novoprotein synthesis. In several organisms, nitrogenaseswitch-off by ammonium is the result of a reversibleADP-ribosylation of the nitrogenase Fe-protein sub-unit carried out by the DraT/DraG system (Zhang etal. 2006; Huergo et al. 2009; Wang et al. 2005;Tremblay and Hallenbeck 2008). Nitrogenase switch-off in response to ammonium also occurs in H.seropedicae (Fu and Burris 1989; Klassen et al. 1997;Noindorf et al. 2006, 2011), but does not involve theDraT/DraG system since neither draT nor draG geneswere found in this organism (Fu and Burris 1989;

202 Plant Soil (2012) 356:197–207

+NH4+

NifA

nif genes

GlnK

-NH4+

NtrC-P NtrC

NtrB

glnB σ70

GlnB-UMP

(active)

AmtB

N2 NH3

N2ase

(active)

(+)

(+)

(+)

σ54

GlnK-UMP

GlnD

nifA σ54

(+)

NtrB

GlnB

NtrC-P NtrC

(inactive)

σ54

nifA σ54

(-)

nlmA glnK amtB (-)

glnB σ70

(+)

UMP UMP UMP

UMP UMP UMP

UMP UMP UMP

σ54 nlmA glnK amtB

GlnD

Fig. 2 Model of transcriptional regulation of nitrogen fixationin H. seropedicae. GlnB is constitutively expressed from a σ70-dependent promoter. At high levels of fixed nitrogen, theincrease in the glutamine concentration induces the deuridyly-lation of GlnB by GlnD. In such conditions, NtrB catalyzes thedephosphorylation of NtrC, and consequently inactivates it(left-hand panel). Under nitrogen-fixing conditions, when thelevel of fixed nitrogen is low, NtrB catalyzes phosphorylation

of NtrC, which activates the transcription of target genes, suchas nifA and the nlmAglnKamtB operon. Under these conditions,the GlnB and GlnK proteins are fully uridylylated by GlnD.Uridylylated GlnK activates NifA, which in turn activates thetranscription of the other nif genes and consequently enablesbiosynthesis of active nitrogenase that reduces atmosphericnitrogen to ammonia (right-hand panel)

-NH4+ NH4

+ shockAmtB

GlnK

AmtB

GlnK

GlnD

GlnK

NlmA NlmA

? ?

N2 NH3

N2ase

Active Nitrogenase

PMU-KnlGPMU-KnlG

N2 NH3

N2ase

Inactive Nitrogenase

UMP UMP UMP UMP UMP UMP

GlnD

cytoplasm cytoplasm

periplasm periplasm

Fig. 3 Model of post-translational control ofnitrogen fixation in H.seropedicae. Undernitrogen-fixing conditions,uridylylated GlnK remainsin its cytosolic form(left-hand panel). After anammonium shock, GlnK israpidly deuridylylated byGlnD and associates withAmtB in the membrane.Under such conditionsnitrogenase is inactive(right-hand panel). Theperiplasmic protein NlmAmay have a role in thisprocess, but its currentfunction is unknown

Plant Soil (2012) 356:197–207 203

GenBank NC_014323). It is not known exactly hownitrogenase activity is inhibited in H. seropedicae, butpresumably the switch-off involves the reallocation ofelectrons and ATP from nitrogenase in order tometabolize the ammonium. However, the signalingpathway for switch-off in response to ammoniuminvolves the GlnK and AmtB proteins (Noindorf et al.2006, 2011) (Fig. 3).

AmtB and/or PII proteins are involved in theammonium-dependent post-translational control ofnitrogenase in several bacteria including H. seropedi-cae (Yakunin and Hallenbeck 2002; Martin andReinhold-Hurek 2002; Huergo et al. 2006; Dodsworthand Leigh 2006). The addition of ammonium to de-repressed cultures of amtB or glnK mutants of H.seropedicae caused only partial inhibition of nitroge-nase activity compared to the wild-type strain,indicating that the amtB and glnK gene productsmay be involved in nitrogenase switch-off by ammo-nium ions in this organism (Noindorf et al. 2006,2011). In contrast, a glnB mutant showed a wild-typephenotype upon ammonium addition. Furthermore,Gusso et al. (2008) showed that nitrogenase switch-off is impaired in an ntrC mutant, which has lowlevels of nlmAglnKamtB operon expression (Noindorfet al. 2006). Recently, Huergo et al. (2010) demon-strated that GlnK of H. seropedicae is directedtowards and binds to the cytoplasmic membranefollowing an ammonium shock. This process isAmtB-dependent since it was not observed in anamtB mutant, suggesting that formation of themembrane-bound GlnK-AmtB complex signals nitro-genase to switch-off upon ammonium addition, asdescribed for Azospirillum brasilense (Huergo et al.2006). These results allowed us to propose the modelpresented in Fig. 3 for post-translational regulation ofnitrogenase. According to this model, under nitrogen-fixing conditions, GlnK is uridylylated and is in thecytoplasm. After ammonium shock, GlnK is deuridy-lylated and is sequestered by the membrane-boundAmtB. These events presumably trigger nitrogenaseinhibition by an unknown mechanism.

Concluding remarks

The mechanism of regulation of nitrogen fixation ofH. seropedicae is highly intricate and occurs at bothtranscriptional and post-translational levels. Regula-

tion of nif gene expression relies on the activity of thetranscriptional activator NifA protein which seems torespond to the level of fixed nitrogen through thesignal-transduction protein GlnK and to the oxygenconcentration through the redox state of a potentialFe–S cluster. Regulation of nifA gene expressionunder control of the Ntr system in H. seropedicae issimilar to that observed in γ-proteobacteria, while themechanism of control of NifA activity in response tofixed nitrogen and oxygen is similar to that of the α-proteobacteria Azospirillum sp. and Rhizobium sp. Onthe other hand, nitrogenase activity is also regulatedby fixed nitrogen in a process involving both GlnKand AmtB. Titration of cytoplasmic unmodified GlnKby AmtB probably signals nitrogenase to switch-offin response to increased external ammonium ionconcentrations. Nitrogen fixation in planta duringthe beneficial plant–bacterial association must nec-essarily be a regulated process, since the bacteriaonly provide fixed nitrogen if enough carbon andenergy is available and if a fixed nitrogen source isnot present. The understanding of this fine regula-tion, also during association with the host, willdecisively contribute to rationally exploiting suchinteractions and provide knowledge for geneticallytailoring bacterial strains with higher potential forthe transfer of fixed nitrogen.

Acknowledgements We are grateful to Dr. Ray Dixon forcritical reading of the manuscript. We are also grateful to RoseliPrado, Valter de Baura, Marilza Lamour and Julieta Pie fortechnical assistance. This work was supported by INCT-FBN/CNPq/MCT, Institutos do Milênio, PRONEX, CAPES, CNPqand Fundação Araucária

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