A novel regulatory role of the Rnf complex of Azoarcus sp. strain BH72

A novel regulatory role of the Rnf complex of Azoarcus sp.strain BH72mmi_7940 408..422

Abhijit Sarkar, Jörg Köhler, Thomas Hurek andBarbara Reinhold-Hurek*University of Bremen, Faculty of Biology, Department ofMicrobe-Plant Interactions, P.O. Box 33 04 40, D-28334Bremen, Germany.

Summary

The superfamily of PII proteins contains the mostwidely distributed signalling proteins in nature.Remarkable is the variety of targets whose activity isaffected by protein–protein interactions. Here we iden-tified as novel partner for interaction with GlnK an Rnfcomplex, known to couple the energy of ion transportto reduce ferredoxins. The endophytic diazotrophicbetaproteobacterium Azoarcus sp. strain BH72 har-bours two rnf-like clusters in the genome, of whichonly the rnf1 cluster was induced under conditions ofN2 fixation under control of the transcriptional activa-tor NifA. Rapid inactivation (‘DraT-independent switchoff’) of nitrogenase activity upon ammonium upshiftwas dependent on the Rnf1 complex. Membranesequestration of GlnK in steady-state N-surplus con-ditions occurred in its unmodified form, signalling N-surplus, and was dependent on presence of the Rnf1complex, suggesting physical interaction. In vitrobinding studies by Far-Western analysis indicatedinteractions of RnfC1 with specifically GlnK but notwith GlnB. As ammonium upshift led to decreasedactivity of the Rnf1 complex in membranes, it might beinactivated by GlnK binding, leading to an interruptionof electron flow to nitrogenase and thus a rapid, DraT-independent nitrogenase switch off. Our data implya hitherto unknown interaction partner for a PII-likeprotein and an additional process under its control.

Introduction

The superfamily of PII proteins contains the most widelydistributed signalling proteins in nature (Sant’Anna et al.,2009). PII-like proteins are ubiquitous among prokaryotesand occur in all domains of life, such as in nitrogen-fixing

Archaea and in chloroplasts of eukaryotes. They arehomotrimeric signal transmitter proteins that by protein–protein interactions co-ordinate the regulation of a widerange of processes related to nitrogen metabolism(Arcondéguy et al., 2001; Forchhammer, 2008). Remark-able for these small proteins is the variety of targetswhose activity is affected, such as several enzymes, tran-scription factors and membrane transport proteins (Forch-hammer, 2008). Even more targets of PII proteins andthus, more PII-controlled processes can yet be expectedto be identified in diverse bacteria (Forchhammer, 2008).

PII proteins are signal transmitters that often have twomodes of signal perception to reflect the nitrogen status ofthe cell as N-sufficient or N-deficient. PII proteins such asGlnK of all studied Proteobacteria are subject to covalentmodification (uridylylation) by the bifunctional enzymeGlnD, which catalyses the modification and demodifica-tion of the PII protein at Tyr51 in the T-loop (Ninfa andJiang, 2005). The modification state reflects the cellularglutamine status, as GlnD activity is regulated by thecellular glutamine pool. The second, conserved mode ofsignal perception consists of the binding of effector mol-ecules ATP, ADP and 2-oxoglutarate (2-OG) (Ninfa andJiang, 2005; Forchhammer, 2008).

Some of the GlnK-mediated regulatory processesinvolve membrane sequestration of these typically solublecytoplasmic proteins (Coutts et al., 2002; Martin andReinhold-Hurek, 2002). When nitrogen-limited cells ofEscherichia coli are exposed to a sudden surplus of com-bined nitrogen, GlnK binds to AmtB, trimeric membraneproteins for high-affinity uptake of ammonia (Javelle et al.,2008), and thereby negatively regulates the transportactivity of AmtB (Coutts et al., 2002). In addition, mem-brane sequestration may deplete the cytoplasm of unmodi-fied GlnK and thereby prevent the interaction with othercytoplasmic targets like NifL (Coutts et al., 2002), or evensequester other regulatory proteins in ternary complexesto the membrane as shown in nitrogen-fixing bacteria(Wang et al., 2005; Huergo et al., 2007; 2009). Recently, astudy on crystal structure of the GlnZ(PII)-DraG complexrevealed even a different form of PII–target interactionwhere its T-loop was not involved, and thereby demon-strated the potential of PII to interact simultaneously withtwo different targets in a ternary complex as in AmtB-GlnZ-DraG (Rajendran et al., 2011). In contrast to this model and

Accepted 29 November, 2011. *For correspondence. [email protected]; Tel. (+49) 421 218 62861; Fax (+49) 421218 62873.

Molecular Microbiology (2012) 83(2), 408–422 � doi:10.1111/j.1365-2958.2011.07940.xFirst published online 21 December 2011

© 2011 Blackwell Publishing Ltd

to observations in other Gram-negative or Gram-positivebacteria (Detsch and Stülke, 2003; Strösser et al., 2004;Wang et al., 2005), membrane association of GlnK is notAmtB-dependent (Martin and Reinhold-Hurek, 2002) inAzoarcus sp. strain BH72, a diazotrophic endophyte ofgrasses (Hurek and Reinhold-Hurek, 2003; Krause et al.,2006).

The process of biological nitrogen fixation catalysed bythe molybdenum-containing nitrogenase is under tightregulation because both nitrogenase components are irre-versibly inactivated by oxygen, and because the process isenergetically costly (Dixon and Kahn, 2004). PII proteinsare involved in both modes of regulation, transcriptionaland post-translational (Drepper et al., 2003). The so-calledinhibition of nitrogenase activity or ‘switch off’ upon additionof ammonium, or in some cases upon energy depletioncaused by anaerobiosis, depends on two different mecha-nisms. In many Alphaproteobacteria such as Rhodospiril-lum rubrum and Azospirillum brasilense, the iron protein ofnitrogenase (NifH) is subject to post-translational modifi-cation, a reversible mono-ADP-ribosylation at a specificarginine residue mediated by DraT/DraG (Pope et al.,1985; Fu et al., 1989; Masepohl et al., 1993). This wasrecently also detected in the betaproteobacterium Azoar-cus sp. strain BH72 (Oetjen and Reinhold-Hurek, 2009;Oetjen et al., 2009). Additionally, rapid DraT-independentswitch-off mechanisms exist, also previously termed‘physiological switch off’ (Martin and Reinhold-Hurek,2002; Oetjen and Reinhold-Hurek, 2009), which do notrequire a covalent modification of nitrogenase, e.g. inRhodobacter capsulatus, A. brasilense, or Azoarcus sp.strain BH72 (Pierrard et al., 1993; Zhang et al., 1996;Martin and Reinhold-Hurek, 2002; Oetjen and Reinhold-Hurek, 2009). An ammonium-induced switch off was alsoreported in Herbaspirillum seropedicae (Fu and Burris,1989) and Azospirillum amazonense (Song et al., 1985),both of which lack a DraT/DraG system. Unlike for theDraT/G-mediated response, details of these mechanismsare not well explored yet.

It is assumed that electron transport might play a role inthe DraT-independent switch off of phototrophic diazotro-phs (Pierrard et al., 1993; Halbleib et al., 2000). Also forAzoarcus sp. strain BH72 there is evidence for involvementof ferredoxin-dependent electron transport. A ferredoxin(FdxN) encoded in the nif operon was found to be the mainbut not essential electron donor for nitrogenase. Moreover,it is essential for the nitrogenase inactivation upon ammo-nium upshift (Egener et al., 2001). As blocking the electrontransport to nitrogenase appeared to be a crucial factor inthe control of nitrogenase activity, we anticipated that theRnf proteins might participate in the regulatory mecha-nism. Rnf membrane complexes (Rhodobacter NitrogenFixation) catalyse electron transfer coupled to ion transportacross the membrane. They show a wide range of func-

tions from energy conservation to driving reverse electronflow (Biegel et al., 2010). In R. capsulatus, the membranecomplex encoded by rnfABCDEGH is required for electrontransport to nitrogenase (Jouanneau et al., 1998; Jeongand Jouanneau, 2000), presumably by driving a reverseelectron flow from NADH to reduce ferredoxin I, whichserves as the electron donor to nitrogenase (Hallenbecket al., 1982; Schmehl et al., 1993; Jouanneau et al., 1998;Jeong and Jouanneau, 2000). In other facultative aerobes,Rnf proteins are required to keep the redox-sensitive tran-scriptional factor SoxR in its reduced state during normalaerobic growth as in E. coli (Koo et al., 2003). In Aceto-bacter woodii, the reverse reaction, Na+-translocatingferredoxin: NAD+-oxidoreductase is used as a new cou-pling site for energy conservation (Imkamp et al., 2007;Müller et al., 2008), a presumably widely distributedmechanism in anaerobes (Biegel et al., 2010).

Here we identified the Rnf complex as novel partner forinteraction with GlnK. In the betaproteobacterium Azoar-cus sp. strain BH72, one of the two rnf gene clusters isrequired for DraT-independent switch off of nitrogenaseupon ammonium shock and for membrane sequestrationof GlnK in response to ammonium as well as anaerobiosisshock. We propose a model in which protein–proteininteraction of GlnK with Rnf1 controls the membrane-associated, Rnf1-catalysed electron transfer, providingreductant to nitrogenase. This previously unknown inter-action may represent a novel regulatory mechanism formembrane ion transport coupled to electron transfer.

Results

Azoarcus sp. strain BH72 possesses two clusters ofrnf-like genes

Two clusters of rnf-like genes were identified in the genomeof Azoarcus sp. strain BH72 (Fig. S1) in different genomiccontexts. Cluster rnf1 consists of seven rnf-like genes(rnfABCDGEH) located adjacent to nifL but with oppositetranscriptional orientation. In cluster rnf2, rnfH wasreplaced by nth (encoding a putative endonuclease III inmost bacteria). Upstream of rnfA2, two ORFs were locatedencoding for a conserved hypothetical hydrolase and aputative nuclease respectively. The equivalent gene prod-ucts of rnf1 and rnf2 clusters exhibited relatively low aminoacid sequence identities ranging from 39% (RnfD1/D2)to 68% (RnfA1/A2). Phylogenetic analysis revealed acommon split into two paralogous gene clusters for manydiazotrophic Proteobacteria (Fig. 1A). Rnf1 clusters thathave in common rnfH are mostly physically linked to nifLA,and represent one of two copies. This suggested that rnf1clusters might be related to N2 fixation in diazotrophs.Single clusters found in Archaea, and mainly in anaerobicFirmicutes, were clearly separated.

rnf gene clusters in Azoarcus sp. BH72 409

© 2011 Blackwell Publishing Ltd, Molecular Microbiology, 83, 408–422

The expression of rnf1 but not of rnf2 is upregulatedunder conditions of N2 fixation

Reporter strains of Azoarcus sp. carrying transcriptionalrnfA1::gusA BH72 (BH::rnfA1-gusA) or rnfA2::gusA

(BH::rnfA2-gusA) fusions were constructed to determinewhether rnf expression was nitrogen-regulated. rnfA1expression was found to be enhanced 13-fold under N2

fixation (1175 � 89 Miller Units) as compared with thepresence of ammonium (85 � 11 Miller Units), whereas

Fig. 1. Phylogenetic distribution of rnf gene clusters in prokaryotes and effects of their respective in-frame deletions on ammonium induced‘switch off’ of nitrogenase and its post-translational modification in Azoarcus sp. strain BH72.A. The concatenated protein tree was derived by Bayesian inference from 2039 aligned amino acid positions and is composed of Rnf CDGEhomologues found in complete genomes. To the right the local rnf operon structures with adjacent nifA and nifL genes are shown. Within theoperon structures, each dash (-) indicates a single open reading frame. Interior branch test support values from a Minimum Evolution analysiswith 100 replicates and Bayesian support values are shown as percentages at each branch (bottom to top).B. Effect of rnf1 or rnf2 in-frame deletions on ammonium-induced ‘switch off’ mediated by adding 2 mM ammonium chloride on nitrogenaseactivity measured by acetylene reduction on wild type, BHDRnf1 or BHDRnf2 respectively. Arrow indicates the time point of ammoniumchloride addition. Data are based on at least two independent experiments.C. Western blot analysis of NifH modification from nitrogen-fixing batch cultures of wild type, BHDRnf1 or BHDRnf2, respectively, challengedwith 2 mM NH4Cl for 5 min. Quantitative estimation of the band intensities of each strain based on the Western blot analysis (lowest panel)given in bar diagrams with the relative percentage of unmodified (dotted) and modified (open) NifH. Error bars indicate the standard deviationfrom three independent experiments.

410 A. Sarkar, J. Köhler, T. Hurek and B. Reinhold-Hurek �


rnfA2 was found to be constitutively expressed at lowlevels regardless of the nitrogen source (ammonium,190 � 8 Miller Units; N2, 200 � 15 Miller Units). Expres-sion of nifH in Azoarcus sp. strain BH72 is found to beelevated in a similar way under growth on N2 (Egener et al.,1999) suggesting a similar regulatory circuit. Accordingly,the upstream region of the rnf1 cluster carried putativeelements typical of regulation of nif gene expression: aputative -12/-24 consensus for s54 –RNA polymerasebinding, a conserved palindrome sequence for integrationhost factor binding and putative UAS (upstream activatingsequences) for NifA and NtrC binding (Fig. S2A). In con-trast, no UAS or s54 binding sequences were identifiedwithin 3 kb upstream of the rnf2 gene cluster.

To investigate the role of the respective transcriptionalactivators putatively involved in regulating rnf1 expres-sion, a transcriptional fusion construct rnfA1::gusA(pK18mob2rnf1-gusA) was chromosomally integrated intowild-type strain BH72, ntrBC- strain BntrBsp or nifLA-

strain BHLAO respectively. Generally, rnf1 expression inall the three strains was low while growing aerobically(complex medium or ammonium) or microaerobically onammonium (Fig. S2B). In the absence of ntrBC, expres-sion of rnf1 was lower by a factor of two (statisticallysignificant, P < 0.05) as compared with wild type orBHLAO under such conditions (except microaerobicallyon ammonium), indicating a role for NtrBC, the typicaltranscriptional activation system for nitrate-related genes.However, with nitrate as nitrogen source rnf1 expressionwas enhanced in the absence of ntrBC (Fig. S2B). ForA. brasilense and R. capsulatus, it has been reported thatNtrX can mediate cross-talk between the NtrBC andNtrYX two component systems (Drepper et al., 2006;Assumpcao et al., 2007). As the genome of strain BH72harbours ntrY- and ntrX-like genes, the alternative tran-scription activator NtrX might complement NtrC functions.However, NifA is likely the major transcriptional activator,as high expression under conditions of nitrogen fixationwas strongly reduced in the nifA mutant (P < 0.05).

Deletion of the rnf1 but not of the rnf2 cluster affectsexponential diazotrophic growth

To understand the physiological role of Rnf proteins inAzoarcus sp. strain BH72, mutant strains were generatedby deleting either the rnf1 cluster or the rnf2 cluster (Fig. S1and Table S1). Unlike R. capsulatus mutants (Schmehlet al., 1993), both the strains were Nif+ and were unaf-fected in ammonium-dependent aerobic growth [genera-tion time (h) of wild type 1.63 � 0.18, of BHDRnf11.68 � 0.25, or of BHDRnf2 1.60 � 0.07, respectively].However, strain BHDRnf1 and not strain BHDRnf2 wasquantitatively affected in diazotrophic growth. Underoxygen-controlled (0.8% oxygen) conditions in a bioreac-

tor, the generation time on N2 of strain BHDRnf1 wasprolonged with 2.85 � 0.14 per hour as compared withwild-type strain with 1.82 � 0.29 per hour. The generationtime of strain BHDRnf2 under identical conditions did notdiffer significantly from the wild type. Interestingly, in-framedeletion of fdxN in strain BH72 affected the diazotrophicgrowth in a similar fashion (59% � 9% of the wild-typegrowth rate). Thus the Rnf1 complex, like FdxN, wasimportant but not essential for diazotrophic growth and wasapparently not functionally complemented by the Rnf2complex.

DraT-independent switch-off response of nitrogenase isabolished in BHDRnf1

Previously, it has been shown that upon addition of ammo-nium to nitrogen-fixing cells of Azoarcus sp. BH72, nitro-genase activity is rapidly inhibited (Egener et al., 2001),independent of the DraTG system (Oetjen and Reinhold-Hurek, 2009). As the electron flow to nitrogenase may berelated to this DraT-independent switch off, the involve-ment of the Rnf complex was analysed. Initial acetylenereduction activity of nitrogen-fixing batch cultures ofBHDRnf1 was slightly decreased as compared with wildtype or BHDRnf2 (Fig. 1B), concordant with the growthexperiments. Addition of 2 mM ammonium chloride tothese cultures (Fig. 1B) led to fast and complete inhibitionof acetylene reduction in strain BH72 and BHDRnf2, whileBHDRnf1 continued to fix nitrogen. Functional complemen-tation by the rnf2 cluster (present in BHDRnf1) was appar-ently not possible. This strongly supported the hypothesisthat gene products of the rnf1 cluster in addition tofdxN are obligatory players of the ammonium-mediatedDraT-independent switch-off response in strain BH72.

The NifH protein of strain BH72 is covalently modified inresponse to ammonium and oxygen deficiency: an ADP-ribosylated NifH protein of lower electrophoretic mobilityaccumulates (difference of approximately 1.5 KD), whichcan be detected by Western blot analysis with antibodiesraised against NifH (Oetjen and Reinhold-Hurek, 2009;Oetjen et al., 2009). Although the rnf1 mutation abolishedDraT-independent switch off of nitrogenase activity, NifHwas still modified in response to ammonium, albeit at aslightly lower level as compared with wild type andBHDRnf2 (Fig. 1C). In the wild type, full modificationrequires one subunit of each NifH dimer to be ADP-ribosylated. Forty-four percent of subunits appeared to bemodified in the wild type, and hence at least 90% of theprotein is expected to be inactivated. By contrast in strainBHDRnf1, only 30% of the NifH subunits were modified,which equates to around 60% of the NifH dimers beinginactivated. It can be therefore deduced that the activityobserved to be insensitive to ammonium shock inBHDRnf1 strain is due to the 40% of NifH that remains



unmodified after the shock. Thus, nitrogenase activity wasstill retained while part of the NifH protein was modified.

Single Amt proteins do not sequester GlnK

In strain BH72 GlnK and AmtB, a high-affinity ammoniumchannel, is required for the signal transduction cascadefor DraT-independent switch off of nitrogenase uponammonium addition (Martin and Reinhold-Hurek, 2002).However, AmtB, unlike in other bacteria, was not respon-sible for membrane sequestration of GlnK (Martin andReinhold-Hurek, 2002). In addition to amtB, the genomeof strain BH72 harbours several similar ORFs encodingputative high-affinity ammonia permeases (AmtY, AmtDand an unusually long AmtE where the N-terminal trans-porter domain is fused with a C-terminal sensory domain),which might participate in GlnK sequestration. GlnK orglnY of strain BH72 are physically linked with amtB andamtY respectively. Chromosomal transcriptional fusionsof amtE or amtD with gusA, respectively, showed that onlyamtE expression was enhanced under conditions of nitro-gen fixation (110 � 13 Miller units versus 41 � 8 Millerunits on ammonium) and not amtD expression (327 � 51Miller units versus 353 � 62 Miller units on ammonium).Therefore, amtB, amtY and amtE were further analysed.

The amtB or the amtE gene was disrupted by markerexchange mutagenesis in strain BHABK or BHDAmtE,while the amtY gene was in-frame deleted in strainBHDAY. The double mutant BHABKDAY carried both,amtB and amtY mutations (Table S1). Cell extracts fromstrains wild-type BH72, BHABK, BHDAY, BHABKDAY andBHDAmtE grown with 10 mM NH4

+ in synthetic medium,were separated by ultracentrifugation into cytoplasmicand membrane fractions and checked for the presence ofGlnB or GlnK in Western blot analysis. Because GlnY isdetectable only in mutants lacking both, GlnB and GlnK(Martin et al., 2000), analysis of GlnY was not pursued. Inall cases, GlnB and GlnK were detected in cytosolic frac-tions, whereas only GlnK and not GlnB was detected inthe membrane fraction in spite of salt treatments of mem-branes (600 mM NaCl) to remove loosely attached pro-teins (Fig. 2A). In all tested amt mutants, however,membrane association of GlnK was retained (Fig. 2A),indicating that none of these ammonium transporters waslikely to specifically bind and sequester GlnK.

Rnf1 cluster is essential for membrane sequestration ofGlnK in strain BH72

Because strain BHDRnf1 was found to be impaired inthe ammonium-induced DraT-independent switch-offresponse and exhibited a nitrogen-regulated expressionpattern, it was tempting to speculate that Rnf1 proteins arepart of the signalling cascade and sequester GlnK to the

membrane. Therefore, membrane association was analy-sed for wild type and mutant strains BHDRnf1 andBHDRnf2 in detail. They were grown either in presence ofammonium chloride as sole N source or under standardN2-fixing conditions in an oxygen-controlled bioreactor.The differential uridylylation status of PII proteins reflectsthe nitrogen status (Martin et al., 2000) and influences theirsignal-transducing ability. Accordingly, GlnB signals werealways detected with either lower electrophoretic mobility(modified) under nitrogen fixation, or with higher mobility

Fig. 2. Determination of cellular localization and covalentmodification of GlnB and GlnK.A. Effect of deletion of ammonium transporter genes (amt) oncellular localization of GlnB and GlnK in Azoarcus sp. strain BH72.After ultracentrifugation, Cytoplasmic (C) and membrane fractions(M) from cells grown in presence of 10 mM ammonium chloridewere subjected to 12.5% SDS-PAGE and followed by Western blotanalysis with either specific anti-GlnB antiserum or anti-GlnKantiserum. Strains used for protein extract: wild-type strain BH72(1), mutants BHABK (amtB) (2), BHDAY (amtY) (3), BHABKDAY(amtBY) (4) and BHDAmtE (amtE) (5); lane 5 not from same gel.B. Determination of cellular localization and covalent modification ofGlnB and GlnK in wild type and mutants BHDRnf1 (Drnf1) orBHDRnf2 (Drnf2), grown under N2 fixation (-N) or in presence ofammonium (+N). Cell extracts were subjected to 18% SDS-PAGEand Western blot analysis with either anti-GlnB antiserum oranti-GlnK antiserum.C. Determination of modification status of membrane-sequesteredGlnK in presence of ammonium (+N) by running in parallel themembrane (M) and whole cell (WC) fractions from wild type andBHDRnf2 (Drnf2) in 18% SDS-PAGE; Western blot analysis withanti-GlnK antiserum.



(unmodified) on ammonium, indicating that the in vivomodification/demodification was complete (Fig. 2B). Themodified GlnK protein was detected under nitrogen fixa-tion, but in presence of ammonium, the unmodified formwas detectable along with the modified protein (Fig. 2B).As in the previous experiment, GlnB was not membraneassociated, while GlnK was detected in membrane frac-tions. Membrane sequestration only occurred in the pres-ence of ammonium, both in wild type and BHDRnf2(Fig. 2B). In contrast, the GlnK signal was completelyabolished in membrane extracts from BHDRnf1. Loading ofmembrane extracts adjacent to whole cell extractsrevealed the modification status of membrane-associatedGlnK as unmodified (Fig. 2C), suggesting that mainly deu-ridylylated GlnK was sequestered to membranes.

The experiments addressing GlnK membrane seques-trations were carried out aerobically under steady-stateconditions in presence of ammonium. To substantiate therole of the Rnf1 complex in the rapid DraT-independentswitch-off response of nitrogenase, the GlnB and GlnKmembrane sequestration status was further analysed forwild type and mutant strain BHDRnf1 both, under ammo-nium shock (+AS) and anaerobic shock (-OS) conditionsrespectively. GlnB was never detected in membrane frac-tions of either of the strains. Modified GlnB was detectedfrom whole cell extracts under N2 fixation, while unmodi-fied GlnB was detected both under (+AS) and (-OS) con-ditions, respectively (Fig. S3A). The pattern of GlnKproteins from whole cell extracts were similar to GlnB;however, both under (+AS) and (-OS) shock, GlnK wasnot completely unmodified. As observed for steady-stateanalysis, GlnK membrane sequestration was detectedonly for wild type under ammonium shock (+AS) andcompletely abolished in mutant BHDRnf1 (Fig. S3A). Sur-prisingly, this was also the case for cells subjected toanaerobiosis (-OS) (Fig. S3A). To reveal the modificationstatus of membrane-bound GlnK more clearly, whole cellextracts were loaded adjacent to membrane extracts(Fig. S3B). Upon anaerobiosis shock, the membrane-bound form of GlnK was apparently completely modified.In cells subjected to ammonium shock (+AS), modified aswell as unmodified GlnK was apparently present in mem-brane fractions (Fig. S3B).

GlnK interacts with RnfC1 in vitro

In order to confirm putative in vivo interactions betweenproteins of the Rnf1 complex and GlnK, in vitro interactionstudies with purified tagged proteins were carried out byFar-Western blot analysis. GlnB and GlnK proteins taggedwith Strep-tag II were overexpressed and subsequentlypurified. To control their uridylylation status, expression ofE. coli cells harbouring petglnKstrep or petglnBstrep wasinduced by IPTG in Luria–Bertani (LB) medium either

supplemented with 20 mM NH4Cl (simulating nitrogensurplus) or with 20 mM a-ketoglutarate (nitrogen limita-tion). The purified proteins GlnB-StrepII showed theexpected electrophoretic mobility and thus modification(Fig. 3A). However, the modification status was less dis-tinct for GlnK-StrepII as shown by a Western blot analysisfrom a native PAGE; particularly when cells were grown inthe presence of ammonium, modified GlnK was stillpresent along with other intermediate uridylylation states(Fig. 3A). Apparently E. coli GlnD did heterologously uridy-lylate Azoarcus GlnB-StrepII and GlnK-StrepII under theseconditions.

Because RnfA1, RnfD1 and RnfE1 are integral mem-brane proteins and reported to be unstable when synthe-sized in E. coli (Jouanneau et al., 1998), while RnfB andRnfC are mainly hydrophilic peripheral membrane pro-teins, RnfC1 and RnfB1 of strain BH72 were selected as aputative interaction partners. However, histidine-taggedRnfB1 (His-RnfB1) was highly unstable and poorlyexpressed, while His-RnfC1 was expressed as a brown,68 kD C-terminally histidine-tagged protein in E. coli(Fig. 3B).

Far-Western analyses were carried out by using His-RnfC1 as prey protein, and then incubating the membranewith GlnK-StrepII as bait protein that was detected withStrep-Tactin HRP conjugate (IBA, Germany). Total proteinextract with overexpressed His-RnfC1 (Fig. 3B, lane 2) orpurified RnfC1-His (Fig. 3B lane 3) were separated on anSDS-PAGE as prey proteins (Fig. 3B upper panel) alongwith a negative control consisting of extract from E. colicarrying the empty vector pET-28a(+) (Fig. 3B, lane 1).Far-Western analyses with purified GlnK-StrepII as bait,obtained from N-surplus (-UMP, together with smallamounts of intermediate uridylylated forms) and N-limitinggrowth (+UMP), revealed specific binding of GlnK to RnfC1(second, third panel). In all cases the negative control(Fig. 3B, Con) did not yield any signal, and unspecificbinding of the Strep-Tactin HRP conjugate on His-taggedprey proteins was ruled out as no signals were obtainedwithout bait (Fig. 3B lowest panel). Incubation with mainlyunmodified GlnK-StrepII yielded more intense signals thanincubation with modified GlnK-StrepII. This concurred withthe in vivo observation that particularly the mainly unmodi-fied form of GlnK-StrepII bound strongly to membranescontaining the Rnf1 complex; however, the modified formof GlnK-StrepII also bound to some extent to His-RnfC1 invitro. Here contamination with unmodified GlnK-StrepIIwas only minor (Fig. 3A), yet might have accounted for thebinding.

A swapping (interchanging the bait and prey of anexperiment) Far-Western analysis was carried out usingunmodified or modified GlnB-StrepII or GlnK-StrepII asprey proteins and His-RnfC1 as bait (Fig. 3C). Proteinsloaded in equal amounts (upper panel) and incubated with



purified His-RnfC1 (second panel) showed a positive andspecific interaction only for GlnK-StrepII and not at all forGlnB-StrepII. However, it appeared that His-RnfC1 inter-acted with both the forms of GlnK-StrepII equally, which isunlikely to be caused by the minor contamination withunmodified GlnK-StrepII present in the purified GlnKpreparation under nitrogen limitation conditions; it may bedue to higher concentrations of prey proteins used for invitro studies. The chances of unspecific interactions wereruled out as no signals were obtained without bait (Fig. 3Clowest panel). By swapping the bait and prey in Far-Western analysis, relatively similar results were obtainedconfirming the fidelity of the interaction. Thus GlnK andnot GlnB interacted specifically with the RnfC1 compo-nent of the Rnf1 complex.

Activity of membrane-bound Rnf1 complex is reducedunder conditions of ammonium-induced switch offof nitrogenase

Assuming that reduction of nitrogenase was mediated byferredoxin FdxN through the Rnf1 complex in the wild-typesituation, we hypothesized that the electron flow shouldbe decreased by binding of deuridylylated GlnK to thecomplex upon ammonium addition, leading to DraT-independent switch off. This should be reflected in areduced activity of the Rnf1 complex. As the activity can bequantified by the oxidation of NADH in vitro with artificialelectron acceptors like hexacyanoferrate III, membranesof Azoarcus sp. strain BH72 were isolated from N2-fixingcultures and from N2-fixing cultures harvested 5 min afteraddition of 2 mM NH4Cl. Ten microgram of membraneproteins of N2-fixing strain BH72 catalysed the oxidation ofNADH with an activity of 19.51 � 3.05 U mg-1. In contrast,a strongly reduced activity of 3.11 � 0.89 U mg-1 wasobtained from membranes after ammonium shock. Mem-branes from N2-fixing cultures of strain BHDRnf1 alsoshowed a reduced activity (7.96 � 1.86 U mg-1) as com-pared with wild type. Because the assays were carried outaerobically, the involvement of anaerobic hydrogenase inthe reaction could be ruled out (Li et al., 2006). Residualactivity might probably arise from membrane-bound Rnf2complex, still functional in BHDRnf1, or other side reac-tions. As electron flow was perturbed by NH4

+ in the wildtype or by the absence of the rnf1 cluster under N2 fixation,the Rnf1 complex of strain BH72 may play a role in theelectron transport towards nitrogenase under nitrogen fixa-tion, regulated by GlnK sequestration.

Discussion

PII signal transduction proteins and Rnf membrane com-plexes were previously not thought to be functionallyrelated. Our following lines of evidence suggest that the

Fig. 3. Determination of physical interaction of heterologouslyexpressed and purified GlnK-StrepII and His-RnfC1 proteins byFar-Western analysis.A. Analysis of GlnB-StrepII and GlnK-StrepII uridylylation status by18% SDS-PAGE and Western blot from 7.5% native PAGE.Overexpression of GlnB-StrepII or GlnK-StrepII was carried outunder N-limitation or N-excess by supplementing LB medium with20 mM a-ketoglutarate (a-KG) or 20 mM ammonium chloride(NH4Cl), respectively, inducing with 500 mM IPTG at 30°C for 2 hand subsequent purification. The upper panel represents aSDS-PAGE of the purified extracts that were run side by side andthen stained with Coomassie Brilliant Blue. About 5 mg of each ofthe samples was loaded for protein staining and analysis. Thelower panel represents a Western blot from the sameoverexpressed and natively purified PII proteins, which were run ona 7.5% native PAGE and subsequently detected by anti-strepantiserum on the blot. The respective growth conditions arelabelled on top of the respective lanes.B. Far-Western analysis showing physical interaction of His-RnfC1used as prey and GlnK-StrepII used as bait. Lane 1: pET-28a(+)vector without insert (Con), Lane 2: overexpressed His-RnfC1 inthe total extract (RnfC1E), Lane 3: His-RnfC1 purified (RnfC1p).Upper panel, Coomassie stained SDS-PAGE; second and thirdpanel, Far-Western analysis baited with GlnK-StrepII underN-excess (GlnK – UMP) (second panel) or N-limitation(GlnK + UMP) (third panel); last panel: Western analysis withoutbait but probed only with anti-strep antiserum (no GlnK).C. Far-Western analysis with GlnB-StrepII or GlnK-StrepII as preyand baited with His-RnfC1. GlnB-StrepII or GlnK-StrepII expressedunder N excess (- UMP) or under N-limitation (+ UMP)respectively. Upper panel, Coomassie stained SDS-PAGE ofStrep-tagged purified GlnB or GlnK proteins; middle panel:Far-Western analysis with His-RnfC1 as bait last panel: Westernanalysis without bait but probed only with anti-His antibody.



signal transmitter protein GlnK controls the Rnf1 complexcatalysed electron transfer by protein–protein interactionand might thereby regulate nitrogenase activity in Azoar-cus sp. strain BH72 (model summarized in Fig. 4): (i)Expression of rnf1 is upregulated under conditions of nitro-gen fixation and under control of the transcriptional activa-tor NifA. (ii) DraT-independent switch off of nitrogenaseactivity upon ammonium upshift is dependent on the Rnf1complex. (iii) Membrane sequestration of GlnK in steady-state N-surplus conditions is Rnf1 complex dependent.(iv) Membrane sequestration of GlnK in response toammonium upshift or anaerobiosis shock is also Rnf1-dependent. (v) Protein–protein interactions betweenRnfC1 and specifically GlnK but not with GlnB were dem-onstrated in vitro. (vi) Ammonium upshift led to decreasedactivity of the Rnf1 complex in membranes. This modelimplies not only a hitherto unknown interaction partner fora PII-like protein and an additional process under itscontrol, but also a novel principle of controlling membrane-bound electron transfer coupled to ion transport.

In Azoarcus sp. BH72, similar to Azotobacter vinelandii,only the rnf1 and not rnf2 cluster is under nif control(Curatti et al., 2005), and indeed slight upregulation ofrnf1 was also detected in Pseudomonas stutzeri (Des-

noues et al., 2003). The conserved physical linkage of thernf cluster genes implies coregulation in one operon,which has indeed been shown for A. woodii (Biegel et al.,2009). In P. stutzeri strain A1501, rnf cluster genes wereas well found to be upregulated under nitrogen fixation inthe range of 2.2- to 17.5-fold (Yan et al., 2010).

In concordance with the expression pattern, only theRnf1 complex affected N2 fixation in Azoarcus sp. Thereduced diazotrophic growth of BHDRnf1 in comparisonwith wild type and BHDRnf2 might be caused bydecreased supply of reductant to nitrogenase similar tothe fdxN mutant of strain BH72 (Egener et al., 2001). Asimilar role of Rnf proteins in electron transport to nitro-genase has been reported in R. capsulatus (Schmehlet al., 1993), and in P. stutzeri (Desnoues et al., 2003).However, in A. vinelandii, Rnf proteins were required forthe early expression of nifHDK during nitrogenase dere-pression and for stable accumulation of the 4Fe4S clusterin NifH, but presumably not for electron transport to nitro-genase (Curatti et al., 2005). Although rnf1 played a majorrole, rnf2 served in a complementary fashion (Curattiet al., 2005).

That the Rnf1 complex is essential for ammonia-mediated fast switch off, is a novel finding in diazotrophs.

Fig. 4. Proposed model for regulation ofnitrogenase DraT-independent switch off byGlnK and the Rnf1 complex. Under nitrogenlimitation (N2 fixation, left side), theproton-motive force or membrane potential isused to drive the ferredoxin:NAD+ : ferredoxin-oxidoreductase activity ofthe Rnf1 complex, serving as the principal lowredox potential electron donor to FdxN as amobile electron carrier to NifH. 2-oxoglutarate(2-OG) levels are high, GlnK is uridylylatedand mainly localized in the cytosol. Inpresence of ammonium (+N) and its import byAmt proteins (right side), the internalglutamine: 2-OG ratio increases by the activityof glutamine synthetase, and GlnK isreversibly deuridylylated by theuridyl-removing (UR) uridyltransferase(UTase). Unmodified GlnK interacts with theRnf1 complex (probably with RnfC1) and isthereby sequestered to the membrane. As aresult, the activity of the Rnf1 complex isperturbed, manifested by decreasedferredoxin reduction and consequent inhibitionof nitrogenase activity. Upon shift of N2-fixingcultures to anaerobiosis (-O2), the cellularnitrogen status remains unaltered like duringnitrogen fixation (modified GlnK); however, adecrease of energy charge (high ADP/ATPratio) facilitates binding of modified GlnK tothe Rnf1 complex, resulting in inhibition ofelectron flow.



Blocking the electron flow to nitrogenase at the level ofgeneration of the reduced FdxN might be an effectivemechanism for a rapid switch off independent of the DraTGsystem. This might involve the membrane-bound Rnf1complex as a target for regulation, mediated by PII proteinas signal transmitter. Indeed, GlnK and not GlnB, wasspecifically membrane sequestered by Rnf1 under steady-state conditions on ammonium as well as in response toammonium upshift during N2 fixation. In several Gram-negative and Gram-positive bacteria, the ammoniumchannel AmtB sequesters GlnK to the inner membraneunder conditions of N-upshift (Coutts et al., 2002; Detschand Stülke, 2003; Strösser et al., 2004; Wang et al., 2005;Huergo et al., 2007; Tremblay et al., 2007). Despite thegenomic conservation, surprisingly an AmtB dependenceof GlnK membrane association was not detected in Azoar-cus sp. strain BH72 (Martin and Reinhold-Hurek, 2002),and neither an involvement of two similar proteins encodedby amtY and amtE was found. The modification status ofmembrane-bound GlnK in vivo was the probably the sameas in steady-state conditions (growth on ammonium), withthe deuridylylated form of GlnK signalling N-surplus, asfound in AmtB-sequestered proteins in other bacteria.There, bound GlnK under N-surplus leads to reducedAmtBtransport activity by blocking ammonia conduction (Couttset al., 2002; Conroy et al., 2007). Analogously GlnKappears to block Rnf1-mediated electron flow underammonium shock, a hypothesis that was supported by adecreased enzymatic Rnf activity of isolated membranesafter ammonium upshift. However, how this is achieved isnot clear at the structural level. This model is also consis-tent with the observation of others (Halbleib et al., 2000)that the Fe protein redox status is important for subsequentcovalent modification by ADP-ribosylation: DraT exhibitedhigher activity with Fe protein in its oxidized form whereasDraG had no activity with oxidized Fe protein.

A shift of N2-fixing cells to anaerobic conditions alsoleads to a rapid, DraT-independent inhibition of nitroge-nase activity (Oetjen and Reinhold-Hurek, 2009). Surpris-ingly, Rnf1-membrane sequestration of GlnK occurredunder these conditions, as well. This suggests that a similarmechanism for nitrogenase switch off also operates duringanaerobiosis shock. Surprisingly, the uridylylated form ofGlnK was detected to be the major membrane associatedform under these conditions in vivo. So far only the non-uridylylated form of GlnK has been reported in AmtB-mediated membrane binding under ammonium surplus(Coutts et al., 2002). The E. coli GlnK-AmtB complexshows a trimer: trimer stoichiometry where the extendedT-loop inserts deeply into the cytoplasmic pore exit of theAmtB channel, thereby blocking ammonia conduction(Conroy et al., 2007). On the other hand the PII-NAGK(N-acetylglutamate kinase) complexes formed both in thecyanobacterium Synechoccocus elongatus and Arabidop-

sis thaliana have a 1:2, NAGK hexamer: PII trimer, stoichi-ometry. Two surfaces of PII are important for NAGKcontacts, one in the PII trimer body and the other in theT-loop (Llácer et al., 2007; Mizuno et al., 2007). The thirdreported PII complex structure is that of S. elongates PII

trimer with three PipX (target) monomer proteins throughthe extended PII T-loops, enhanced by ADP binding (Lláceret al., 2010). However, as mentioned earlier, a recent studyon GlnZ-DraG crystal structure shows that some interac-tions of PII protein with its targets in a ternary complex canoccur even without the involvement of its T-loop region(Rajendran et al., 2011). The membrane sequestration ofGlnK by Rnf1-component(s) in Azoarcus sp. BH72 may beconformationally different and distinct from conventionalGlnK-AmtB membrane associations. Moreover, in vitroFar-Western analysis indicated that both forms of overex-pressed GlnK proteins may bind to RnfC1. The binding ofmodified as well as unmodified form of GlnK with Rnf1complex in strain BH72 adds further to the list of reporteddifferent forms of PII–target interactions.

It has been shown that factors other than the PII modifi-cation status can drive its association with the membrane,e.g. in R. capsulatus (Tremblay et al., 2007). Partiallymodified PII protein has been detected in the membranefraction of ammonium-upshifted cultures of R. capsulatus(Tremblay et al., 2007). In Bacillus subtilis, membraneassociation of GlnK occurs irrespective of modification andis possibly influenced by ATP levels (Heinrich et al., 2006).In A. brasilense, in vitro uridylylation of GlnB and GlnZ byGlnD is differentially dependent onATP and 2-OG whereasglutamine stimulated deuridylylation (Araujo et al., 2008).Although the integration of nitrogen and carbon signals byPII proteins (uridylylation and 2-OG binding) has beenclearly established, the same is not true for energysensing. In R. rubrum, theADP/ATP ratio overrides the C/Nsignal and influences complex formation of GlnJ withAmtB1 (Teixeira et al., 2008). Interestingly, T-loop residueGln39 of R. rubrum GlnJ, which is required for targetinteraction involving the metabolite (ATP and 2-OG)-boundconformations is also conserved in GlnK of strain BH72.Cells of strain BH72 with strictly respiratory energymetabolism, are likely to experience energy depletionwhen shifted to anaerobiosis. Sensing of cellular energydepletion by GlnK and its subsequent association with theRnf1 complex in the membrane is probably independent ofits uridylylation status. The occurrence of modified GlnKproteins in membrane fractions upon ammonium upshift(+AS) was likely to arise from simultaneous anaerobicshock inferred by sampling from a microoxic N2-fixingculture, masking the effect of ammonium addition.

In vitro protein interaction studies further corroboratedthe specific association of RnfC1 with GlnK and not withGlnB. This hydrophilic RnfC1 protein component harboursmotifs for (4Fe–4S) centre as well as for NADH binding



(Kumagai et al., 1997), which might represent putativebinding sites for ferredoxin and NADH, respectively,acting as putative targets for blockage by GlnK. From thein vivo membrane association studies it appears that inresponse to ammonium probably mainly the unmodifiedform of GlnK is membrane sequestered. However, theuridylylated form of GlnK might also be associated withRnfC1 in in vitro studies, concurring with the observationsof in vivo anaerobiosis shock.

Based on our results we propose a working model(Fig. 4) illustrating an alternative mode of GlnK seques-tration by Rnf proteins occurring in our model diazotrophAzoarcus sp. upon ammonium upshift and anaerobiosis.The observed protein–protein interaction has not yet beendescribed, but might occur also in other species, given thewide distribution of the respective genes in prokaryotes. Apossible control of enzyme catalysed electron transfer bynon-enzymatic protein–protein interactions might repre-sent a novel role for versatile signal transmitter proteins.

Experimental procedures

Bacterial strains and plasmids

Bacterial strains and plasmids used in this study are listed inTable S1. Oligonucleotides used for PCR amplifications arelisted in Table S2.

Media and growth conditions

Unless otherwise stated, E. coli was grown in LB or on LB agarplates (Ausubel et al., 1987) at 37°C following standard pro-tocols and using antibiotics when necessary. Azoarcus sp.strain BH72 was routinely grown aerobically at 37°C inVM-ethanol medium (Hurek et al., 1995). Growth of Azoarcussp. strain BH72 for triparental conjugation was carried out inVM-Malate medium (Reinhold-Hurek et al., 1993). Triparentalconjugation was carried out by mixing the receiver, donor andhelper [E. coli (pRK2013)] strains at OD578 1 in the ratio of20:1:1, respectively, on KON agar plates, which are based onsynthetic malate medium (SM) (Reinhold et al., 1985) supple-mented with 5 g of yeast extract and 1 g of NaCl per litre. Afterincubation overnight at 37°C cells were scraped from KONplates, suspended in SM medium and eventually plated asdiluted fractions on VM-ethanol plates containing chloram-phenicol (12.5 mg ml-1). For mutant selection and cultivation,antibiotics were added at 30, 40, 20 and 30 mg ml-1 for ampi-cillin, spectinomycin, streptomycin and kanamycin respec-tively. For selection of double recombinants, singlerecombinant strains were plated on VM-Ethanol agar platescontaining 6% sucrose and without antibiotic. Growth on syn-thetic medium supplemented with either 10 mM NH4Cl or10 mM KNO3 or 10 mM glutamate as sole nitrogen source wasperformed in 1 l Erlenmeyer flasks with a culture volume of50 ml on a rotary shaker at 200 r.p.m. To obtain nitrogen-fixingbatch cultures, cells were washed twice with N-free syntheticmedium, adjusted to an optical density at 578 nm of 0.05 in50 ml of the same medium or with 10 mM glutamate (nitrogen-

fixing condition) and incubated microaerobically (1.6% headspace oxygen) in rubber-sealed 1 l Erlenmeyer flasks (Kargand Reinhold-Hurek, 1996), with rotary shaking at 150 r.p.m.To induce ammonium-switch off in nitrogen-fixing batch cul-tures, 2 mM NH4Cl was added and cells harvested at 5 or10 min after addition. For large-scale controlled growth,nitrogen-fixing Azoarcus sp. was grown on nitrogen-free SMmedium (Reinhold et al., 1986) in a 2 l bioreactor (Biostat-B; B.Braun Biotech) at 0.6% dissolved O2 (Egener et al., 2001). Toinduce anaerobic shock (-OS) on nitrogen-fixing cultures,20 ml cultures at OD578 of 0.5 were removed from the bioreac-tor, transferred to flasks containing an N2 atmosphere, andincubated for 15 min with shaking at 37°C before harvest (fourreplicates). To induce ammonium shock (+AS), half of the cellsfrom the bioreactor were removed and treated as (-N) extract.To the other half, NH4Cl was added to a final concentration of2 mM and incubated further for 15 min before harvest.

DNA manipulations

Isolation of chromosomal DNA was carried out as describedpreviously (Hurek et al., 1993). Other DNA and RNA tech-niques followed standard protocols (Ausubel et al., 1987).Genomic clones were characterized by restriction mappingand Southern blot analyses with digoxigenin-labelled DNAprobes. The enzymes for DNA manipulation were supplied byFermentas (St. Leon-Rot, Germany).

DNA sequence analysis

For DNA sequencing the didesoxynucleotide chain termina-tion method was used with the ALFexpress automatedsequencer (GE Healthcare, Freiburg, Germany) by standardprocedures (Hurek et al., 1997). Sequence comparisonswere carried out using the Blast program (Altschul et al.,1990). Protein domains were predicted using Pfam (Batemanet al., 2004).

Generation of deletion mutants, marker exchangemutants and transcriptional reporter gene fusion strains

Knock-out mutants for rnf1 and rnf2 were constructed byin-frame deletion of respective gene clusters. To delete rnf1-gene cluster, the 0.8 kb upstream region of rnfA1 was ampli-fied by oligonucleotides Rnf1_upFW and Rnf1_upREV havingXbaI and PstI sites at their respective 5′ ends, and the frag-ment cloned into XbaI-PstI sites of pPCR-Script AmpSK(+)(pJRnf1UP). Similarly, the 1 kb downstream region of azo0511(probable rnfH1) was amplified (Rnf1_downFW andRnf1_downREV) and cloned into PstI and HindIII sites ofpJRnf1UP to generate pJRnf1UD containing the deletion. Theinsert was further subcloned into XbaI-HindIII sites ofpK18mobsacB to generate pJRnf1UD-MSB. In order to deleternf2-gene cluster, the 0.9 kb region upstream of rnfA2 wasamplified using Rnf2upfor and Rnf2uprev and cloned intoXbaI-EcoRI sites of pPCR-Script AmpSK(+) (pRnf2UP). The1.03 kb downstream fragment of rnfE2 was amplified usingRrnf2dwnfor and Rnf2dwnrev was into the EcoRI-HindIII siteof pRnf2UP to generate deletion fragment pRnf2UD, which



was subcloned into XbaI-HindII sites of pK18mobsacB(pRnf2UD-MSB). Both plasmids, pJRnf1UD-MSB orpRnf2UD-MSB, respectively, were transferred to Azoarcus sp.strain BH72 by triparental conjugation with helper strain E. coli(pRK2013). Individual single recombinants were furtherselected for double recombination on 6% sucrose inVM-Ethanol agar. Strains showing deletions of rnf1 or rnf2gene clusters, respectively, were distinguished from the wildtype by colony PCR and the deletion further confirmed bySouthern blot hybridization (data not shown).

In a similar way, to inactivate amtY in strain wild-type BH72and amtB – mutant BHABK, plasmid pJKDaY was conjugatedinto the respective strains and finally selected on 6% sucroseon VM ethanol-agar for double recombinants carrying anin-frame deletion of amtY.

For inactivation of amtE by marker exchange mutagenesis,a 0.72 kb fragment upstream of amtE (using primersAmtEUP-Fw and AmtEUP-Rv) and a 0.72 kb fragment down-stream (AmtEDN-Fw and AmtEDN-Rv) were amplified,cloned and joined, and a resistance cartridge (W cassette)inserted to generate pJKa203WpPCR as described inTable S1. The inactivation of amtE was achieved by conju-gating pDAE-MSBW into the wild-type strain BH72.Kanamycin-sensitive but Sp/Sm-resistant colonies were con-firmed as amtE marker exchange mutants BHDAmtE bySouthern blot hybridization (data not shown).

Fragments carrying the 5′ ends of rnfA1 and rnfA2 that hadbeen generated for deletion mutants were subcloned intovectors carrying promoterless uidA genes to generatetranscriptional fusions (plasmids pK18mob2rnf1-gusA andpMSBrnf2-gusA). After plasmid transfer by conjugation intostrain BH72, single recombinants were selected and con-trolled for correct genomic integration by Southern blothybridization.

Overexpression and purification of Strep-tagII or His-tagfusion proteins

GlnB and GlnK of Azoarcus sp. strain BH72 were expressedas C-terminal Strep-tagII proteins in E. coli (BL21-DE3). glnBand glnK were PCR amplified using GlnBstrep-Fw andGlnBstrep-Rv or GlnKstrep-Fw and GlnKstrep-Rv, respec-tively, from genomic DNA of Azoarcus sp. strain BH72. Ineach case the forward primer for glnB or glnK had a startcodon for the respective amplified gene flanked by PscI orNcoI at their respective 5′ ends, while the reverse primerharboured a sequence encoding the Strep-TagII (WSH-PQFEK) followed by a stop codon and a HindIII site at its 5′end. PscI-HindIII digested 0.382 kb glnB-, or NcoI-HindIIIdigested 0.377 kp glnK- PCR-products, respectively, werecloned into NcoI-HindIII sites of pET-28a(+) to generate pET-glnBstrep and pETglnKstrep.

rnfC1 from strain BH72 was amplified using primersRnfC1his-Fw and RnfC1his-Rv carrying a PscI site or a HindIIIsite, respectively, at the 5′ ends. The PscI- and HindIII-digested amplicon was cloned into NcoI-HindIII digested pET-28a(+) vector in E. coli DH5a to generate pet28aRnfC1His.

For protein expression, E. coli BL21(DE3) was transformedwith plasmids petglnBstrep, petglnKstrep or pet28aRnfC1Hisrespectively. To generate nitrogen-rich or nitrogen limitingconditions, LB medium was supplemented either with 20 mM

NH4Cl or 2 mM a ketoglutarate. Each of the former twostrains was grown in LB medium at 37°C until optical densityof the culture at 600 nm reached a value of 0.6. IPTG wasadded to induce at 500 mM, and cultures were incubated foranother 1–2 h at 30°C before harvest. For the latter strain, LBmedium was supplemented with 50 mm FeSO4, and cultureswere grown until an OD600 of 1 was attained at 37°C. IPTGwas added to a final concentration of 500 mM and induced for2 h at room temperature. All the further extraction steps werecarried out at 4°C.

Strep-tagII GlnB and GlnK or 6X Histidine-tagged RnfC1were purified under native conditions. Usually 150 mg ofpellet was suspended in 1 ml lysis buffer (50 mM NaH2PO4,300 mM NaCl, 1 mM EDTA at pH 8) and incubated on ice for30 min with 1 mg ml-1 of lysozyme. Following three timessonication on ice, the lysate was centrifuged for 20 min at 4°Cand the cleared lysate used for purification. The Strep-tagIIfusion proteins were purified over Strep-tactin sepharosebeads according to manufacturers instruction (Strep-tagstarter kit No. 2–1201-55, IBA, Biotagnology, Göttingen). 6XHistidine-tagged proteins were purified using Ni-NTAAgarose(No. 30210, Qiagen, Hilden) according to manufacturer’sinstructions.

Membrane isolation, SDS-PAGE, native PAGE andWestern blotting

Cytoplasmic and membrane protein fractions were isolatedas described previously (Martin and Reinhold-Hurek, 2002).For the analysis of NifH modification, 1 ml aliquots of cellsuspensions were removed from the culture, and proteinswere precipitated immediately on ice with 100 ml of trichloro-acetic acid (TCA) solution (1 g of TCA per ml) as describedpreviously (Zhang et al., 1993; Oetjen and Reinhold-Hurek,2009). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Hureket al., 1994). For separation of the two forms of NifH protein,a 10% (wt/vol) acrylamide low cross-linker gel with a ratio ofacrylamide to N,N’-methylene bisacrylamide of 172:1 wasused (Kanemoto and Ludden, 1984). For separation of modi-fied and unmodified forms of PII, protein extracts were run on18% SDS-PAGE using a 37.5:1 acrylamide : bisacrylamidemix from Rotiphorese 30 (Roth)(Johansson and Nordlund,1997).

To resolve the uridylylation states of purified PII proteinsunder native conditions, native PAGE (7.5%) was preparedusing acrylamide to bisacrylamide of 37.5:1 in 375 mM Tris-HCl buffer, pH 8.8, without detergents. Purified proteinsamples up to 5 mg in 50 mM sodium phosphate pH 7 wereloaded with NSB buffer (Native Sample Loading buffer:125 mM Tris, 20% glycerol and Bromophenol blue) withoutprior heat denaturation; gels were run at 150 V in 1¥ Tankbuffer (Tris 25 mM, glycine 180 mM) until the blue dye reachedthe bottom of the gel.

Electro-transfer of proteins onto nitrocellulose membraneswas performed as described previously (Hurek et al., 1994) for45 min at 8 V with a semidry electroblotter (Bio-Rad, Munich,Germany). The NifH protein of Azoarcus sp. strain BH72 wasdetected using antiserum against NifH of R. rubrum, kindlyprovided by R. Ludden (Berkeley, California) as outlined pre-viously. GlnB and GlnK, were immunodetected using antisera



raised against purified fusion proteins (Martin et al., 2000).Proteins were visualized using ECL Western blotting detectionreagents (GE Healthcare). Protein concentrations were deter-mined by the Bio-Rad protein assay based on the method ofBradford (Bradford, 1976). The relative of NifH band intensitieswere quantified using LAS-3000 mini (Fujifilm). Multipleimages of ECL-treated blots with varying exposure times weresubsequently analysed with the help of the Image readerprogramme.

In vitro Far-Western protein–protein interaction assay

In vitro binding assays were performed as described byAndrade et al. (2006) with some modifications. Briefly,approximately 5 mg of purified recombinant proteins or totallysates of E. coli cultures expressing recombinant proteinswere resolved by SDS-PAGE and transferred to nitrocellulosemembranes. The membranes were blocked with TBS-TT-milk[140 mM NaCl, 20 mM TrisHCl (pH 7.4), 0.1% Triton X-100,0.1% Tween-20, 5% skimmed milk powder] for 1 h and thenprobed with His-RnfC1 or GlnK-StrepII (50 mg ml-1) in thesame buffer for 12 h at room temperature. Membranes werethen washed four times with TBST-TT buffer and incubatedeither with Penta-His Antibody for 2 h, followed by washingand subsequent incubation with rabbit anti-mouse immuno-globulin HRP (1:10000) (DAKO) for 1 h or incubated withStrep-Tactin HRP conjugate according to manufacturer’sinstruction (IBA, Göttingen, Germany). Proteins were finallyvisualized using the ECL Western blotting detection kit asdescribed above.

Determination of nitrogenase and b-glucuronidaseactivity

Nitrogenase activity of batch cultures was determined byusing the acetylene reduction method (Egener et al., 1998).Activity of b-glucuronidase was measured quantitativelyusing the method described earlier (Jefferson et al., 1987) asmodified (Egener et al., 1999) and expressed in Miller units,defined as E420 ¥ 1000/t (min) OD600.

Determination of activity of membrane-associatedNAD reductase

For isolation of membrane proteins, cell pellets were sus-pended in phosphate buffer pH 7, lysed by French Press andcentrifuged to remove the cell debris. The clear superna-tant was subjected to ultracentrifugation (30 min, 4°C,2 000 000 g) for pelleting membranes, which were washedonce with phosphate buffer supplemented with 600 mM NaCl.Finally, the pellet was suspended in phosphate buffer andprotein concentration estimated. Ten microgram of membraneprotein in 10 ml was used per assay. In a reaction volume of1 ml in a cuvette, 100 mM potassium phosphate pH 7, 1 mMhexacyanoferrate(III) and 1 mM NADH was added. After addi-tion of membrane proteins, the absorbance was measuredspectrophotometrically by following the reduction of hexacy-anoferrate(III) at 20°C by NADH at 420 nm for 12 min,e = 1.02 mM-1 cm-1 (Boiangiu et al., 2005; Imkamp et al.,2007).

Statistical and phylogenetic analysis

The GraphPad InStat software package (GraphPad software,San Diego, CA, USA) was used for statistical analysis. Forphylogenetic analysis of Rnf proteins, multiple sequencealignments of each gene were constructed with MAFFT usingthe G-INS-i setting at default (Katoh et al., 2005). Bayesiananalyses of the concatenated alignment were conducted atPhylogeny.fr (http://www.phylogeny.fr) using standard set-tings. For minimum evolution analyses MEGA version 5(Tamura et al., 2011) was used. Minimum evolution trees wereinferred from maximum likelihood distances using the Jones-Taylor-Thornton (JTT) model (Jones et al., 1992) with agamma shape parameter of 1.76, which was estimated fromthe data set.

Acknowledgements

We would like to acknowledge financial support from theDeutsche Forschungsgemeinschaft (DFG) to B.R.-H. (GrantsRE 756/5-3 and RE 756/14-1). We would like to thank ArshadA. Syed and Sarang Limaye for participating in the construc-tion of the amtE or rnf mutants, respectively, and to Prof.Wolfgang Buckel, Philipps-Universität Marburg, for directionson the Rnf enzyme assay, and Prof. P. W. Ludden, Universityof California, Berkeley for kindly providing the antibodiesagainst NifH.

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A novel regulatory role of the Rnf complex of Azoarcus sp. strain BH72