1 Ruhr-Universität Bochum, Lehrstuhl für Biologie der Mikroorganismen, D-44780 Bochum, Germany
2 Université de Montréal, Département de microbiologie et immunologie, CP 6128, succursale Centre-ville, Montréal, Québec, Canada H3C 3J7
Correspondence
Thomas Drepper
thomas.drepper{at}ruhr-uni-bochum.de
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ABSTRACT |
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INTRODUCTION |
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In Escherichia coli and many other bacteria, GlnB and GlnD (uridylyltransferase/uridylyl removing enzyme) are key elements of the regulatory networks controlling nitrogen assimilation (Merrik & Edwards, 1995; Ninfa & Atkinson, 2000
; Arcondéguy et al., 2001
). Both GlnD and GlnB are involved in sensing the nitrogen status of the cells by direct interaction with glutamine and 2-oxoglutarate, respectively. In E. coli, GlnB controls the activity of glutamine synthetase and the sensor kinase NtrB (Ninfa & Atkinson, 2000
), whereas the GlnB proteins of Azospirillum brasilense, Herbaspirillum seropedicae and Rhodospirillum rubrum also play essential roles in nitrogen fixation, since glnB mutations lead to synthesis of inactive NifA in the latter three organisms (Benelli et al., 1997
; de Zamaroczy et al., 1993
; Liang et al., 1992
; Souza et al., 1999
; Zhang et al., 2000
). In addition, many bacteria contain a second glnB-like gene designated glnK (for a review, see Thomas et al., 2000
; Arcondéguy et al., 2001
). In most cases, glnK appears to be co-transcribed with amtB, which is located downstream of glnK, and encodes a high-affinity (methyl-) ammonium transporter, and expression of glnKamtB is activated by NtrC. In contrast, Azoarcus sp. BH72 and Rs. rubrum contain three PII-encoding genes, namely a glnB gene and two different glnK-like genes forming bicistronic operons with amtB-like genes (Martin et al., 2000
; Zhang et al., 2001b
). The additional copies of glnK are designated glnY (in Azoarcus) and glnJ (in Rs. rubrum).
In E. coli, GlnB and GlnK are structurally and functionally similar (Atkinson & Ninfa, 1998, 1999
; van Heeswijk et al., 1996
). However, recent studies demonstrated that in Klebsiella pneumoniae, only GlnK is involved in transmission of the nitrogen status to the NifL/NifA regulatory system, suggesting functional differences between GlnB and GlnK in this organism (Arcondéguy et al., 1999
; He et al., 1998
; Holtel & Merrick, 1989
; Jack et al., 1999
). In addition, PII-like proteins also have distinct roles in mediating nitrogen control of nitrogenase activity in some diazotrophic organisms (Klassen et al., 2001
; Martin & Reinhold-Hurek, 2002
; Zhang et al., 2001b
). In Rs. rubrum as well as in Azoarcus sp. BH72, GlnB and one of the two GlnK-like proteins (GlnJ and GlnK, respectively) are involved in regulation of DraT/DraG activity, whereas the second GlnK-like protein apparently has no function in this regulatory mechanism (Martin & Reinhold-Hurek, 2002
; Zhang et al., 2001b
).
In this report, we characterized the glnKamtB operon in R. capsulatus and analysed the roles of GlnB and GlnK in the regulation of nitrogen fixation by ammonium. We demonstrated that both GlnB and GlnK are involved in the control of the synthesis and activity of both the molybdenum and the iron-only nitrogenase. Most remarkably, only in a glnBglnK double mutant strain are all levels of ammonium control of molybdenum nitrogenase completely circumvented, whereas at least the activity of the transcriptional activator of the alternative nitrogenase, AnfA, was still affected by ammonium in this mutant.
Preliminary results concerning the role of R. capsulatus GlnB and GlnK in the regulation of nitrogen fixation were presented at the 12th and 13th International Congress on Nitrogen Fixation (Drepper et al., 2000; Groß et al., 2002
; Hallenbeck et al., 2002
).
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METHODS |
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Construction of glnK and ntrC interposon mutants.
To construct a defined glnK interposon mutant, a 2·6 kb XhoI cassette carrying the gentamicin resistance gene from pWKR440 was inserted into the XhoI site located within the glnK coding region. The resulting hybrid plasmid, designated pSG5II.b1, was subsequently used to create R. capsulatus glnK single mutant strain SG26. In order to generate a glnBglnK double mutant (TD166), the 264 bp PCR fragment encompassing an internal glnK fragment was cloned into a mobilizable derivative of pUC18, resulting in hybrid plasmid pSG2.1, which subsequently was integrated into the genome of R. capsulatus mutant strain PHU332 (glnB) by single cross-over recombination. Plasmid integration of pSG2.1 resulted in an R. capsulatus mutant strain containing a 3'-truncated glnK gene, which was under control of the wild-type promoter. Expression of the truncated glnK gene resulted in a mutant GlnK protein in which the 28 C-terminal amino acid residues (encoding the C-loop and part of the B-loop) are substituted by 22 vector-encoded amino acid residues.
To construct a defined ntrC interposon mutant, the 2·6 kb SalI gentamicin resistance cassette from plasmid pWKR202 was inserted into the XhoI site of the mobilizable suicide hybrid plasmid pPBK1, resulting in hybrid plasmid pTD8I (ntrC : : [Gm]).
Construction of hybrid plasmid pTD6-5I constitutively expressing anfA (anfAc).
A 1756 bp KasIEcoRI fragment encompassing R. capsulatus anfA was cloned into the mobilizable broad-host-range vector pPHU231. Subsequently, a 1489 bp HindIIISalI cassette carrying the kanamycin resistance gene (aphII) from transposon Tn5 was inserted at the KasI site upstream of the anfA gene. In the resulting hybrid plasmid pTD6-5I expression of anfA was driven by the constitutively expressed aphII promoter (anfAc).
-Galactosidase assays.
R. capsulatus strains carrying lacZ fusions were cultured photoheterotrophically as batch cultures in RCV (for strains carrying the glnKlacZ fusion) or AK-NL (for strains carrying the anfAlacZ fusion) minimal medium with either 15 mM ammonium or 10 mM serine as sole nitrogen source until the late-exponential growth phase. -Galactosidase activities were determined by the SDS/chloroform method as described previously (Miller, 1972
; Hübner et al., 1991
).
Western analysis.
Cell-free protein extracts of R. capsulatus were isolated as described before (Masepohl et al., 1988). Proteins were separated on SDS-polyacrylamide gels with an acrylamide concentration gradient of 1015 %, and subsequently blotted onto PVDF membranes (Roth). Detection of NifA1, NifH and AnfH proteins was performed using the ECL kit (Amersham Pharmacia Biotech).
Nitrogenase ADP-ribosylation and in vivo activity assays.
R. capsulatus strains were grown photoheterotrophically in 3 ml cultures (in 15 ml Hungate tubes) in either RCV or AK-NL minimal medium supplemented with 5 mM serine under an argon atmosphere. Nitrogenase activity was measured in whole cells by the acetylene reduction method using a Hewlett Packard gas chromatograph model 5890 series II with a Chrompack alumina GC column for the acetylene/ethylene/ethane separation. The formation of ethane was routinely monitored as an indication of the activity of the alternative nitrogenase (Wang et al., 1993). Nitrogenase-mediated H2 evolution was determined as described previously (Klein et al., 1991
). For the results shown in Fig. 2
, in vivo nitrogenase activity was analysed by the acetylene reduction method as described previously (Yakunin et al., 1999
). Culture samples (2550 µl) were removed from the vials at the times indicated in Fig. 2
as described previously (Yakunin & Hallenbeck, 2002
) and equal amounts of total protein (1 µg per well) were loaded onto polyacrylamide gels. Immunoblotting with chemiluminescence detection was used to monitor the modification state of Fe-protein essentially as described previously (Yakunin & Hallenbeck, 1998
).
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RESULTS AND DISCUSSION |
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Ammonium control of synthesis and activity of molybdenum nitrogenase is completely abolished in a glnBglnK double mutant
To study the function of R. capsulatus GlnB and GlnK in the regulation of nitrogen fixation, single glnB and glnK mutant strains (PHU332 and SG26) as well as a glnBglnK double mutant (TD166) were constructed (Methods). In contrast to the situation in glnK single mutant strain SG26 (based on interposon mutagenesis; glnK : : Gm), the glnK gene in the double mutant strain TD166 was disrupted by plasmid integration mutagenesis using pSG2.1 (Methods). At this point it should be noted that the phenotypes of single glnK interposon and plasmid integration mutants were essentially the same (B. Masepohl, B. Lucas & T. Drepper, unpublished results). The influence of glnB and glnK single and double mutations on the nif-encoded nitrogenase system was analysed at three different levels of regulation, namely the ammonium-dependent regulation of nifA expression (level 1), the post-translational ammonium control of NifA activity (level 2) and the post-translational ammonium control of nitrogenase activity (level 3).
Ammonium-dependent regulation of nifA expression
First, we examined accumulation of the transcription activator NifA1 in R. capsulatus wild-type and in the mutant strains PHU332, SG26 and TD166 (Fig. 1a). For this purpose, cells were cultured under nitrogenase-derepressing (-N) or -repressing (+N) conditions prior to protein extraction and Western analysis using a NifA1-specific antiserum raised against a synthetic oligopeptide corresponding to the N-terminal 17 amino acid residues (Paschen et al., 2001
). In the wild-type strain, NifA1 accumulated only in N-limited cells, which is consistent with NtrC-mediated transcriptional control of the nifA1 gene (Fig. 1a
, lanes 1 and 2). As expected, inactivation of glnB led to accumulation of a low level of NifA1 protein in ammonium-grown cells (Fig. 1a
, lanes 3 and 4). In contrast, the glnK mutant SG26 (Fig. 1a
, lanes 5 and 6) showed a pattern of NifA1 accumulation that was similar to the wild-type. These data are corroborated by analysis of a nifA1lacZ fusion in the respective mutant backgrounds (Hübner et al., 1993
; A. Paschen, S. Groß & W. Klipp, unpublished results). In the glnBglnK double mutant TD166, synthesis and/or accumulation of NifA1 was greatly enhanced under both N-limiting and N-sufficient conditions (Fig. 1a
, lanes 7 and 8). Thus it seems likely that R. capsulatus GlnB is the predominant signal transducing protein for the first regulatory level (Ntr-mediated control of nifA transcription), while GlnK is unable to fully substitute for GlnB in regulating activity of the sensor kinase NtrB.
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The role of GlnB and GlnK in the regulation of NifA activity has previously been examined in several diazotrophic bacteria, including Azospirillum brasilense, H. seropedicae, Rs. rubrum, K. pneumoniae, Azotobacter vinelandii and Azorhizobium caulinodans. There seem to be three different mechanisms of NifA regulation by PII-like proteins. (i) In Azospirillum brasilense, H. seropedicae and Rs. rubrum, GlnB is essential for NifA activity (Benelli et al., 1997; de Zamaroczy et al., 1993
; Liang et al., 1992
; Zhang et al., 2000
), most likely involving a GlnB-mediated activation of preformed NifA under N-limiting conditions. (ii) In K. pneumoniae and Azotobacter vinelandii, GlnK functions as a signal transduction protein modulating the activity of NifL, which inactivates NifA under nitrogen-sufficient conditions (Arcondéguy et al., 1999
; He et al., 1998
; Holtel & Merrick, 1989
; Jack et al., 1999
; Little et al., 2000
, 2002
; Reyes-Ramirez et al., 2001
; Rudnick et al., 2002
). (iii) In contrast, neither GlnB nor GlnK is required for NifA activity under ammonium depletion in Azorhizobium caulinodans (Michel-Reydellet & Kaminski, 1999
) and R. capsulatus (this study), but they seem to play a role in inactivation of NifA in the presence of ammonium.
Post-translational ammonium control of nitrogenase activity
Since a glnBglnK double mutation led to constitutive expression of molybdenum nitrogenase, we asked whether nitrogenase was active in the presence of ammonium. This question was addressed by examination of the in vivo nitrogenase activity of R. capsulatus strain TD166 via the acetylene reduction assay and by measurement of H2 production (Table 2). Indeed, mutant strain TD166 exhibited high levels of nitrogenase activity in the presence of fixed nitrogen, demonstrating that a glnBglnK double mutation leads to circumvention of all ammonium-dependent regulatory mechanisms inhibiting synthesis and activity of molybdenum nitrogenase. Furthermore, nitrogenase activity in TD166 was clearly enhanced under nitrogen-limiting conditions compared to the values obtained for the wild-type under the corresponding conditions. It is worth mentioning that in the glnBglnK mutant the acetylene reduction activity of nitrogenase was four times higher than in the wild-type, whereas H2 production was only slightly (1·5-fold) enhanced. This observation might be explained by increased H2 consumption via uptake hydrogenase, since expression of the R. capsulatus hupSL genes (encoding the small and large subunit of the uptake hydrogenase) is induced by H2 via the HupT/HupR two-component system (Colbeau & Vignais, 1992
; Toussaint et al., 1997
; Dischert et al., 1999
).
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Similar results concerning the role of PII-like proteins in controlling DraT/DraG activity have recently been described for several other diazotrophic bacteria, including Rs. rubrum, Azoarcus sp. strain BH72 and Azospirillum brasilense (Klassen et al., 2001; Martin & Reinhold-Hurek, 2002
; Zhang et al., 2000
, 2001a
, b
). In all cases, the PII signal transduction proteins play an important role in regulating not only synthesis of nitrogenase but also activity of the enzyme complex in response to the cellular nitrogen status.
Recent studies demonstrated that R. capsulatus strains disrupted for the amtB gene (which is located immediately downstream of glnK) were defective in regulating in vivo molybdenum nitrogenase activity in response to ammonium but not to darkness (Yakunin & Hallenbeck, 2002). Since glnK mutations used in this study are thought to be polar onto amtB expression, we cannot exclude that loss of ammonium control of nitrogenase in the glnBglnK double mutant is partially due to the absence of AmtB. However, at least GlnB also appears to be involved in the regulation of DraT/DraG activity, since ammonium-dependent nitrogenase control was also absent in a single glnB mutation background (T. Drepper, B. Masepohl, A. F. Yakunin & P. C. Hallenbeck, unpublished results). To elucidate the specific roles of GlnB, GlnK and AmtB in control of nitrogenase activity in more detail, further studies based on defined mutant strains are required. For this purpose, a glnK mutant strain carrying a marker-less in-frame deletion, which should be non-polar onto amtB expression, has been constructed (B. Masepohl and coworkers, unpublished results). Analysis of this deletion strain in comparison with appropriate glnB and amtB mutants is currently under investigation.
The molybdenum and alternative nitrogenases are regulated differently in response to ammonium
Since a glnBglnK double mutation led to complete loss of ammonium control of molybdenum nitrogenase, we asked whether this also holds true for the alternative nitrogenase. For this purpose, appropriate glnB and glnK single and double mutants were constructed in an nifHDK deletion background (KS36) to avoid any interference of molybdenum nitrogenase during analysis of the alternative nitrogenase. The resulting mutant strains were grown phototrophically in molybdenum-free medium (AK-NL) in the absence or presence of ammonium prior to determination of in vivo nitrogenase activity via the acetylene reduction assay. Under nitrogen limitation, all tested mutant strains exhibited nitrogenase activities comparable to that of the parental strain KS36 (data not shown). Unlike the situation with molybdenum nitrogenase, no activity of the alternative nitrogenase was detectable in the glnBglnK double mutant under +N conditions (data not shown). Therefore, in contrast to ammonium control of the nif-encoded nitrogenase, absence of both GlnB and GlnK was not sufficient for synthesis of an active alternative nitrogenase in the presence of ammonium.
To determine at which regulatory level this PII-independent ammonium control of the alternative nitrogenase occurs, we first analysed expression of the anfA gene under nitrogen-sufficient and nitrogen-limiting conditions. For this purpose, the reporter plasmid pKS131A carrying an anfAlacZ fusion (Kutsche et al., 1996) was introduced into R. capsulatus strains KS94A (anfA), TD52 (ntrC anfA), TD53 (glnB anfA) and TD120 (glnK anfA). As shown before, transcription of anfA was strongly induced under -N compared to +N conditions, and expression of anfA was strictly dependent on NtrC (Table 3
; Kutsche et al., 1996
). A mutation in glnB resulted in constitutive expression of anfA regardless of the absence or presence of ammonium in the growth medium. In contrast, the glnK mutant TD120 showed a pattern of anfA expression similar to the wild-type. These findings are in agreement with the current regulatory model where most of the NtrC molecules are permanently in the phosphorylated, active form when GlnB is absent, since GlnK appears to be unable to substitute for GlnB in regulating activity of NtrB.
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GlnB and GlnK are key elements in the control of synthesis and activity of both nitrogenases in R. capsulatus
As summarized in Fig. 4, ammonium regulation of both nitrogenase systems occurs on at least three different levels: transcriptional activation of nifA and anfA expression controlled by the Ntr system, post-translational regulation of NifA and AnfA activity, and post-translational regulation of both nitrogenases. At one level, the activity of the sensor kinase NtrB would be primarily under the control of GlnB. Under nitrogen-limited conditions, NtrB phosphorylates NtrC, and NtrC-P in turn activates transcription of nifA and anfA. At a second level, regulation of NifA activity is mediated by GlnB and GlnK. Consequently, ammonium control of NifA activity is completely abolished in a glnBglnK double mutant. In contrast to NifA, even in a glnBglnK double mutant, AnfA activity is still inhibited by ammonium. At a third level, the PII-like proteins are involved in the control of the DraT/DraG system. This regulatory model has been corroborated by preliminary yeast two-hybrid studies (Pawlowski et al.,
2003). Proteinprotein interactions were demonstrated for GlnBNtrB, GlnBNifA1, GlnBNifA2, GlnBDraT, GlnKDraT, GlnKNifA1, GlnKNifA2 and GlnKDraT. In accordance with this model, we could not detect interaction of GlnK with NtrB.
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ACKNOWLEDGEMENTS |
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Received 14 January 2003;
revised 9 April 2003;
accepted 9 May 2003.