Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus

Thomas Drepper1, Silke Groß1, Alexander F. Yakunin2, Patrick C. Hallenbeck2, Bernd Masepohl1 and Werner Klipp1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
In most bacteria, nitrogen metabolism is tightly regulated and PII proteins play a pivotal role in the regulatory processes. Rhodobacter capsulatus possesses two genes (glnB and glnK) encoding PII-like proteins. The glnB gene forms part of a glnBglnA operon and the glnK gene is located immediately upstream of amtB, encoding a (methyl-) ammonium transporter. Expression of glnK is activated by NtrC under nitrogen-limiting conditions. The synthesis and activity of the molybdenum and iron nitrogenases of R. capsulatus are regulated by ammonium on at least three levels, including the transcriptional activation of nifA1, nifA2 and anfA by NtrC, the regulation of NifA and AnfA activity by two different NtrC-independent mechanisms, and the post-translational control of the activity of both nitrogenases by reversible ADP-ribosylation of NifH and AnfH as well as by ADP-ribosylation independent switch-off. Mutational analysis revealed that both PII-like proteins are involved in the ammonium regulation of the two nitrogenase systems. A mutation in glnB results in the constitutive expression of nifA and anfA. In addition, the post-translational ammonium inhibition of NifA activity is completely abolished in a glnBglnK double mutant. However, AnfA activity was still suppressed by ammonium in the glnBglnK double mutant. Furthermore, the PII-like proteins are involved in ammonium control of nitrogenase activity via ADP-ribosylation and the switch-off response. Remarkably, in the glnBglnK double mutant, all three levels of the ammonium regulation of the molybdenum (but not of the alternative) nitrogenase are completely circumvented, resulting in the synthesis of active molybdenum nitrogenase even in the presence of high concentrations of ammonium.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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The phototrophic non-sulfur purple bacterium Rhodobacter capsulatus is able to fix atmospheric dinitrogen by either a conventional molybdenum-containing (nif-encoded) nitrogenase or an alternative heterometal-free (anf-encoded) nitrogenase. Synthesis and activity of both nitrogenase systems are tightly controlled at different levels in response to ammonium availability (for a review, see Masepohl & Klipp, 1996; Masepohl et al., 2002). At one level, the ammonium-dependent transcriptional control of the nifA1, nifA2 and anfA genes (which encode specific transcriptional activators of all the other nif and anf genes) is mediated by a regulatory cascade which is similar to the Ntr system of enteric bacteria. The R. capsulatus Ntr system consists of the signal transduction protein GlnB (see below) and the two-component regulatory system NtrB/NtrC (Kranz & Foster-Hartnett, 1990). Under nitrogen-limiting conditions, the sensor kinase NtrB phosphorylates, and thereby activates, the response regulator NtrC (Cullen et al., 1996; Kranz & Foster-Hartnett, 1990). In addition to the NtrC-dependent activation, anfA transcription is repressed by MopA and MopB in the presence of molybdenum (Kutsche et al., 1996). At a second regulatory level, the activity of NifA1 and NifA2 is inhibited in response to ammonium (Hübner et al., 1993; Paschen et al., 2001). The characterization of R. capsulatus NifA1 mutants which are able to activate nif gene transcription in the presence of ammonium revealed that the N-terminal domain of NifA is involved in this post-translational control mechanism (Paschen et al., 2001). Finally, at a third level of control, the activity of both nitrogenases is regulated in response to ammonium and darkness via reversible ADP-ribosylation of the dinitrogenase reductases NifH and AnfH mediated by DraT (dinitrogenase reductase ADP-ribosyltransferase) and DraG (dinitrogenase-reductase-activating glycohydrolase) and by a DraT/DraG-independent switch-off mechanism (Masepohl et al., 1993; Pierrard et al., 1993; Yakunin & Hallenbeck, 1998). Recently it has been shown that the (methyl-) ammonium transporter AmtB, but not its homologue AmtY, may act as an ammonium sensor which is involved in controlling nitrogenase activity via DraT/DraG and also via the DraT/DraG-independent mechanism (Yakunin & Hallenbeck, 2002).

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).


   METHODS
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METHODS
RESULTS AND DISCUSSION
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Strains, media and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. The growth conditions, media and antibiotic concentrations used to cultivate E. coli and R. capsulatus strains were as described previously (Klipp et al., 1988; Masepohl et al., 1988). R. capsulatus cultures were grown either in RCV minimal medium in order to derepress molybdenum nitrogenase, or in molybdenum-free AK-NL minimal medium for the expression of the alternative nitrogenase. To remove traces of molybdenum, the media were treated with activated carbon as described by Schneider et al. (1991). Conjugational plasmid transfer from E. coli S17-1 into R. capsulatus via filter mating was carried out as described previously (Klipp et al., 1988).


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Table 1. Bacterial strains and plasmids used in this study

 
Isolation of the R. capsulatus glnK gene and construction of a glnKlacZ fusion plasmid.
The R. capsulatus glnK gene was isolated by a PCR-based strategy by amplification of a glnK internal 264 bp DNA fragment using degenerate primers based on conserved regions of GlnB proteins of different bacteria (5'-GCSATCATCAARCCSTTCAARCTB-3' and 5'-CACRAAGATCTTSCCRTCSCCRAT-3', where B is C, G or T, R is A or G, and S is C or G). Subsequently, a hybrid plasmid (pSG5II) carrying a 2127 bp SalI fragment was isolated by screening a size-fractionated gene bank by Southern hybridization using the amplified glnK fragment as a probe. As a basis for analysing glnK expression, a 1669 bp SalI–XhoI fragment (glnK') from pSG5II was cloned into the broad-host-range vector pML5B-, resulting in hybrid plasmid pSG9II (glnKlacZ).

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 KasI–EcoRI fragment encompassing R. capsulatus anfA was cloned into the mobilizable broad-host-range vector pPHU231. Subsequently, a 1489 bp HindIII–SalI 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).

{beta}-Galactosidase assays.
R. capsulatus strains carrying lacZ fusions were cultured photoheterotrophically as batch cultures in RCV (for strains carrying the glnK–lacZ fusion) or AK-NL (for strains carrying the anfA–lacZ fusion) minimal medium with either 15 mM ammonium or 10 mM serine as sole nitrogen source until the late-exponential growth phase. {beta}-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 10–15 %, 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 (25–50 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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Fig. 2. Ammonium-induced nitrogenase switch-off and ADP-ribosylation of NifH in the wild-type strain B10S (a) and the glnBglnK mutant strain TD166 (b). HNL cultures were inoculated and grown photosynthetically in modified liquid RCV medium. Samples were withdrawn at the indicated times for the measurement of in vivo nitrogenase activity and the ADP-ribosylation status of Fe-protein as described by Yakunin et al. (1999). Results are presented as the total amounts of ethylene produced per vial. Ammonium was added as an anoxic solution to a final concentration of 200 µM at the time indicated by the arrow.

 

   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Regulation of glnK expression
Like many proteobacteria, R. capsulatus contains two PII-encoding genes, glnB and glnK (Drepper et al., 2000; Masepohl et al., 2002). The R. capsulatus glnK gene region was isolated by a PCR-based strategy (Methods). The glnK gene is associated with amtB, encoding a high-affinity (methyl-) ammonium transporter (Yakunin & Hallenbeck, 2002), while the previously described glnB gene is cotranscribed with glnA, encoding glutamine synthetase (Kranz et al., 1990). The deduced R. capsulatus GlnK protein consists of 112 amino acid residues and is 59 % identical to GlnB of R. capsulatus. An even higher degree of identity (88 %) exists for the GlnK proteins of R. capsulatus and Rhodobacter sphaeroides (Qian & Tabita, 1998). The presence of two DNA sequences 137 and 154 bp upstream of the R. capsulatus glnK translational start codon exhibiting strong similarity to NtrC-binding sites (GC-N7-T-N3-GC; Foster-Hartnett & Kranz, 1994) suggested that expression of glnK–amtB is regulated by NtrC in dependence on ammonium availability. To verify this assumption, regulation of glnK expression was examined in R. capsulatus wild-type (B10S) and an ntrC mutant strain (TD50) containing the reporter plasmid pSG9II carrying a glnKlacZ fusion. Expression of glnK was eightfold higher in wild-type cells grown under nitrogen limitation (2125 Miller units) compared to ammonium-grown cells (256 Miller units). This transcriptional activation was found to depend on NtrC, since disruption of the ntrC gene led to a drastic decrease of expression of the glnKlacZ fusion in the absence of ammonium (246 Miller units). Similarly, NtrC-mediated activation of glnK has been described for other bacteria, including E. coli, K. pneumoniae and Azospirillum brasilense (Atkinson & Ninfa, 1998; de Zamaroczy, 1998; He et al., 1998; Jack et al., 1999; van Heeswijk et al., 1996). In addition to NtrC-mediated glnK activation under N-limiting conditions, low but significant glnK expression occurred in an ntrC mutant background in both the presence and absence of ammonium, whereas no {beta}-galactosidase activity was detectable in the strains containing the vector plasmid pML5 carrying the promoter-less lacZ gene (data not shown). These findings suggest that transcription of glnK might start at a second NtrC-independent promoter leading to a constitutive low level expression of the glnKamtB operon. This would be similar to the situation found for the glnK-like glnZ gene in Azospirillum brasilense (de Zamaroczy, 1998), where transcription of glnZ starts from two different promoters (P1 and P2). Expression analysis of the glnZ gene revealed that only transcription from the major P1 promoter (but not from the weaker P2 promoter) is regulated by NtrC in dependence on the cellular nitrogen status.

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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Fig. 1. Immunodetection of NifA1 and NifH in glnB and glnK mutant strains. R. capsulatus wild-type and mutant strains were grown in RCV minimal medium under nitrogenase-derepressing (10 mM serine as N-source; odd-numbered lanes) or repressing (20 mM ammonium as N-source; even-numbered lanes) conditions, before protein extracts were analysed by Western blots using antibodies against NifA1 (a) or NifH (b). The arrowhead marks the presence of a faint NifA1 band in mutant strain PHU332 (glnB) under ammonium-sufficient conditions (lane 4). Lanes: 1 and 2, B10S (wild-type); 3 and 4, PHU332 (glnB); 5 and 6, SG26 (glnK); 7 and 8, TD166 (glnBglnK).

 
Post-translational ammonium control of NifA activity
Previous studies demonstrated that constitutive (NtrC-independent) expression of nifA1 leads to a high-level accumulation of the NifA1 protein in both the absence and the presence of ammonium, but NifA1-mediated nifH transcription was still inhibited by ammonium (Paschen et al., 2001). To further analyse the roles of GlnB and GlnK in this post-translational ammonium control of NifA activity, we examined the same protein extracts which were prepared for the NifA1 detection (Fig. 1a) by Western blot analysis using a NifH-specific antiserum (Fig. 1b). Most remarkably, extremely high amounts of NifH accumulated in the glnBglnK double mutant TD166 in both the absence and presence of ammonium (Fig. 1b, lanes 7 and 8), suggesting that ammonium control of synthesis and activity of NifA is completely abolished in the absence of both PII-like proteins. In contrast to the situation described for TD166, in the glnB mutant strain PHU332 (where only GlnK is present), no accumulation of NifH was found under ammonium-sufficient conditions (Fig. 1b, lane 4), although significant amounts of NifA1 were present in this mutant strain under +N conditions (Fig. 1a, lane 4). These data clearly demonstrate that GlnK is sufficient for ammonium inhibition of NifA activity. Since mutant strain SG26 (glnK- glnB+) did not accumulate NifA1 and consequently did not synthesize NifH under +N conditions (Fig. 1a, b, lane 6), it remains speculative whether GlnB (like GlnK) might also be sufficient for ammonium control of NifA activity.

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 glnB–glnK 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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Table 2. Activity of molybdenum nitrogenase in the glnB–glnK mutant strain TD166

 
As described above, the glnBglnK double mutant strain TD166 accumulated extremely large amounts of NifH protein and exhibited high levels of nitrogenase activity in the presence of ammonium. However, a protein band corresponding to the ADP-ribosylated form of NifH was clearly visible in extracts of all tested strains (even under -N conditions; Fig. 1b). Since harvesting of cells for protein extraction involved a centrifugation step (leading to darkness-induced ADP-ribosylation of NifH in the wild-type), we asked whether the ADP-ribosylated form of NifH was already present in the ammonium-grown cultures or appeared upon exposure of the cells to darkness. In order to discriminate between these two possibilities, we used highly nitrogen-limited (HNL) cultures, obtained by growing cultures to early stationary phase in RCV medium with N2 as sole nitrogen source. These growth conditions have previously been shown to be optimal for analysis of the ammonium-dependent switch-off response (Yakunin & Hallenbeck, 1998). To avoid darkness-induced ADP-ribosylation of NifH during cell harvesting, protein extracts were prepared using the rapid boiling method as described previously (Yakunin & Hallenbeck, 2002). Under these experimental conditions, switch-off of nitrogenase activity with a concomitant ADP-ribosylation of Fe-protein was readily observed in wild-type cells upon the addition of 200 µM ammonium to the culture (Fig. 2a). By contrast, the glnBglnK double mutant strain TD166 was incapable of carrying out either nitrogenase modification or switch-off of in vivo nitrogenase activity (Fig. 2b). Essentially the same results were obtained with these strains when a higher concentration (1 mM) of ammonium was used (data not shown). These results clearly demonstrate that in the glnBglnK double mutant, both ammonium-induced short-term responses (ADP-ribosylation of NifH as well as the DraT/DraG-independent switch-off) are completely abolished. Therefore, ADP-ribosylation of NifH in the glnBglnK mutant, as shown in Fig. 1(b), is probably caused by darkness-induced switch-off, indicating that this response is independent of the GlnB/GlnK-signalling system.

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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Table 3. Expression of anfAlacZ (pKS131A) in different genetic backgrounds

 
In addition, protein extracts isolated from the R. capsulatus strains KS36 ({Delta}nifHDK), TD202 (glnB {Delta}nifHDK), TD178 (glnK {Delta}nifHDK) and TD205 (glnBglnK {Delta}nifHDK) were examined by Western analysis using a NifH antiserum, known to recognize AnfH (Fig. 3a). All cultures grown in the absence of ammonium contained comparable amounts of the unmodified and modified forms of dinitrogenase reductase, designated AnfH and AnfHADP-R, respectively (in all cases an additional protein band with a lower molecular mass was detectable in these strains, which might correspond to a degradation product of AnfH). These data indicate that different mutations in glnB and/or glnK do not significantly affect expression of the alternative nitrogenase under nitrogen-limiting conditions. In contrast to the molybdenum nitrogenase system, the alternative nitrogenase was not expressed in the presence of ammonium in any of the tested mutant backgrounds including the glnBglnK double mutant (Fig. 3a, lane 8). These data were further corroborated by examination of appropriate R. capsulatus strains carrying plasmid pTD6-5I (anfAc). Constitutive expression of anfA driven by the aphII promoter was verified by analysis of strains harbouring an anfAclacZ fusion (data not shown). Subsequently, activation of the anfH promoter mediated by anfAc was examined by immunodetection of AnfH (Fig. 3b). In all tested strains, including the glnBglnK double mutant (TD186), significant amounts of AnfH protein were detectable only under ammonium-deficient conditions. These experiments strongly suggest that ammonium inhibition of AnfA activity involves an as-yet-unidentified regulatory mechanism. At present it remains speculative whether this new mechanism acts completely independently of GlnB and/or GlnK. However, ammonium control of NifA and AnfA activity is clearly different.



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Fig. 3. Immunodetection of AnfH in glnB and glnK mutant strains. The parental strain KS36 ({Delta}nifHDK) and selected mutant strains were grown in molybdenum-free AK-NL minimal medium under nitrogenase-derepressing (5 mM serine as N-source; odd-numbered lanes) or repressing (20 mM ammonium as N-source; even-numbered lanes) conditions, before protein extracts were analysed by Western blots using cross-reacting antiserum against NifH. Accumulation of AnfH was tested for strains carrying wild-type anfA (a) or a constitutively expressed anfA gene (anfAc, pTD6-5I; b), respectively. Lanes: 1 and 2, KS36 ({Delta}nifHDK); 3 and 4, TD176 ({Delta}nifHDK/glnB); 5 and 6, TD178 ({Delta}nifHDK/glnK); 7 and 8, TD186 ({Delta}nifHDK/glnB/glnK).

 
As might be expected, the glnBglnK double mutant strain TD205 was also impaired in the DraT-mediated ammonium-dependent post-translational modification of AnfH (data not shown), reflecting that the PII-like proteins generally transmit the ammonium signal to the DraT/DraG system, which in turn controls the activity of both the molybdenum nitrogenase and the alternative nitrogenase.

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). Protein–protein interactions were demonstrated for GlnB–NtrB, GlnB–NifA1, GlnB–NifA2, GlnB–DraT, GlnK–DraT, GlnK–NifA1, GlnK–NifA2 and GlnK–DraT. In accordance with this model, we could not detect interaction of GlnK with NtrB.



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Fig. 4. Model of signal transduction by PII-like proteins in R. capsulatus. GlnB and GlnK are involved in different levels of regulation of synthesis and activity of both nitrogenase systems by ammonium. For details, see text.

 


   ACKNOWLEDGEMENTS
 
This work was supported by financial grants from the Deutsche Forschungsgemeinschaft, Studienstiftung des Deutschen Volkes, the Fonds der Chemischen Industrie, and the Natural Sciences and Engineering Research Council of Canada (OGP0036584).


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Arcondéguy, T., van Heeswijk, W. C. & Merrick, M. (1999). Studies on the role of GlnK and GlnB in regulating Klebsiella pneumoniae NifL-dependent nitrogen control. FEMS Microbiol Lett 180, 263–270.[CrossRef][Medline]

Arcondéguy, T., Jack, R. & Merrick, M. (2001). PII signal transduction proteins, pivotal players in microbial nitrogen control. Microbiol Mol Biol Rev 65, 80–105.[Abstract/Free Full Text]

Atkinson, M. R. & Ninfa, A. J. (1998). Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli. Mol Microbiol 29, 431–447.[CrossRef][Medline]

Atkinson, M. R. & Ninfa, A. J. (1999). Characterization of the GlnK protein of Escherichia coli. Mol Microbiol 32, 301–313.[CrossRef][Medline]

Benelli, E. M., Souza, E. M., Funayama, S., Rigo, L. U. & Pedrosa, F. O. (1997). Evidence for two possible glnB-type genes in Herbaspirillum seropedicae. J Bacteriol 179, 4623–4626.[Abstract]

Colbeau, A. & Vignais, P. M. (1992). Use of hupS : : lacZ gene fusion to study regulation of hydrogenase expression in Rhodobacter capsulatus: stimulation by H2. J Bacteriol 174, 4258–4264.[Abstract]

Cullen, P. J., Bowman, W. C. & Kranz, R. G. (1996). In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system. J Biol Chem 271, 6530–6536.[Abstract/Free Full Text]

de Zamaroczy, M. (1998). Structural homologues PII and PZ of Azospirillum brasilense provide intracellular signalling for selective regulation of various nitrogen-dependent functions. Mol Microbiol 29, 449–463.[CrossRef][Medline]

de Zamaroczy, M., Paquelin, A. & Elmerich, C. (1993). Functional organization of the glnB-glnA cluster of Azospirillum brasilense. J Bacteriol 175, 2507–2515.[Abstract]

Dischert, W., Vignais, P. M. & Colbeau, A. (1999). The synthesis of Rhodobacter capsulatus HupSL hydrogenase is regulated by the two-component HupT/HupR system. Mol Microbiol 34, 995–1006.[CrossRef][Medline]

Drepper, T., Groß, S., Masepohl, B. & Klipp, W. (2000). Ammonium and molybdenum regulation of the molybdenum nitrogenase and the alternative nitrogenase in Rhodobacter capsulatus. In Nitrogen Fixation: From Molecules to Crop Productivity, p. 129. Edited by F. O. Pedrosa, M. Hungria, G. Yates & W. E. Newton. Dordrecht, Boston, London: Kluwer.

Foster-Hartnett, D. & Kranz, R. G. (1994). The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters. J Bacteriol 176, 5171–5176.[Abstract]

Groß, S., Drepper, T., Masepohl, B., Rehman, T., Yakunin, A. F., Hallenbeck, P. C. & Klipp, W. (2002). Regulatory circuits controlling both nitrogenases in Rhodobacter capsulatus. In Nitrogen Fixation: Global Perspectives, p. 419. Edited by T. M. Finan, M. R. O'Brian, D. B. Layzell, J. K. Vessey & W. Newton. New York & Wallingford: CABI.

Hallenbeck, P. C., Yakunin, A. F., Drepper, T., Gross, S., Masepohl, B. & Klipp, W. (2002). Regulation of nitrogenase in the photosynthetic bacterium, Rhodobacter capsulatus. In Nitrogen Fixation: Global Perspectives, pp. 223–227. Edited by T. M. Finan, M. R. O'Brian, D. B. Layzell, J. K. Vessey & W. Newton. New York & Wallingford: CABI.

Hanahan, D. (1983). Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166, 557–580.[Medline]

He, L., Soupene, E., Ninfa, A. & Kustu, S. (1998). Physiological role for the GlnK protein of enteric bacteria: relief of NifL inhibition under nitrogen-limiting conditions. J Bacteriol 180, 6661–6667.[Abstract/Free Full Text]

Holtel, A. & Merrick, M. J. (1989). The Klebsiella pneumoniae PII protein (glnB gene product) is not absolutely required for nitrogen regulation and is not involved in NifL-mediated nif gene regulation. Mol Gen Genet 217, 474–480.[Medline]

Hübner, P., Willison, J. C., Vignais, P. M. & Bickle, T. A. (1991). Expression of regulatory nif genes in Rhodobacter capsulatus. J Bacteriol 173, 2993–2999.[Medline]

Hübner, P., Masepohl, B., Klipp, W. & Bickle, T. A. (1993). nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations. Mol Microbiol 10, 123–132.[Medline]

Jack, R., de Zamaroczy, M. & Merrick, M. (1999). The signal transduction protein GlnK is required for NifL-dependent nitrogen control of nif gene expression in Klebsiella pneumoniae. J Bacteriol 181, 1156–1162.[Abstract/Free Full Text]

Klassen, G., de Souza, E. M., Yates, M. G., Rigo, L. U., Inaba, J. & Pedrosa, F. O. (2001). Control of nitrogenase reactivation by the GlnZ protein in Azospirillum brasilense. J Bacteriol 183, 6710–6713.[Abstract/Free Full Text]

Klein, G., Klipp, W., Jahn, A., Steinborn, B. & Oelze, J. (1991). The relationship of biomass, polysaccharide and H2 formation in the wild-type and nifA/nifB mutants of Rhodobacter capsulatus. Arch Microbiol 155, 477–482.

Klipp, W., Masepohl, B. & Pühler, A. (1988). Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: duplication of a nifA-nifB region. J Bacteriol 170, 693–699.[Medline]

Kranz, R. G. & Foster-Hartnett, D. (1990). Transcriptional regulatory cascade of nitrogen-fixation genes in anoxygenic photosynthetic bacteria: oxygen- and nitrogen-responsive factors. Mol Microbiol 4, 1793–1800.[Medline]

Kranz, R. G., Pace, V. M. & Caldicott, I. M. (1990). Inactivation, sequence, and lacZ fusion analysis of a regulatory locus required for repression of nitrogen fixation genes in Rhodobacter capsulatus. J Bacteriol 172, 53–62.[Medline]

Kutsche, M., Leimkühler, S., Angermüller, S. & Klipp, W. (1996). Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of {sigma}54, and repressed by molybdenum. J Bacteriol 178, 2010–2017.[Abstract]

Labes, M., Pühler, A. & Simon, R. (1990). A new family of RSF1010-derived expression and lacZ-fusion broad-host-range vectors for Gram-negative bacteria. Gene 89, 37–46.[CrossRef][Medline]

Liang, Y. Y., de Zamaroczy, M., Arsene, F., Paquelin, A. & Elmerich, C. (1992). Regulation of nitrogen fixation in Azospirillum brasilense Sp7: involvement of nifA, glnA and glnB gene products. FEMS Microbiol Lett 79, 113–120.[Medline]

Little, R., Reyes-Ramirez, F., Zhang, Y., van Heeswijk, W. C. & Dixon, R. (2000). Signal transduction to the Azotobacter vinelandii NifL-NifA regulatory system is influenced directly by interaction with 2-oxoglutarate and the PII regulatory protein. EMBO J 19, 6041–6050.[Abstract/Free Full Text]

Little, R., Colombo, V., Leech, A. & Dixon, R. (2002). Direct interaction of the NifL regulatory protein with the GlnK signal transducer enables the Azotobacter vinelandii NifL-NifA regulatory system to respond to conditions replete for nitrogen. J Biol Chem 277, 15472–15481.[Abstract/Free Full Text]

Martin, D. E. & Reinhold-Hurek, B. (2002). Distinct roles of PII-like signal transmitter proteins and amtB in regulation of nif gene expression, nitrogenase activity, and posttranslational modification of NifH in Azoarcus sp. strain BH72. J Bacteriol 184, 2251–2259.[Abstract/Free Full Text]

Martin, D. E., Hurek, T. & Reinhold-Hurek, B. (2000). Occurrence of three PII-like signal transmitter proteins in the diazotrophic proteobacterium Azoarcus sp. BH72. Mol Microbiol 38, 276–288.[CrossRef][Medline]

Masepohl, B. & Klipp, W. (1996). Organization and regulation of genes encoding the molybdenum nitrogenase and the alternative nitrogenase in Rhodobacter capsulatus. Arch Microbiol 165, 80–90.[CrossRef]

Masepohl, B., Klipp, W. & Pühler, A. (1988). Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus. Mol Gen Genet 212, 27–37.[Medline]

Masepohl, B., Krey, R. & Klipp, W. (1993). The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of the molybdenum and the alternative nitrogenase. J Gen Microbiol 139, 2667–2675.[Medline]

Masepohl, B., Drepper, T., Paschen, A., Gross, S., Pawlowski, A., Raabe, K., Riedel, K. U. & Klipp, W. (2002). Regulation of nitrogen fixation in the phototrophic purple bacterium Rhodobacter capsulatus. J Mol Microbiol Biotechnol 4, 243–248.[Medline]

Merrick, M. J. & Edwards, R. A. (1995). Nitrogen control in bacteria. Microbiol Rev 59, 604–622.[Medline]

Michel-Reydellet, N. & Kaminski, P. A. (1999). Azorhizobium caulinodans PII and GlnK proteins control nitrogen fixation and ammonia assimilation. J Bacteriol 181, 2655–2658.[Abstract/Free Full Text]

Miller, J. H. (1972). Assay of {beta}-galactosidase. In Experiments in Molecular Genetics, pp. 352–355. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

Ninfa, A. J. & Atkinson, M. R. (2000). PII signal transduction proteins. Trends Microbiol 8, 172–179.[CrossRef][Medline]

Paschen, A., Drepper, T., Masepohl, B. & Klipp, W. (2001). Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium. FEMS Microbiol Lett 200, 207–213.[CrossRef][Medline]

Pawlowski, A., Riedel, K.-U., Klipp, W., Dreiskemper, P., Groß, S., Bierhoff, H., Drepper, T. & Masepohl, B. (2003). Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus. J Bacteriol (in press).

Pierrard, J., Ludden, P. W. & Roberts, G. P. (1993). Posttranslational regulation of nitrogenase in Rhodobacter capsulatus: existence of two independent regulatory effects of ammonium. J Bacteriol 175, 1358–1366.[Abstract]

Qian, Y. & Tabita, F. R. (1998). Expression of glnB and a glnB-like gene (glnK) in a ribulose bisphosphate carboxylase/oxygenase-deficient mutant of Rhodobacter sphaeroides. J Bacteriol 180, 4644–4649.[Abstract/Free Full Text]

Reyes, F., Roldan, M. D., Klipp, W., Castillo, F. & Moreno-Vivian, C. (1996). Isolation of periplasmatic nitrate reductase genes from Rhodobacter sphaeroides DSM 158: structural and functional differences among prokaryotic nitrate reductases. Mol Microbiol 19, 1307–1318.[Medline]

Reyes-Ramirez, F., Little, R. & Dixon, R. (2001). Role of Escherichia coli nitrogen regulatory genes in the nitrogen response of the Azotobacter vinelandii NifL-NifA complex. J Bacteriol 183, 3076–3082.[Abstract/Free Full Text]

Rudnick, P., Kunz, C., Gunatilaka, M. K., Hines, E. R. & Kennedy, C. (2002). Role of GlnK in NifL-mediated regulation of NifA activity in Azotobacter vinelandii. J Bacteriol 184, 812–820.[Abstract/Free Full Text]

Schneider, K., Müller, A., Johannes, K.-U., Diemann, E. & Kottmann, J. (1991). Selective removal of molybdenum traces from growth media of N2-fixing bacteria. Anal Biochem 193, 292–298.[CrossRef][Medline]

Simon, R., Priefer, U. & Pühler, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Bio/Technology 1, 784–791.

Souza, E. M., Pedrosa, F. O., Drummond, M., Rigo, L. U. & Yates, M. G. (1999). Control of Herbaspirillum seropedicae NifA activity by ammonium ions and oxygen. J Bacteriol 181, 681–684.[Abstract/Free Full Text]

Thomas, G., Coutts, G. & Merrick, M. (2000). The glnKamtB operon. Trends Genet 16, 11–14.[CrossRef][Medline]

Toussaint, B., de Sury d'Aspremont, R., Delic-Attree, I., Berchet, V., Elsen, S., Colbeau, A., Dischert, W., Lazzaroni, Y. & Vignais, P. M. (1997). The Rhodobacter capsulatus hupSLC promoter: identification of cis-regulatory elements and of trans-activating factors involved in H2 activation of hupSLC transcription. Mol Microbiol 26, 927–937.[CrossRef][Medline]

van Heeswijk, W. C., Hoving, S., Molenaar, D., Stegeman, B., Kahn, D. & Westerhoff, H. V. (1996). An alternative PII protein in the regulation of glutamine synthetase in Escherichia coli. Mol Microbiol 21, 133–146.[Medline]

Vieira, J. & Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259–268.[CrossRef][Medline]

Wang, G., Angermüller, S. & Klipp, W. (1993). Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum-pterin-binding proteins. J Bacteriol 175, 3031–3042.[Abstract]

Yakunin, A. F. & Hallenbeck, P. C. (1998). Short-term regulation of nitrogenase activity by NH+4 in Rhodobacter capsulatus: multiple in vivo nitrogenase response to NH+4 addition. J Bacteriol 180, 6392–6395.[Abstract/Free Full Text]

Yakunin, A. F. & Hallenbeck, P. C. (2002). AmtB is necessary for NH+4-induced nitrogenase switch-off and ADP-ribosylation in Rhodobacter capsulatus. J Bacteriol 184, 4081–4088.[Abstract/Free Full Text]

Yakunin, A. F., Laurinavichene, T. V., Tsygankov, A. A. & Hallenbeck, P. C. (1999). The presence of ADP-ribosylated Fe-protein of nitrogenase in Rhodobacter capsulatus is correlated with cellular nitrogen status. J Bacteriol 181, 1994–2000.[Abstract/Free Full Text]

Zhang, Y., Pohlman, E. L., Ludden, P. W. & Roberts, G. P. (2000). Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum. J Bacteriol 182, 983–992.[Abstract/Free Full Text]

Zhang, Y., Pohlmann, E. L., Halbleib, C. M., Ludden, P. W. & Roberts, G. P. (2001a). Effect of PII and its homolog GlnK on reversible ADP-ribosylation of dinitrogenase reductase by heterologous expression of the Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyl transferase-dinitrogenase reductase-activating glycohydrolase regulatory system in Klebsiella pneumoniae. J Bacteriol 183, 1610–1620.[Abstract/Free Full Text]

Zhang, Y., Pohlmann, E. L., Ludden, P. W. & Roberts, G. P. (2001b). Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status. J Bacteriol 183, 6159–6168.[Abstract/Free Full Text]

Received 14 January 2003; revised 9 April 2003; accepted 9 May 2003.