Abstract

The Rhodobacter capsulatus nrfA gene product exhibits extensive similarity to the nif (nitrogen fixation) regulatory factor NrfA of Azorhizobium caulinodans and the nucleoid-associated protein Hfq of Escherichia coli. Mutational analysis revealed that, in contrast to the situation in A. caulinodans, NrfA is not essential for diazotrophic growth of R. capsulatus, but it is required for maximal growth rates with N2 as sole nitrogen source via either molybdenum nitrogenase or the alternative nitrogenase. NrfA was shown to control N2 fixation in R. capsulatus at the level of expression of the regulatory genes nifA1, nifA2 and anfA, encoding the transcriptional activators of all the other nitrogen fixation genes.

Introduction

The phototrophic purple bacterium Rhodobacter capsulatus is able to fix atmospheric dinitrogen either via molybdenum nitrogenase (nif-encoded) or via an alternative heterometal-free nitrogenase (anf-encoded). Synthesis and activity of both nitrogenases are tightly controlled at different regulatory levels by ammonium and other environmental factors (for a review, see[14]).

R. capsulatus harbors a general nitrogen regulation (Ntr) system similar to the enteric Ntr system that senses and responds to the cellular nitrogen status[14]. Under conditions of nitrogen starvation, the sensor kinase NtrB promotes the phosphorylation of the response regulator NtrC, and in turn, NtrC-P can function as a transcriptional activator of its target genes. Among these are nifA1, nifA2 and anfA, which encode the transcriptional activators of all the other nif and anf genes. In addition to ammonium control, transcription of anfA is inhibited by molybdenum via the ModE-like repressor proteins MopA and MopB 13,14].

In contrast to other diazotrophic bacteria, R. capsulatus contains two almost identical copies of nifA, which can functionally substitute for each other [5,15,21]. NifA-dependent transcriptional nif gene activation is controlled by a histone-like protein, called HvrA [10,14,24]. Here, HvrA acts as a negative modulator of transcription of selected nif genes, including nifH and nifB, whereas transcription of nifA1 and nifA2 is not affected by mutations in hvrA. In addition, HvrA is long known to be involved in low-light activation of the photosynthetic apparatus[2].

In the present study we analyzed the role of an until now unnoticed small protein, NrfA, in control of the nitrogen fixation process in R. capsulatus. NrfA exhibits strong similarity to the nucleoid-associated protein Hfq (HF-I) of Escherichia coli1,7]. Originally, Hfq has been described as a site-specific RNA-binding protein involved in replication of the RNA bacteriophage Qβ[27], but it also binds sequence-non-specific to DNA[1]. Disruption of hfq in E. coli causes pleiotropic phenotypes, indicating that Hfq is involved in regulation of a great variety of cellular processes including control of rpoS expression [18,19,28,29]. In contrast to the situation in E. coli, Azorhizobium caulinodans mutants defective in the hfq-like nrfA gene show growth properties similar to the wild-type under nitrogen-replete conditions, but exhibit a Nif phenotype, since NrfA is absolutely required for nifA expression 8,9]. However, A. caulinodans nrfA restores the defect in rpoS translation to an E. coli hfq mutant, indicating that NrfA and Hfq have similar activities in both bacteria[9].

In contrast to the situation in A. caulinodans, the R. capsulatus nrfA gene product is not essential for growth on N2, but it is required for maximal diazotrophic growth not only via the classical molybdenum nitrogenase but also via the alternative nitrogenase. NrfA-mediated control of both nitrogenase systems was shown to occur at the level of expression of the respective regulatory genes, nifA1, nifA2 and anfA.

Materials and methods

Bacterial strains, plasmids, and growth conditions

The bacterial strains and plasmids used in this study are listed in Table 1. Methods for conjugational plasmid transfer between E. coli S17–1 and R. capsulatus, and the selection of mutants, growth media, growth conditions, and antibiotic conditions were as previously described [11,15,25].

1

Bacterial strains and plasmids

Strain or plasmid Genotype and/or relevant characteristicsa Reference or source 
E. coli 
DH5α host for pUC plasmids [4
S17–1 RP4–2 (Tc::Mu) (Km::Tn7) integrated into the chromosome [26
R. capsulatus 
B10S spontaneous Smr mutant of R. capsulatus B10 [11
DG7 nrfA::[SpcrThis work 
TD22 ΔnifHDK::[GmrThis work 
TD22-DG7 ΔnifHDK::[Gmr]; nrfA::[SpcrThis work 
KS36-KS94 ΔnifHDK::[Spcr]; anfA::[KmrThis work 
Plasmids 
pK18 Kmr; lacZα [23
pUC18 Apr; lacZα [31
pBBR1MCS-2 broad host range, mob, Kmr [12
pHP45Ω Apr, Spcr [22
pWKR459 pK18 derivative carrying the 8.3-kb Xho I fragment (Tcr, mob) from pJQ18 This work 
pDG1 pK18 derivative carrying a 1040-bp PCR fragment (nrfAThis work 
pDG2 pUC18 derivative carrying the 0.95-kb Sal I fragment (nrfA) from pDG1 This work 
pDG3 pDG2 derivative carrying the 2-kb Bam HI fragment [Spcr] from pHP45Ω This work 
pDG7 pDG3 derivative carrying the 8.3-kb Eco RI fragment (Tcr, mob) from pWKR459 This work 
pFS2 pSUP401 derivative carrying a nifH-lacZ fusion F. Simon, Bochum, Germany 
pKS131A pPHU236 derivative carrying an anfA-lacZ fusion [13
pNAP1 pBBR1MCS-2 derivative carrying the 0.95-kb Bam HI–Sal I fragment (nrfA) from pDG1 This work 
pPHU282 pPHU234 derivative carrying a nifA2-lacZ fusion [6
pPHU284 pPHU234 derivative carrying a nifA1-lacZ fusion [6
pTD7-2I pSUP401 derivative carrying an anfH-lacZ fusion This work 
Strain or plasmid Genotype and/or relevant characteristicsa Reference or source 
E. coli 
DH5α host for pUC plasmids [4
S17–1 RP4–2 (Tc::Mu) (Km::Tn7) integrated into the chromosome [26
R. capsulatus 
B10S spontaneous Smr mutant of R. capsulatus B10 [11
DG7 nrfA::[SpcrThis work 
TD22 ΔnifHDK::[GmrThis work 
TD22-DG7 ΔnifHDK::[Gmr]; nrfA::[SpcrThis work 
KS36-KS94 ΔnifHDK::[Spcr]; anfA::[KmrThis work 
Plasmids 
pK18 Kmr; lacZα [23
pUC18 Apr; lacZα [31
pBBR1MCS-2 broad host range, mob, Kmr [12
pHP45Ω Apr, Spcr [22
pWKR459 pK18 derivative carrying the 8.3-kb Xho I fragment (Tcr, mob) from pJQ18 This work 
pDG1 pK18 derivative carrying a 1040-bp PCR fragment (nrfAThis work 
pDG2 pUC18 derivative carrying the 0.95-kb Sal I fragment (nrfA) from pDG1 This work 
pDG3 pDG2 derivative carrying the 2-kb Bam HI fragment [Spcr] from pHP45Ω This work 
pDG7 pDG3 derivative carrying the 8.3-kb Eco RI fragment (Tcr, mob) from pWKR459 This work 
pFS2 pSUP401 derivative carrying a nifH-lacZ fusion F. Simon, Bochum, Germany 
pKS131A pPHU236 derivative carrying an anfA-lacZ fusion [13
pNAP1 pBBR1MCS-2 derivative carrying the 0.95-kb Bam HI–Sal I fragment (nrfA) from pDG1 This work 
pPHU282 pPHU234 derivative carrying a nifA2-lacZ fusion [6
pPHU284 pPHU234 derivative carrying a nifA1-lacZ fusion [6
pTD7-2I pSUP401 derivative carrying an anfH-lacZ fusion This work 

aAbbreviations: Apr, ampicillin resistance; Gmr, gentamicin resistance; Kmr, kanamycin resistance; Smr, streptomycin resistance; Spcr, spectinomycin resistance; Tcr, tetracycline resistance.

To determine diazotrophic growth of R. capsulatus strains, 3-ml cultures were inoculated in either RCV or molybdenum-free AK-NL minimal medium in 15-ml Hungate tubes. Subsequently, the gas atmosphere was exchanged to pure N2. Cultures were incubated under phototrophic conditions, and growth was analyzed by following the optical density at 660 nm.

Construction of R. capsulatus nrfA mutant strain DG7

A 1040-bp DNA fragment carrying the R. capsulatus nrfA gene region was PCR-amplified using synthetic primers (5′-TCGGCGAAGATTTCCAGGATCAAC-3′ and 5′-GGATCGTGCTCGGCGGTCAG-3′). PCR amplification was carried out in a RoboCycler Gradient 40 (Stratagene) using Pfu polymerase. The PCR fragment was blunt-end-cloned into the Sma I site of pK18 resulting in hybrid plasmid pDG1. Plasmid pDG1 contains two Sal I sites flanking the nrfA gene (Fig. 2); one Sal I site comes from the PCR-amplified DNA fragment, whereas the other Sal I site descends from the multiple cloning site of vector pK18. The corresponding 0.95-kb Sal I fragment (nrfA) was subcloned from pDG1 into pUC18 resulting in hybrid plasmid pDG2. To disrupt the nrfA gene, a 2-kb Bam HI spectinomycin cartridge from pHP45Ω was inserted into the Bcl I site of pDG2 resulting in hybrid plasmid pDG3. Finally, an 8.3-kb Eco RI fragment (containing a tetracycline resistance gene and the mop locus of RP4) from pWKR459 was inserted into pDG3 leading to the mobilizable hybrid plasmid pDG7 (Fig. 2). Conjugational transfer of pDG7 from E. coli S17–1 into R. capsulatus and selection for marker rescue were carried out as described earlier 11,15].

2

Mutational analysis of R. capsulatus nrfA. Plasmid pDG1 contains a 1040-bp PCR fragment (nrfA) cloned into pK18. Filled squares symbolize the synthetic primers used for PCR amplification (Section 2). The Sal I and Bam HI sites upstream of nrfA descend from the multiple cloning site of pK18. The mobilizable hybrid plasmid pDG7 was used to create R. capsulatus nrfA mutant strain DG7. The mobilizable hybrid plasmid pNAP1 (used for complementation of DG7) is based on the broad host range vector pBBR1MCS-2, which is able to replicate in R. capsulatus.

2

Mutational analysis of R. capsulatus nrfA. Plasmid pDG1 contains a 1040-bp PCR fragment (nrfA) cloned into pK18. Filled squares symbolize the synthetic primers used for PCR amplification (Section 2). The Sal I and Bam HI sites upstream of nrfA descend from the multiple cloning site of pK18. The mobilizable hybrid plasmid pDG7 was used to create R. capsulatus nrfA mutant strain DG7. The mobilizable hybrid plasmid pNAP1 (used for complementation of DG7) is based on the broad host range vector pBBR1MCS-2, which is able to replicate in R. capsulatus.

As a basis for complementation of the phenotype of R. capsulatus nrfA mutant strain DG7, hybrid plasmid pNAP1 was constructed. For this purpose, the 0.95-kb Bam HI–Sal I fragment from pDG1 (Fig. 2; the Bam HI site comes from the multiple cloning site of the vector part of pDG1) was cloned into the mobilizable broad host range vector plasmid pBBR1MCS-2 resulting in pNAP1.

β-Galactosidase and in vivo nitrogenase assays

To determine the β-galactosidase activities of R. capsulatus strains carrying nif-lacZ or anf-lacZ reporter gene fusions, cultures were grown either in RCV minimal medium or in molybdenum-free AK-NL minimal medium with tetracycline 13,17]. For growth under nitrogen-limiting conditions, serine was added to final concentrations of 9.5 mM. Nitrogen-sufficient conditions were achieved by addition of 20 mM NH4Cl to the medium. Following growth to the mid-exponential phase, β-galactosidase activities of R. capsulatus strains were determined by the sodium dodecyl sulfate–chloroform method 6,16].

Nitrogenase activities of whole cells were determined by the acetylene reduction assay as described by Wang et al.[30]. In addition to ethylene production, formation of ethane was routinely monitored as an indication of the activity of the alternative nitrogenase.

Results and discussion

Diazotrophic growth and nitrogenase activity of R. capsulatus nrfA mutant strains

The R. capsulatus nrfA gene is predicted to encode a small basic protein consisting of 77 amino acid residues, which is highly homologous to A. caulinodans NrfA (75% identity, 91% similarity). The two NrfA proteins exhibit much higher similarity to each other than to their E. coli counterpart, Hfq, which in addition contains a C-terminal extension (Fig. 1). Since the nrfA gene is essential for diazotrophic growth in A. caulinodans[8], we carried out mutational analysis to examine the role of the nrfA product in control of the N2 fixation process in R. capsulatus.

1

Comparison of R. capsulatus NrfA, A. caulinodans NrfA and E. coli Hfq. Protein sequences were aligned for maximal matching. The C-terminal extension (X20) of E. coli Hfq has no counterpart in either of the two NrfA proteins.

1

Comparison of R. capsulatus NrfA, A. caulinodans NrfA and E. coli Hfq. Protein sequences were aligned for maximal matching. The C-terminal extension (X20) of E. coli Hfq has no counterpart in either of the two NrfA proteins.

The R. capsulatus nrfA gene is part of the ntr gene region (Fig. 2). Analysis of the DNA sequence and complementation studies (see below) imply that the nrfA gene is transcribed from a promoter located within the 196-bp ntrX-nrfA intergenic region. Similar to the situation in A. caulinodans, the R. capsulatus nrfA gene might be co-transcribed with an hflX-like gene.

Based on a PCR fragment, containing the entire nrfA coding sequence and the putative promoter, hybrid plasmid pDG7 was constructed (Section 2; Fig. 2). In pDG7, the nrfA gene is disrupted by a spectinomycin cartridge (omega cassette). Plasmid pDG7 was introduced into R. capsulatus wild-type strain B10S and the nifHDK deletion strain TD22, respectively, and marker rescue resulting in polar nrfA mutant strains was selected for as described earlier[15]. The resulting strains were called DG7 (nrfA) and TD22-DG7 (ΔnifHDK, nrfA), respectively. Analysis of strain TD22-DG7 allows to examine the influence of an nrfA mutation onto the alternative nitrogenase system without interference of molybdenum nitrogenase (see below).

Since mutations in E. coli hfq cause pleiotropic effects [18,19,28,29], we first analyzed growth of R. capsulatus mutant strains DG7 (nrfA) and TD22-DG7 (ΔnifHDK, nrfA) under N-sufficient conditions. For this purpose, the nrfA mutant strains were grown in minimal medium with ammonium as an N source. Similar to an A. caulinodans nrfA mutant[8], these strains exhibited growth properties comparable to those of the parental strains B10S (wild-type) and TD22 (ΔnifHDK), indicating that at least under N-sufficient conditions, mutations in nrfA do not significantly influence growth properties of R. capsulatus (data not shown). The global regulatory function of E. coli Hfq is largely due to its role in expression of the σS subunit of RNA polymerase[19]. However, examination of the almost completed genome sequence of R. capsulatus[20] did not reveal the presence of a σS-like protein. Therefore, the apparent absence of a pleiotropic phenotype in an R. capsulatus nrfA mutant might be explained by σS-independent regulation of stationary phase processes.

To analyze the role of NrfA in the nitrogen fixation process, R. capsulatus wild-type and mutant strains were grown under diazotrophic conditions either in RCV minimal medium (conditions in which molybdenum nitrogenase is synthesized; Fig. 3A) or in molybdenum-free AK-NL minimal medium (conditions in which the alternative nitrogenase is synthesized; Fig. 3B). The parental strains B10S (wild-type) and TD22 (ΔnifHDK) showed normal diazotrophic growth via either molybdenum nitrogenase or via the alternative nitrogenase, respectively. Both nrfA mutant strains, DG7 (nrfA) and TD22-DG7 (ΔnifHDK, nrfA), were also able to grow with N2 as sole nitrogen source, albeit at significantly decreased levels compared to the corresponding parental strains. Therefore, in contrast to the situation in A. caulinodans, NrfA is not essential for diazotrophic growth in R. capsulatus, but it is required for maximal growth rates via both nitrogenase systems.

3

Diazotrophic growth of R. capsulatus wild-type and selected mutant strains. To analyze diazotrophic growth via either molybdenum nitrogenase or via the alternative nitrogenase, R. capsulatus strains were grown in RCV (A) or in molybdenum-free AK-NL minimal medium (B), respectively. B10S (wild-type), DG7 (nrfA), TD22 (ΔnifHDK), TD22-DG7 (ΔnifHDK, nrfA), KS36-KS94 (ΔnifHDK, anfA).

3

Diazotrophic growth of R. capsulatus wild-type and selected mutant strains. To analyze diazotrophic growth via either molybdenum nitrogenase or via the alternative nitrogenase, R. capsulatus strains were grown in RCV (A) or in molybdenum-free AK-NL minimal medium (B), respectively. B10S (wild-type), DG7 (nrfA), TD22 (ΔnifHDK), TD22-DG7 (ΔnifHDK, nrfA), KS36-KS94 (ΔnifHDK, anfA).

In addition to these studies on diazotrophic growth, we analyzed the role of NrfA on in vivo nitrogenase activity (Table 2). R. capsulatus wild-type and mutant strains were grown until the mid-exponential growth phase, under conditions in which either molybdenum nitrogenase or the alternative nitrogenase operates, prior to determination of in vivo nitrogenase activity by the acetylene reduction assay (Section 2). As expected, R. capsulatus strains defective for nrfA showed significantly lower nitrogenase activities compared to the corresponding parental strains. Since the omega cassette used to disrupt the nrfA gene might be polar onto expression of the hflX gene (Fig. 2), we wanted to rule out that the observed phenotype was due to the failure to synthesize the HflX protein. For this purpose, the broad host range hybrid plasmid pNAP1, which carries the entire nrfA gene including the putative promoter region, but only the 5′ part of hflX (Fig. 2), was introduced into nrfA mutant strain DG7. As shown in Table 2, plasmid pNAP1 complemented the NrfA phenotype of mutant strain DG7 to wild-type levels, strongly suggesting that the decreased diazotrophic growth rates of mutant DG7 can be attributed to the absence of NrfA rather than the failure to synthesize HflX. In line with this finding, it is worth to mention that an E. coli strain unable to express hflX does not exhibit an obvious phenotype[29]. Furthermore, since pNAP1 replicates autonomously in R. capsulatus, these experiments corroborate the assumption that transcription of the nrfA gene is driven by a promoter located in the ntrX-nrfA intergenic region (Fig. 2).

2

Activity of molybdenum nitrogenase and the alternative nitrogenase in an R. capsulatus nrfA mutant background

Strain Relevant characteristics Mediuma Nitrogenase activityb 
   +N −N 
B10S wild-type RCV 3.1±1.4 637.4±24.0 
DG7 nrfA RCV 0.4±0.1 86.3±16.1 
TD6 ΔnifHDK, anfA RCV n.d.c 0.9±0.2 
B10S (pNAP1) wild-type, +nrfA RCV 1.7±0.1 861.5±88.0 
DG7 (pNAP1) nrfA, +nrfA RCV 4.2±1.4 614.2±20.2 
TD22 ΔnifHDK AK-NL 0.6±0.1 112.9±28.1 
DG9 ΔnifHDK, nrfA AK-NL 0.6±0.2 45.4±10.7 
TD6 ΔnifHDK, anfA AK-NL 0.5±0.1 0.8±0.2 
Strain Relevant characteristics Mediuma Nitrogenase activityb 
   +N −N 
B10S wild-type RCV 3.1±1.4 637.4±24.0 
DG7 nrfA RCV 0.4±0.1 86.3±16.1 
TD6 ΔnifHDK, anfA RCV n.d.c 0.9±0.2 
B10S (pNAP1) wild-type, +nrfA RCV 1.7±0.1 861.5±88.0 
DG7 (pNAP1) nrfA, +nrfA RCV 4.2±1.4 614.2±20.2 
TD22 ΔnifHDK AK-NL 0.6±0.1 112.9±28.1 
DG9 ΔnifHDK, nrfA AK-NL 0.6±0.2 45.4±10.7 
TD6 ΔnifHDK, anfA AK-NL 0.5±0.1 0.8±0.2 

aCultivation of R. capsulatus strains under phototrophic conditions in RCV medium allows formation of molybdenum nitrogenase, whereas cultivation in molybdenum-free AK-NL medium leads to derepression of the alternative nitrogenase.

bIn vivo nitrogenase activity was determined by the acetylene reduction assay and values given are expressed as nmol ethylene produced per (h×mg protein).

cNot determined.

Expression of nif and anf genes in an R. capsulatus nrfA mutant background

As described above, diazotrophic growth properties based on either molybdenum nitrogenase or the alternative nitrogenase were significantly decreased in an nrfA mutant background. Western blot analysis using NifH-specific antibodies demonstrated that nrfA mutant strain DG7 contained lower levels of nitrogenase reductase than the wild-type (data not shown), suggesting that NrfA is required for maximal synthesis of nitrogenase rather than playing a role in regulation of specific activity of nitrogenase. To determine at which regulatory level NrfA influences synthesis of both nitrogenases, we examined expression of selected nif-lacZ and anf-lacZ reporter gene fusions in wild-type and nrfA mutant backgrounds (Section 2; Table 3). These studies were based on hybrid plasmids pPHU284 (nifA1-lacZ), pPHU282 (nifA2-lacZ) and pKS131A (anfA-lacZ) containing translational (in-frame) fusions, and pFS2 (nifH-lacZ) and pTD7-2I (anfH-lacZ) carrying transcriptional fusions (Table 1). In all five constructs, lacZ expression is driven by the authentic nif and anf promoters. The results shown in Table 3 may be summarized as follows. (i) Expression of nifH was significantly decreased in nrfA mutant strain DG7 compared to the wild-type, suggesting that the decreased level of in vivo molybdenum nitrogenase activity in DG7 was due to decreased transcription of the structural genes of nitrogenase. (ii) Expression of both nifA1 and nifA2 was decreased to about 40% in DG7, indicating that disruption of nrfA has a similar effect onto expression of both nifA copies. Therefore, as in A. caulinodans, NrfA seems to control nitrogen fixation in R. capsulatus at the level of nifA expression. However, at present it remains speculative whether NrfA is required for the initiation of nifA transcription or, as discussed for A. caulinodans, has an effect on nifA mRNA stability and/or translation[9]. Furthermore, we cannot rule out that in R. capsulatus, NrfA somehow might affect the level of NtrC, which is essential for transcriptional activation of nifA (and anfA). In contrast to the situation in A. caulinodans, NrfA was not absolutely required for synthesis of NifA in R. capsulatus. In this context it is worth to mention that database searches in the almost completed genome sequence of R. capsulatus[20] did not reveal the presence of a second nrfA-like gene, which might substitute for the nrfA gene disrupted in mutant strain DG7. (iii) Similar to the situation concerning the nif-encoded nitrogenase, NrfA was found to be required for maximal expression of the anf-encoded nitrogenase. NrfA-mediated control of the alternative nitrogenase also occurred at the level of expression of the regulatory gene, anfA, encoding the anf-specific transcriptional activator.

3

Expression of selected nif-lacZ and anf-lacZ fusions in R. capsulatus wild-type and nrfA mutant strain DG7

Strain (plasmid) Relevant characteristics Mediuma β-Galactosidase activityb 
   +N −N 
B10S (pFS2) wild-type, nifH-lacZ RCV 7.5±2.6 2 879.4±290.9 
DG7 (pFS2) nrfA, nifH-lacZ RCV 1 251.0±113.8 
B10S (pTD7-2I) wild-type, anfH-lacZ AK-NL 16.0±16.0 1 437.7±60.2 
DG7 (pTD7-2I) nrfA, anfH-lacZ AK-NL 4.7±4.2 549.4±97.7 
B10S (pPHU284) wild-type, nifA1-lacZ RCV 11 895.3±531.5 
DG7 (pPHU284) nrfA, nifA1-lacZ RCV 4 616.3±107.0 
B10S (pPHU282) wild-type, nifA2-lacZ RCV 3 083.1±38.0 
DG7 (pPHU282) nrfA, nifA2-lacZ RCV 1 227.2±284.8 
B10S (pKS131A) wild-type, anfA-lacZ AK-NL 130.5±97.6 2 033.8±141.3 
DG7 (pKS131A) nrfA, anfA-lacZ AK-NL 24.6±4.2 438.0±21.4 
Strain (plasmid) Relevant characteristics Mediuma β-Galactosidase activityb 
   +N −N 
B10S (pFS2) wild-type, nifH-lacZ RCV 7.5±2.6 2 879.4±290.9 
DG7 (pFS2) nrfA, nifH-lacZ RCV 1 251.0±113.8 
B10S (pTD7-2I) wild-type, anfH-lacZ AK-NL 16.0±16.0 1 437.7±60.2 
DG7 (pTD7-2I) nrfA, anfH-lacZ AK-NL 4.7±4.2 549.4±97.7 
B10S (pPHU284) wild-type, nifA1-lacZ RCV 11 895.3±531.5 
DG7 (pPHU284) nrfA, nifA1-lacZ RCV 4 616.3±107.0 
B10S (pPHU282) wild-type, nifA2-lacZ RCV 3 083.1±38.0 
DG7 (pPHU282) nrfA, nifA2-lacZ RCV 1 227.2±284.8 
B10S (pKS131A) wild-type, anfA-lacZ AK-NL 130.5±97.6 2 033.8±141.3 
DG7 (pKS131A) nrfA, anfA-lacZ AK-NL 24.6±4.2 438.0±21.4 

aSee footnote of Table 2.

bβ-Galactosidase activity was determined by the method described by Miller[16].

Conclusion

As shown above, NrfA has to be added to the list of proteins involved in control of nitrogen fixation genes in R. capsulatus[14]. In detail, expression of the regulatory genes nifA1, nifA2 and anfA appears to involve both NtrC and NrfA, but only NtrC is essential for transcriptional activation of these genes. Expression of nifA2 (but not nifA1) is co-activated by RegA, known to be involved in regulation of the global redox switch between aerobic and anaerobic growth in Rhodobacter species[3]. Even under nitrogen-deficient conditions, transcription of anfA is inhibited by the Mo-dependent repressor proteins MopA and MopB. In contrast to NrfA, the nucleoid-associated protein HvrA does not influence expression of nifA1, nifA2 or anfA, but acts as a negative modulator of nitrogen fixation genes by binding to selected nif promoters, including PnifH.

Acknowledgements

The authors thank F. Simon for construction of hybrid plasmid pFS2. This work was supported by financial grants from the Deutsche Forschungsgemeinschaft, Germany (SFB 480).

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