Rhodobacter capsulatus AnfA is essential for production of Fe-nitrogenase proteins but dispensable for cofactor biosynthesis and electron supply.

The photosynthetic α-proteobacterium Rhodobacter capsulatus reduces and thereby fixes atmospheric dinitrogen (N2 ) by a molybdenum (Mo)-nitrogenase and an iron-only (Fe)-nitrogenase. Differential expression of the structural genes of Mo-nitrogenase (nifHDK) and Fe-nitrogenase (anfHDGK) is strictly controlled and activated by NifA and AnfA, respectively. In contrast to NifA-binding sites, AnfA-binding sites are poorly defined. Here, we identified two highly similar AnfA-binding sites in the R. capsulatus anfH promoter by studying the effects of promoter mutations on in vivo anfH expression and in vitro promoter binding by AnfA. Comparison of the experimentally determined R. capsulatus AnfA-binding sites and presumed AnfA-binding sites from other α-proteobacteria revealed a consensus sequence of dyad symmetry, TAC-N6 -GTA, suggesting that AnfA proteins bind their target promoters as dimers. Chromosomal replacement of the anfH promoter by the nifH promoter restored anfHDGK expression and Fe-nitrogenase activity in an R. capsulatus strain lacking AnfA suggesting that AnfA is required for AnfHDGK production, but dispensable for biosynthesis of the iron-only cofactor and electron delivery to Fe-nitrogenase, pathways activated by NifA. These observations strengthen our model, in which the Fe-nitrogenase system in R. capsulatus is largely integrated into the Mo-nitrogenase system.

INTRODUCTION

Molecular nitrogen (N2) is highly abundant in air, but only diazotrophic bacteria and archaea are capable of utilizing this source of nitrogen, while non‐diazotrophic prokaryotes and all eukaryotes depend on more reduced nitrogen forms like ammonium, nitrate, or organic nitrogen compounds (Dos Santos, Fang, Mason, Setubal, & Dixon, 2012; Zhang & Gladyshev, 2008; Zhang, Rump, & Gladyshev, 2011). In a process called biological nitrogen fixation, diazotrophs reduce N2 to ammonia (NH3) by three structurally and functionally similar nitrogenase isoenzymes, each composed of two components, a catalytic dinitrogenase and a dinitrogenase reductase. All diazotrophs possess a molybdenum nitrogenase consisting of the NifDK and NifH components. Also, some diazotrophs encode either a vanadium nitrogenase (VnfDGK, VnfH) or an iron‐only nitrogenase (AnfDGK, AnfH), or both Mo‐free isoenzymes (McRose, Zhang, Kraepiel, & Morel, 2017). Since Mo‐nitrogenase is more efficient than the other nitrogenases in terms of ATP consumption per N2 reduced, diazotrophs preferentially synthesize Mo‐nitrogenase when ammonium is limiting (Eady, 2003; Mus, Alleman, Pence, Seefeldt, & Peters, 2018; Schneider, Gollan, Dröttboom, Selsemeier‐Voigt, & Müller, 1997; Sippel & Einsle, 2017; Thiel & Pratte, 2014). Many diazotrophs repress the production of Mo‐free nitrogenases by one‐component ModE regulators, which directly sense and respond to molybdate availability (reviewed by Demtröder, Narberhaus, & Masepohl, 2019). The Mo‐free nitrogenases take over under Mo‐limiting conditions.

Mo‐nitrogenase contains a complex iron‐molybdenum cofactor, FeMoco, whose synthesis requires the NifUS, NifH, NifB, NifEN, NifQ, and NifV proteins (reviewed by Burén, Jiménez‐Vicente, Echavarri‐Erasun, & Rubio, 2020; Curatti & Rubio, 2014; Hu & Ribbe, 2016). The NifUS, NifB, and NifV proteins are also involved in biosynthesis of the iron‐vanadium cofactor of V‐nitrogenase, FeVco, and the iron‐only cofactor of Fe‐nitrogenase, FeFeco (Drummond, Walmsley, & Kennedy, 1996; Hamilton et al., 2011; Hu & Ribbe, 2016; Kennedy & Dean, 1992; Sippel & Einsle, 2017; Yang, Xie, Wang, Dixon, & Wang, 2014). Electron transfer to the nitrogenases involves species‐specific proteins like NifF, FdxN, RnfABCDGEH, and FixABC (Boyd, Costas, Hamilton, Mus, & Peters, 2015; Dos Santos et al., 2012; Oldroyd, 2013; Poudel et al., 2018).

In proteobacteria, expression of nif, vnf, and anf genes depends on the alternative sigma factor RpoN and the structurally and functionally related transcription activators NifA, VnfA, and AnfA, respectively (Bush & Dixon, 2012; Dixon & Kahn, 2004; Drummond et al., 1996; Fischer, 1994; Hamilton et al., 2011; Heiniger, Oda, Samanta, & Harwood, 2012; Hübner, Masepohl, Klipp, & Bickle, 1993; Joerger, Jacobson, & Bishop, 1989; Kutsche, Leimkühler, Angermüller, & Klipp, 1996; Merrick, 1993; Mus et al., 2018; Oda et al., 2005; Oliveira et al., 2012; Sarkar & Reinhold‐Hurek, 2014; Souza, Pedrosa, Rigo, Machado, & Yates, 2000; Walmsley, Toukdarian, & Kennedy, 1994; Zhang, Pohlmann, Ludden, & Roberts, 2000; Zou et al., 2008). These activators encompass three modules, namely an N‐terminal GAF, a central AAA+, and a C‐terminal HTH domain, involved in environmental sensing, ATP‐dependent activation of target gene transcription, and DNA binding, respectively. Many NifA proteins contain a conserved cysteine motif in an interdomain linker connecting the central and C‐terminal domains, while VnfA and AnfA proteins contain conserved cysteine motifs in their GAF domains (Figure A1 in Appendix 2). The cysteine residues are known or presumed to bind iron‐sulfur clusters making these proteins sensitive to oxygen (Austin & Lambert, 1994; Fischer, 1994; Nakajima et al., 2010; Yoshimitsu, Takatani, Miura, Watanabe, & Nakajima, 2011). This prevents inappropriate induction of nitrogen fixation, a process not compatible with oxygen.

FIGURE A1

Comparison of AnfA proteins

RpoN proteins bind promoters with the consensus CTGG–N8–TTGC (N stands for any nucleotide) located at position –24/–12 upstream of the transcription start site (Buck, Miller, Drummond, & Dixon, 1986; Bush & Dixon, 2012; Fischer, 1994; Merrick, 1993; Zhang & Buck, 2015). NifA proteins bind cis‐regulatory elements with the consensus TGT–N10–ACA, which is typically found within a distance of 150 base‐pairs upstream of the transcription start site (Buck et al., 1986; Demtröder, Pfänder, Schäkermann, Bandow, & Masepohl, 2019; Fischer, 1994). NifA‐binding sites are well‐characterized in many α‐, β‐, and γ‐proteobacteria (Barrios, Grande, Olvera, & Morett, 1998; Buck et al., 1986; González, Olvera, Sobero, & Morett, 1998; Gubler, 1989; Lee, Berger, & Kustu, 1993; Monteiro et al., 1999; Wang, Kolb, Cannon, & Buck, 1997), whereas VnfA‐ and AnfA‐binding sites have been studied so far only in the γ‐proteobacterium Azotobacter vinelandii, which is one of few diazotrophs having both Mo‐free nitrogenases (Austin & Lambert, 1994; Drummond et al., 1996; Frise, Green, & Drummond, 1994; Woodley, Buck, & Kennedy, 1996). In this species, the sequences GTAC–N6–GTAC and C–N–GG–N3–GGTA have been suggested as binding sites for VnfA and AnfA, respectively (Austin & Lambert, 1994; Woodley et al., 1996).

To determine AnfA‐binding site requirements in a diazotroph distantly related to A. vinelandii, we examined the regulation of Fe‐nitrogenase genes in the photosynthetic α‐proteobacterium Rhodobacter capsulatus, which fixes nitrogen by the Mo‐ and Fe‐nitrogenases (Schneider, Müller, Schramm, & Klipp, 1991; Schüddekopf, Hennecke, Liese, Kutsche, & Klipp, 1993; Strnad et al., 2010). Differential expression of the corresponding nif and anf genes is tightly regulated. While NifA activates the promoters upstream of multiple nif genes, AnfA activates the anfH promoter as indicated by reporter fusions and proteome profiling (Demtröder, Pfänder, et al., 2019; Figure 1a). Since the sigma factor RpoN is encoded by the nifU2‐rpoN operon, NifA indirectly controls AnfA‐mediated expression of the Fe‐nitrogenase genes (Demtröder, Pfänder, et al., 2019).

FIGURE 1

Model of the nitrogen fixation regulon in Rhodobacter capsulatus. (a) Production of Mo‐ and Fe‐nitrogenases in the wild type. In the absence of ammonium (–NH4 +), the superior regulator NtrC activates transcription of nifA and anfA in concert with the housekeeping sigma factor RpoD (Foster‐Hartnett, Cullen, Monika, & Kranz, 1994; Kutsche et al., 1996). MopA and MopB independently repress anfA in the presence of molybdate (+MoO4 2–; Wiethaus et al., 2006). NifA and AnfA activate their target genes by partnering with the alternative sigma factor RpoN. Noteworthy, NifA indirectly controls AnfA‐mediated anfHDGK expression by controlling RpoN production (Demtröder, Pfänder, et al., 2019). Involvement of NifA‐activated genes in biosynthesis of the iron‐molybdenum cofactor (FeMoco) of Mo‐nitrogenase and the iron‐only cofactor (FeFeco) of Fe‐nitrogenase and electron transfer to both nitrogenases is indicated. (b) Production of active Fe‐nitrogenase in a strain lacking AnfA. In this study, we constructed strain YP515‐BS85 containing mutations in the anfA and nifD genes (marked by red crosses) and a chromosomal substitution of the anfH promoter (P) by the nifH promoter (P) thereby putting anfHDGK expression under NifA control. This strain grew under N2‐fixing conditions (Figure 4b) suggesting that AnfA is dispensable for FeFeco biosynthesis and electron supply to Fe‐nitrogenase. For further details, see text

FIGURE 4

Effect of P → P substitution on anfH‐lacZ expression and diazotrophic growth. (a) Effect of P → P substitution on anfH‐lacZ expression. R. capsulatus strains carrying a chromosomally integrated transcriptional anfH‐lacZ fusion based on plasmid pMH187 (Demtröder, Pfänder, et al., 2019) were phototrophically grown in RCV minimal medium (no Mo added) with either 10 mM serine or 10 mM ammonium. The strains used were as follows: B10S:pMH187 (anfA +, anfH‐lacZ), YP516:pMH187 (P → P, anfA +, anfH‐lacZ), and YP515:pMH187 (P → P ΔanfA, anfH‐lacZ). LacZ (β‐galactosidase) activity is given in Miller units (Miller, 1972). The results represent the means and standard deviations of five independent experiments. (b) Effect of P → P substitution on Fe‐nitrogenase activity in R. capsulatus strains lacking AnfA. R. capsulatus strains were phototrophically grown in RCV minimal medium (no Mo added) with N2 as the sole nitrogen source. The strains used were as follows: B10S (wild type), BS85 (anfA +, ΔnifD), YP516‐BS85 (P → P, anfA +, ΔnifD), YP515‐BS85 (P → P, ΔanfA, ΔnifD), and KS94A‐YP415 (ΔanfA‐ΔnifD). The results represent the means and standard deviations of three independent measurements

In this study, we show that R. capsulatus AnfA binds two highly similar palindromic sites in the anfH promoter. Based on conserved sequences in various α‐proteobacteria, we define a general AnfA‐binding site consensus, TAC–N6–GTA. Besides, we present evidence that the anfH promoter is the only Fe‐nitrogenase‐related promoter in R. capsulatus strictly depending on AnfA.

MATERIALS AND METHODS

Strains, plasmids, and growth conditions

Bacterial strains and plasmids used in this study are listed in Table A1 in Appendix 1. Rhodobacter capsulatus minimal medium V (RCV) was prepared as previously described (Demtröder, Pfänder, et al., 2019). In this medium, a fixed nitrogen source and molybdate (Mo) have been omitted. Traces of Mo arising from impurities of the chemicals used support residual Mo‐nitrogenase activity but are low enough to permit the production of Fe‐nitrogenase. To examine diazotrophic growth, cultures were inoculated in 3 ml RCV medium in screw‐capped 17‐ml Hungate tubes before the exchange of headspace air for pure N2 gas and incubation in the light. When required, 10 mM serine was added as a fixed nitrogen source, which (in contrast to ammonium) does not inhibit nitrogen fixation.

TABLE A1

Rhodobacter capsulatus strains and plasmids

Strain or plasmid	Relevant characteristics	Source or reference
Strains
B10S	R. capsulatus wild type; Smr	Klipp, Masepohl, and Pühler (1988)
BS85	nifD::Sp mutant (ΔnifD) of B10S; Smr, Spr	Hoffmann et al. (2014)
KS94A	anfA::Sp mutant (ΔanfA) of B10S; Smr, Spr	Wang, Angermüller, and Klipp (1993)
KS94A‐YP415	anfA::Sp, nifD::Gm mutant (ΔanfA, ΔnifD) of B10S; Gmr, Smr, Spr	This study
YP515‐BS85	PanfH → PnifH, ΔanfA::Gmr, ΔnifD::Spr mutant of B10S; Gmr, Smr, Spr	This study
YP516‐BS85	PanfH → PnifH, anfA +, ΔnifD::Spr mutant of B10S; Gmr, Smr, Spr	This study
Vector plasmids
pASK_IBA45+	AHT‐inducible expression vector; Apr	IBA GmbH Göttingen, Germany
pBBR1MCS	Mobilizable broad‐host‐range vector; Cmr	Kovach et al. (1995)
pUC18	Narrow‐host‐range vector; Apr	Norrander, Kempe, and Messing (1983)
pYP35	lacTeT cassette donor; Apr, Ter, oriT	Gisin et al. (2010)
Hybrid plasmids
pBBR_F1‐lacZ to pBBR_F6‐lacZ	pBBR1MCS derivatives carrying PanfH fragments F1 to F6 (Figure 2a,b) fused to lacZ; Cmr, Ter	This study
pBBR_Mut1‐lacZ to pBBR_Mut7‐lacZ; pBBR_Mut2/6‐lacZ; pBBR_Mut2/7‐lacZ	pBBR_F1‐lacZ variants carrying mutations Mut1 to Mut7, Mut2/6, and Mut2/7 (Figure 3a); Cmr, Ter	This study
pMH187	Mobilizable narrow‐host‐range plasmid carrying anfH‐lacZ; Ter, oriT	Demtröder, Pfänder, et al. (2019)
pYP409	pASK_IBA45 + derivative encoding AnfA_DBD; Apr	This study
pYP515	pUC18 derivative carrying rcc00583‐Gmr‐PnifH‐anfH; Apr, Ter, oriT	This study
pYP516	pUC18 derivative carrying anfA‐Gmr‐PnifH‐anfH; Apr, Ter, oriT	This study

Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Gm, gentamicin; Sm, streptomycin; Sp, spectinomycin; Te, tetracycline.

Construction of Rhodobacter capsulatus anfH‐lacZ reporter strains and β‐galactosidase assays

The anfH promoter was narrowed down by nested deletions. For this, appropriate primer pairs were used to PCR‐amplify promoter variants F1 to F6 (Figure 2a,b), thereby adding BamHI and HindIII sites. Corresponding BamHI‐HindIII fragments were cloned into the broad‐host‐range vector pBBR1MCS (Kovach et al., 1995) before insertion of a lacTeT cassette (carrying a promoterless lacZ gene, a tetracycline resistance gene, and an oriT transfer origin) from plasmid pYP35 (Gisin et al., 2010) into the HindIII site. The resulting reporter plasmids carrying transcriptional lacZ fusions were designated pBBR_F1‐lacZ to pBBR_F6‐lacZ.

FIGURE 2

Effect of nested deletions in the R. capsulatus anfH promoter on anfH‐lacZ expression. (a) Cis‐regulatory elements in the anfA‐anfH intergenic region. The DNA sequence encompasses the AnfA translation stop codon (TGA), the Rho‐independent anfA transcription terminator, two AnfA‐binding sites (AnfA_BS), the RpoN‐binding site (RpoN_BS), and the AnfH translation start codon (ATG). Arrowheads mark inverted repeat sequences. The start sites of anfH promoter deletion variants F1 to F6 are indicated. (b) Reporter fusions between anfH promoter deletion variants and lacZ. Promoter variants F1 to F6 were cloned into a broad‐host‐range vector, before insertion of a lacZ cassette (designed for transcriptional fusions) immediately downstream of the anfH start codon resulting in reporter plasmids pBBR_F1‐lacZ to pBBR_F6‐lacZ (Materials and Methods). (c) Expression of anfH‐lacZ fusions. R. capsulatus reporter strains carrying pBBR_F1‐lacZ to pBBR_F6‐lacZ were phototrophically grown in RCV minimal medium with 10 mM serine but without Mo addition, conditions allowing anfHDGKOR3 expression. LacZ (β‐galactosidase) activity is given in Miller units (Miller, 1972). The results represent the means and standard deviations of five independent experiments

To generate site‐directed substitution mutations in the anfH promoter (Figure 3a), plasmid pBBR_F1‐lacZ served as a template. Base‐pair substitutions were introduced using the QuikChange protocol (Stratagene). The resulting pBBR_F1‐lacZ derivatives carrying transcriptional lacZ fusions to different promoter variants were designated pBBR_Mut1‐lacZ to pBBR_Mut7‐lacZ, pBBR_Mut2/6‐lacZ, and pBBR_Mut2/7‐lacZ.

FIGURE 3

Effect of base substitutions in the anfH promoter on anfH‐lacZ expression and AnfA binding. (a) Base substitutions in the AnfA‐binding sites. Plasmid pBBR_F1‐lacZ (carrying the wild‐type anfH promoter fragment F1 fused to lacZ shown in Figure 2b) served as a template for base substitution mutations Mut1 to Mut7 (highlighted in red). (b) Expression of anfH‐lacZ fusions. R. capsulatus reporter strains carrying pBBR_F1‐lacZ (WT) and its variants (Mut1 to Mut7, Mut2/6, and Mut2/7) were phototrophically grown in RCV minimal medium (no Mo added) with 10 mM serine. LacZ (β‐galactosidase) activity is given in Miller units (Miller, 1972). Data for WT control are the same as in Figure 2c. The results represent the means and standard deviations of five independent experiments. (c) Binding of AnfA_DBD to the anfH promoter. In vitro binding of the separated DNA‐binding domain of AnfA (AnfA_DBD) to the anfH promoter was examined by EMSA. PCR fragments carrying the wild‐type (WT; F1 fragment) anfH promoter and its variants Mut1 to Mut7, Mut2/6, and Mut2/7 were 32P‐labeled before incubation with the indicated amounts of AnfA protein. AnfA‐promoter complexes and free promoter fragments (labeled C and F, respectively) were electrophoretically separated and detected by autoradiography. EMSA analyses were done in duplicate with one representative result shown in (c)

The reporter plasmids were conjugationally transferred into the R. capsulatus wild‐type strain B10S. Following phototrophic growth of the R. capsulatus reporter strains in RCV medium with 10 mM serine (no Mo added) until the late logarithmic phase, LacZ (β‐galactosidase) activity was determined (Miller, 1972).

Examination of AnfA binding to the anfH promoter

In vitro binding of the DNA‐binding domain of AnfA (AnfA_DBD) to the anfH promoter was examined by electrophoretic mobility shift assays (EMSA) as previously described (Müller et al., 2010). To overexpress AnfA_DBD, appropriate primers were used to PCR‐amplify a DNA fragment coding for the C‐terminal 72 amino acid residues of AnfA (Figure A1 in Appendix 2) thereby adding SacII and NcoI sites. The corresponding SacII‐NcoI fragment was cloned into the expression vector pASK_IBA45+ (IBA GmbH Göttingen) resulting in hybrid plasmid pYP409. For purification of the Strep‐tagged AnfA_DBD, Escherichia coli BL21 (DE) carrying pYP409 was cultivated with AHT induction before cell disruption and Strep‐Tactin affinity chromatography as previously described (Hoffmann, Ali, et al., 2016).

The F1 fragment (carrying the wild‐type anfH promoter) and its variants (Figure 3a) were labeled with γ‐32P‐ATP, and free γ‐32P‐ATP was removed by gel filtration using Illustra ProbeQuant G‐50 Micro Columns (GE‐Healthcare). After 20 min incubation of labeled promoter variants with increasing amounts of the purified AnfA_DBD protein, bound and free DNAs were separated in 6% polyacrylamide gels. Radioactive bands were detected by phosphor screen exposure.

Substitution of the Rhodobacter capsulatus anfH promoter by the nifH promoter

To replace the anfH promoter (P) by the nifH promoter (P), we exchanged the 266 bp anfA‐anfH intergenic region by the 267 bp fdxD‐nifH intergenic region. For this purpose, we constructed mutagenesis plasmid pYP516 containing the 3′ end of anfA (including the translation stop codon, TGA), a gentamicin (Gm) resistance cassette, P, and the 5′ end of anfH (starting with the translation start codon, ATG). To replace the anfH promoter and to delete the anfA gene in a single step, we constructed mutagenesis plasmid pYP515 containing an anfA upstream fragment (but lacking the anfA coding region), a Gm cassette, P, and the 5′ end of anfH. Plasmids pYP516 and pYP515 were conjugationally introduced into the R. capsulatus strain BS85 (ΔnifD), in which the nifD gene is disrupted by a spectinomycin (Sp) cassette. BS85 does not exhibit Mo‐nitrogenase activity, and hence, any nitrogenase activity observed in this background can be assigned to Fe‐nitrogenase (Demtröder, Pfänder, et al., 2019). Promoter replacement mutants were identified by selection for Gm resistance and screening for loss of vector‐encoded tetracycline resistance indicating marker rescue by double cross‐over events. The resulting mutants were called YP516‐BS85 (P → P, anfA +, ΔnifD) and YP515‐BS85 (P → P, ΔanfA, ΔnifD).

In contrast to strain YP515‐BS85 (P → P, ΔanfA, ΔnifD), strain KS94A‐YP415 (ΔanfA, ΔnifD) contains the wild‐type anfH promoter upstream of the anfHDGKOR3 operon. In this strain, the anfA and nifD genes are disrupted by Sp and Gm cassettes, respectively.

RESULTS

Localization of the Rhodobacter capsulatus anfH promoter by nested deletions

Rhodobacter capsulatus AnfA is essential for the expression of the anfHDGKOR3 operon (Demtröder, Pfänder, et al., 2019), but the anfH promoter has not been investigated. The coding regions of anfH and its upstream gene, anfA, are separated by 266 bp (Figure 2a). This intergenic region includes three conspicuous sequences, namely (a) a GC‐rich inverted repeat sequence followed by a T‐rich stretch likely acting as Rho‐independent terminator of anfA transcription, (b) two 17 bp direct repeats each encompassing inverted repeat sequences, which are promising candidates as AnfA‐binding sites, and (c) a highly conserved RpoN‐binding site. For clarity, the 17 bp sequences will from now on be called distal and proximal AnfA‐binding sites.

To localize the anfH promoter (P), we analyzed the effects of nested promoter deletions on anfH expression. For this purpose, we generated transcriptional fusions between P fragments, F1 to F6, and a promoterless lacZ gene (Figure 2b). R. capsulatus strains carrying the corresponding reporter plasmids were grown in RCV minimal medium with serine as a nitrogen source without molybdate addition, conditions compatible with the synthesis of Fe‐nitrogenase (Demtröder, Pfänder, et al., 2019; Hoffmann, Wagner, et al., 2016), before determination of LacZ (β‐galactosidase) activity.

Fragments F1 and F2 mediated considerable LacZ activity (Figure 2c) indicating that the anfA‐anfH intergenic region and in particular the region downstream of the putative anfA transcription terminator contain all cis‐regulatory elements required for P activation. F3‐based LacZ activity was reduced to 29% of the F1 value probably due to the absence of one half‐site of the distal AnfA‐binding site (see below). As expected, fragments F4 to F6 resulted in only background LacZ activity consistent with the absence of both AnfA‐binding sites.

Effects of AnfA‐binding site mutations on anfH expression

To dissect the function of the distal and proximal AnfA‐binding sites in P, we generated pBBR_F1‐lacZ variants carrying site‐directed mutations Mut1 to Mut7 (Figure 3a) and the combinations Mut2/6 and Mut2/7. Mut1 mediated clear LacZ activity (72% of the F1 value) suggesting that this sequence plays only a minor role in anfH expression consistent with our findings on nested promoter deletions (Figure 2). LacZ activity of Mut2 to Mut7 dropped to 20%–40% as compared to F1 (carrying the wild‐type anfH promoter), while combined mutations Mut2/6 and Mut2/7 abolished LacZ activity (Figure 3b). These observations suggest that either of the two AnfA‐binding sites mediates P activation to some extent and that full activation requires both sites.

Effects of AnfA‐binding site mutations on promoter binding by AnfA

The AnfA protein encompasses three domains, namely a GAF, an AAA+, and a HTH domain, involved in environmental sensing, activation of RNA polymerase, and promoter binding, respectively. To test the direct binding of AnfA to P, we performed electrophoretic mobility shift assays (EMSA). For this, the radiolabeled F1 fragment (carrying the wild‐type anfH promoter) and its variants (Mut1 to Mut7, Mut2/6, and Mut2/7) were incubated with increasing amounts of the Strep‐tagged DNA‐binding domain of AnfA (AnfA_DBD) encompassing the C‐terminal 72 amino acid residues of the full‐length regulator (Figure A1 in Appendix 2). AnfA_DBD was expected to bind the same target sequence as the full‐length regulator, but to be more stable in solution according to findings on Azotobacter vinelandii AnfA and NifA, and Herbaspirillum seropedicae NifA (Austin & Lambert, 1994; Lee et al., 1993; Monteiro, Souza, Yates, Pedrosa, & Chubatsu, 2003).

AnfA_DBD bound the F1 fragment (carrying the wild‐type anfH promoter) demonstrating that the isolated C‐terminal domain indeed functions in promoter binding independent of the other AnfA domains (Figure 3c). Mut1 was bound comparably well as F1 indicating that this sequence is dispensable for AnfA binding. AnfA_DBD also bound promoter variants Mut2 to Mut7 albeit less effectively than F1, and the combined Mut2/6 and Mut2/7 variants were barely shifted. These observations suggest that recognition of the distal or proximal sites by AnfA is essentially unbiased. Taken together, the in vitro binding studies (Figure 3c) are well in line with the in vivo data on anfH expression (Figure 3b) supporting the hypothesis that the two 17 bp repeat sequences (Figure 2a) serve as distal and proximal AnfA‐binding sites.

NifA‐driven anfHDGKOR expression restores Fe‐nitrogenase activity in a strain lacking AnfA

Productive nitrogen fixation by the Fe‐nitrogenase requires more than the expression of the anfHDGKOR3 operon. Current knowledge suggests that AnfA is required only for anfHDGKOR3 expression and has little impact on the expression of NifA‐activated genes like nifB and rnfA, which are essential for FeFeco biosynthesis and electron transfer to Fe‐nitrogenase, respectively (Demtröder, Pfänder, et al., 2019; Schüddekopf et al., 1993). We, therefore, speculated that AnfA might be dispensable for the production of active (N2‐fixing) Fe‐nitrogenase as long as the anfHDGKOR3 operon is adequately expressed. To achieve AnfA‐independent anfHDGKOR3 expression, we replaced the anfH promoter (P) by the NifA‐activated nifH promoter (P). Both promoters mediate comparably strong expression of their downstream genes at least under Mo‐limiting conditions (Demtröder, Pfänder, et al., 2019). If the anfHDGKOR3 operon is the only member of the AnfA regulon, nitrogen fixation by Fe‐nitrogenase should become entirely NifA‐dependent in the P → P strain (Figure 1b).

To determine the effect of P → P substitution on anfHDGKOR3 expression, we generated two R. capsulatus strains, which carry the same P → P promoter substitution, but either in the wild‐type (anfA +) or ΔanfA background. Subsequently, we introduced transcriptional anfH‐lacZ reporter fusions in these strains and, as a control, in the wild‐type strain B10S by chromosomal integration of plasmid pMH187 as previously described (Demtröder, Pfänder, et al., 2019). The resulting reporter strains were grown with either serine or ammonium as a nitrogen source before LacZ activity was determined.

The wild‐type control (containing the native AnfA‐dependent anfH promoter) showed the expected high anfH‐lacZ expression in serine cultures, while no expression was observed in ammonium cultures (Figure 4a; Demtröder, Pfänder, et al., 2019; Kutsche et al., 1996; Wiethaus, Wirsing, Narberhaus, & Masepohl, 2006). Both P → P strains expressed anfH‐lacZ to comparable levels as the wild‐type control showing that P → P substitution mediated effective anfHDGKOR3 expression independent of AnfA.

To test whether nitrogen fixation by Fe‐nitrogenase was entirely NifA‐dependent in the P → P strains, we introduced a polar nifD mutation (ΔnifD) in these strains, before examination of diazotrophic growth. Regardless of the nifD mutation, these strains were expected to express all the other NifA‐dependent nitrogen fixation genes involved in cofactor biosynthesis and electron transport (Figure 1b). Since ΔnifD strains lack Mo‐nitrogenase, diazotrophic growth of these strains depends entirely on Fe‐nitrogenase (Demtröder, Pfänder, et al., 2019). As controls, we included the wild type, the parental ΔnifD strain, and a ΔanfA‐ΔnifD strain lacking both nitrogenases.

The wild‐type and ΔnifD strains grew well with N2 as the sole nitrogen source, while the ΔanfA‐ΔnifD strain failed to grow diazotrophically (Figure 4b) consistent with earlier studies (Demtröder, Pfänder, et al., 2019; Kutsche et al., 1996). Both P → P strains grew almost as well as the parental ΔnifD strain suggesting that P → P substitution indeed decouples production of functional Fe‐nitrogenase from AnfA.

Taken together, our findings show that substitution of the AnfA‐dependent anfH promoter by the NifA‐activated nifH promoter restores anfHDGKOR expression and Fe‐nitrogenase activity in a strain lacking AnfA. In other words, AnfA appears to be dispensable for FeFeco biosynthesis and electron delivery to Fe‐nitrogenase.

DISCUSSION

Despite the wide distribution of Fe‐nitrogenases (McRose et al., 2017), our knowledge of Fe‐nitrogenase‐related promoters was so far limited to one species, the γ‐proteobacterium Azotobacter vinelandii (Austin & Lambert, 1994; Drummond et al., 1996). Here, we characterized two AnfA‐binding sites in the anfH promoter in the α‐proteobacterium Rhodobacter capsulatus by in vivo and in vitro studies. Each of these binding sites mediated anfH expression to some extent, but maximal expression required both AnfA‐binding sites.

Sequences similar to the R. capsulatus AnfA‐binding sites are conserved in α‐proteobacterial promoters preceding potential anfHDGK operons (Figure 5a; McRose et al., 2017). Sequence logo representation (based on all distal and proximal AnfA‐binding sites shown in Figure 5a) revealed a conserved sequence of dyad symmetry, TAC–N6–GTA, as the AnfA‐binding site consensus (Figure 5b). Binding sites of dyad symmetry are typically bound by dimeric regulators (Kim & Little, 1992; Klose, North, Stedman, & Kustu, 1994) suggesting that AnfA proteins also bind target promoters as dimers. The AnfA‐binding site consensus previously defined for A. vinelandii, C–N–GG–N3–GGTA (Austin & Lambert, 1994), and our consensus share the strictly conserved GTA motif (Figure 5a). The A. vinelandii AnfA‐binding sites, which were identified by footprint experiments, lack the TAC motif indicating that this motif is dispensable for promoter recognition by A. vinelandii AnfA. In R. capsulatus, individual TAC (Mut2, Mut5) and GTA mutations (Mut4, Mut7) reduced anfH‐lacZ expression by 60%–80%, while the Mut2/Mut7 combination abolished expression (Figure 3b). Together these findings suggest that AnfA proteins in different bacteria require the GTA motif, but differ in their dependence on the TAC motif to activate their target promoters.

FIGURE 5

AnfA‐binding sites in proteobacterial anfH promoters. (a) Comparison of AnfA‐binding sites. Binding of AnfA to distal and proximal sites has been experimentally shown for R. capsulatus (this study) and A. vinelandii (Austin & Lambert, 1994). Affiliation of bacterial strains to the α‐ and γ‐proteobacteria, and the numbers of nucleotides (N) between cis‐regulatory elements are indicated. Known and presumed AnfA‐binding sites encompass strictly conserved GTA and partially conserved TAC motifs (highlighted in red). Lower and upper case lettering in the consensus sequences indicates conservation in at least four or five of the respective sequences, respectively. (b) AnfA‐binding site logo. The AnfA‐binding site consensus based on all distal and proximal sites shown in (a) was generated using the weblogo.berkeley.edu program

Rhodobacter capsulatus AnfA is essential for anfHDGKOR3 expression and consequently, for nitrogen fixation by Fe‐nitrogenase (Demtröder, Pfänder, et al., 2019). Remarkably, a strain lacking AnfA but driving anfHDGKOR3 expression by NifA regained the capacity to grow diazotrophically via Fe‐nitrogenase (Figure 4b). This means that AnfA in the wild type is required for AnfHDGKOR3 production, but dispensable for FeFeco biosynthesis and electron delivery to Fe‐nitrogenase. For a regulatory model, see Figure 1b. Our findings do not necessarily exclude other AnfA targets than the anfH promoter. Indeed, AnfA affects the expression of different nitrogen fixation genes including iscN, nifE, fprA, and nifB (Demtröder, Pfänder, et al., 2019). None of these genes, however, is preceded by an obvious AnfA‐binding site (or just a GTA motif) suggesting that AnfA control of these genes is indirect.

In contrast to the R. capsulatus nifB promoter, we found potential AnfA‐binding sites in the nifB promoters of Rhodospirillum rubrum (TAC–N6–GTA) and Rhodomicrobium vannielii (CAC–N6–GTA) suggesting direct nifB activation by AnfA in these strains. In line with the requirement of NifB for the activity of all three nitrogenases in A. vinelandii, the nifB promoter can be activated by NifA, VnfA, or AnfA, which bind to overlapping sites in the nifB promoter (Drummond et al., 1996).

Our finding that only a single Fe‐nitrogenase‐related target, the anfH promoter, strictly requires activation by AnfA in R. capsulatus, raises the question of why this diazotroph needs AnfA. One explanation is that AnfA contributes (indirectly) to fine regulation of NifA‐dependent genes (Demtröder, Pfänder, et al., 2019). Also, Mo repression of anfA introduces a regulatory level to prevent the production of Fe‐nitrogenase under Mo‐replete conditions. This guarantees the exclusive activity of Mo‐nitrogenase, which exhibits higher N2‐reducing activity than Fe‐nitrogenase (Hoffmann, Wagner, et al., 2016; Wiethaus et al., 2006).

CONFLICT OF INTERESTS

None declared.

AUTHOR CONTRIBUTION

Lisa Demtröder: Conceptualization (supporting); Investigation (lead); Writing‐original draft (equal). Yvonne Pfänder: Investigation (supporting). Bernd Masepohl: Conceptualization (lead); Funding acquisition (lead); Writing‐original draft (equal).

ETHICS STATEMENT

None required.