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

Abstract

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.

Keywords: Rhodobacter; AnfA; Fe-nitrogenase; Mo-nitrogenase; NifA.

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_anfH) by the nifH promoter (P_nifH) 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 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

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)

FIGURE 4

Effect of P_anfH → P_nifH substitution on anfH‐lacZ expression and diazotrophic growth. (a) Effect of P_anfH → P_nifH 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_anfH → P_nifH, anfA ⁺, anfH‐lacZ), and YP515:pMH187 (P_anfH → P_nifH, 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_anfH → P_nifH 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_anfH → P_nifH, anfA ⁺, ΔnifD), YP515‐BS85 (P_anfH → P_nifH, ΔanfA, ΔnifD), and KS94A‐YP415 (ΔanfA‐ΔnifD). The results represent the means and standard deviations of three independent measurements

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

FIGURE A1

Comparison of AnfA proteins