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.

A Red Fluorescent Protein Reporter System Developed for Measuring Gene Expression in Photosynthetic Bacteria under Anaerobic Conditions

“…These results demonstrate that the RFP reporter system has an obvious advantage over qRT-PCR in sensitivity. Compared with the previously used lacZ reporter system in the photosynthetic bacterium Rhodobacter capsulatus [9,10], the RFP reporter system is operationally simple, time saving, and highly sensitive.…”

“…Given that RNA molecules can be easily degraded and the integrity of RNA molecules is quite important for subsequent real-time PCR experiment (which reflects the gene expression levels at the moment of sampling), it requires to take a lot of time and effort to reduce the RNA degradation during the preparation of RNA samples [8]. The lacZ gene encoding β-galactosidase is another reporter gene that is commonly used in photosynthetic bacteria [9][10][11]. A colorimetric assay is used to determine the activity of β-galactosidase, which is responsible for the degradation of β-galactosyl linkages [12,13].…”

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides

“…While the FmdA_AmdA family of proteins is primarily known for hydrolysis of acetate and formate, some members of this family have activity on other amide-containing compounds, including lactams and aliphatic amides . Interestingly, the cluster of 610 amidase-associated operons that we identified is dominated by proteins from Gram-negative bacteria that are specialized in the uptake nitrogen-containing compounds, such as Rhizobiales , Mesorhizobium , and Rhodobacteraceae − (Figure A,C). Notably, no other clusters within the SBP_bac_5 family were associated with a FmdA_AmdA protein, and the structure of these amidase-associated operons was clearly distinct from previously well-studied amidase operons such as the Pseudomonas aeruginosa amidase operon, , Rhodococcus sp.…”

Engineering of a photosynthetic diazotroph as biofertilizer for efficient nitrogen fixation powered by sunlight

“…The conserved binding motifs for AnfA and NifA are predicted to be 'TAC-N 6 -GTA' and 'TGT-N 10 -ACA', respectively [27][28][29]. To test the binding a nity of AnfA for the predicted binding site upstream of anfH gene, we carried out the EMSA to detect the interactions between the AnfA and the motif 'TAC-N 7 -GTA' upstream of anfH.…”