Proteome Profiling of the Rhodobacter capsulatus Molybdenum Response Reveals a Role of IscN in Nitrogen Fixation by Fe-Nitrogenase

Abstract

Rhodobacter capsulatus is capable of synthesizing two nitrogenases, a molybdenum-dependent nitrogenase and an alternative Mo-free iron-only nitrogenase, enabling this diazotroph to grow with molecular dinitrogen (N 2 ) as the sole nitrogen source. Here, the Mo responses of the wild type and of a mutant lacking ModABC, the high-affinity molybdate transporter, were examined by proteome profiling, Western analysis, epitope tagging, and lacZ reporter fusions. Biological nitrogen fixation (BNF) is a central process in the global nitrogen cycle, which converts chemically inert atmospheric dinitrogen to ammonia, a bioavailable form of nitrogen. BNF is catalyzed by complex metalloenzymes called nitrogenases, which are exclusively found in diazotrophic bacteria and archaea but not in eukaryotes (1). All diazotrophs synthesize a molybdenum-dependent nitrogenase containing an iron-molybdenum cofactor, FeMoco. In addition to Mo-nitrogenases, some bacteria synthesize alternative Mo-free nitrogenases, which contain either an iron-vanadium cofactor, FeVco, or an iron-only cofactor, FeFeco (2). Alternative nitrogenases are less efficient than Monitrogenases in terms of consumption of reducing power and ATP per molecule of N 2 fixed (3, 4). Consequently, diazotrophs preferentially utilize Mo-nitrogenase as long as sufficient molybdate, the only bioavailable form of molybdenum, is available. To support Mo-nitrogenase activity under Mo-limiting conditions, most diazotrophs synthesize a high-affinity molybdate transporter, ModABC (1).The purple nonsulfur alphaproteobacterium Rhodobacter capsulatus is known for its metabolic versatility, and it has been used for decades as a model organism to study photosynthesis, hydrogen production, and nitrogen fixation (5-9). In particular, it is capable of using light energy to generate the ATP required for the energetically demanding nitrogen fixation process. R. capsulatus synthesizes two nitrogenases, namely, a Mo-nitrogenase and a Fenitrogenase but no V-nitrogenase (10, 11). The synthesis and activity of the two nitrogenases are controlled at the transcriptional, translational, and posttranslational levels by a regulatory cascade responding to ammonium and Mo availability (8, 9). Upon ammonium limitation, the nitrogen regulatory protein NtrC becomes activated by phosphorylation. In turn, NtrC-P activates transcription of nifA and anfA, which encode the transcription activators of Mo-nitrogenase and Fe-nitrogenase genes, respectively. At high Mo concentrations, anfA transcription is repressed by two structurally and functionally related Mo-responsive regu-

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

“…For this purpose, we generated transcriptional fusions between P anfH 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.…”

“…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 N 2 ‐reducing activity than Fe‐nitrogenase (Hoffmann, Wagner, et al, 2016; Wiethaus et al, 2006).…”

Molybdate uptake by Agrobacterium tumefaciens correlates with the cellular molybdenum cofactor status

“…Agrobacterium tumefaciens reporter strains carrying FLAG‐fusions were constructed as recently described (Hoffmann et al ., ). Briefly, the modA , moaA , moaE and moeA genes were PCR‐amplified (for primers, see Supporting Information Table S2) with their stop codons exchanged against three cytidine residues (CCC).…”

Coordinated regulation of nitrogen fixation and molybdate transport by molybdenum

“…In R. capsulatus , nifHDK transcription increases about two‐fold upon Mo addition (Hoffmann et al , ). This regulation involves AnfA, the transcriptional activator of the Fe‐nitrogenase genes (our unpublished results).…”

“…). As a consequence, transcription of nitrogen fixation genes increases considerably and nitrogenase proteins accumulate to nearly 10% of soluble proteins in nitrogen‐fixing cells (Dingler et al , ; Schneider et al , ; Hamilton et al , ; Hoffmann et al , ). Preference for Mo‐nitrogenase, the most efficient enzyme, is provided by molybdate‐repressed transcription of V‐ and Fe‐nitrogenase genes as shown for A. variabilis , A. vinelandii and R. capsulatus (Kutsche et al , ; Hamilton et al , ; Thiel and Pratte, ).…”