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 (N2) 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. Many Mo-controlled proteins identified in this study have documented or presumed roles in nitrogen fixation, demonstrating the relevance of Mo control in this highly ATP-demanding process. The levels of Mo-nitrogenase, NifHDK, and the Mo storage protein, Mop, increased with increasing Mo concentrations. In contrast, Fe-nitrogenase, AnfHDGK, and ModABC, the Mo transporter, were expressed only under Mo-limiting conditions. IscN was identified as a novel Mo-repressed protein. Mo control of Mop, AnfHDGK, and ModABC corresponded to transcriptional regulation of their genes by the Mo-responsive regulators MopA and MopB. Mo control of NifHDK and IscN appeared to be more complex, involving different posttranscriptional mechanisms. In line with the simultaneous control of IscN and Fe-nitrogenase by Mo, IscN was found to be important for Fe-nitrogenase-dependent diazotrophic growth. The possible role of IscN as an A-type carrier providing Fe-nitrogenase with Fe-S clusters is discussed.

Importance: Biological nitrogen fixation is a central process in the global nitrogen cycle by which the abundant but chemically inert dinitrogen (N2) is reduced to ammonia (NH3), a bioavailable form of nitrogen. Nitrogen reduction is catalyzed by nitrogenases found in diazotrophic bacteria and archaea but not in eukaryotes. All diazotrophs synthesize molybdenum-dependent nitrogenases. In addition, some diazotrophs, including Rhodobacter capsulatus, possess catalytically less efficient alternative Mo-free nitrogenases, whose expression is repressed by Mo. Despite the importance of Mo in biological nitrogen fixation, this is the first study analyzing the proteome-wide Mo response in a diazotroph. IscN was recognized as a novel member of the molybdoproteome in R. capsulatus. It was dispensable for Mo-nitrogenase activity but supported diazotrophic growth under Mo-limiting conditions.

FIG 1

Accumulation of Mo-nitrogenase proteins and nifK-lacZ expression. R. capsulatus strains were grown in RCV minimal medium with the indicated molybdate concentrations. The strains used were as follows: B10S, the wild-type strain (A and C); R438, the ΔmodABC mutant (B and D); B10S::pYP348, the wild-type strain carrying nifK-lacZ (E); and R438::pYP348, the ΔmodABC mutant carrying nifK-lacZ (F). (A to D) Western analyses were performed using antisera against NifH (A and B) or NifDK (C and D). “NifH*” marks the ADP-ribosylated form of NifH. Molecular weight data are given in thousands. (E and F) LacZ (β-galactosidase) activity is given in Miller units (MU) (23). The results represent the means and standard deviations of the results of at least three independent measurements.

FIG 2

Expression of mop-lacZ and Mop-FLAG accumulation. R. capsulatus strains were grown in RCV minimal medium with the indicated molybdate concentrations. (A and B) The strains used were B10S::pEW18, the wild-type strain carrying mop-lacZ (A), and B10S::pEW10, the wild-type strain carrying mop-FLAG (B). (A) LacZ (β-galactosidase) activity is given in Miller units (23). The results represent the means and standard deviations of the results of at least three independent measurements. (B) Western analysis was performed using FLAG antibodies. Molecular weight data are given in thousands.

FIG 3

Expression of iscN-FLAG, iscN-lacZ, nifU1-FLAG, and nifU1-lacZ reporter fusions. R. capsulatus strains were grown in RCV minimal medium with the indicated molybdate concentrations. The strains used were as follows: B10S::pEW13, the wild-type strain carrying iscN-FLAG (A); B10S::pEW58, the wild-type strain carrying iscN-lacZ (B); B10S::pEW51, the wild-type strain carrying nifU1-FLAG (C); and B10S::pEW53, the wild-type strain carrying nifU1-lacZ (D). (A and C) Western analysis was performed using FLAG antibodies. Molecular weight data are given in thousands. (B and D) LacZ (β-galactosidase) activity is given in Miller units (23). The results represent the means and standard deviations of the results of at least three independent measurements.

FIG 4

Diazotrophic growth of iscN mutant strains. (A and B) To examine diazotrophic growth, R. capsulatus strains were grown in RCV minimal medium under a pure N₂ atmosphere. The strains used in the experiments represented by panel A were B10S (wild type; open circles) and YP340 (ΔiscN mutant; filled circles). The strains used in the experiments represented by panel B were BS85 (ΔnifDK mutant; open squares) and BS85-YP340 (ΔnifDK-ΔiscN mutant; filled squares). Growth experiments were done three times, and one representative data set is shown. In addition, the ΔnifDK and ΔnifDK-ΔiscN strains were grown in RCV medium containing serine. (C) Samples taken at the logarithmic phase (Log phase) and the stationary phase (Stat phase) were examined by Western analysis. Antibodies raised against R. capsulatus NifDK were used to detect AnfD and AnfK proteins. Molecular weight data are given in thousands.