Dataset Information

Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii.

ABSTRACT: Rnf proteins are proposed to form membrane-protein complexes involved in the reduction of target proteins such as the transcriptional regulator SoxR or the dinitrogenase reductase component of nitrogenase. In this work, we investigate the role of rnf genes in the nitrogen-fixing bacterium Azotobacter vinelandii. We show that A. vinelandii has two clusters of rnf-like genes: rnf1, whose expression is nif-regulated, and rnf2, which is expressed independently of the nitrogen source in the medium. Deletion of each of these gene clusters produces a time delay in nitrogen-fixing capacity and, consequently, in diazotrophic growth. Deltarnf mutations cause two distinguishable effects on the nitrogenase system: (i), slower nifHDK gene expression and (ii), impairment of nitrogenase function. In these mutants, dinitrogenase reductase activity is lowered, whereas dinitrogenase activity remains essentially unaltered. Further analysis indicates that deltarnf mutants accumulate an inactive and iron-deficient form of NifH because they have lower rates of incorporation of [4Fe-4S] into NifH. Deltarnf mutations also cause a noticeable decrease in aconitase activity; however, they do not produce general oxidative stress or modification of Fe metabolism in A. vinelandii. Our results suggest the existence of a redox regulatory mechanism in A. vinelandii that controls the rate of expression and maturation of nitrogenase by the activity of the Rnf protein complexes. rnf1 plays a major and more specific role in this scheme, but the additive effects of mutations in rnf1 and rnf2 indicate the existence of functional complementation between the two homologous systems.

SUBMITTER: Curatti L

PROVIDER: S-EPMC1088376 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii.

Curatti Leonardo L Brown Carolyn S CS Ludden Paul W PW Rubio Luis M LM

Proceedings of the National Academy of Sciences of the United States of America 20050421 18

Rnf proteins are proposed to form membrane-protein complexes involved in the reduction of target proteins such as the transcriptional regulator SoxR or the dinitrogenase reductase component of nitrogenase. In this work, we investigate the role of rnf genes in the nitrogen-fixing bacterium Azotobacter vinelandii. We show that A. vinelandii has two clusters of rnf-like genes: rnf1, whose expression is nif-regulated, and rnf2, which is expressed independently of the nitrogen source in the medium. D ...[more]

PMID: 15845763

Similar Datasets

Project description:Biological nitrogen fixation, the microbial reduction of atmospheric nitrogen to bioavailable ammonia, represents both a major limitation on biological productivity and a highly desirable engineering target for synthetic biology. However, the engineering of nitrogen fixation requires an integrated understanding of how the gene regulatory dynamics of host diazotrophs respond across sequence-function space of its central catalytic metalloenzyme, nitrogenase. Here, we interrogate this relationship by analyzing the transcriptome of Azotobacter vinelandii engineered with a phylogenetically inferred ancestral nitrogenase protein variant. The engineered strain exhibits reduced cellular nitrogenase activity but recovers wild-type growth rates following an extended lag period. We find that expression of genes within the immediate nitrogen fixation network is resilient to the introduced nitrogenase sequence-level perturbations. Rather the sustained physiological compatibility with the ancestral nitrogenase variant is accompanied by reduced expression of genes that support trace metal and electron resource allocation to nitrogenase. Our results spotlight gene expression changes in cellular processes adjacent to nitrogen fixation as productive engineering considerations to improve compatibility between remodeled nitrogenase proteins and engineered host diazotrophs. IMPORTANCE Azotobacter vinelandii is a key model bacterium for the study of biological nitrogen fixation, an important metabolic process catalyzed by nitrogenase enzymes. Here, we demonstrate that compatibilities between engineered A. vinelandii strains and nitrogenase variants can be modulated at the regulatory level. The engineered strain studied here responds by adjusting the expression of proteins involved in cellular processes adjacent to nitrogen fixation, rather than that of nitrogenase proteins themselves. These insights can inform future strategies to transfer nitrogenase variants to non-native hosts.

Project description:The functional versatility of the Fe protein, the reductase component of nitrogenase, makes it an appealing target for heterologous expression, which could facilitate future biotechnological adaptations of nitrogenase-based production of valuable chemical commodities. Yet, the heterologous synthesis of a fully active Fe protein of Azotobacter vinelandii (AvNifH) in Escherichia coli has proven to be a challenging task. Here, we report the successful synthesis of a fully active AvNifH protein upon co-expression of this protein with AvIscS/U and AvNifM in E. coli. Our metal, activity, electron paramagnetic resonance, and X-ray absorption spectroscopy/extended X-ray absorption fine structure (EXAFS) data demonstrate that the heterologously expressed AvNifH protein has a high [Fe4S4] cluster content and is fully functional in nitrogenase catalysis and assembly. Moreover, our phylogenetic analyses and structural predictions suggest that AvNifM could serve as a chaperone and assist the maturation of a cluster-replete AvNifH protein. Given the crucial importance of the Fe protein for the functionality of nitrogenase, this work establishes an effective framework for developing a heterologous expression system of the complete, two-component nitrogenase system; additionally, it provides a useful tool for further exploring the intricate biosynthetic mechanism of this structurally unique and functionally important metalloenzyme. IMPORTANCE The heterologous expression of a fully active Azotobacter vinelandii Fe protein (AvNifH) has never been accomplished. Given the functional importance of this protein in nitrogenase catalysis and assembly, the successful expression of AvNifH in Escherichia coli as reported herein supplies a key element for the further development of heterologous expression systems that explore the catalytic versatility of the Fe protein, either on its own or as a key component of nitrogenase, for nitrogenase-based biotechnological applications in the future. Moreover, the "clean" genetic background of the heterologous expression host allows for an unambiguous assessment of the effect of certain nif-encoded protein factors, such as AvNifM described in this work, in the maturation of AvNifH, highlighting the utility of this heterologous expression system in further advancing our understanding of the complex biosynthetic mechanism of nitrogenase.

Project description:The nitrogen-fixing microbe Azotobacter vinelandii has the ability to produce three genetically distinct, but mechanistically similar, components that catalyze nitrogen fixation. For two of these components, the Mo-dependent and V-dependent components, their corresponding metal-containing active site cofactors, designated FeMo-cofactor and FeV-cofactor, respectively, are preformed on separate molecular scaffolds designated NifEN and VnfEN, respectively. From prior studies, and the present work, it is now established that neither of these scaffolds can replace the other with respect to their in vivo cofactor assembly functions. Namely, a strain inactivated for NifEN cannot produce active Mo-dependent nitrogenase nor can a strain inactivated for VnfEN produce an active V-dependent nitrogenase. It is therefore proposed that metal specificities for FeMo-cofactor and FeV-cofactor formation are supplied by their respective assembly scaffolds. In the case of the third, Fe-only component, its associated active site cofactor, designated FeFe-cofactor, requires neither the NifEN nor VnfEN assembly scaffold for its formation. Furthermore, there are no other genes present in A. vinelandii that encode proteins having primary structure similarity to either NifEN or VnfEN. It is therefore concluded that FeFe-cofactor assembly is completed within its cognate catalytic protein partner without the aid of an intermediate assembly site. IMPORTANCE Biological nitrogen fixation is a complex process involving the nitrogenases. The biosynthesis of an active nitrogenase involves a large number of genes and the coordinated function of their products. Understanding the details of the assembly and activation of the different nitrogen fixation components, in particular the simplest one known so far, the Fe-only nitrogenase, would contribute to the goal of transferring the necessary genetic elements of bacterial nitrogen fixation to cereal crops to endow them with the capacity for self-fertilization. In this work, we show that there is no need for a scaffold complex for the assembly of the FeFe-cofactor, which provides the active site for Fe-only nitrogenase. These results are in agreement with previously reported genetic reconstruction experiments using a non-nitrogen-fixing microbe. In aggregate, these findings provide a high degree of confidence that the Fe-only system represents the simplest and, therefore, most attractive target for mobilizing nitrogen fixation into plants.

Dataset Information

Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii.

Publications

Genes required for rapid expression of nitrogenase activity in Azotobacter vinelandii.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets