Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii

Hamilton, Trinity L.; Ludwig, Marcus; Dixon, Ray; Boyd, Eric S.; Santos, Patricia C. Dos; Setúbal, João Carlos; Bryant, Donald A.; Dean, Dennis R.; Peters, John W.

doi:10.1128/jb.05099-11

Cited by 97 publications

(142 citation statements)

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“…However, there are other nitrogenase-like sequences encoded in the R. capsulatus genome, which could potentially perform this function (23). In contrast, the increase of vnfENX expression in iron-only conditions in A. vinelandii (14), coupled with the absence of diazotrophic growth in double mutants lacking nifEN and vnfE (13), strongly supports a role for the VnfEN scaffold in FeFe nitrogenase maturation in this organism. Although it is difficult to rationalize the discrepancy between our results and the results observed in A. vinelandii, it is important to emphasize that the strictly aerobic lifestyle of Azotobacter is very different from the facultative anaerobe E. coli, which can only support nitrogen fixation under anaerobic conditions.…”

Section: Discussionmentioning

confidence: 94%

“…Furthermore, some nif genes (e.g., electron transfer components) are specifically adapted to support nitrogenase activity in accordance with the physiological requirements of the host organism. Notably, alternative nitrogenases are only found in species that also encode a molybdenum-dependent nitrogenase (14,23).…”

Section: Resultsmentioning

confidence: 99%

“…These genes provide scaffolds and carriers for the final maturation of the cofactor, enabling insertion of the heterometal (Mo or V, respectively) and homocitrate in a reaction catalyzed by dinitrogenase reductase (Fe protein). Genetic analysis and transcriptome profiling of A. vinelandii indicate that the anf system is dependent on vnfEN for maturation of the FeFe cofactor in the iron-only nitrogenase (13,14). Finally, nifM, which potentially encodes a peptidyl-prolyl cis-trans isomerase required for proper folding of the Fe protein (15), is thought to be required for all three types of nitrogenase (16).…”

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Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Yang

Xie

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium-or irondependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Yang

Xie

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Genome sequences were screened for the presence of homologs of nifNXZYQVSUFMWALI 1 I 2 OT and nafY, iscA lrv, and clpX (associated with nif), and manual assignments were verified by using reciprocal BLASTp (see Table S1 in the supplemental material). The composition of gene clusters associated with alternative nitrogenases, which have yet to be identified in a genome that does not also encode nif (5, 6), were not included in phylogenetic and evolutionary analyses, since they appear to utilize components of the nif system to synthesize their active site metalloclusters (19). The FixABCX protein sequences (encoded by fixABCX) from A. vinelandii were used as BLASTp queries in order to determine if the 189 genomes of taxa harboring homologs of nifHDKEB also encoded Fix.…”

Section: Methodsmentioning

confidence: 99%

Evolution of Molybdenum Nitrogenase during the Transition from Anaerobic to Aerobic Metabolism

Boyd¹,

Costas²,

Hamilton³

et al. 2015

J. Bacteriol.

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Molybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Phylogenetic evidence indicates that oxygen (O 2 )-sensitive Nif emerged in an anaerobic archaeon and later diversified into an aerobic bacterium. Aerobic bacteria that fix N 2 have adapted a number of strategies to protect Nif from inactivation by O 2 , including spatial and temporal segregation of Nif from O 2 and respiratory consumption of O 2 . Here we report the complement of Nif-encoding genes in 189 diazotrophic genomes. We show that the evolution of Nif during the transition from anaerobic to aerobic metabolism was accompanied by both gene recruitment and loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes and their phylogenetic distribution are strongly correlated with adaptation to utilize O 2 in metabolism, the increase is not correlated with any of the known O 2 protection mechanisms. Rather, gene recruitment appears to have been in response to selective pressure to optimize Nif synthesis to meet fixed N demands associated with aerobic productivity and to more efficiently regulate Nif under oxic conditions that favor protein turnover. Consistent with this hypothesis, the transition of Nif from anoxic to oxic environments is associated with a shift from posttranslational regulation in anaerobes to transcriptional regulation in obligate aerobes and facultative anaerobes. Given that fixed nitrogen typically limits ecosystem productivity, our observations further underscore the dynamic interplay between the evolution of Earth's oxygen, nitrogen, and carbon biogeochemical cycles. IMPORTANCEMolybdenum nitrogenase (Nif), which catalyzes the reduction of dinitrogen to ammonium, has modulated the availability of fixed nitrogen in the biosphere since early in Earth's history. Nif emerged in an anaerobe and later diversified into aerobes. Here we show that the transition of Nif from anaerobic to aerobic metabolism was accompanied by both gene recruitment and gene loss, resulting in a substantial increase in the number of nif genes. While the observed increase in the number of nif genes is strongly correlated with adaptation to utilize O 2 in metabolism, the increase is not correlated with any of the known O 2 protective mechanisms. Rather, gene recruitment was likely a response to more efficiently regulate Nif under oxic conditions that favor protein turnover.A ll life requires fixed nitrogen (N), and its availability often limits ecosystem productivity (1, 2). Most of the N on Earth is in the form of dinitrogen (N 2 ), which is unreactive, bio-unavailable, and must be chemically reduced to ammonium (NH 4 ϩ ) before it can be incorporated into biological molecules, such as proteins or nucleic acids. The primary enzyme that catalyzes the reduction of N 2 to NH 4 ϩ is the molybdenum (Mo)-dependent nitrogenase Nif (3, 4). Our recent phylogenetic studies...

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“…In line with these observations, Northern blot hybridization analyses have indicated differential expression of the nif structural proteins (7), and immunoblotting has shown that the Fe protein occurs in significant excess compared to MoFe protein levels in cultures grown under diazotrophic conditions (2, 7). Moreover, global transcriptional analyses indicate that nifH mRNA accumulates at ϳ3-fold-higher levels than nifD and nifK messages in A. vinelandii grown diazotrophically (4). Because the genes encoding the nitrogenase Fe protein and MoFe protein are cotranscribed in A. vinelandii, a mechanism must exist to control differential nif structural gene transcript abundance.…”

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Differential Accumulation of nif Structural Gene mRNA in Azotobacter vinelandii

et al. 2011

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Northern analysis was employed to investigate mRNA produced by mutant strains of Azotobacter vinelandii with defined deletions in the nif structural genes and in the intergenic noncoding regions. The results indicate that intergenic RNA secondary structures effect the differential accumulation of transcripts, supporting the high Fe protein-to-MoFe protein ratio required for optimal diazotrophic growth.Biological nitrogen fixation occurs by the activity of nitrogenase, which exists as a complex metalloenzyme composed of two easily separable components. The Fe protein component (encoded by nifH) serves as the obligate electron donor to the MoFe protein component (encoded by nifDK), which contains the active site for dinitrogen reduction (12). In addition to the structural proteins, the nitrogenase enzyme requires extensive biosynthetic machinery consisting of enzymes, scaffolds, and carrier proteins to assemble the metalloclusters required for catalysis (3,15). nif genes are located in two clusters in the model diazotroph, Azotobacter vinelandii. The major nif cluster contains the structural genes and those for most of the biosynthetic machinery, which are organized in several contiguous operons (6), and a minor nif cluster contains the nif regulatory elements and the remaining, necessary biosynthetic genes (8). The major nif cluster in A. vinelandii was sequenced more than 3 decades ago and has been the subject of a number of gene deletion and mutation studies, which laid the groundwork for our current understanding of the operon structure and regulation of nif (6). The genes encoding the nitrogenase structural proteins, nifH, nifD, and nifK, are located in the major nif operon and are cotranscribed from a single promoter, the nifH promoter, along with nifT, nifY, orf1, and lrv (6). The nifH promoter is efficient and is effectively regulated, driving the rapid and abundant expression of nitrogenase in the absence of fixed N and conversely an abrupt decline in nitrogenase component transcription in the presence of fixed N (16).

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Transcriptional Profiling of Nitrogen Fixation in Azotobacter vinelandii

Cited by 97 publications

References 71 publications

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Evolution of Molybdenum Nitrogenase during the Transition from Anaerobic to Aerobic Metabolism

Differential Accumulation of nif Structural Gene mRNA in Azotobacter vinelandii

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