Kinetics of
            <i>nif</i>
            Gene Expression in a Nitrogen-Fixing Bacterium

Poza‐Carrión, César; Jiménez-Vicente, Emilio; Navarro-Rodríguez, Mónica; Echávarri-Erasun, Carlos; Rubio, Luis M.

doi:10.1128/jb.00942-13

Cited by 68 publications

(70 citation statements)

References 48 publications

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“…20 and Supplementary Note 10). To our knowledge, this was the first transcriptomics investigation of nitrogen fixation in K. oxytoca, although the cluster has been investigated in other organisms [28][29][30] . The different operons within the nif cluster were transcribed to different levels (Fig.…”

Section: Resultsmentioning

confidence: 96%

Functional optimization of gene clusters by combinatorial design and assembly

et al. 2014

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Large microbial gene clusters encode useful functions, including energy utilization and natural product biosynthesis, but genetic manipulation of such systems is slow, difficult and complicated by complex regulation. We exploit the modularity of a refactored Klebsiella oxytoca nitrogen fixation (nif) gene cluster (16 genes, 103 parts) to build genetic permutations that could not be achieved by starting from the wild-type cluster. Constraint-based combinatorial design and DNA assembly are used to build libraries of radically different cluster architectures by varying part choice, gene order, gene orientation and operon occupancy. We construct 84 variants of the nifUSVWZM operon, 145 variants of the nifHDKY operon, 155 variants of the nifHDKYENJ operon and 122 variants of the complete 16-gene pathway. The performance and behavior of these variants are characterized by nitrogenase assay and strand-specific RNA sequencing (RNA-seq), and the results are incorporated into subsequent design cycles. We have produced a fully synthetic cluster that recovers 57% of wild-type activity. Our approach allows the performance of genetic parts to be quantified simultaneously in hundreds of genetic contexts. This parallelized design-build-test-learn cycle, which can access previously unattainable regions of genetic space, should provide a useful, fast tool for genetic optimization and hypothesis testing.

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Section: Resultsmentioning

confidence: 96%

Functional optimization of gene clusters by combinatorial design and assembly

et al. 2014

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“…However, NifH levels were slightly higher in the Δ fdxN mutant, and it is known that nifHDK gene expression is driven by a common promoter upstream nifH . Apart from NifH, the Δ fdxN mutant accumulated higher levels of other proteins involved in FeMo‐co synthesis, such as NifU, NifB, NifQ and NifEN [40]. This phenotype can be interpreted as a compensatory mechanism to increase nitrogenase activity.…”

Section: Discussionmentioning

confidence: 96%

Role of Azotobacter vinelandii FdxN in FeMo‐co biosynthesis

et al. 2013

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a b s t r a c tBiosynthesis of metal clusters for the nitrogenase component proteins NifH and NifDK involves electron donation events. Yet, electron donors specific to the biosynthetic pathways of the [4Fe-4S] cluster of NifH, or the P-cluster and the FeMo-co of NifDK, have not been identified. Here we show that an Azotobacter vinelandii mutant lacking fdxN was specifically impaired in FeMo-co biosynthesis. The DfdxN mutant produced 5-fold less NifB-co, an early FeMo-co biosynthetic intermediate, than wild type. As a consequence, it accumulated FeMo-co-deficient apo-NifDK and was impaired in Nif-DK activity. We conclude that FdxN plays a role in FeMo-co biosynthesis, presumably by donating electrons to support NifB-co synthesis by NifB. This is the first role in nitrogenase biosynthesis unequivocally assigned to any A. vinelandii ferredoxin.

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“…9−11 To add more complexity, Nif protein stoichiometry has been shown to be dynamic and strongly regulated. 12 …”

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confidence: 99%

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

et al. 2017

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Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell.

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Kinetics of nif Gene Expression in a Nitrogen-Fixing Bacterium

Cited by 68 publications

References 48 publications

Functional optimization of gene clusters by combinatorial design and assembly

Functional optimization of gene clusters by combinatorial design and assembly

Role of Azotobacter vinelandii FdxN in FeMo‐co biosynthesis

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

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