Overview of physiological, biochemical, and regulatory aspects of nitrogen fixation in <i>Azotobacter vinelandii</i>

Campo, Julia S. Martín del; Rigsbee, Jack; Batista, Marcelo Bueno; Mus, Florence; Rubio, Luis M.; Einsle, Oliver; Peters, John W.; Dixon, Ray; Dean, Dennis R.; Santos, Patricia C. Dos

doi:10.1080/10409238.2023.2181309

Cited by 20 publications

(20 citation statements)

References 294 publications

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“…Asterisks indicate adjusted P < 0.05. Gene and transcriptional unit annotations from Del Campo et al ( 10 ), the latter which mirrors operon predictions based on the transcriptional data presented here (Supplemental file 4).…”

Section: Resultssupporting

confidence: 73%

“…Regulatory precision is necessary due to the high metabolic cost of nitrogen fixation as well as the complex nature of its supporting protein network. For example, the aerobic, diazotrophic model gammaproteobacterium Azotobacter vinelandii has more than 50 proteins that support three nitrogenase isozyme systems, with molecular functions including nitrogenase regulation and assembly, electron transport, and cofactor synthesis ( 8 , 10 , 11 ). The oxygen sensitivity of the nitrogenase metalloenzyme demands that aerobic microbial hosts (desirable models for nitrogen fixation transfer to crop plants such as cereals) utilize particular strategies to ensure metabolic compatibility, including temporal/spatial regulation of nitrogen fixation activity or protection via heightened respiratory rates ( 9 , 12 ).…”

Section: Introductionmentioning

confidence: 99%

“…In this study, we probe the regulatory response of A. vinelandii to a synthetic, ancestral variant of the nitrogenase catalytic protein NifD, previously resurrected from within the direct A. vinelandii evolutionary lineage ( 20 ). The establishment of the A. vinelandii genetic system, which has detailed the nitrogenase regulon structure and expression mechanisms, and the detailed physiological information available from decades of work, makes A. vinelandii an ideal model organism for the transformation of synthetic nitrogenase genes and downstream transcriptional characterization ( 1 , 8 , 10 , 22 , 23 ). The nitrogenase variants of the engineered A. vinelandii strain have reduced N 2 reduction activity, also reflected by reduced cellular nitrogenase substrate reduction rates.…”

Section: Introductionmentioning

confidence: 99%

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Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Rivier,

Myers,

Garcia

et al. 2023

Microbiol Spectr

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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.

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Section: Resultssupporting

confidence: 73%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Rivier,

Myers,

Garcia

et al. 2023

Microbiol Spectr

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“…Relative to known diazotrophs, A. vinelandii possesses a highly complex genetic system for N-fixation, hosting 52 genes within clusters that support the Mo-dependent nitrogenase system (Nif), as well as additional genes that support the alternative V-(Vnf)and Fe-only (Anf) nitrogenases 4, 47 . By comparison, some diazotrophs, namely certain thermophilic archaea, can perform N-fixation with less than ten genes with known counterparts in A. vinelandii 48 .…”

Section: Discussionmentioning

confidence: 99%

A CRISPR interference system for the nitrogen-fixing bacteriumAzotobacter vinelandii

Russell,

Garcia,

Kaçar

2023

Preprint

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A grand challenge for the next century can be found in mitigating the effects of changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a particularly vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium,Azotobacter vinelandii(A. vinelandii). Here we report the first CRISPR interference (CRISPRi) system for targeted gene silencing inA. vinelandiithat integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by ∼60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system extends the utility ofA. vinelandiiand will aid efforts to engineer microbial nitrogen fixation for desired purposes.For Table of Contents Only

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“…So far, three genetically distinct but mechanistically similar nitrogenase isoforms have been described. Among the nitrogenase isoforms, the Mo-dependent enzyme, which is produced by all diazotrophs, has the highest catalytic efficiency making it a favored target for ultimate expression in crop plants ( 9 ). Mo-dependent nitrogenase is a complex two-component metalloenzyme.…”

Section: Introductionmentioning

confidence: 99%

Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of Saccharomyces cerevisiae

Dobrzyńska,

Pérez-González,

Echavarri-Erasun

et al. 2024

mBio

Self Cite

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Biological nitrogen fixation, the conversion of inert N 2 to metabolically tractable NH 3 , is only performed by certain microorganisms called diazotrophs and is catalyzed by the nitrogenases. A [7Fe-9S-C-Mo- R -homocitrate]-cofactor, designated FeMo-co, provides the catalytic site for N 2 reduction in the Mo-dependent nitrogenase. Thus, achieving FeMo-co formation in model eukaryotic organisms, such as Saccharomyces cerevisiae , represents an important milestone toward endowing them with a capacity for Mo-dependent biological nitrogen fixation. A central player in FeMo-co assembly is the scaffold protein NifEN upon which processing of NifB-co, an [8Fe-9S-C] precursor produced by NifB, occurs. Prior work established that NifB-co can be produced in S. cerevisiae mitochondria. In the present work, a library of nifEN genes from diverse diazotrophs was expressed in S. cerevisiae , targeted to mitochondria, and surveyed for their ability to produce soluble NifEN protein complexes. Many such NifEN variants supported FeMo-co formation when heterologously produced in the diazotroph A. vinelandii . However, only three of them accumulated in soluble forms in mitochondria of aerobically cultured S. cerevisiae . Of these, two variants were active in the in vitro FeMo-co synthesis assay. NifEN, NifB, and NifH proteins from different species, all of them produced in and purified from S. cerevisiae mitochondria, were combined to establish successful FeMo-co biosynthetic pathways. These findings demonstrate that combining diverse interspecies nitrogenase FeMo-co assembly components could be an effective and, perhaps, the only approach to achieve and optimize nitrogen fixation in a eukaryotic organism. IMPORTANCE Biological nitrogen fixation, the conversion of inert N2 to metabolically usable NH3, is a process exclusive to diazotrophic microorganisms and relies on the activity of nitrogenases. The assembly of the nitrogenase [7Fe-9S-C-Mo- R -homocitrate]-cofactor (FeMo-co) in a eukaryotic cell is a pivotal milestone that will pave the way to engineer cereals with nitrogen fixing capabilities and therefore independent of nitrogen fertilizers. In this study, we identified NifEN protein complexes that were functional in the model eukaryotic organism Saccharomyces cerevisiae . NifEN is an essential component of the FeMo-co biosynthesis pathway. Furthermore, the FeMo-co biosynthetic pathway was recapitulated in vitro using only proteins expressed in S. cerevisiae . FeMo-co biosynthesis was achieved by combining nitrogenase FeMo-co assembly components from different species, a promising strategy to engineer nitrogen fixation in eukaryotic organisms.

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Overview of physiological, biochemical, and regulatory aspects of nitrogen fixation in Azotobacter vinelandii

Cited by 20 publications

References 294 publications

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

Regulatory response to a hybrid ancestral nitrogenase in Azotobacter vinelandii

A CRISPR interference system for the nitrogen-fixing bacteriumAzotobacter vinelandii

Nitrogenase cofactor biosynthesis using proteins produced in mitochondria of Saccharomyces cerevisiae

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