A methodology for markerless genetic modifications in
            <i>Azotobacter vinelandii</i>

Eberhart, Lauren J.; Knutson, Carolann M.; Barney, Brett M.

doi:10.1111/jam.13091

Cited by 12 publications

(9 citation statements)

References 37 publications

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“…The construction of A. vinelandii strain AZBB163, which is deregulated for nitrogen fixation and produces enhanced levels of ammonium in the growth medium, was previously described (4,10,12). The construction of a strain lacking the ability to synthesize polyhydroxybutyrate (AZBB131) was described previously (38). A. vinelandii strains AZBB326 and AZBB328 were constructed as detailed in Table 1, by transforming AZBB163 with the plasmids described in Table 3.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

Barney

Plunkett

Velmurugan

et al. 2017

Appl Environ Microbiol

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Biological nitrogen fixation is accomplished by a diverse group of organisms known as diazotrophs and requires the function of the complex metalloenzyme nitrogenase. Nitrogenase and many of the accessory proteins required for proper cofactor biosynthesis and incorporation into the enzyme have been characterized, but a complete picture of the reaction mechanism and key cellular changes that accompany biological nitrogen fixation remain to be fully elucidated. Studies have revealed that specific disruptions of the antiactivator-encoding gene result in the deregulation of the transcriptional activator NifA in the nitrogen-fixing bacterium , triggering the production of extracellular ammonium levels approaching 30 mM during the stationary phase of growth. In this work, we have characterized the global patterns of gene expression of this high-ammonium-releasing phenotype. The findings reported here indicated that cultures of this high-ammonium-accumulating strain may experience metal limitation when grown using standard Burk's medium, which could be amended by increasing the molybdenum levels to further increase the ammonium yield. In addition, elevated levels of nitrogenase gene transcription are not accompanied by a corresponding dramatic increase in hydrogenase gene transcription levels or hydrogen uptake rates. Of the three potential electron donor systems for nitrogenase, only the gene cluster showed a transcriptional correlation to the increased yield of ammonium. Our results also highlight several additional genes that may play a role in supporting elevated ammonium production in this aerobic nitrogen-fixing model bacterium. The transcriptional differences found during stationary-phase ammonium accumulation show a strong contrast between the deregulated (-disrupted) and wild-type strains and what was previously reported for the wild-type strain under exponential-phase growth conditions. These results demonstrate that further improvement of the ammonium yield in this nitrogenase-deregulated strain can be obtained by increasing the amount of available molybdenum in the medium. These results also indicate a potential preference for one of two ATP synthases present in as well as a prominent role for the membrane-bound hydrogenase over the soluble hydrogenase in hydrogen gas recycling. These results should inform future studies aimed at elucidating the important features of this phenotype and at maximizing ammonium production by this strain.

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Section: Methodsmentioning

confidence: 99%

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

Barney

Plunkett

Velmurugan

et al. 2017

Appl Environ Microbiol

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“…We hypothesized that if the amtB gene were deleted, it might result in a higher rate of ammonium accumulation within the supernatant. Using a strain from a previous report [11,26], the nifL deletion that results in extracellular ammonium accumulation was added to a strain containing the amtB deletion, creating A. vinelandii strain AZBB281. When evaluated for ammonium production, our results demonstrated that AZBB281 accumulated ammonium in the supernatant at a faster rate as compared to AZBB163 at both day 2 and day 3 ( Fig.…”

Section: Disruption Of a Futile Cycle Associated With Ammonium Importmentioning

confidence: 99%

“…Detailed methods for the transformation of A. vinelandii have been described previously [26,42]. Transformation of A. vinelandii DJ in order to modify target genes utilizing double homologous recombination with the pyrF counter-selection technique were implemented as described previously [11,26]. Details for strains constructed for use in this study are provided in Table 3.…”

Section: Genetic Constructs Gene Deletion and Confirmationmentioning

confidence: 99%

“…For experiments measuring the levels of sucrose in the medium, the Sigma-Aldrich kit (Part No: MAK013-1KT) was used as directed by the manufacturer. Strains indicated by an asterisk (*) are completed and were used in ammonium production experiments a As described previously [26] b As described previously [11] c As described previously [6] A…”

Section: Sucrose Quantificationmentioning

confidence: 99%

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Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation

2020

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Background: The obligate aerobe Azotobacter vinelandii is a model organism for the study of biological nitrogen fixation (BNF). This bacterium regulates the process of BNF through the two component NifL and NifA system, where NifA acts as an activator, while NifL acts as an anti-activator based on various metabolic signals within the cell. Disruption of the nifL component in the nifLA operon in a precise manner results in a deregulated phenotype that produces levels of ammonium that far surpass the requirements within the cell, and results in the release of up to 30 mM of ammonium into the growth medium. While many studies have probed the factors affecting growth of A. vinelandii, the features important to maximizing this high-ammonium-releasing phenotype have not been fully investigated. Results: In this work, we report the effect of temperature, medium composition, and oxygen requirements on sustaining and maximizing elevated levels of ammonium production from a nitrogenase deregulated strain. We further investigated several pathways, including ammonium uptake through the transporter AmtB, which could limit yields through energy loss or futile recycling steps. Following optimization, we compared sugar consumption and ammonium production, to attain correlations and energy requirements to drive this process in vivo. Ammonium yields indicate that between 5 and 8% of cellular protein is fully active nitrogenase MoFe protein (NifDK) under these conditions. Conclusions: These findings provide important process optimization parameters, and illustrate that further improvements to this phenotype can be accomplished by eliminating futile cycles.

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“…vinelandii DJ is a very well-studied obligate aerobe with an unusual metabolism that allows nitrogen fixation at atmospheric conditions . It is an exceptional model due to its natural competency under metal-starved conditions as well as expression of three different nitrogenase enzymatic complexes (MoFe, VFe, and FeFe cores). − It was used previously to elucidate the structure , of the nitrogenase enzyme complex. Using homologous recombination with antibiotic selection, as well as sacB counterselection, A.…”

Section: Introductionmentioning

confidence: 99%

Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes

Venkataraman,

Yñigez-Gutierrez,

Infante

et al. 2023

ACS Synth. Biol.

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A methodology for markerless genetic modifications in Azotobacter vinelandii

Abstract: This study demonstrates the successful application of a counter-selection approach to yield markerless genetic modifications to A. vinelandii, which should be of interest for a range of applications in this important model bacterium.

Cited by 12 publications

References 37 publications

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

Transcriptional Analysis of an Ammonium-Excreting Strain of Azotobacter vinelandii Deregulated for Nitrogen Fixation

Key factors affecting ammonium production by an Azotobacter vinelandii strain deregulated for biological nitrogen fixation

Synthetic Biology Toolbox for Nitrogen-Fixing Soil Microbes

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