Genomic Manipulations of the Diazotroph Azotobacter vinelandii

Santos, Patricia C. Dos

doi:10.1007/978-1-4939-8864-8_6

Cited by 13 publications

(21 citation statements)

References 79 publications

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“…Since its discovery more than 100 years ago (Lipman, 1903), A. vinelandii has served a variety of scientific and industrial applications. These include the study of aerobic respiration (Poole & Hill, 1997), hydrogen uptake and production (Kow & Burris, 1984;Noar, Loveless, Navarro-Herrero, Olson, & Bruno-Barcena, 2015), polymer production (Clementi, 1997;Galindo, Pena, Nunez, Segura, & Espin, 2007), and, importantly, the genetics and biochemistry of nitrogenasecatalyzed nitrogen fixation (Dos Santos, 2019;Hoffman et al, 2014;Noar & Bruno-Barcena, 2018). Due to the challenge of heterologous expression of functional nitrogenases A. vinelandii has been used for their expression, purification, and biochemical characterization (Hoffman et al, 2014;Lee, Ribbe, & Hu, 2019;Solomon et al, 2020).…”

Section: Commentary Background Informationmentioning

confidence: 99%

“…Azotobacter vinelandii ( A. vinelandii ), a Gram‐negative, obligately aerobic, diazotrophic Gamma proteobacterium, has been an important model organism since its discovery more than a century ago (Lipman, 1903). One of the most significant contributions of the A. vinelandii model has been toward understanding the evolution, genetics, and biochemistry of nitrogenase‐catalyzed nitrogen fixation (Dos Santos, 2019; Hoffman, Lukoyanov, Yang, Dean, & Seefeldt, 2014; Hu, Fay, Lee, Wiig, & Ribbe, 2010; Noar & Bruno‐Barcena, 2018). A. vinelandii growth assays are utilized to evaluate the physiological impact of genomic modifications and varied environmental conditions on diazotrophic growth (Arragain et al., 2017; McRose, Baars, Morel, & Kraepiel, 2017; Mus, Tseng, Dixon, & Peters, 2017; Plunkett, Knutson, & Barney, 2020).…”

Section: Introductionmentioning

confidence: 99%

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Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii

et al. 2021

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Azotobacter vinelandii (A. vinelandii) is a commonly used model organism for the study of aerobic respiration, the bacterial production of several industrially relevant compounds, and, perhaps most significantly, the genetics and biochemistry of biological nitrogen fixation. Laboratory growth assessments of A. vinelandii are useful for evaluating the impact of environmental and genetic modifications on physiological properties, including diazotrophy. However, researchers typically rely on manual growth methods that are oftentimes laborious and inefficient. We present a protocol for the automated growth assessment of A. vinelandii on a microplate reader, particularly well-suited for studies of diazotrophic growth. We discuss common pitfalls and strategies for protocol optimization, and demonstrate the protocol's application toward growth evaluation of strains carrying modifications to nitrogen-fixation genes.

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Section: Commentary Background Informationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii

et al. 2021

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“…Nucleotide sequences for ancestral nitrogenase proteins were codon-optimized for expression in A. vinelandii , synthesized, and cloned into plasmids for subsequent genomic engineering and protein purification (see Materials and Methods). We leveraged a two-step strategy for markerless integration of ancestral nitrogenase genes into the A. vinelandii genome, following Dos Santos (24). Native nifD or nifHDK genes were first knocked out by a selective marker followed by replacement of the marker with the synthetic ancient gene(s), yielding three engineered strains: AK013, AK023, and AK014.…”

Section: Resultsmentioning

confidence: 99%

“…Engineering of A. vinelandii strains followed Dos Santos (24). A. vinelandii WT and DJ2278 (Δ nifD:: KmR) strains were generously provided by Dennis Dean (Virginia Tech).…”

Section: Methodsmentioning

confidence: 99%

Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

Garcia

Harris

Rivier

et al. 2022

Preprint

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Understanding the coevolutionary dynamics between proteins, cellular networks, and environmental pressures remains an outstanding challenge in biology. The effects of molecular constraints on the development of metabolic pathways that shape the biosphere remain underexplored. Extant genes, relics of a >3-billion-year history of life, offer the means to experimentally reconstruct protein evolutionary trajectories. Surveying life s extinct genetic repertoire may unlock adaptive states to environmental conditions unobserved today. However, the lack of suitable experimental systems poses a hurdle for such strategies, particularly for the reconstruction of biogeochemically critical ancient proteins and their supporting networks. To fill this gap, here we developed an evolutionarily guided, systems biology and biochemistry approach for the resurrection and functional characterization of ancient nitrogen fixation machinery in Azotobacter vinelandii. We report the recovery of active, ancestral nitrogenase enzymes and observe the conservation of core, catalytic features that are robust to numerous residue-level changes and modular incorporation of ancestral protein components. These results highlight the preservation of protein features vital for primary function over long timescales. An ancient-modern hybrid experimental strategy enables the reconstruction and historical characterization of essential biosystems, providing an evolutionarily vetted toolbox for the study, resurrection, and engineering of life s key metabolic innovations.

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“…A. vinelandii , mutants were generated either by transformation or conjugation as indicated in Table S2. The transformation was carried out essentially as described previously (Bueno Batista et al, 2021; Dos Santos, 2011, 2019). Indirect selection of recombinants was achieved by the congression procedure (Brigle et al, 1987; Robinson et al, 1986) in which co‐transformation with plasmid pDB303 harbouring the mutation rpoB113 conferring rifampicin resistance is used for the initial selection of transformants.…”

Section: Methodsmentioning

confidence: 99%

Interactions between paralogous bacterial enhancer‐binding proteins enable metal‐dependent regulation of alternative nitrogenases in Azotobacter vinelandii

Appia-Ayme

Little

Chandra

et al. 2022

Molecular Microbiology

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The ability to carry out biological nitrogen fixation, a key process in the nitrogen cycle that underpins sustainable agriculture, is conferred in diverse bacteria and archaea by nitrogenase enzymes that catalyse the reduction of gaseous dinitrogen to ammonia. All diazotrophs that have been sequenced so far encode molybdenum nitrogenase with an active site co-factor designated as FeMo-co that contains a complex inorganic cluster [7Fe-9S-C-Mo] in which the molybdenum atom is bound to a molecule of R-homocitrate (Burén et al., 2020;Einsle & Rees, 2020). However, in addition to expressing the molybdenum nitrogenase enzyme, some diazotrophs also

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Genomic Manipulations of the Diazotroph Azotobacter vinelandii

Cited by 13 publications

References 79 publications

Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii

Automated Laboratory Growth Assessment and Maintenance of Azotobacter vinelandii

Nitrogenase resurrection and the evolution of a singular enzymatic mechanism

Interactions between paralogous bacterial enhancer‐binding proteins enable metal‐dependent regulation of alternative nitrogenases in Azotobacter vinelandii

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