Mechanism of Ammonia Release from nifL Interposon Mutants of Azotobacter vinelandii

Brewin, Brett; Woodley, Paul; Drummond, Martin

doi:10.1007/978-94-011-5159-7_54

Cited by 16 publications

(68 citation statements)

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“…Another improvement was achieved by Brewin and colleagues (1999), resulting in production of greater quantities of ammonium. Again, the wild type did not excrete ammonium (Brewin et al, 1999).…”

Section: Biological Nitrogen Fixationmentioning

confidence: 90%

“…Considering the possibility of replacing industrially produced ammonia fertilisers, many attempts to modify two species of this diazotroph-A. beijerinckii and A. vinelandii-were undertaken with the aim of producing an environmentally friendly bacterial fertiliser (Brewin et al, 1999). Generally, regulation of ammonia producichtion by Azotobacter, especially A. vinelandii, is similar to that achieved by using Klebsiella pneumoniae, being regulated by nifL and nifA.…”

Section: Mutation Of Azotobacter Nif Genes For Ammonia Accumulationmentioning

confidence: 99%

“…The NifL protein binds to and inactivates NifA when ammonium is present where even at relatively low levels. At higher levels of ammonium, expression of the nifLA operon does not occur, and so NifA is not synthesized (Brewin et al, 1999). An idea to mutate nifL for enhancing ammonia production by Klebsiella pneumoniae for agricultural purposes generated many studies to generate a mutant with a damaged nifL gene.…”

Section: Mutation Of Azotobacter Nif Genes For Ammonia Accumulationmentioning

confidence: 99%

See 2 more Smart Citations

Ammonia Accumulation of Novel Nitrogen-Fixing Bacteria

Iwata¹,

Yu²,

Azlan³

et al. 2012

Biotechnology - Molecular Studies and Novel Applications for Improved Quality of Human Life

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Section: Biological Nitrogen Fixationmentioning

confidence: 90%

Section: Mutation Of Azotobacter Nif Genes For Ammonia Accumulationmentioning

confidence: 99%

Section: Mutation Of Azotobacter Nif Genes For Ammonia Accumulationmentioning

confidence: 99%

See 1 more Smart Citation

Ammonia Accumulation of Novel Nitrogen-Fixing Bacteria

Iwata¹,

Yu²,

Azlan³

et al. 2012

Biotechnology - Molecular Studies and Novel Applications for Improved Quality of Human Life

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“…vinelandii Transformation and Analysis of Transformants. Mutations were introduced into the A. vinelandii genome by using plasmid pIM32, which contains a 1-kb region upstream of nifL, a spectinomycin-resistance cassette in the SmaI site upstream of the nifL promoter (33), and wild-type nifL and nifA sequences. This plasmid also contains an engineered EcoRI site close to the SmaI site within nifL to facilitate identification of recombinants by PCR.…”

Section: Methodsmentioning

confidence: 99%

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

Martínez‐Argudo

Little

Dixon

2004

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

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NifL is an antiactivator that tightly regulates transcription of genes required for nitrogen fixation in Azotobacter vinelandii by controlling the activity of its partner protein NifA, a member of the family of 54 -dependent transcriptional activators. Although the C-terminal region of A. vinelandii NifL shows homology to the transmitter domains of histidine protein kinases, signal transduction between NifL and NifA is conveyed by means of proteinprotein interactions rather than by phosphorylation. Binding of the ligand 2-oxoglutarate to NifA plays a crucial role in preventing inhibition by NifL under conditions appropriate for nitrogen fixation. We have used a suppressor screen to identify a critical arginine residue (R306) in NifL that is required to release NifA from inhibition under appropriate environmental conditions. Amino acid substitutions at position 306 result in constitutive inhibition of NifA activity by NifL, thus preventing nitrogen fixation. Biochemical studies with one of the mutant proteins demonstrate that the substitution alters the conformation of NifL significantly and prevents the response of NifA to 2-oxoglutarate. We propose that arginine 306 is critical for the propagation of signals perceived by A. vinelandii NifL in response to the redox and fixed-nitrogen status and is required for a conformational switch that inactivates the inhibitory function of NifL under conditions appropriate for nitrogen fixation.2-oxoglutarate ͉ signal transduction ͉ antiactivator ͉ redox control ͉ nitrogen regulation S tructural rearrangements of sensor proteins in response to environmental cues provide a fundamental mechanism for signal propagation within cells. However, although conformational changes have been well characterized in isolated signaling domains, mechanisms for signal transmission by means of interdomain interactions are frequently less well understood. For example, structural studies have identified ligand-induced conformational changes in the sensor domains of histidine protein kinases (HPKs), but it is not known how these changes are communicated to the kinase domain to control phosphoryl transfer.The Azotobacter vinelandii NifL regulatory protein is a histidine kinase-like protein that controls the expression of the genes required for nitrogen fixation in response to the redox, nitrogen, and carbon status. NifL is an antiactivator that tightly regulates the activity of its partner protein NifA, a member of the family of 54 -transcriptional activators (1, 2), by means of the formation of an inhibitory complex (3-5). The domain architecture of NifL is similar to that of some HPKs, with an N-terminal Per-ArntSim (PAS) domain (6, 7) containing a flavin adenine dinucleotide (FAD) cofactor that senses the redox status (8, 9) and a C-terminal domain containing conserved residues corresponding to the N, G1, F, and G2 boxes that constitute the ATPbinding domain of the GHKL superfamily of ATPases (10-14) (Fig. 1). Unlike the HPKs, the GHKL domain of NifL does not exhibit ATP hydrolysis or transphos...

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“…In response to incubation in an acidic, sugar-free medium, the saprobe is induced to release ammonium ions and ammonia in quantities which increase the pH of its extracellular environment (8). Parasite-phase cultures similarly respond to acidification of their external environment by secretion of NH 4 ϩ and NH 3 . A source of intracellular ammonia is urea hydrolysis, and Coccidioides has been reported to produce an enzymatically active urease (25,39).…”

mentioning

confidence: 99%

Urease Produced by Coccidioides posadasii Contributes to the Virulence of This Respiratory Pathogen

et al. 2006

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Urease activity during in vitro growth in the saprobic and parasitic phases of Coccidioides spp. is partly responsible for production of intracellular ammonia released into the culture media and contributes to alkalinity of the external microenvironment. Although the amino acid sequence of the urease of Coccidioides posadasii lacks a predicted signal peptide, the protein is transported from the cytosol into vesicles and the central vacuole of parasitic cells (spherules). Enzymatically active urease is released from the contents of mature spherules during the parasitic cycle endosporulation stage. The endospores, together with the urease and additional material which escape from the ruptured parasitic cells, elicit an intense host inflammatory response. Ammonia production by the spherules of C. posadasii is markedly increased by the availability of exogenous urea found in relatively high concentrations at sites of coccidioidal infection in the lungs of mice. Direct measurement of the pH at these infection sites revealed an alkaline microenvironment. Disruption of the urease gene of C. posadasii resulted in a marked reduction in the amount of ammonia secreted in vitro by the fungal cells. BALB/c mice challenged intranasally with the mutant strain showed increased survival, a wellorganized granulomatous response to infection, and better clearance of the pathogen than animals challenged with either the parental or the reconstituted (revertant) strain. We conclude that ammonia and enzymatically active urease released from spherules during the parasitic cycle of C. posadasii contribute to host tissue damage, which exacerbates the severity of coccidioidal infection and enhances the virulence of this human respiratory pathogen.

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Mechanism of Ammonia Release from nifL Interposon Mutants of Azotobacter vinelandii

Cited by 16 publications

References 1 publication

Ammonia Accumulation of Novel Nitrogen-Fixing Bacteria

Ammonia Accumulation of Novel Nitrogen-Fixing Bacteria

A crucial arginine residue is required for a conformational switch in NifL to regulate nitrogen fixation in Azotobacter vinelandii

Urease Produced by Coccidioides posadasii Contributes to the Virulence of This Respiratory Pathogen

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