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Nitrogen oxyanion-dependent dissociation of a two-component complex that regulates bacterial nitrate assimilation.

Luque-Almagro VM, Lyall VJ, Ferguson SJ, Roldán MD, Richardson DJ, Gates AJ - J. Biol. Chem. (2013)

Bottom Line: The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT.NasT has been shown to bind the leader RNA for nasA.Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.

View Article: PubMed Central - PubMed

Affiliation: From the Centre for Molecular and Structural Biochemistry and.

ABSTRACT
Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.

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Related in: MedlinePlus

Expression and co-purification of NasS and NasT. Shown are the results from overexpression of recombinant P. denitrificans NasS and NasT proteins in E. coli BL21(DE3) (A), Ni2+ IMAC affinity purification of the NasS-NasT complex from the soluble (sol.) cell extract (B), and further purification of the NasS-NasT complex by anion-exchange (C) and size-exclusion (D) chromatography. Protein expression and purification were assessed by SDS-PAGE using Coomassie Brilliant Blue staining. IPTG, isopropyl β-d-thiogalactopyranoside; mAU, milli-absorbance units.
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Figure 2: Expression and co-purification of NasS and NasT. Shown are the results from overexpression of recombinant P. denitrificans NasS and NasT proteins in E. coli BL21(DE3) (A), Ni2+ IMAC affinity purification of the NasS-NasT complex from the soluble (sol.) cell extract (B), and further purification of the NasS-NasT complex by anion-exchange (C) and size-exclusion (D) chromatography. Protein expression and purification were assessed by SDS-PAGE using Coomassie Brilliant Blue staining. IPTG, isopropyl β-d-thiogalactopyranoside; mAU, milli-absorbance units.

Mentions: The mechanism by which the putative NO3− sensor NasS and the ANTAR-type protein NasT cooperate to control nas gene expression in bacteria is unclear but may involve a protein-protein interaction (2). To investigate whether NasS and NasT interact, the expression construct pET-24a/nasTS was produced, which would not only yield high levels of recombinant forms of P. denitrificans NasS and NasT proteins but would also provide a means of immobilizing NasT via a polyhistidine tag during affinity purification. SDS-PAGE analysis of soluble protein extracts prepared from E. coli host cells containing the pET-24a/nasTS construct showed clear overexpression of two proteins at ∼22 and 42 kDa when isopropyl β-d-thiogalactopyranoside was added to the cell cultures (Fig. 2A).


Nitrogen oxyanion-dependent dissociation of a two-component complex that regulates bacterial nitrate assimilation.

Luque-Almagro VM, Lyall VJ, Ferguson SJ, Roldán MD, Richardson DJ, Gates AJ - J. Biol. Chem. (2013)

Expression and co-purification of NasS and NasT. Shown are the results from overexpression of recombinant P. denitrificans NasS and NasT proteins in E. coli BL21(DE3) (A), Ni2+ IMAC affinity purification of the NasS-NasT complex from the soluble (sol.) cell extract (B), and further purification of the NasS-NasT complex by anion-exchange (C) and size-exclusion (D) chromatography. Protein expression and purification were assessed by SDS-PAGE using Coomassie Brilliant Blue staining. IPTG, isopropyl β-d-thiogalactopyranoside; mAU, milli-absorbance units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795266&req=5

Figure 2: Expression and co-purification of NasS and NasT. Shown are the results from overexpression of recombinant P. denitrificans NasS and NasT proteins in E. coli BL21(DE3) (A), Ni2+ IMAC affinity purification of the NasS-NasT complex from the soluble (sol.) cell extract (B), and further purification of the NasS-NasT complex by anion-exchange (C) and size-exclusion (D) chromatography. Protein expression and purification were assessed by SDS-PAGE using Coomassie Brilliant Blue staining. IPTG, isopropyl β-d-thiogalactopyranoside; mAU, milli-absorbance units.
Mentions: The mechanism by which the putative NO3− sensor NasS and the ANTAR-type protein NasT cooperate to control nas gene expression in bacteria is unclear but may involve a protein-protein interaction (2). To investigate whether NasS and NasT interact, the expression construct pET-24a/nasTS was produced, which would not only yield high levels of recombinant forms of P. denitrificans NasS and NasT proteins but would also provide a means of immobilizing NasT via a polyhistidine tag during affinity purification. SDS-PAGE analysis of soluble protein extracts prepared from E. coli host cells containing the pET-24a/nasTS construct showed clear overexpression of two proteins at ∼22 and 42 kDa when isopropyl β-d-thiogalactopyranoside was added to the cell cultures (Fig. 2A).

Bottom Line: The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT.NasT has been shown to bind the leader RNA for nasA.Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.

View Article: PubMed Central - PubMed

Affiliation: From the Centre for Molecular and Structural Biochemistry and.

ABSTRACT
Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.

Show MeSH
Related in: MedlinePlus