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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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Size-exclusion chromatography analysis of NO3−-dependent dissociation of purified NasS-NasT. Representative chromatograms for protein elution recorded in the absence (upper trace) and presence (lower trace) of 1 mm NO3− are shown. SDS-PAGE analysis of protein content for selected column fractions is shown below each chromatogram. Protein bands were visualized by Coomassie Brilliant Blue staining. mAU, milli-absorbance units.
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Figure 6: Size-exclusion chromatography analysis of NO3−-dependent dissociation of purified NasS-NasT. Representative chromatograms for protein elution recorded in the absence (upper trace) and presence (lower trace) of 1 mm NO3− are shown. SDS-PAGE analysis of protein content for selected column fractions is shown below each chromatogram. Protein bands were visualized by Coomassie Brilliant Blue staining. mAU, milli-absorbance units.

Mentions: Additional experiments involving analytical size-exclusion chromatography were performed to investigate the result of NO3−-dependent dissociation of the NasS-NasT complex in more detail (Fig. 6). In the absence of NO3−, NasS-NasT eluted at ∼14 ml as a single symmetrical peak. SDS-PAGE analysis revealed equivalent amounts of NasS and NasT in all fractions across this peak. An apparent molecular mass of 134 ± 10 kDa could be assigned to NasS-NasT by comparison with various protein standards applied to the same column under identical conditions. When the column equilibration buffer was supplemented with NO3−, an asymmetric protein elution profile was observed that was clearly different from that observed for the protein in the absence of NO3−.


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)

Size-exclusion chromatography analysis of NO3−-dependent dissociation of purified NasS-NasT. Representative chromatograms for protein elution recorded in the absence (upper trace) and presence (lower trace) of 1 mm NO3− are shown. SDS-PAGE analysis of protein content for selected column fractions is shown below each chromatogram. Protein bands were visualized by Coomassie Brilliant Blue staining. mAU, milli-absorbance units.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Size-exclusion chromatography analysis of NO3−-dependent dissociation of purified NasS-NasT. Representative chromatograms for protein elution recorded in the absence (upper trace) and presence (lower trace) of 1 mm NO3− are shown. SDS-PAGE analysis of protein content for selected column fractions is shown below each chromatogram. Protein bands were visualized by Coomassie Brilliant Blue staining. mAU, milli-absorbance units.
Mentions: Additional experiments involving analytical size-exclusion chromatography were performed to investigate the result of NO3−-dependent dissociation of the NasS-NasT complex in more detail (Fig. 6). In the absence of NO3−, NasS-NasT eluted at ∼14 ml as a single symmetrical peak. SDS-PAGE analysis revealed equivalent amounts of NasS and NasT in all fractions across this peak. An apparent molecular mass of 134 ± 10 kDa could be assigned to NasS-NasT by comparison with various protein standards applied to the same column under identical conditions. When the column equilibration buffer was supplemented with NO3−, an asymmetric protein elution profile was observed that was clearly different from that observed for the protein in the absence of NO3−.

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