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Effect of point mutations on Herbaspirillum seropedicae NifA activity.

Aquino B, Stefanello AA, Oliveira MA, Pedrosa FO, Souza EM, Monteiro RA, Chubatsu LS - Braz. J. Med. Biol. Res. (2015)

Bottom Line: Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins.However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium.This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.

ABSTRACT
NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

No MeSH data available.


Related in: MedlinePlus

Transcriptional activity of indicated Herbaspirillumseropedicae strains carrying a chromosomal nifH:lacZfusion. +G25E indicates cells carrying a pLAFR3.18-based plasmid expressing theG25E NifA mutant; (-) indicates absence of plasmid. Cells were grown in NFbHPmedium supplemented with 10 mM NH4Cl under aerobic conditions at 30°C.Cells were then centrifuged (1700 g for 2 min), resuspended inNFbHP (nitrogen-free) medium, and de-repressed for 7 h under 1.5% oxygen.β-galactosidase was determined as described. Data are reported as the mean±SD ofat least 3 independent experiments. β-galactosidase activity is reported as Millerunits.
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f04: Transcriptional activity of indicated Herbaspirillumseropedicae strains carrying a chromosomal nifH:lacZfusion. +G25E indicates cells carrying a pLAFR3.18-based plasmid expressing theG25E NifA mutant; (-) indicates absence of plasmid. Cells were grown in NFbHPmedium supplemented with 10 mM NH4Cl under aerobic conditions at 30°C.Cells were then centrifuged (1700 g for 2 min), resuspended inNFbHP (nitrogen-free) medium, and de-repressed for 7 h under 1.5% oxygen.β-galactosidase was determined as described. Data are reported as the mean±SD ofat least 3 independent experiments. β-galactosidase activity is reported as Millerunits.

Mentions: The G25E mutation was also tested in the NifAdel and NifAdel/GlnKdel strains carrying anifH::lacZ chromosomal fusion, which allowed assessment oftranscriptional NifA activity in the presence of high ammonium concentrations (Figure 4). The wild-type H.seropedicae strain (SmR1) carrying the nifH::lacZ fusiononly showed β-galactosidase activity at low ammonium concentrations. Conversely, theG25E mutant showed nifH::lacZ transcription in both the NifAdel andNifAdel/GlnKdel strains, regardless of ammonium concentration. However, comparison ofβ-galactosidase activity at both low and high ammonium concentrations indicated that theG25E mutant protein was partially regulated by ammonium, as transcriptional activity washigher at low ammonium concentrations. This result suggested that the G25E mutant didnot depend on GlnK for activity, but could still detect ammonium concentration.


Effect of point mutations on Herbaspirillum seropedicae NifA activity.

Aquino B, Stefanello AA, Oliveira MA, Pedrosa FO, Souza EM, Monteiro RA, Chubatsu LS - Braz. J. Med. Biol. Res. (2015)

Transcriptional activity of indicated Herbaspirillumseropedicae strains carrying a chromosomal nifH:lacZfusion. +G25E indicates cells carrying a pLAFR3.18-based plasmid expressing theG25E NifA mutant; (-) indicates absence of plasmid. Cells were grown in NFbHPmedium supplemented with 10 mM NH4Cl under aerobic conditions at 30°C.Cells were then centrifuged (1700 g for 2 min), resuspended inNFbHP (nitrogen-free) medium, and de-repressed for 7 h under 1.5% oxygen.β-galactosidase was determined as described. Data are reported as the mean±SD ofat least 3 independent experiments. β-galactosidase activity is reported as Millerunits.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4541686&req=5

f04: Transcriptional activity of indicated Herbaspirillumseropedicae strains carrying a chromosomal nifH:lacZfusion. +G25E indicates cells carrying a pLAFR3.18-based plasmid expressing theG25E NifA mutant; (-) indicates absence of plasmid. Cells were grown in NFbHPmedium supplemented with 10 mM NH4Cl under aerobic conditions at 30°C.Cells were then centrifuged (1700 g for 2 min), resuspended inNFbHP (nitrogen-free) medium, and de-repressed for 7 h under 1.5% oxygen.β-galactosidase was determined as described. Data are reported as the mean±SD ofat least 3 independent experiments. β-galactosidase activity is reported as Millerunits.
Mentions: The G25E mutation was also tested in the NifAdel and NifAdel/GlnKdel strains carrying anifH::lacZ chromosomal fusion, which allowed assessment oftranscriptional NifA activity in the presence of high ammonium concentrations (Figure 4). The wild-type H.seropedicae strain (SmR1) carrying the nifH::lacZ fusiononly showed β-galactosidase activity at low ammonium concentrations. Conversely, theG25E mutant showed nifH::lacZ transcription in both the NifAdel andNifAdel/GlnKdel strains, regardless of ammonium concentration. However, comparison ofβ-galactosidase activity at both low and high ammonium concentrations indicated that theG25E mutant protein was partially regulated by ammonium, as transcriptional activity washigher at low ammonium concentrations. This result suggested that the G25E mutant didnot depend on GlnK for activity, but could still detect ammonium concentration.

Bottom Line: Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins.However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium.This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brasil.

ABSTRACT
NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain.

No MeSH data available.


Related in: MedlinePlus