Limits...
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 NifA variant proteins in Escherichiacoli JM109 (DE3) carrying pRT22 (nifH::lacZ). (-)indicates cells carrying pET-29a. Full-length NifA was expressed from pRAM1.ΔN-NifA indicates an N-truncated form of NifA expressed from pRAM2. Full-lengthNifA mutants, as indicated, were expressed from pET-29a-based plasmids.β-galactosidase expression experiments were performed in NFDM medium (31) with 20 mM of ammonium chloride (+N) or0.2% casamino acids (-N), in the absence of O2. Data are reported asthe mean±SD of 3 independent assays. β-galactosidase activity is reported asMiller units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4541686&req=5

f02: Transcriptional activity of NifA variant proteins in Escherichiacoli JM109 (DE3) carrying pRT22 (nifH::lacZ). (-)indicates cells carrying pET-29a. Full-length NifA was expressed from pRAM1.ΔN-NifA indicates an N-truncated form of NifA expressed from pRAM2. Full-lengthNifA mutants, as indicated, were expressed from pET-29a-based plasmids.β-galactosidase expression experiments were performed in NFDM medium (31) with 20 mM of ammonium chloride (+N) or0.2% casamino acids (-N), in the absence of O2. Data are reported asthe mean±SD of 3 independent assays. β-galactosidase activity is reported asMiller units.

Mentions: The ability of the H. seropedicae NifA mutants to activatenif promoters was determined in E. coli JM109(DE3)carrying plasmid pRT22 (K. pneumoniae nifH::lacZ fusion) (Figure 2). Full-length NifA, expressed from plasmidpRAM1, showed no β-galactosidase activity, consistent with previous descriptions, mainlybecause of lower expression of endogenous E. coli PII, which isnecessary to relieve the negative control of the N-terminal GAF domain on the catalyticdomain of NifA (7). In contrast, the N-terminaltruncated NifA protein (ΔN-NifA) expressed from pRAM2 was fully functional in E.coli regardless of the ammonium concentration (22). These results confirmed the regulatory role of the N-terminalGAF domain on H. seropedicae NifA that has been described previously:in the presence of ammonium or the absence of PII, the N-terminal GAF inhibits NifAtranscriptional activity (6,23). The constructed NifA point mutants were analyzed under the sameconditions and showed no activity, except for NifA G25E, which partially activatednifH transcription in E. coli. G25E also appearedto retain some nitrogen control, as activation of transcription was higher under lowammonium concentrations. This result indicated that the G25E substitution affected theneed for PII for NifA activity in H. seropedicae.


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 NifA variant proteins in Escherichiacoli JM109 (DE3) carrying pRT22 (nifH::lacZ). (-)indicates cells carrying pET-29a. Full-length NifA was expressed from pRAM1.ΔN-NifA indicates an N-truncated form of NifA expressed from pRAM2. Full-lengthNifA mutants, as indicated, were expressed from pET-29a-based plasmids.β-galactosidase expression experiments were performed in NFDM medium (31) with 20 mM of ammonium chloride (+N) or0.2% casamino acids (-N), in the absence of O2. Data are reported asthe mean±SD of 3 independent assays. β-galactosidase activity is reported asMiller units.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f02: Transcriptional activity of NifA variant proteins in Escherichiacoli JM109 (DE3) carrying pRT22 (nifH::lacZ). (-)indicates cells carrying pET-29a. Full-length NifA was expressed from pRAM1.ΔN-NifA indicates an N-truncated form of NifA expressed from pRAM2. Full-lengthNifA mutants, as indicated, were expressed from pET-29a-based plasmids.β-galactosidase expression experiments were performed in NFDM medium (31) with 20 mM of ammonium chloride (+N) or0.2% casamino acids (-N), in the absence of O2. Data are reported asthe mean±SD of 3 independent assays. β-galactosidase activity is reported asMiller units.
Mentions: The ability of the H. seropedicae NifA mutants to activatenif promoters was determined in E. coli JM109(DE3)carrying plasmid pRT22 (K. pneumoniae nifH::lacZ fusion) (Figure 2). Full-length NifA, expressed from plasmidpRAM1, showed no β-galactosidase activity, consistent with previous descriptions, mainlybecause of lower expression of endogenous E. coli PII, which isnecessary to relieve the negative control of the N-terminal GAF domain on the catalyticdomain of NifA (7). In contrast, the N-terminaltruncated NifA protein (ΔN-NifA) expressed from pRAM2 was fully functional in E.coli regardless of the ammonium concentration (22). These results confirmed the regulatory role of the N-terminalGAF domain on H. seropedicae NifA that has been described previously:in the presence of ammonium or the absence of PII, the N-terminal GAF inhibits NifAtranscriptional activity (6,23). The constructed NifA point mutants were analyzed under the sameconditions and showed no activity, except for NifA G25E, which partially activatednifH transcription in E. coli. G25E also appearedto retain some nitrogen control, as activation of transcription was higher under lowammonium concentrations. This result indicated that the G25E substitution affected theneed for PII for NifA activity in H. seropedicae.

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