Purification and characterisation of Azospirillum brasilense N-truncated NtrX protein.
The NtrX protein has been identified as a transcriptional activator of genes involved in the metabolic control of alternative nitrogen sources, acting as a member of a two-component regulatory system. The in silico analysis of the NtrX amino acid sequence shows that this protein contains an N-terminal receiver domain, a central AAA+ superfamily domain and a C-terminal DNA binding domain. To over-express and purify this protein, the ntrX gene of Azospirillum brasilense lacking the first eight codons was cloned into the vector pET29a+. The NtrX protein was over-expressed as an S.Tag fusion protein induced by l-arabinose in the Escherichia coli strain BL21AI and purified by ion exchange and affinity chromatography. The ATPase activity of NtrX was measured by coupling the ATP conversion to ADP with NADH oxidation. The ATPase activity of NtrX was stimulated in the presence of A. brasilense sigma(54)/NtrC-dependent promoter of the glnBA gene. Phosphorylation by carbamyl-phosphate also stimulated ATPase, in a manner similar to the NtrC protein. Together our results suggest that NtrX is active in the phosphorylated form and that there may be a cross-talk between the NtrYX and NtrBC regulatory systems in A. brasilense.
Assumpção
Marcelo Constantino
MC
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, Brazil.
de Souza
Emanuel Maltempi
EM
Yates
M Geoffrey
MG
de Oliveira Pedrosa
Fábio
F
Benelli
Elaine Machado
EM
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D015806:Azospirillum brasilense / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D000251:Adenosine Triphosphatases