Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Abstract

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

“…Also, the activity was similar in cells assayed in the presence or absence of ammonium, confirming that removal of the N-terminus of NifA leads to loss of ammonium regulation. Nishikawa et al ( 11 ) have reported similar results. In addition, we observed that the N-truncated NifA was active and non-regulated regardless of the presence of genes encoding endogenous P II proteins ( glnK or glnB ) in the E. coli backgrounds, although the β-galactosidase activity was approximately 2-fold higher in strains RB9060 ( glnB ) and UNF3435 ( glnB/glnK ) ( Figure 1 ).…”

“…The DNA fragment encoding the N-truncated NifA protein was excised from the plasmid pCNpETCCT ( 11 ) as an Xba I/ Hin dIII fragment and cloned into pDK5 ( 12 ), yielding the plasmid pCNK5CCT. The sequence encoding the N-terminal GAF domain of the NiFA protein was amplified using the primers NifA5′NT (5′-GGTGTCG CATATG CCGGTG-3′) and NifA3′Cent (5′-CATG AAGCTT TACTCCTCGGCC-3′), which introduced Nde I and Hin dIII restriction sites, respectively (underlined), and cloned into the pCR2.1 cloning vector (Invitrogen), yielding the plasmid pLANTTA.…”

Purification and biochemical characterization of the DNA binding domain of the nitrogenase transcriptional activator NifA fromGluconacetobacter diazotrophicus

“…When oxygen levels are high, NifL interacts with NifA, inhibiting ATP hydrolysis in the AAA + domain which prevents transcriptional activation [1,12]. In contrast, in α-and β-proteobacteria, redox sensing takes place on NifA itself [13,14]. NifA contains four conserved cysteine residues in the AAA + domain and at the start of the IDL (Figure 1A) [14,15].…”

“…In contrast, in α-and β-proteobacteria, redox sensing takes place on NifA itself [13,14]. NifA contains four conserved cysteine residues in the AAA + domain and at the start of the IDL (Figure 1A) [14,15]. In addition, NifA in many α-proteobacterial species contain a single Cys or two Cys residues upstream of the DNA binding domain (Figure 1C) at the C-terminal end of the IDL.…”

Purification and Biochemical Characterization of the DNA Binding Domain of the Nitrogenase Transcriptional Activator NifA from Gluconacetobacter diazotrophicus

“…When oxygen levels are high, NifL interacts with NifA, inhibiting ATP hydrolysis in the AAA + domain which prevents transcriptional activation [ 1 , 12 ]. In contrast, in α- and β-proteobacteria, redox sensing takes place on NifA itself [ 13 , 14 ]. NifA contains four conserved cysteine residues in the AAA + domain and at the start of the IDL (Fig.…”