Expression and functional analysis of an N‐truncated NifA protein of Herbaspirillum seropedicae

Abstract

In Herbaspirillum seropedicae, an endophytic diazotroph, nif gene expression is under the control of the transcriptional activator NifA. We have over-expressed and purified a protein containing the central and C-terminal domains of the H. seropedicae NifA protein, N-truncated NifA, fused to a His-Tag sequence. This fusion protein was found to be partially soluble and was purified by affinity chromatography. Band shift and footprinting assays showed that the N-truncated NifA protein was able to bind specifically to the H. seropedicae nifB promoter region. In vivo analysis showed that this protein activated the nifH promoter of Klebsiella pneumoniae in Escherichia coli only in the absence of oxygen and this activation was not negatively controlled by ammonium ions.z 1999 Federation of European Biochemical Societies.

Fnr Is Involved in Oxygen Control of Herbaspirillum seropedicae N-Truncated NifA Protein Activity in Escherichia coli

“…coli strains harboring plasmids pIMA217 and pRAM7 were analyzed for transcriptional activation of the nifHDK:: lacZ fusion. N-truncated NifA (with the first 202 amino acid residues deleted and without the N-terminal domain and the Q-linker) activated transcription of the H. seropedicae nifH promoter in E. coli under low-oxygen conditions (Table 2) regardless of the ammonium levels, as shown previously (25). However, in fnr strain JRG1728 of E. coli, the N-truncated NifA protein failed to activate the H. seropedicae nifH promoter either in the presence or in the absence of oxygen.…”

“…A wild-type behavior was observed when strain JRG1728 was transformed with a plasmid carrying the fnr gene (31) expressed from its own promoter. We could not use the fulllength NifA protein in these experiments since it has no activity in E. coli (25,26). On the other hand, as expected, the K. pneumoniae NifA protein was fully active under all conditions tested, including in the fnr mutant (Table 2), since it is not directly sensitive to oxygen or directly dependent on fnr (10).…”

“…Although the in vivo NifL-reducing and -oxidizing species have not been defined yet (10,18,23), it has been suggested that a heme protein may be involved (13). The NifA proteins from rhizobia, A. brasilense, and H. seropedicae are not active in the presence of high oxygen concentrations and require iron for in vivo activation of nif gene promoters, suggesting that the NifA proteins of this class may sense oxygen directly (2,32,9,25). It has been suggested that the oxygen sensitivity of these NifA proteins involves a cysteine motif located at the end of the central domain and a linker region for the C-terminal domain, which resembles an iron-sulfur cluster-binding motif (9).…”

Identification of the essential histidine residue for high-affinity binding of AlbA protein to albicidin antibiotics

“…It shows a low level of sequence similarity to the DNA-binding proteins encoded by ntrC from Acidithiobacillus ferrooxidans (Salazar et al, 2001) and nifA from several bacterial species (Monteiro et al, 1999;Gu et al, 2000;Souza et al, 2000). There is no significant similarity beyond that previously noted at the N terminus between AlbA and AlbB from Alcaligenes denitrificans, which also encodes an albicidin-binding protein (Basnayake & Birch, 1995).…”

History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience

“…Dr. Pedrosa's group also demonstrated that the NifA protein of H. seropedicae, similar to those from the α-Proteobacteria (including A. brasilense), is sensitive to oxygen. Besides, it requires Fe for in vivo activity and the interdomain region between the Cterminal domain and the central domain is involved in oxygen sensitivity (Monteiro et al 1999a). In addition, Dr. Pedrosa's group isolated and characterized the glnB gene of H. seropedicae and determined the three-dimensional structure of the purified protein product, a transducer protein of the PII signal.…”