S. Funayama

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

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.

Structural organization and regulation of the nif genes of Herbaspirillum seropedicae

Abstract The promoter regions of nifA and nifH genes of Herbaspirillom seropedicae have been isolated and investigated, and the nifA promoter has been characterized in detail. Both regions contain a −24 −12 type promoter and NifA-binding sites; the nifA promoter also has an NtrC-binding site. The nifA expression is activated by NtrC and repressed by ammonium ions but not by oxygen. When the NtrC-binding site was deleted nifA expression was NifA-dependent. The native NifA protein of H. seropedicae was unable to activate nifH expression in Escherichia coli whereas a truncated protein, lacking the N-terminal domain was able to activate nifH expression in the absence of oxygen and presence of iron, indicating that the N-terminal domain regulates NifA activity. The truncated protein also activates nifH in Azospirillum brasilense in the presence or absence of ammonium ions. This suggests that the N-terminal domain senses the nitrogen status of the cell.

Cloning of the nifA and nifB Genes of Azospirillum Brasilense Strain FP2

A genomic library of Azospirillum brasilense was constructed and used to isolate the nifA gene by complementation of a nifA - mutant of A. brasilense (FP10). A recombinant plasmid, pMAK7, was isolated and found to contain a functional nifA gene and to hybridize with nifA and nifB probes from Herbaspirillum seropedicae.

Sequential Analysis of the NifA Gene Promoter Region of Azospirillum brasilense

The factors involved in the regulation of the nifA gene in Azospirillum brasilense are not fully established, and its promoter has not yet been characterized. The nifA promoter of A. brasilense is constitutively expressed in the presence of ammonium ions and O2 (Liang et al., 1991). We have identified the promoter region of the nifA gene of A. brasilense and confirmed that its expression is independent of σN, NifA and NtrC.

Purification and Characterization of the PII Protein from H. seropedicae

Nitrogen metabolism in enterobacteriaceae is controlled by a regulatory cascade in which the PII protein is a transducer of nitrogen levels and modulates the activities of NtrB and GlnE proteins. In Herbaspirillum seropedicae a glnB mutant has a Nif negative phenotype. The results suggest that in H. seropedicae the PII protein has a role in the positive control of NifA activity as described in Azospirillum brasilense (de Zamaroczy et al., 1996).