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.
Monteiro
R A
RA
Department of Biochemistry, Universidade Federal do Paraná, Curitiba PR, Brazil.
Souza
E M
EM
Funayama
S
S
Yates
M G
MG
Pedrosa
F O
FO
Chubatsu
L S
LS
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019117:Burkholderia / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D007711:Klebsiella pneumoniae / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; C061338:NifA protein, Bacteria; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors