Role of conserved cysteine residues in Herbaspirillum seropedicae NifA activity.
Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that associates with economically important crops. NifA protein, the transcriptional activator of nif genes in H. seropedicae, binds to nif promoters and, together with RNA polymerase-sigma(54) holoenzyme, catalyzes the formation of open complexes to allow transcription initiation. The activity of H. seropedicae NifA is controlled by ammonium and oxygen levels, but the mechanisms of such control are unknown. Oxygen sensitivity is attributed to a conserved motif of cysteine residues in NifA that spans the central AAA+ domain and the interdomain linker that connects the AAA+ domain to the C-terminal DNA binding domain. Here we mutagenized this conserved motif of cysteines and assayed the activity of mutant proteins in vivo. We also purified the mutant variants of NifA and tested their capacity to bind to the nifB promoter region. Chimeric proteins between H. seropedicae NifA, an oxygen-sensitive protein, and Azotobacter vinelandii NifA, an oxygen-tolerant protein, were constructed and showed that the oxygen response is conferred by the central AAA+ and C-terminal DNA binding domains of H. seropedicae NifA. We conclude that the conserved cysteine motif is essential for NifA activity, although single cysteine-to-serine mutants are still competent at binding DNA.
Oliveira
Marco A S
MA
Department of Biochemistry and Molecular Biology, Universidade Federal do Parana, Curitiba, PR 81531-990, Brazil.
Baura
Valter A
VA
Aquino
Bruno
B
Huergo
Luciano F
LF
Kadowaki
Marco A S
MA
Chubatsu
Leda S
LS
Souza
Emanuel M
EM
Dixon
Ray
R
Pedrosa
Fábio O
FO
Wassem
Roseli
R
Monteiro
Rose A
RA
D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D042803:Herbaspirillum / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D014157:Transcription Factors; D003545:Cysteine; D010100:Oxygen