Effects of over-expression of the regulatory enzymes DraT and DraG on the ammonium-dependent post-translational regulation of nitrogenase reductase in Azospirillum brasilense.
Nitrogen fixation in Azospirillum brasilense is regulated at transcriptional and post-translational levels. Post-translational control occurs through the reversible ADP-ribosylation of dinitrogenase reductase (Fe Protein), mediated by the dinitrogenase reductase ADP-ribosyltransferase (DraT) and dinitrogenase reductase glycohydrolase (DraG). Although the DraT and DraG activities are regulated in vivo, the molecules responsible for such regulation remain unknown. We have constructed broad-host-range plasmids capable of over-expressing, upon IPTG induction, the regulatory enzymes DraT and DraG as six-histidine-N-terminal fused proteins (His). Both DraT-His and DraG-His are functional in vivo. We have analyzed the effects of DraT-His and DraG-His over-expression on the post-translational modification of Fe Protein. The DraT-His over-expression led to Fe Protein modification in the absence of ammonium addition, while cells over-expressing DraG-His showed only partial ADP-ribosylation of Fe Protein by adding ammonium. These results suggest that both DraT-His and DraG-His lose their regulation upon over-expression, possible by titrating out negative regulators.
Huergo
Luciano F
LF
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP 19046, 81531-990 Curitiba, PR, Brazil.
Souza
Emanuel M
EM
Steffens
Maria B R
MB
Yates
M Geoffrey
MG
Pedrosa
Fábio O
FO
Chubatsu
Leda S
LS
D036002:ADP Ribose Transferases / Q000235:genetics / Q000502:physiology; D015806:Azospirillum brasilense / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000502:physiology; D010088:Oxidoreductases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*
D001426:Bacterial Proteins; D000644:Quaternary Ammonium Compounds; C110078:draTG protein, Bacteria; D010088:Oxidoreductases; C029130:nitrogenase reductase; D036002:ADP Ribose Transferases; C057637:dinitrogenase reductase ADP-ribosyltransferase; D009699:N-Glycosyl Hydrolases; C036091:ADP-ribosyl-(dinitrogen reductase) hydrolase