The FixL protein of Rhizobium meliloti can be separated into a heme-binding oxygen-sensing domain and a functional C-terminal kinase domain.
Transcription of nitrogen fixation (nif and fix) genes in Rhizobium meliloti is induced by a decrease in oxygen concentration. The products of two genes, fixL and fixJ, are responsible for sensing and transmitting the low-oxygen signal. The proteins encoded by fixL and fixJ (FixL and FixJ, respectively) are homologous to a family of bacterial proteins that transduce environmental signals through a common phosphotransfer mechanism [David, M., Daveran, M., Batut, J., Dedieu, A., Domergue, O., Ghai, J., Hertig, C., Boistard, P. & Khan, D. (1988) Cell 54, 671-683]. FixL, the oxygen sensor, is a membrane protein. It has previously been shown that a soluble derivative of FixL, FixL*, is an oxygen-binding hemoprotein and a kinase that autophosphorylates and also phosphorylates FixJ [Gilles-Gonzalez, M. A., Ditta, G. S. & Helinski, D. R. (1991) Nature (London) 350, 170-172]. In this work, deletion derivatives of fixL* were constructed and overexpressed in Escherichia coli, and the truncated proteins were purified. We show that a fragment of FixL from amino acid residue 127 to residue 260 binds heme, retains the ability to bind oxygen, and has no detectable kinase activity. A C-terminal fragment of FixL, beginning at residue 260, fails to bind heme but is active as a kinase. We also demonstrate that anaerobiosis results in an enhancement of FixL* autophosphorylation and FixJ phosphorylation activities in vitro. Finally, we show that the heme-binding region of FixL is required in vitro for oxygen regulation of its kinase activities.
Monson
E K
EK
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Weinstein
M
M
Ditta
G S
GS
Helinski
D R
DR
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006418:Heme / Q000378:metabolism; D006420:Hemeproteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000071677:Histidine Kinase; D008970:Molecular Weight; D009586:Nitrogen Fixation / Q000235:genetics*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006420:Hemeproteins; D011994:Recombinant Proteins; C069289:FixJ protein, Bacteria; D006418:Heme; D011494:Protein Kinases; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase