Mutants of the two-component regulatory protein FixJ of Rhizobium meliloti that have increased activity at the nifA promoter.
FixL and FixJ belong to a two-component regulatory system in Rhizobium meliloti that induces the expression of numerous nitrogen-fixation genes during symbiosis with alfalfa. FixJ is a positive activator required for transcription of the regulatory genes nifA and fixK, while FixL is an oxygen-binding hemoprotein capable of regulating the phosphorylation status of both itself and FixJ, in response to oxygen availability. In this study, we isolated four FixJ mutants that display increased activity at the nifA promoter (PnifA) in Escherichia coli. All four mutants possess amino acid changes in a domain of FixJ that is conserved in other response regulator proteins, and all exhibit increased activity at PnifA in R. meliloti that is dependent on the presence of FixL. One of the mutant proteins, while less efficient at accepting phosphate from a truncated derivative of FixL (FixL*), nevertheless has a phosphorylated form that is more stable than the phosphorylated form of wild-type (wt) FixJ and is more resistant to the phosphatase activity of FixL*. The wt FixJ-phosphate was found to have a half-life of approximately 4 h, which makes it an unusually long-lived response regulator protein. The exceptional stability of wt FixJ-phosphate and the altered phosphorylation properties observed for the mutant are discussed in relation to signal transduction in the FixLJ system.
Weinstein
M
M
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Lois
A F
AF
Ditta
G S
GS
Helinski
D R
DR
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D009154:Mutation; D009586:Nitrogen Fixation / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016962:Sinorhizobium meliloti / Q000235:genetics*; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; C061338:NifA protein, Bacteria; D011994:Recombinant Proteins; D014157:Transcription Factors; C069289:FixJ protein, Bacteria