Isolation of phosphorylation-deficient mutants of the Rhizobium meliloti two-component regulatory protein, FixJ.
Rhizobium meliloti FixL and FixJ are members of a symbiotically essential two-component system that regulates nitrogen-fixation genes in response to environmental oxygen concentrations. FixL is a membrane protein that is thought to relay information about oxygen availability to FixJ via a phosphotransfer mechanism. FixJ increases expression of the nifA and fixK genes by activating transcription of the nifA and fixK promoters (p-nifA and p-fixK, respectively). In this study, we examined the relationship between the in vivo activity of FixJ as a transcriptional regulator and its ability to be phosphorylated in vitro by the sensor FixL. FixJ mutants were isolated that showed decreased activity on p-nifA in Escherichia coli. Most of the FixJ mutant proteins also showed decreased activity on the fixK promoter. These mutants were analysed in R. meliloti for activity on p-nifA during vegetative growth, where similarities and differences were observed when compared with their phenotypes in E. coli. Three mutants showing significantly less activity in R. meliloti were examined for symbiotic activity in planta and were found to be ineffective. When these three mutant FixJ proteins were examined in vitro for their ability to be phosphorylated by FixL, two mutants were found to have a significantly decreased ability to accept phosphate from FixL. These findings 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
Monson
E K
EK
Ditta
G S
GS
Helinski
D R
DR
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism; D000071677:Histidine Kinase; D009154:Mutation; D010766:Phosphorylation; D010944:Plants / Q000235:genetics / Q000382:microbiology; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D006420:Hemeproteins; C069289:FixJ protein, Bacteria; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase