The uncoupling of oxygen sensing, phosphorylation signalling and transcriptional activation in oxygen sensor FixL and FixJ mutants.
The rhizobial FixL/FixJ system, a member of the superfamily of bacterial two-component signal transducing systems, regulates the expression of nitrogen fixation-related genes by sensing environmental oxygen tension. Oxygen-free (deoxy) FixL is autophosphorylated at an invariant histidine residue with ATP, and the phosphoryl group is transferred to FixJ, leading to an enhancement in transcriptional activity at low oxygen tensions, but the histidine kinase activity of the oxygen-bound (oxy) form is inhibited. To investigate the mechanism of oxygen sensing, we established a FixL/FixJ-mediated PfixK-lacZ reporter system in Escherichia coli, and isolated FixL and FixJ mutations conferring an upregulation of lacZ gene expression on the reporter cells even under aerobic conditions. FixL mutant proteins, which contain single amino acid changes near the autophosphorylation site, showed elevated levels of autophosphorylation and a concomitant phosphoryl transfer to FixJ in the presence of oxygen, although their oxygen-binding affinities were unimpaired. These mutational analyses suggest that the autophosphorylation domain plays a crucial role in regulatory coupling between oxygen binding and kinase activity. FixJ mutants in helix alpha1 and strand beta5 of the N-terminal half exhibited the formation of a stable acyl phosphate bond. In contrast, those in helices alpha4 and alpha5 constitutively bound to the fixK promoter in a monomeric form, suggesting that the alpha4 and alpha5 helices may be involved in the post-phosphorylation/dimerization signal transfer to liberate the DNA-binding activity of the C-terminal domain, not only serving as a dimerization interface.
Saito
Ken
K
RIKEN Harima Institute/SPring-8, Mikazuki, Hyogo 679-5148, Japan.
Ito
Eiichi
E
Hosono
Kaito
K
Nakamura
Kayako
K
Imai
Kiyohiro
K
Iizuka
Tetsutaro
T
Shiro
Yoshitsugu
Y
Nakamura
Hiro
H
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006420:Hemeproteins / Q000235:genetics / Q000378:metabolism*; D000071677:Histidine Kinase; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010100:Oxygen / Q000378:metabolism*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000502:physiology*; D016962:Sinorhizobium meliloti / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D006420:Hemeproteins; C069289:FixJ protein, Bacteria; C067929:FixL protein, Bacteria; D000071677:Histidine Kinase; D010100:Oxygen