The uncoupling of oxygen sensing, phosphorylation signalling and transcriptional activation in oxygen sensor FixL and FixJ mutants

Abstract

SummaryThe 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 P fixK -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 a a a a 1 and strand b b b b 5 of the N-terminal half exhibited the formation of a stable acyl phosphate bond. In contrast, those in helices a a a a 4 and a a a a 5 constitutively bound to the fixK promoter in a monomeric form, suggesting that the a a a a 4 and a a a a 5 helices may be involved in the postphosphorylation/dimerization signal transfer to liberate the DNA-binding activity of the C-terminal domain, not only serving as a dimerization interface.

Heme-based sensors: defining characteristics, recent developments, and regulatory hypotheses

“…This is clearly not the case. Multiple observations of stringent regulation of FixJ turnover in air have been reported, and one of those reports has come from the same research group [6,7,40,52].…”

The Signaling Pathway in Histidine Kinase and the Response Regulator Complex Revealed by X-ray Crystallography and Solution Scattering

“…38 The kinase buffer was composed of 50 mM Tris-HCl (pH 7.8), 50 mM KCl, 0.2 mM MgCl 2 , and 10 mM DTT. Each stock solution of ThkA was diluted with kinase buffer to a final concentration of 5.56 μM.…”

ADP reduces the oxygen-binding affinity of a sensory histidine kinase, FixL: The possibility of an enhanced reciprocating kinase reaction

“…The expression and purification of the His 6 -tag FixL and FixJ derived from Sinorhizobium meliloti have been described (6,16). FixL mutants, N403Q, N403E, N403C, and H285V, were constructed by PCR-based nucleotide changes, AAC to CAG, AAC to GAA, AAC to TGC, and CAC to GTT, respectively.…”

“…FixL is an orthodox sensory histidine kinase consisting of a heme-based sensor domain as an oxygen-binding site and a histidine kinase domain, which are aligned in a single polypeptide (3). Deoxy FixL is autophosphorylated at a conserved histidine residue by using ATP, and the phosphoryl group is then transferred to FixJ; but the kinase activity is down-regulated when molecular oxygen binds to the heme moiety of FixL (4)(5)(6). Unphosphorylated FixL preferably exists in the form of a quaternary complex of FixL 2 ͞ FixJ 2 (7) like the EnvZ 2 ͞OmpR 2 complex (8), but on oxygenregulated phosphorylation, phospho-FixJ is liberated to bind to the promoter regions of the nifA and fixK genes (9,10).…”