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Bacterial cells possess phosphotransfer signaling mechanisms known as ‘two-component regulatory systems’ that elicit a variety of adaptive responses to the cells’ environments.Each
of these systems generally consists of a histidine sensor kinase and a response regulator.The
sensor kinase senses extra- and intracellular stimuli and regulates the function of its cognate
response regulator through a phosphorylation reaction. Accordingly, the response regulator mediates certain changes in gene expression or cell behavior.
A typical sensor kinase has a histidine kinase (HK) domain containing an invariant His residue that is autophosphorylated in an ATP-dependent manner, whereas a typical response regulator has a receiver domain containing a conserved Asp residue that can acquire a phosphoryl group from its cognate sensor kinase. Most two-component systems have this type of a simple His–Asp phosphorelay scheme.
However, some histidine sensor kinases, including BarA, known as tripartite sensor kinases (TSKs), have a more complex type of phosphorelay consisting of two additional domains: a receiver domain containing a conserved Asp residue, and a histidine-containing phosphotransmitter (HPt) domain. In such systems, signals are transmitted through a more sophisticated threestep phosphorelay. First, a phosphoryl group moves from ATP to the HK domain (His residue); secondly, it moves to the receiver domain (Asp residue); and finally it moves to the HPt domain (His residue). Subsequently, the HPt-phosphorylated TSKs phosphorylate the receiver domain (Asp residue) of response regulators.
A typical sensor kinase demonstrated that phosphate group of ATP bound to one monomer subunit transferd to the His residue in the other subunit in the homodimer; this confirmed
that the autophosphorylation reaction of E. coli EnvZ occurs in an intermolecular (trans)
mode.
The mode of the three-step phosphorelay from ATP to the terminal HPt domain in TSKs
remains unclear in most cases. In this study, we introduce Phos-tag SDS-PAGE as a simple method for identifying whether the reactions of autophosphorylation and phosphoryl transfer in TSKs occur in a cis or in a trans mode.
We performed a complementation assay between the BarA G2(ATP binding site was mutated) and H302A (pHis site was mutated) mutants to confirm the intramolecular nature of the autophosphorylation of BarA. It has been demonstrated that heterodimers of BarA are also formed by mixing the both mutants under the conditions for the in vitro complementation assay.The reaction products were analyzed by Phos-tag SDS-PAGE.
The WT protein was successfully autophosphorylated, and Phos-tag SDS-PAGE permitted us
to detect two upshifted bands corresponding to the phosphorylated forms H302–P and D718–
P.There was only a small difference between the degrees of migration of the phosphorylated BarA of H302–P and that of the nonphosphorylated form (non-P).
The G2 and H302A mutants did not autophosphorylate as described above. In the autophosphorylation reaction of an equal mixture of G2 and H302A mutants, on the other hand, we observed a clear upshifted band corresponding to the phosphorylated form D718–P, showing
that a complementary autophosphorylation reaction occurs as an intermolecular reaction and
the following phosphoryl-transfer reaction from the H302 residue to the D718 residue occurs
as an intra- or intermolecular reaction [(i)]. The complementation assay therefore demonstrated
that BarA autophosphorylates in a trans manner.
To confirm and identify the mode of the subsequent multistep phosphorelay, we performed
three more complementation assays between the BarA H302A and D718A mutants, the
H302A and H861A mutants, and the D718A and H861A mutants, respectively, in the presence
of UvrY (see the lanes for the BarA/UvrY phosphorelay). We observed a single
upshifted band corresponding to the phosphorylated UvrY (UvrY–P) in the phosphorelay
reaction with the BarA WT, showing that a phosphoryl-transfer reaction occurs from the BarA
WT to UvrY. In the phosphorelay reaction using an equal mixture of H302A and D718A
mutants in the presence of UvrY, a clear upshifted band corresponding to the phosphorylated
BarA of D718–P was observed, and the phosphoryl group was transferred successfully to UvrY.
This result confirmed that the complementary phosphotransfer reaction from the H302 residue
to the D718 residue proceeds as an intermolecular reaction (trans mode) [(ii)]. However,
in the phosphorelay reaction using an equal mixture of H302A and H861A mutants,
although a clear upshifted band corresponding to the phosphorylated BarA of D718–P was
observed, no upshifted band for UvrY was visible. Furthermore, in the phosphorelay reaction
using an equal mixture of D718A and H861A mutants, we detected a single upshifted band of
UvrY. These results indicate that the phosphoryl-transfer reaction from the D718 residue to
the H861 residue occurs in a trans mode [(iii) and (iv)]. We therefore concluded that
the three-step phosphorelay of BarA proceeds in a trans-trans-trans mode (
Related data
Sinorhizobium meliloti FixL/FixJ, in vitro kinase assay
E.coli EvgS/EvgA,in vitro kinase assay -1
E.coli EvgS/EvgA,in vitro kinase assay -2
E.coli BarA/UvrY, in vitro kinase assay
E.coli ,PhoR/PhoB, in vitro kinase assay
E.coli EnvZ, in vitro kinase assay