Signaling fragments of a bacterial chemoreceptor
by Peter Ames and Sandy Parkinson
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Bacteria employ transmembrane chemoreceptors known as methyl-
accepting
chemotaxis proteins (MCPs) to mediate a variety of adaptive
behaviors. MCP
molecules have an external ligand-binding domain for monitoring
attractant
or repellent levels in the environment and a cytoplasmic signaling
domain
for communicating with the flagellar rotary motors (Fig. 1). MCPs form
ternary complexes with two cytoplasmic proteins, CheA, a histidine
kinase,
and CheW, which couples CheA activity to chemoreceptor control. To
explore
the structure and function of the MCP signaling complex, we identified
soluble fragments of the serine chemoreceptor, Tsr, that could
activate the
CheA kinase, both in vivo and in vitro.
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Figure 1. Structure and signaling properties of MCPs. MCP
molecules are
homodimers, nearly 400 Å in length, of interwound, largely alpha-helical
subunits (yellow and red). The MCP
signaling domain modulates CheA kinase activity in response to
changes in
ligand occupancy. CheA in turn passes phosphoryl groups to two
effector
proteins, CheY, which controls the direction of flagellar rotation, and
CheB, which regulates MCP methylation state to bring about sensory
adaptation. The methylation sites (blue) are located along the dimer
interface and might control signal output by modulating the
interactions
between MCP subunits.
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To identify active signaling fragments of Tsr, we inserted random
segments
of the tsr coding region into an inducible expression vector and
screened
for recombinant plasmids that could inhibit or "jam" the chemotactic
behavior of a wild-type host strain (Fig. 2, left). We reasoned that Tsr
fragments that could bind CheA or CheW would jam chemotactic
signaling by competitively titrating these signaling components away
from
the cell's other chemoreceptors. Moreover, fragments that
assemble a
functional signaling complex should also interfere with chemotactic
behavior by transmitting inappropriate signals to the flagella.
Actively signaling fragments obtained from wild-type Tsr caused
excessive
clockwise (CW) rotation of the flagellar motors. CW-signaling
fragments
invariably included the highly conserved cytoplasmic "tip" of the Tsr
molecule, indicating that CW signals emanate from that region (Fig. 2,
right). Some CW fragments lacked the methylation sites involved in
sensory
adaptation, demonstrating that MCP methylation is not essential for
CW
signaling. However, longer fragments containing the methylation sites
only
caused CW rotation if methylated, implying that the unmethylated
form of
MCP molecules actively interferes with assembly or function of the CW
signaling complex.
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Figure 2. Tsr signaling fragments. (Left) Scheme for
identifying active
fragments. (1) Random pieces of the tsr coding region were
cloned into an
inducible expression vector. (2) Recombinant plasmids were screened
in a
wild-type host for ability to jam chemotaxis upon induction. (Right)
Fragments that jam chemotaxis and enhance CW flagellar rotation.
Methylation sites (blue) are not essential for these activities. The
shortest active fragment (350-438) requires a point mutation
(black)
to stabilize its CW-signaling conformation.
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Cells that lack MCPs cannot generate CW signals and, consequently,
rotate
their flagella exclusively in the counter-clockwise direction. Tsr
fragments with CW signaling activity cause such cells to spread faster
through soft agar, a behavior we call "pseudotaxis" (Fig. 3, left). Using
the pseudotaxis effect to monitor CW signaling activity, we
progressively
trimmed our shortest Tsr fragment to determine the minimum size of
the CW
signaling segment. The current record holder is an 89-residue
fragment
centered on the signaling tip (Fig. 2). Several larger CW fragments
were
purified and shown in vitro to activate the CheA
kinase. CW fragments acted in a highly cooperative manner, with
perhaps
8-10 fragments needed to activate a single CheA molecule (Fig. 3,
right).
Activation also required the CheW coupling protein, suggesting that Tsr
fragments assemble ternary signaling complexes comparable to
those made by full-length MCP molecules in vivo.
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Figure 3. Assays for CW signaling. (Left) Induction of
pseudotactic
spreading by a Tsr fragment in vivo. The host cells lack other
MCPs and
spread faster through soft agar upon expression of a fragment that
enhances
CW flagellar rotation. (Right) Stimulation of CheA activity by a
CW-signaling Tsr fragment in vitro. Fragment action is CheW-
dependent and
highly cooperative.
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Our studies of Tsr fragments have provided important new insights
into the
signaling mechanisms of bacterial chemoreceptors. For example, it
now
appears that several MCP molecules must cooperate in the assembly
and
function of ternary signaling complexes. In addition, the in vivo
and in
vitro assays that we developed for Tsr signaling fragments have
enabled us
to identify the binding contacts and other determinants of
chemoreceptor
function, as described in other posters.
Ames, P. & J.S. Parkinson (1994) Constitutively signaling fragments of
Tsr,
the E. coli serine chemoreceptor. J. Bacteriol. 176:
6340-6348.
Ames, P., Y.A. Yu & J.S. Parkinson (1996) Methylation segments are
not
required for chemotactic signaling by cytoplasmic fragments of Tsr,
the
methyl-accepting serine chemoreceptor of Escherichia coli.
Mol. Microbiol.,
19: 737-746.
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