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					MicroReview: C-di-GMP: the dawning of a novel
bacterial signaling system

   C-di-GMP is emerging as a novel global second
    messenger.
           Recent findings show involvement in cell
            metabolism and biological processes in the cell.
   GGDEF domain protein involved in c-di-GMP
    synthesis.
   EAL domain protein involved in c-di-GMP
    degradation.
   These two proteins affect cell differentiation,
    multicellular behavior, and interactions between
    microorganisms and their eukaryotic hosts.
   GGDEF and EAL domain found across many
    branches of the phylogenetic tree of bacteria.

   1 GGDEF domain has ½ GTP binding catalytic site,
    so dimer of 2 GGDEF form active diguanylate
    cyclase activity (DGC).
   In E. coli, S. enterica, Pseudomonas
    fluorescence, activated GGDEF domain proteins
    and resulting elevated c-di-GMP concentrations
    favour the production of adhesive matrix
    components.

   Also, GGDEF protein is found to play a role in
    stimulation of motility in E. coli.

   In C. crescentus, the GGDEF domain protein
    named PleD controls cell morphology.
Cell cycle-dependent dynamic localization of a
bacterial response regulator with a novel di-
guanylate cyclase output domain
Background

   Developmental transitions depend
    on adaptation in levels and
    arrangement of proteins.
       Relies on transcriptional regulators
       Establishment of gradients through the
        compartmentalization of signaling
        complexes.
   Two component regulatory system
       Sensor kinase
       Soluble response regulator
   C. crescentus has obligate developmental transition that
    allows switch between sessile, adhesive, and motile cell
    during cell cycle.
       Cell poles are continuously remodeled during cell
        differentiation.

    In the predivisional cell, a pili being assembled at one
    pole while the opposite pole consists of a stalk (an
    adhesive organelle).
Purpose of study

   Investigate the function and regulation
    of the PleD response regulator in
    C.crescentus polar development.

   Investigate whether polar kinases DivJ
    and PleC involved in modulating the
    phosphorylation state of PleD.

   Propose PleD contains an intrinsic
    nucleotide cyclase activity that
    converts two molecules of GTP into
    cycli diguanylic acid (c-di-GMP).
DivJ and PleC directly control PleD
phosphyrylation

   Previous studies
    established a role
    of polar kinases
    DivJ and PleC in
    PleD control
    (Aldridge et. Al.
    2003).
   Use in vitro
    phosphorylation
    assays.
The PleD response regulator localizes
to the stalked pole

   PleD is a soluble
    cytoplasmic
    protein.
   DivJ and PleC are
    sensor kinases
    membrane bound.
   Hypothesis: PleD
    might perform its
    regulatory function
    at the pole.
Test PleD perform its regulatory
function locally
   Analyzed the
    subcellular distribution
    of PleD during the C.
    crescentus cell cycle.
   A PleD-GFP fusion was
    introduced into mutant
    pleD strain.
   Analysis showed that
    most stalked and
    predivisional cells have
    PleD-GFP concentrated
    at the stalked pole.
       Absent from the
        flagellated swarmer pole
- Data for the insertion into mutant pleD strain was not shown in
the table.
What is the spatial distribution of PleD-GFP during
the cell cycle?




   Perform time lapse fluorescence
    microscopy with isolated swarmer
    cells of WT strain.
   First, PleD protein evenly distributed within C.
    crescentus swarmer cells.

   As time passes then it concentrates at the
    emerging stalked pole during the swarmer-to-
    stalked cell differentiation.

   With increasing time signal at the stalked pole
    increases in strengths.
Localization of activated PleD


   PleD might exist in two different
    forms.

   Test whether phosphorylation of
    PleD is required for dynamic
    localization.
   Fused GFP to an inactive mutant
    PleD at position 53.
       Mutated site is Aspartic acid phosphoryl
        acceptor
   Immunoblot analysis showed PleD-
    D53N-GFP mutant was distributed
    evenly in all cells.
   Also, PleD-GFP in absence of DivJ
    and PleC kinases failed to localize at
    the pole
   So phosphorylation is important in
    sequestering PleD to the pole.
   Test to distinguish between:
       Phosphorylation might constitute the
        targeting signal
       PleD prefers binding to the cell pole in
        its active confirmation
   Analyzed constitutively active PleD
    mutant protein, PleD* D53N (lacks
    the phosphoryl acceptor site @
    position 53).
   PleD* D53N –GFP localizes almost
    exclusively to the pole in WT and
    divJ, pleC double mutant.
   Analysis of
    PleDGG368DE-GFP
    fusion protein,
    which lacks an
    active C-terminal
    output domain.
       Results:
        localization of PleD
        at the pole.
   Conclusion: An activated
    conformation of PleD, rather than
    the PleD readout itself, is required
    for polar sequestration of the
    regulator.
What is the output signal of PleD?
   We know now PleD accumulates at the
    old pole of the cell only in its activated
    state.
   We know from genetic data (Aldridge et.
    Al. 2003) phosphorylated PleD is required
    for the differentiation of a flagellated into
    a stalked pole.
   We know that PleD could coordinate the
    developmental events involved in pole
    remodeling.
   Past research (Ross et. Al. 1991, Tal et.
    Al. 1998) showed a link between:
       GGDEF domain
       Synthesis of cyclic di-GMP


   To examine possibilities that the PleD
    output domain harbors di-guanylate
    cyclase activity.
            Performed a biochemical assay to test PleD’s
             ability to convert GTP into c-di-GMP.
   Extracts of CB15N ∆pleD showed no
    activity.




                   5 10 15 20 30
                       minutes
   To demonstrate that PleD was
    responsible for the activity.
       PleD with a C-terminal His-tag was
        overexpressed in E. Coli.




                     30 45 60 300
                     seconds
Confirm indeed it is cyclic GMP

   The reaction product of PleD and GTP
    was analyzed by mass spectrometry.




                      Exactly matches the molecular
                      weight of c-di-GMP
      C-di-GMP Competing?

         When synthesized c-di-GMP was
          added to the reaction mix to a
          similar concentration of the GTP
          substrate.
Each reaction mix
contained:
25µg PleD
100µM of GTP
   Suggest that c-di-GMP competes
    with GTP for the binding site.
         Does PleD function as a
         phosphodiesterase (PDE)?
            C-di-GMP concentration was measured quantatively by High-
             pressured Liquid Chromatography (HPLC) after incubation with
             purified PleD-His6 protein for several hours.

                       HPLC analysis of
                                           Reaction Products
                         Reactants




2 minute intervals
   This suggests that PleD has a di-
    guanylate cyclase activity but lacks
    phosphodiesterase activity.
       The di-guanylate cyclase activity
        constitutes as the signaling output of
        the PleD response regulator.
   Cyclases activity is GTP specific




Unlabeled nucleotides
Formation of c-di-GMP

   Structure of PleD response
    regulator suggests:
       Receiver Domain (N-terminal)
        information input
       Regulatory output domain (C-terminal)
        contains GGDEF domain
   Test whether the guanylate cyclase
    activity is indeed localized in the
    GGDEF domain.
       Determined activity of WT PleD
        through:
            Examining the activities of two mutant
             proteins in the GGDEF domain.
                 Mutant alleles pleD∆368-372 (lacking the entire
                  GGDEF motif)
                 pleDGG368DE (2 Gly replaced by Asp and Glu)
   Results showed both mutant
    proteins lacked di-guanylate cyclase
    activity in vitro.
       Thus, requires an intact GGDEF output
        domain.
Discussion

   Activated confirmation of the
    protein provides the information for
    polar localization.
       Not, phosphorylation itself.
   Two different mechanisms can be
    envisioned to explain the coupling
    between activity and polar
    localization of PleD.
       PleD could auto-catalytically controls its
        own subcellular positioning
       Pole recognition might be restricted to
        the activated form of PleD.
   Observations of inactive PleDD53N-GFP
       Present throughout the cell




   Observations of GFP fused to PleDGG368DE, unable to generate
    c-di-GMP.
       Present at the pole




   Better favors the 2nd mechanism.
More conclusions

   GGDEF domain of PleD posseses
    DGC, cyclase activity but not PDE
    activity.

   GGDEF domains represent a signal
    output of a complex bacterial signal
    transduction network, in which
    leads to the production of c-di-GMP.
GGDEF domain and different sensory
inputs
   Presence of a large number of
    potential DGCs in single bacterial
    species raises the question of how
    the output specificity of parallel
    signaling pathways might be
    achieved?

				
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