24 research outputs found

    Potential Role of a Bistable Histidine Kinase Switch in the Asymmetric Division Cycle of <i>Caulobacter crescentus</i>

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    <div><p>The free-living aquatic bacterium, <i>Caulobacter crescentus</i>, exhibits two different morphologies during its life cycle. The morphological change from swarmer cell to stalked cell is a result of changes of function of two bi-functional histidine kinases, PleC and CckA. Here, we describe a detailed molecular mechanism by which the function of PleC changes between phosphatase and kinase state. By mathematical modeling of our proposed molecular interactions, we derive conditions under which PleC, CckA and its response regulators exhibit bistable behavior, thus providing a scenario for robust switching between swarmer and stalked states. Our simulations are in reasonable agreement with <i>in vitro</i> and <i>in vivo</i> experimental observations of wild type and mutant phenotypes. According to our model, the kinase form of PleC is essential for the swarmer-to-stalked transition and to prevent premature development of the swarmer pole. Based on our results, we reconcile some published experimental observations and suggest novel mutants to test our predictions.</p></div

    Two scenarios for the function of PleC (kinase or phosphatase) in the early predivisional cell.

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    <p>Spatiotemporal dynamics of PleC (green) and DivL (dark blue) during the cell cycle under two scenarios for the functional transition of PleC. In scenario 1, PleC is a kinase in early PD cells, as suggested in [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004348#pcbi.1004348.ref054" target="_blank">54</a>–<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004348#pcbi.1004348.ref056" target="_blank">56</a>]. In scenario 2, PleC is a phosphatase in early PD cells, as described in the protection by dephosphorylation model [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004348#pcbi.1004348.ref045" target="_blank">45</a>,<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004348#pcbi.1004348.ref053" target="_blank">53</a>].</p

    PleC kinase conformation is required to establish replicative asymmetry.

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    <p>Concentration gradients are color coded as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004348#pcbi.1004348.g005" target="_blank">Fig 5</a>. <b>(A)</b> In Δ<i>pleC</i> mutant cells (K<sup>-</sup>P<sup>-</sup>B<sup>-</sup>), free active form of DivL is lower than in wild-type cells, resulting in loss of CtrA~P in the predivisional cell. <b>(B)</b> In <i>pleC</i><sub>H610A</sub> mutant cells (K<sup>-</sup>P<sup>-</sup>B<sup>+</sup>), an elevated level of DivK~P results in less active form of DivL and reduced CtrA~P. <b>(C)</b> In <i>pleC</i><sub>F778L</sub> mutant cells (K<sup>-</sup>P<sup>+</sup>B<sup>+</sup>), inhibitor sequestration is retained, resulting in a normal CtrA~P gradient.</p

    Dynamical Localization of DivL and PleC in the Asymmetric Division Cycle of <i>Caulobacter crescentus</i>: A Theoretical Investigation of Alternative Models

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    <div><p>Cell-fate asymmetry in the predivisional cell of <i>Caulobacter crescentus</i> requires that the regulatory protein DivL localizes to the new pole of the cell where it up-regulates CckA kinase, resulting in a gradient of CtrA~P across the cell. In the preceding stage of the cell cycle (the “stalked” cell), DivL is localized uniformly along the cell membrane and maintained in an inactive form by DivK~P. It is unclear how DivL overcomes inhibition by DivK~P in the predivisional cell simply by changing its location to the new pole. It has been suggested that co-localization of DivL with PleC phosphatase at the new pole is essential to DivL’s activity there. However, there are contrasting views on whether the bifunctional enzyme, PleC, acts as a kinase or phosphatase at the new pole. To explore these ambiguities, we formulated a mathematical model of the spatiotemporal distributions of DivL, PleC and associated proteins (DivJ, DivK, CckA, and CtrA) during the asymmetric division cycle of a <i>Caulobacter</i> cell. By varying localization profiles of DivL and PleC in our model, we show how the physiologically observed spatial distributions of these proteins are essential for the transition from a stalked cell to a predivisional cell. Our simulations suggest that PleC is a kinase in predivisional cells, and that, by sequestering DivK~P, the kinase form of PleC enables DivL to be reactivated at the new pole. Hence, co-localization of PleC kinase and DivL is essential to establishing cellular asymmetry. Our simulations reproduce the experimentally observed spatial distribution and phosphorylation status of CtrA in wild-type and mutant cells. Based on the model, we explore novel combinations of mutant alleles, making predictions that can be tested experimentally.</p></div

    Localization and function of the DivK-PleC-DivK and DivL-CckA-CtrA signaling networks, as suggested by the model calculations reported here.

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    <p>Localization and function of the DivK-PleC-DivK and DivL-CckA-CtrA signaling networks, as suggested by the model calculations reported here.</p

    Point mutations in CckA suppress the <i>ΔdivJ</i> mutant phenotype by affecting CckA kinase activity.

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    <p>(<b>A</b>) <i>ΔdivJ</i> cells show higher CtrA∼P level than their wild type counterpart. Point mutations in CckA and DivL are known to suppress the mutant phenotype. To understand how, we plot the steady-state level of CtrA∼P as a function of rate constants governing (<b>B</b>) DivL activating CckA kinase (<i>k</i><sub>cp-ck</sub>), (<b>C</b>) CckA kinase activity (<i>k</i><sub>ck-ck1</sub>), and (<b>D</b>) CckA phosphatase activity (<i>k</i><sub>cp-ch1</sub>). The vertical green lines indicate the values of the rate constants in the <i>ΔdivJ</i> background. Reducing the value of <i>k</i><sub>cp-ck</sub> or <i>k</i><sub>ck_ck1</sub> causes a corresponding reduction in CtrA∼P level. However, increasing <i>k</i><sub>cp_ch1</sub> does not cause CtrA∼P to fall to its wild-type level.</p

    Over-expressing DivK causes a drop in PleD phosphorylation.

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    <p>The steady state level of PleD∼P is plotted against increasing amount of total DivK for (<b>A</b>) <i>ΔdivJ divK<sub>D53N</sub></i>, (<b>B</b>) <i>ΔdivJ</i>, and (<b>C</b>) wild type background. Although the absolute levels vary among the three cell types, in each case PleD∼P level shows an initial increase followed by a drop at high DivK.</p

    Swarmer-to-stalked transition is uncoupled from G1-to-S transition in DivL mislocalization mutants.

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    <p>Concentration gradients are color coded as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1004348#pcbi.1004348.g005" target="_blank">Fig 5</a>. <b>(A)</b> DivL is uniformly distributed at all times in the cell cycle. Even though PleC transitions to a kinase, DivL remains deactivated (bound to DivK~P) in the predivisional cell, resulting in no gradient of CtrA~P. <b>(B)</b> DivL (and CckA) are localized at the old pole (<i>t</i> = 0–90 min), before switching to the new pole in the predivisional cell (<i>t</i> = 90–120 min). Even after PleC transitions to a kinase, DivL is not deactivated in stalked cells, delaying the dephosphorylation of CtrA~P until PleC delocalizes from the old pole (at <i>t</i> = 50 min). <b>(C)</b> DivL and PleC are co-localized at one of the poles during all stages of the cell cycle. Hence, CtrA is phosphorylated through all stages of the cell cycle (G1-arrest).</p

    Following cytokinesis, PleC reverts to the phosphatase form.

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    <p><b>(A)</b> The total concentration of CckA (blue curve) remains constant during the cell cycle. However, the proportions of phosphatase (red curve) and kinase (green curve) forms of CckA change for each stage of the cell cycle. After cytokinesis (compartmentalization), the concentrations of the kinase and phosphatase fractions of CckA in the swarmer and stalked compartments (<i>t</i> = 120–150 min) are similar to their concentration in the non-compartmentalized swarmer (<i>t</i> = 0–30 min) and stalked cell stages (<i>t</i> = 30–90 min), respectively. <b>(B)</b> Spatiotemporal distribution of PleC kinase, DivK~P and CtrA~P after compartmentalization (at <i>t</i> = 120 min). Color indicates concentration gradients from minimum (blue) to maximum (red).</p

    Comparison of <i>pleC</i><sub>F778L</sub> and <i>divK</i><sub>D90G</sub> mutants reveals the importance of the PleC kinase state.

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    <p>For wild-type cells (<b>A</b>) and three different mutants (<b>B–D</b>), we plot the steady-state fractions of four variables: PleC kinase, DivK∼P, CtrA∼P and PleD∼P. DivK<sub>X</sub> is a hypothetical mutant form of DivK which is still phosphorylated and dephosphorylated by PleC but does not induce the conformational change of PleC from phosphatase to kinase.</p
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