We established that the sensor histidine kinase DivJ has an important role in the regulation of C. crescentus cell cycle period and noise. This was accomplished by designing and conducting single-cell experiments to probe the dependence of cell cycle noise on divJ expression and constructing a simplified cell cycle model that captures the dependence of cell cycle noise on DivJ with molecular details.In addition to its role in regulating the cell cycle, DivJ also affects polar cell development in C. crescentus, regulating swarming motility and surface adhesion. We propose that pleiotropic control of polar cell development by the DivJ–DivK–PleC signaling pathway underlies divJ-dependent tuning of cell swarming and adhesion behaviors.We have integrated the study of single-cell fluorescence dynamics with a kinetic model simulation to provide direct quantitative evidence that the DivJ histidine kinase is localized to the cell pole through a dynamic diffusion-and-capture mechanism during the C. crescentus cell cycle.
Temporally-coordinated localization of various structural and signaling proteins is critical for proper cell cycle regulation and polar cell development in the bacterium, Caulobacter crescentus. Included among these dynamically-localized regulatory proteins is the sensor histidine kinase, DivJ (Wheeler and Shapiro, 1999). Co-localized with DivJ in the early stalked phase is the phosphorylated response regulator DivK∼P (Jacobs et al, 2001), and the protease ClpXP (McGrath et al, 2006), which degrades the master cell cycle regulator, CtrA (Jenal and Fuchs, 1998). Recent single-cell measurements of surface attached C. crescentus cells have revealed an intriguing role for DivJ in the control of noise in cell division period (Siegal-Gaskins and Crosson, 2008). The noise of the cell cycle increases significantly upon disruption of the divJ gene, with a relatively small accompanying increase in the mean cell cycle time. The deterministic nature of the existing cell cycle models (Li et al, 2008, 2009; Shen et al, 2008) cannot explain the measured increase in cell cycle period and noise in a divJ null strain. Moreover, mechanistic descriptions of how DivJ and its signaling partners are localized and how these proteins underlie the control of polar cell development and cell adhesion in C. crescentus remain immature.
The single-cell experiments and analysis presented herein reveal that C. crescentus cell cycle period and noise can be tuned by DivJ (Figure 2). Specifically, in the case of low (or no) divJ expression the cell cycle is perturbed, and this is quantified by way of the (measured) noise in the cell cycle period. The level of noise is readily controlled through regulated expression of the divJ gene (Figure 2B). A simplified protein interaction network of stalked C. crescentus cell cycle regulation involving minimal components (CtrA, CtrA∼P, DivK, DivK∼P, and DivJ) was constructed to explore such tunability at the molecular level. The agreement of our model with our (and other) experiments suggests this simplified protein regulatory network is sufficient to explain the major features of the C. crescentus cell cycle. Indeed, stochastic simulations of this model using the Gillespie method (Gillespie, 1976) establish the importance of robust DivJ-mediated phosphorylation of its cognate receiver protein, DivK, in regulating the variance of cell cycle oscillations. Increased variability in the concentration of DivK∼P at the single cell level under divJ depletion subsequently leads to increased noise in the regulation of CtrA phosphorylation and degradation. Our experiments and simulations provide evidence that the steady state level of DivK∼P at the single-cell level (as maintained by DivJ) is essential in maintaining regular timing of the cell division period in C. crescentus.
In addition to its role in regulating cell cycle, divJ expression also affects polar cell development in C. crescentus. Specifically, the capacity of swarmer cells to adhere to a glass surface is suppressed at high levels of divJ expression. The effect of elevated divJ expression on the adhesive capacity of the cell is reflected in a reduced rate of two-dimensional biofilm formation. This effect is quantitatively captured by our mathematical model that relates single-cell surface adhesion physiology and biofilm formation dynamics. This result, and our observation that divJ expression tunes swarming motility in semi-solid growth medium, suggests a model in which increased DivJ concentration in the swarmer compartment (due to constitutive overexpression) ultimately results in improper development of polar organelles that are required for adhesion of swarming motility.
Despite the appreciated significance of protein localization for bacterial physiological functions, the molecular mechanism of how polar protein localization is achieved has only been tested in a few cases (Shapiro et al, 2002; Thanbichler and Shapiro, 2008). Mechanisms such as the polar insertion model and diffusion-and-capture have been proposed but the community's knowledge is limited to very few examples (Charles et al, 2001; Rudner et al, 2002). We provide direct evidence from experiments and simulations that the DivJ histidine kinase becomes localized to the cell pole through a dynamic diffusion-and-capture mechanism during the C. crescentus cell cycle (Figure 7). We show that a kinetic model based on a Langmuir adsorption/desorption relationship (Figure 7D) is sufficient to explain the time evolution of the single cell fluorescence time traces (Figure 7C and E) and allows establishing quantitative correspondences between the simulated dynamics and experimentally determined DivJ–EGFP dynamics. This localization mechanism is consistent with a diffusion-and-capture model. In short, the model posits that proteins are randomly distributed and are freely diffusing until they are captured at the site where they ultimately reside (Rudner et al, 2002; Shapiro et al, 2002; Bardy and Maddock, 2007). With a diffusion-and-capture pathway, it has been argued that proteins can be adsorbed either dynamically or statically (Shapiro et al, 2009). Our analysis of DivJ–EGFP in single cells supports a dynamic diffuse-and-capture mechanism for DivJ localization.
Sensor histidine kinases underlie the regulation of a range of physiological processes in bacterial cells, from chemotaxis to cell division. In the gram-negative bacterium Caulobacter crescentus, the membrane-bound histidine kinase, DivJ, is a polar-localized regulator of cell cycle progression and development. We show that DivJ localizes to the cell pole through a dynamic diffusion and capture mechanism rather than by active localization. Analysis of single C. crescentus cells in microfluidic culture demonstrates that controlled expression of divJ permits facile tuning of both the mean and noise of the cell division period. Simulations of the cell cycle that use a simplified protein interaction network capture previously measured oscillatory protein profiles, and recapitulate the experimental observation that deletion of divJ increases the cell cycle period and noise. We further demonstrate that surface adhesion and swarming motility of C. crescentus in semi-solid media can also be tuned by divJ expression. We propose a model in which pleiotropic control of polar cell development by the DivJ–DivK–PleC signaling pathway underlies divJ-dependent tuning of cell swarming and adhesion behaviors.