Regulatory cohesion of cell cycle and cell differentiation through interlinked phosphorylation and second messenger networks

Abstract

In Caulobacter crescentus, phosphorylation of key regulators is coordinated with the second messenger cyclic di-GMP to drive cell-cycle progression and differentiation. The diguanylate cyclase PleD directs pole morphogenesis, while the c-di-GMP effector PopA initiates degradation of the replication inhibitor CtrA by the AAA+ protease ClpXP to license S phase entry. Here, we establish a direct link between PleD and PopA reliant on the phosphodiesterase PdeA and the diguanylate cyclase DgcB. PdeA antagonizes DgcB activity until the G1-S transition, when PdeA is degraded by the ClpXP protease. The unopposed DgcB activity, together with PleD activation, upshifts c-di-GMP to drive PopA-dependent CtrA degradation and S phase entry. PdeA degradation requires CpdR, a response regulator that delivers PdeA to the ClpXP protease in a phosphorylation-dependent manner. Thus, CpdR serves as a crucial link between phosphorylation pathways and c-di-GMP metabolism to mediate protein degradation events that irreversibly and coordinately drive bacterial cell-cycle progression and development.

Figure 1. DgcB, PleD and PdeA antagonistically regulate C. crescentus polar development

(A) Behavior of C. crescentus wild type and c-di-GMP signaling mutants (see also Figs. S1 and S2). Surface attachment (black bars), fraction of cells producing a holdfast (dark grey), colony size on motility plates (light grey), and cellular concentration of c-di-GMP (white) are indicated. Representative examples of pictures of DIC images with overlayed fluorescent holdfast staining (black spots) are shown underneath the graph for all strains. Error bars represent the standard error of the mean. Note that pleD mutants form smaller colonies on semisolid agar plates, despite of their reported hypermotility phenotype in liquid media (Aldridge et al., 2003; Burton et al., 1997). All deletions were complemented by providing a copy of the respective gene in trans (Fig. S4 and data not shown). (B) Timing of holdfast synthesis during the C. crescentus cell cycle. Fluorescently labeled holdfast structures are shown as in (A). Cell cycle progression is shown schematically above the micrographs. Small white arrows highlight holdfasts; black arrows indicate the time point of holdfast appearance.

Figure 2. Cell cycle dependent degradation of PdeA by the ClpXP protease

(A) Immunoblots of synchronized cultures of C. crescentus were stained with anti-DgcB, anti-PleD, anti-PdeA or anti-CtrA antibodies as indicated. (B) Subcellular localization of PdeA during the C. crescentus cell cycle. DIC and fluorescence images of synchronized C. crescentus ΔpdeA cells expressing an N-terminal Venus-PdeA fusion protein. Black arrows mark the old cell pole; white arrows indicate polar PdeA (see also Fig. S3 and Movie S1). The relative numbers of fluorescent foci at the old pole of the primary cell (black) and the old pole of the newbore secondary cell (grey) are depicted over time (n = 58). (C) Quantification of cellular PdeA levels as determined by immunoblots from C. crescentus wild type and mutant strains. The dominant negative clpX allele (clpX^ATP*) was induced for 3 h, ClpX and ClpP depletion strains (P_X∷clpX, P_X∷clpP) were grown in the absence of xylose for 10 h prior to sample harvest. Data were normalized to wild type PdeA levels. Experiments were performed as independent triplicates. Error bars represent the standard error of the mean. (D) Analysis of ClpX-dependent degradation of PdeA during the cell cycle. Strains containing the dominant negative clpX allele (clpX^ATP*) under the control of the xylose inducible promoter P_xyl were analyzed during the cell cycle in the presence (induced) and absence (uninduced) of xylose. Specific antibodies were used to determine levels of ClpXP (PdeA and CtrA) and ClpAP substrates (FliF). (E) PdeA stability as determined by pulse/chase. The dominant negative clpX allele (clpX^ATP*) was induced with xylose for 3 hours prior to the radioactive labeling. All samples were normalized to radiolabeled PdeA present immediately after adding the chase solution.

Figure 3. Polar localization and degradation of PdeA requires CpdR

(A) Cell cycle-dependent degradation of PdeA requires CpdR but not PopA or RcdA. Synchronized cultures of C. crescentus wild type and mutant strains were analyzed by immunoblots with anti-PdeA antibodies. (B) Localization of PdeA to the cell pole requires CpdR but not PopA or RcdA. PdeA-YFP localization was analyzed in C. crescentus wild type and mutant strains (see also Figs. S4 and S5). White arrows in the DIC images mark old cell poles, arrows in the YFP channel highlight polar PdeA foci. The relative number of cells with polar foci is shown below the corresponding micrographs.

Figure 4. CpdR is a phosphorylation-dependent adaptor for PdeA degradation

(A) CpdR is required for ClpXP mediated PdeA degradation in vitro. Purifed PdeA (1 μM) was incubated with 0.4 μM ClpX and 0.8 μM ClpP, either in the absence of CpdR, in the presence of 1 μM CpdR without addition of an ATP regeneration system, or in the presence of both 1 μM CpdR and an ATP regeneration system. (B-C) Quantification of the soluble peptide release upon degradation of ³⁵S labeled PdeA as a function of time. In addition to 2 μM PdeA, all reactions contain 0.2 μM ClpX, 0.4 μM ClpP, 1 mM GTP, and an ATP regeneration system (see also Figure S6). (B) Only unphosphorylated CpdR stimulates PdeA degradation by ClpXP. CpdR: contains unphosphorylated CpdR without the phosphorelay; CpdR + CckA: contains unphosphorylated CpdR and the histidine kinase CckA without the phosphor-transfer protein ChpT; CpdR∼P + CckA/ChpT: CpdR was pre-incubated with CckA/ChpT for 10 min prior to PdeA addition; CckA/ChpT: contains the phosphorelay, but no CpdR. (C) The non-phosphorylateable CpdR_D51A stimulates PdeA degradation even in the presence of a phosphodonor. D51A: contains the non-phosphorylatable CpdR_D51A variant without phosphorelay; D51A + CckA: contains CpdR_D51A and the histidine kinase without the phosphor-transfer protein; D51A + CckA/ChpT: contains CpdRD51A and the CckA/ChpT phosphorelay. (D) Dephosphorylation of CpdR drives PdeA degradation. Phosphorylation of CpdR (CpdR∼P) with the CckA/ChpT phosphorelay deactivates delivery of PdeA. Addition of the CckA_H322A phosphatase (arrow) reactivates PdeA degradation. Degradation was monitored by following loss of fluorescence of a GFP-PdeA fusion protein substrate.

Figure 5. PdeA and DgcB directly interact and co-localize at the cell pole

(A) Bacterial two-hybrid assays depicting PdeA and DgcB inteaction partners (Karimova et al., 1998). The loading scheme is indicated in the lower right corner. (B) Schematic summary of the interactions shown in (A). Individual protein domains are indicated. Arrows connect interaction partners as defined in (A).

Figure 6. Pole development and cell cycle progression requires PdeA degradation

(A) Attachment and motility of C. crescentus wild type and mutant strains expressing a stabilized form of PdeA. The mean of eight (attachment) and four (motility) independent colonies is depicted. Data are presented as relative values of the wild type. Error bars represent the standard error of the mean. (B) Cell cycle dependent holdfast formation in strains expressing a stabilized form of PdeA. Small white arrows highlight labeled holdfasts; black arrows indicate the time point of holdfast appearance. Distribution of stabilized PdeA-FLAG during the cell cycle is indicated in the immunoblot stained with anti-PdeA antibodies. (C) Cell cycle-dependent degradation of CtrA in strains with altered c-di-GMP metabolism. Synchronized swarmer cells of wild type and mutants were followed throughout the cell cycle. CtrA protein levels were analyzed in immunoblots. Immunoblots with an anti-CcrM antibody are shown as control for cell cycle progression (see also Figure S7).

Figure 7. Model for the integration of protein phosphorylation, degradation and c-di-GMP pathways to coordinate C. crescentus pole morphogenesis with cell cycle progression

(A) Regulatory network controlling C. crescentus pole morphogenesis and cell cycle progression. Blue lines indicate phosphorylation reactions; yellow lines indicate processes involved in the regulation of proteolysis; green lines indicate signaling via c-di-GMP. Postulated diguanylate cyclases (DGC) and c-di-GMP effector proteins (E) are indicated. Red and green protein names specify ClpXP substrates. (B) Spatial arrangement at the incipient stalked pole of proteins involved in cell cycle control and development. PdeA and CtrA are recruited to the cell pole by CpdR and PopA. CpdR-mediated degradation of PdeA together with PleD activation increases the concentration of c-di-GMP to activate PopA as well as yet unknown effector-proteins (E) required pole morphogenesis.