A phospho-signaling pathway controls the localization and activity of a protease complex critical for bacterial cell cycle progression

Abstract

Temporally and spatially controlled master regulators drive the Caulobacter cell cycle by regulating the expression of >200 genes. Rapid clearance of the master regulator, CtrA, by the ClpXP protease is a critical event that enables the initiation of chromosome replication at specific times in the cell cycle. We show here that a previously unidentified single domain-response regulator, CpdR, when in the unphosphorylated state, binds to ClpXP and, thereby, causes its localization to the cell pole. We further show that ClpXP localization is required for CtrA proteolysis. When CpdR is phosphorylated, ClpXP is delocalized, and CtrA is not degraded. Both CtrA and CpdR are phosphorylated via the same CckA histidine kinase phospho-signaling pathway, providing a reinforcing mechanism that simultaneously activates CtrA and prevents its degradation by delocalizing the CpdR/ClpXP complex. In swarmer cells, CpdR is in the phosphorylated state, thus preventing ClpXP localization and CtrA degradation. As swarmer cells differentiate into stalked cells (G1/S transition), unphosphorylated CpdR accumulates and is localized to the stalked cell pole, where it enables ClpXP localization and CtrA proteolysis, allowing the initiation of DNA replication. Dynamic protease localization mediated by a phospho-signaling pathway is a novel mechanism to integrate spatial and temporal control of bacterial cell cycle progression.

Fig. 1.

The CpdR response regulator is localized to the cell pole. (A) Swarmer cells of a strain expressing cpdR-yfp from a low copy-number plasmid in a wild-type background were harvested, resuspended on an agarose pad, and, thereafter, imaged by DIC and fluorescence microscopy every 45 min. Arrows indicate CpdR-YFP foci. (B and C) Schematic of CtrA localization (B; ref. 8) and localization of the ClpXP protease and the RcdA degradation factor as a function of the cell cycle (C; ref. 3).

Fig. 2.

Two ClpXP substrates, CtrA and the polar McpA chemoreceptor, depend on CpdR for their proteolysis. (A) Immunoblots of synchronized populations of wild-type, ΔcpdR::Ω, ΔcpdR::Ω with the complementing plasmid pcpdR, and cpdRD51A strains by using antibodies to CtrA and McpA. A cell cycle schematic is shown. (B and C) Pulse–chase experiments showing the half-lives of CtrA (B) and McpA (C) in wild-type, ΔcpdR::Ω, and cpdRD51A strains.

Fig. 3.

CpdR directly interacts with ClpXP and is required for its polar localization. (A) Wild-type (Upper) or ΔcpdR::Ω (Lower) strains, containing a chromosomal xylose-inducible clpX-gfp fusion were treated for 1 h with xylose and were observed by DIC and fluorescence microscopy. Arrows indicate ClpX-GFP foci. (B) Samples from coimmunoprecipitation experiments performed with anti-M2 antibody and whole-cell extract were probed with anti-ClpX and anti-ClpP antisera. The strains used in these experiments are indicated at the right of the figure.

Fig. 4.

The CpdR phosphorylation state affects ClpX localization in a CckA-dependent manner. (A) CpdR and CpdR∼P accumulation in wild-type and mutant cells, revealing that CpdR depends on CckA for its phosphorylation. Cells were labeled with 30 μCi [γ-³²P]-ATP per ml of culture for 3 min, lysed, and immunoprecipitated with anti-CpdR antibodies. (B Upper) A cpdRD51A strain with xylose-inducible clpX-gfp expression was treated for 1 h with xylose. Swarmer cells were isolated, resuspended on an agarose pad, and imaged by DIC and fluorescence microscopy at 30-min intervals over the course of the cell cycle. Arrows indicate ClpX-GFP foci. (B Lower) Schematic of ClpX-GFP localization during the wild-type cell cycle (3). (C) Proportion of swarmer cells containing ClpX-GFP or CpdR-YFP polar foci in wild-type and cpdRD51A strains. The total number of observed cells is indicated as “n,” the proportion of cells with polar foci as “polar,” and the proportion of cells with no polar foci as “non-polar.”

Fig. 5.

The regulation of CtrA proteolysis by cell cycle-dependent phosphorylation of CpdR. (A) Schematic model of CpdR phosphorylation in the presence of [γ-³²P] mediated by CckA (Left). Aliquots of a population of synchronized wild-type cells (Center) were labeled with 30 μCi [γ-³²P]-ATP per ml of culture for 3 min at the indicated intervals. Samples were lysed, and immunoprecipitated with anti-CpdR antibodies. Green arrows represent the time point of the [γ-³²P]-ATP pulse. Phosphorylated CpdR is detected in predivisional cells, coincident with the time when CckA is localized and active (9, 10). As a control, additional samples were taken at 20-min intervals for immunoblot analysis with CpdR antibodies to assess total CpdR accumulation. A mixed population of wild-type cells was grown in 500 ml of M5G media to an OD₆₆₀ of 0.25, resuspended in 20 ml of M5G, labeled with 15 μCi [³²P]-ATP per ml of culture for 45 min, and swarmer cells were isolated and allowed to proceed synchronously through the cell cycle. Samples were taken at 0, 20, and 40 min, lysed, and immunoprecipitated with anti-CpdR antibodies, showing that CpdR∼P is present in the swarmer cells (Right). Immunoblots using antibodies to CpdR and CtrA showed no change in CpdR levels and the expected decrease in CtrA at the swarmer-to-stalked cell transition. (B) Model of CckA phospho-signaling pathway mediated proteolysis of the CtrA master regulator. A detailed description is given in Discussion.