Dynamics of two Phosphorelays controlling cell cycle progression in Caulobacter crescentus

Abstract

In Caulobacter crescentus, progression through the cell cycle is governed by the periodic activation and inactivation of the master regulator CtrA. Two phosphorelays, each initiating with the histidine kinase CckA, promote CtrA activation by driving its phosphorylation and by inactivating its proteolysis. Here, we examined whether the CckA phosphorelays also influence the downregulation of CtrA. We demonstrate that CckA is bifunctional, capable of acting as either a kinase or phosphatase to drive the activation or inactivation, respectively, of CtrA. By identifying mutations that uncouple these two activities, we show that CckA's phosphatase activity is important for downregulating CtrA prior to DNA replication initiation in vivo but that other phosphatases may exist. Our results demonstrate that cell cycle transitions in Caulobacter require and are likely driven by the toggling of CckA between its kinase and phosphatase states. More generally, our results emphasize how the bifunctional nature of histidine kinases can help switch cells between mutually exclusive states.

FIG. 1.

Cell cycle abundance of ChpT. Wild-type cells were synchronized and allowed to proceed through a single cell cycle, with lysates collected every 20 min and used for immunoblotting with anti-CtrA or anti-ChpT serum. The cell cycle diagram above indicates the approximate cell cycle stage at each time point.

FIG. 2.

Full-length CckA can drive the phosphorylation and dephosphorylation of CtrA and CpdR. (A) Phosphorelay reconstitutions using the purified components indicated by pluses; minuses indicate components not used. The components indicated were mixed together without ATP. Phosphotransfer reactions were started with the addition of ATP and incubated for 30 min at 30°C. The position of each phosphorylated component is marked with an arrowhead on the right. (B, C) Dephosphorylation of CtrA and CpdR. Purified CtrA∼P (B) or CpdR∼P (C) was incubated with the components indicated but without ATP to test for backtransfer and dephosphorylation to yield inorganic phosphate (P_i). Phosphatase reactions were started with the addition of CckA and, if indicated, ChpT. Each reaction was allowed to proceed for 0, 10, 30, or 60 min before being stopped. Note that the faint bands appearing in the first lane of panels B and C correspond to the components used to phosphorylate CtrA and CpdR (see Materials and Methods). In each panel, CckA-HK-RD and CckA-HK contain His₆-MBP tags; CckA-RD, ChpT, CpdR, and CtrA contain TRX-His₆ tags; and ChpT contains a His₆ tag. Different tags were used for ChpT to optimize band separation.

FIG. 3.

Identification of mutants that uncouple kinase and phosphatase activity in CckA. (A) Summary of CckA mutations tested. For mutations reported previously to produce K⁺ P⁻ or K⁻ P⁺ EnvZ, the same amino acid substitutions were introduced at the corresponding sites of CckA, based on an alignment of EnvZ and CckA sequences. Mutations are listed below the domain of CckA in which they were constructed. Domains include TM, DHp, CA, and RD. Phosphorylation sites (H322 and D623) are indicated above their respective locations. (B) Each mutant version of CckA-HK-RD was tested for autophosphorylation (top) and phosphotransfer to ChpT (bottom). Autophosphorylation reactions were started with the addition of ATP to preincubated mixtures of the indicated mutant CckA and reaction buffer. Reaction mixtures were incubated for 30 min at 30°C. Phosphotransfer reactions were performed using autophosphorylated CckA. These reactions were started with the addition of ChpT and allowed to proceed for 30 min before being stopped. (C) Each mutant version of CckA-HK-RD was tested for dephosphorylation of CtrA∼P. Purified CtrA∼P was isolated, and reactions were started with the addition of ChpT and mutant versions of CckA and then allowed to proceed for 30 min at 30°C. The positions of phosphorylated components in each panel are marked with arrowheads on the left. P_i, inorganic phosphate.

FIG. 4.

Biochemical characterization of CckA(V366P). (A) Time course of CtrA dephosphorylation by CckA-HK-RD constructs. Point mutations are indicated above the time courses. WT, wild type. (B) Quantification of CckA-HK-RD band intensities in time courses shown in panel A. The intensities for each construct were normalized to the percent maximum. (C) Quantification of inorganic phosphate band intensities in time courses shown in panel A. P_i, inorganic phosphate.

FIG. 5.

Complementation analysis of mutant alleles of cckA. Cellular morphology (left) and flow cytometry (right) analysis of strains in which the chromosomal copy of cckA was deleted and the cckA allele indicated on the far left was driven by the native cckA promoter on a low-copy-number plasmid. Scale bar, 4 μm; WT, wild type.

FIG. 6.

CckA contributes to but is not essential for the inactivation of CtrA and CpdR. Cells expressing various combinations of cckA and ctrA alleles were analyzed by flow cytometry to assess chromosomal content as a readout of CtrA activity. Each strain harbored a cckA chromosomal deletion and expressed either cckA (light gray) or cckA(V366P) (dark gray) from a low-copy-number plasmid using the native cckA promoter. Each strain expressed one of the ctrA alleles indicated above the panels from a medium-copy-number plasmid using the xylose-inducible promoter P_xyl. High levels of CtrA(D51E) partially mimic CtrA∼P. CtrAΔ3Ω is a nonproteolyzable version of CtrA. All strains were grown in M2G to mid-exponential phase (OD₆₀₀, ∼0.2 to 0.4) in the presence of xylose for 8 h, followed by the addition of rifampin for 3 additional hours, and then analyzed by flow cytometry. (A) Representative flow cytometry profiles. (B) Quantification of the percentage of cells with one chromosome in the flow cytometry profiles. Error bars represent the standard errors of the means (n = 3).

FIG. 7.

Overproducing CckA inactivates CtrA. (A) The effect of overproducing various CckA constructs was examined by light microscopy and flow cytometry. A diagram of the overexpression constructs used is shown at the top. Cells harbored the construct indicated above each micrograph and flow cytometry profile pair. Each construct was expressed from a xylose-inducible promoter on a medium-copy-number plasmid. Cultures were grown to mid-exponential phase (OD₆₀₀, ∼0.2 to 0.4) in the presence of xylose for 4 hours and then fixed for microscopy and flow cytometry analysis. (B) Genetic interactions between pleC and cckA. Cells harbored a transposon insertion in pleC and carried a full-length copy of cckA or cckA(V366P) under the control of a xylose-inducible promoter. Note that in the flow cytometry profiles, the far right edge of the profile includes an integration of all cells that have chromosome accumulation beyond the range shown, if any. Scale bar, 4 μm.

FIG. 8.

Regulation of the balance between CckA kinase and phosphatase activities controls the cell cycle. (A) Left, summary of regulatory pathway controlling CtrA activity. Right, schematic of Caulobacter cell cycle indicating temporal pattern of CtrA activity. (B) Summary of cell cycle phosphorelays. Net phosphate flow depends on the activity of CckA. As a kinase, CckA drives the phosphorylation of CtrA and CpdR. As a phosphatase, CckA drives the dephosphorylation of CtrA and CpdR. Cell cycle transitions and changes in CtrA activity are thus driven by changes in the kinase/phosphatase balance of CckA. DivK influences CckA's switching between kinase and phosphatase states. P_i, inorganic phosphate.